US20020025517A1 - Methods and compositions for cellular and metabolic engineering - Google Patents

Methods and compositions for cellular and metabolic engineering Download PDF

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US20020025517A1
US20020025517A1 US09/188,777 US18877798A US2002025517A1 US 20020025517 A1 US20020025517 A1 US 20020025517A1 US 18877798 A US18877798 A US 18877798A US 2002025517 A1 US2002025517 A1 US 2002025517A1
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Jeremy Minshull
Willem P. C. Stemmer
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Definitions

  • Metabolic engineering is the manipulation of intermediary metabolism through the use of both classical genetics and genetic engineering techniques.
  • Cellular engineering is generally a more inclusive term referring to the modification of cellular properties.
  • Cameron et al. ( Applied Biochem. Biotech. 38:105-140 (1993)) provide a summary of equivalent terms to describe this type of engineering, including “metabolic engineering”, which is most often used in the context of industrial microbiology and bioprocess engineering, “in vitro evolution” or “directed evolution”, most often used in the context of environmental microbiology, “molecular breeding”, most often used by Japanese researchers, “cellular engineering”, which is used to describe modifications of bacteria, animal, and plant cells, “rational strain development”, and “metabolic pathway evolution”.
  • the terms “metabolic engineering” and “cellular engineering” are used preferentially for clarity; the term “evolved” genes is used as discussed below.
  • Metabolic engineering can be divided into two basic categories: modification of genes endogenous to the host organism to alter metabolite flux and introduction of foreign genes into an organism. Such introduction can create new metabolic pathways leading to modified cell properties including but not limited to synthesis of known compounds not normally made by the host cell, production of novel compounds (e.g. polymers, antibiotics, etc.) and the ability to utilize new nutrient sources.
  • Specific applications of metabolic engineering can include the production of specialty and novel chemicals, including antibiotics, extension of the range of substrates used for growth and product formation, the production of new catabolic activities in an organism for toxic chemical degradation, and modification of cell properties such as resistance to salt and other environmental factors.
  • Bailey Science 252:1668-1674 (1991) describes the application of metabolic engineering to the recruitment of heterologous genes for the improvement of a strain, with the caveat that such introduction can result in new compounds that may subsequently undergo further reactions, or that expression of a heterologous protein can result in proteolysis, improper folding, improper modification, or unsuitable intracellular location of the protein, or lack of access to required substrates. Bailey recommends careful configuration of a desired genetic change with minimal perturbation of the host.
  • Liao Curr. Opin. Biotech. 4:211-216 (1993) reviews mathematical modelling and analysis of metabolic pathways, pointing out that in many cases the kinetic parameters of enzymes are unavailable or inaccurate.
  • Stephanopoulos et al. ( Trends. Biotechnol. 11:392-396 (1993)) describe attempts to improve productivity of cellular systems or effect radical alteration of the flux through primary metabolic pathways as having difficulty in that control architectures at key branch points have evolved to resist flux changes. They conclude that identification and characterization of these metabolic nodes is a prerequisite to rational metabolic engineering. Similarly, Stephanopoulos ( Curr. Opin. Biotech. 5:196-200 (1994)) concludes that rather than modifying the “rate limiting step” in metabolic engineering, it is necessary to systematically elucidate the control architecture of bioreaction networks.
  • the present invention is generally directed to the evolution of new metabolic pathways and the enhancement of bioprocessing through a process herein termed recursive sequence recombination.
  • Recursive sequence recombination entails performing iterative cycles of recombination and screening or selection to “evolve” individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes (Stemmer, Bio/Technology 13:549-553 (1995)). Such techniques do not require the extensive analysis and computation required by conventional methods for metabolic engineering.
  • Recursive sequence recombination allows the recombination of large numbers of mutations in a minimum number of selection cycles, in contrast to traditional, pairwise recombination events.
  • RSR recursive sequence recombination
  • One aspect of the invention is a method of evolving a biocatalytic activity of a cell, comprising:
  • Another aspect of the invention is a method of evolving a gene to confer ability to catalyze a reaction of interest, the method comprising:
  • a further aspect of the invention is a method of generating a new biocatalytic activity in a cell, comprising:
  • Another aspect of the invention is a modified form of a cell, wherein the modification comprises a metabolic pathway evolved by recursive sequence recombination.
  • a further aspect of the invention is a method of optimizing expression of a gene product, the method comprising:
  • a further aspect of the invention is a method of evolving a biosensor for a compound A of interest, the method comprising:
  • FIG. 1 is a drawing depicting a scheme for in vitro recursive sequence, recombination.
  • the invention provides a number of strategies for evolving metabolic and bioprocessing pathways through the technique of recursive sequence recombination.
  • One strategy entails evolving genes that confer the ability to use a particular substrate of interest as a nutrient source in one species to confer either more efficient use of that substrate in that species, or comparable or more efficient use of that substrate in a second species.
  • Another strategy entails evolving genes that confer the ability to detoxify a compound of interest in one or more species of organisms.
  • Another strategy entails evolving new metabolic pathways by evolving an enzyme or metabolic pathway for biosynthesis or degradation of a compound A related to a compound B for the ability to biosynthesize or degrade compound B, either in the host of origin or a new host.
  • a further strategy entails evolving a gene or metabolic pathway for more efficient or optimized expression of a particular metabolite or gene product.
  • a further strategy entails evolving a host/vector system for expression of a desired heterologous product. These strategies may involve using all the genes in a multi-step pathway, one or several genes, genes from different organisms, or one or more fragments of a gene.
  • the strategies generally entail evolution of gene(s) or segment(s) thereof to allow retention of function in a heterologous cell or improvement of function in a homologous or heterologous cell. Evolution is effected generally by a process termed recursive sequence recombination. Recursive sequence recombination can be achieved in many different formats and permutations of formats, as described in further detail below. These formats share some common principles. Recursive sequence recombination entails successive cycles of recombination to generate molecular diversity, i.e., the creation of a family of nucleic acid molecules showing substantial sequence identity to each other but differing in the presence of mutations.
  • Each recombination cycle is followed by at least one cycle of screening or selection for molecules having a desired characteristic.
  • the molecule(s) selected in one round form the starting materials for generating diversity in the next round.
  • recombination can occur in viva or in vitro.
  • diversity resulting from recombination can be augmented in any cycle by applying prior methods of mutagenesis (e.g., error-prone PCR or cassette mutagenesis, passage through bacterial mutator strains, treatment with chemical mutagens) to either the substrates for or products of recombination.
  • FIG. 1 One format for recursive sequence recombination in vitro is illustrated in FIG. 1.
  • the initial substrates for recombination are a pool of related sequences.
  • the X's in FIG. 1, panel A show where the sequences diverge.
  • the sequences can be DNA or RNA and can be of various lengths depending on the size of the gene or DNA fragment to be recombined or reassembled. Preferably the sequences are from 50 bp to 100 kb.
  • the pool of related substrates can be fragmented, usually at random, into fragments of from about 5 bp to 5 kb or more, as shown in FIG. 1, panel B.
  • the size of the random fragments is from about 10 bp to 1000 bp, more preferably the size of the DNA fragments is from about 20 bp to 500 bp.
  • the substrates can be digested by a number of different methods, such as DNAseI or RNAse digestion, random shearing or restriction enzyme digestion.
  • the concentration of nucleic acid fragments of a particular length or sequence is often less than 0.1% or 1% by weight of the total nucleic acid.
  • the number of different specific nucleic acid fragments in the mixture is usually at least about 100, 500 or 1000.
  • the mixed population of nucleic acid fragments are denatured by heating to about 80° C. to 100° C., more preferably from 90° C. to 96° C., to form single-stranded nucleic acid fragments and then reannealed.
  • Single-stranded nucleic acid fragments having regions of sequence identity with other single-stranded nucleic acid fragments can then be reannealed by cooling to 20° C. to 75° C., and preferably from 40° C. to 65° C. Renaturation can be accelerated by the addition of polyethylene glycol (“PEG”) or salt.
  • the salt concentration is preferably from 0 mM to 600 mM, more preferably the salt concentration is from 10 mM to 100 mM.
  • the salt may be such salts as (NH 4 ) 2 SO 4 , KCl, or NaCl.
  • concentration of PEG is preferably from 0% to 20%, more preferably from 5% to 10%.
  • the fragments that reanneal can be from different substrates as shown in FIG. 1, panel C.
  • the annealed nucleic acid fragments are incubated in the presence of a nucleic acid polymerase, such as Taq or Klenow, and dNTP's (i.e. DATP, dCTP, dGTP and dTTP). If regions of sequence identity are large, Iaq or other high-temperature polymerase can be used with an annealing temperature of between 45-65° C. If the areas of identity are small, Klenow or other low-temperature polymerases can be used with an annealing temperature of between 20-30° C.
  • the polymerase can be added to the random nucleic acid fragments prior to annealing, simultaneously with annealing or after annealing.
  • the cycle of denaturation, renaturation and incubation of random nucleic acid fragments in the presence of polymerase is sometimes referred to as “shuffling” of the nucleic acid in vitro.
  • This cycle is repeated for a desired number of times. Preferably the cycle is repeated from 2 to 100 times, more preferably the sequence is repeated from 10 to 40 times.
  • the resulting nucleic acids are a family of double-stranded polynucleotides of from about 50 bp to about 100 kb, preferably from 500 bp to 50 kb, as shown in FIG. 1, panel D.
  • the population represents variants of the starting substrates showing substantial sequence identity thereto but also diverging at several positions. The population has many more members than the starting substrates.
  • the population of fragments resulting from recombination is preferably first amplified by PCR, then cloned into an appropriate vector and the ligation mixture used to transform host cells.
  • subsequences of recombination substrates can be generated by amplifying the full-length sequences under conditions which produce a substantial fraction, typically at least 20 percent or more, of incompletely extended amplification products.
  • the amplification products, including the incompletely extended amplification products are denatured and subjected to at least one additional cycle of reannealing and amplification.
  • This variation wherein at least one cycle of reannealing and amplification provides a substantial fraction of incompletely extended products, is termed “stuttering.”
  • the incompletely extended products anneal to and prime extension on different sequence-related template species.
  • At least one cycle of amplification can be conducted using a collection of overlapping single-stranded DNA fragments of related sequence, and different lengths. Each fragment can hybridize to and prime polynucleotide chain extension of a second fragment from the collection, thus forming sequence-recombined polynucleotides.
  • single-stranded DNA fragments of variable length can be generated from a single primer by Vent DNA polymerase on a first DNA template. The single stranded DNA fragments are used as primers for a second, Kunkel-type template, consisting of a uracil-containing circular single-stranded DNA. This results in multiple substitutions of the first template into the second (see Levichkin et al. Mol. Biology 29:572-577 (1995)).
  • Gene clusters such as those involved in polyketide synthesis (or indeed any multi-enzyme pathways catalyzing analogous metabolic reactions) can be recombined by recursive sequence recombination even if they lack DNA sequence homology. Homology can be introduced using synthetic oligonucleotides as PCR primers. In addition to the specific sequences for the gene being amplified, all of the primers used to amplify one type of enzyme (for example the acyl carrier protein in polyketide synthesis) are synthesized to contain an additional sequence of 20-40 bases 5′ to the gene (sequence A) and a different 20-40 base sequence 3′ to the gene (sequence B).
  • the adjacent gene (in this case the keto-synthase) is amplified using a 5′ primer which contains the complementary strand of sequence B (sequence B′), and a 3′ primer containing a different 20-40 base sequence (C).
  • primers for the next adjacent gene contain sequences C′ (complementary to C) and D. If 5 different polyketide gene clusters are being shuffled, all five acyl carrier proteins are flanked by sequences A and B following their PCR amplification. In this way, small regions of homology are introduced, making the gene clusters into site-specific recombination cassettes.
  • the amplified genes can then be mixed and subjected to primerless PCR.
  • Sequence B at the 3′ end of all of the five acyl carrier protein genes can anneal with and prime DNA synthesis from sequence B′ at the 5′ end of all five keto reductase genes. In this way all possible combinations of genes within the cluster can be obtained.
  • Oligonucleotides allow such recombinants to be obtained in the absence of sufficient sequence homology for recursive sequence recombination described above. Only homology of function is required to produce functional gene clusters.
  • This method is also useful for exploring permutations of any other multi-subunit enzymes.
  • An example of such enzymes composed of multiple polypeptides that have shown novel functions when the subunits are combined in novel ways are dioxygenases. Directed recombination between the four protein subunits of biphenyl and toluene dioxygenases produced functional dioxygenases with increased activity against trichloroethylene (Furukawa et. al. J. Bacteriol. 176: 2121-2123 (1994)). This combination of subunits from the two dioxygenases could also have been produced by cassette-shuffling of the dioxygenases as described above, followed by selection for degradation of trichloroethylene.
  • the separate functions of the acyl carrier protein, keto-synthase, keto-reductase, etc. reside in a single polypeptide.
  • domains within the single polypeptide may be shuffled, even if sufficient homology does not exist naturally, by introducing regions of homology as described above for entire genes. In this case, it may not be possible to introduce additional flanking sequences to the domains, due to the constraint of maintaining a continuous open reading frame.
  • groups of oligonucleotides are synthesized that are homologous to the 3′ end of the first domain encoded by one of the genes to be shuffled, and the 5′ ends of the second domains encoded by all of the other genes to be shuffled together. This is repeated with all domains, thus providing sequences that allow recombination between protein domains while maintaining their order.
  • the cassette-based recombination method can be combined with recursive sequence recombination by including gene fragments (generated by DNase, physical shearing, DNA stuttering, etc.) for one or more of the genes.
  • gene fragments generated by DNase, physical shearing, DNA stuttering, etc.
  • individual genes can be shuffled at the same time (e.g., all acyl carrier protein genes can also be provided as fragmented DNA), allowing a more thorough search of sequence space.
  • the initial substrates for recombination are a collection of polynucleotides comprising variant forms of a gene.
  • the variant forms usually show substantial sequence identity to each other sufficient to allow homologous recombination between substrates.
  • the diversity between the polynucleotides can be natural (e.g., allelic or species variants), induced (e.g., error-prone PCR or error-prone recursive sequence recombination), or the result of in vitro recombination. Diversity can also result from resynthesizing genes encoding natural proteins with alternative codon usage. There should be at least sufficient diversity between substrates that recombination can generate more diverse products than there are starting materials.
  • the diverse substrates are incorporated into plasmids.
  • the plasmids are often standard cloning vectors, e.g., bacterial multicopy plasmids. However, in some methods to be described below, the plasmids include mobilization (MOB) functions.
  • the substrates can be incorporated into the same or different plasmids.
  • plasmids having different types of selectable markers are used to allow selection for cells containing at least two types of vector.
  • the different plasmids can come from two distinct incompatibility groups to allow stable co-existence of two different plasmids within the cell. Nevertheless, plasmids from the same incompatibility group can still co-exist within the same cell for sufficient time to allow homologous recombination to occur.
  • Plasmids containing diverse substrates are initially introduced into cells by any method (e.g., chemical transformation, natural competence, electroporation, biolistics, packaging into phage or viral systems). Often, the plasmids are present at or near saturating concentration (with respect to maximum transfection capacity) to increase the probability of more than one plasmid entering the same cell.
  • the plasmids containing the various substrates can be transfected simultaneously or in multiple rounds. For example, in the latter approach cells can be transfected with a first aliquot of plasmid, transfectants selected and propagated, and then infected with a second aliquot of plasmid.
  • the conditions for electroporation are the same as those conventionally used for introducing exogenous DNA into cells (e.g., 1,000-2,500 volts, 400 ⁇ F and a 1-2 mM gap). Under these conditions, plasmids are exchanged between cells allowing all substrates to participate in recombination. In addition the products of recombination can undergo further rounds of recombination with each other or with the original substrate. The rate of evolution can also be increased by use of conjugative transfer. To exploit conjugative transfer, substrates can be cloned into plasmids having MOB genes, and tra genes are also provided in cis or in trans to the MOB genes.
  • the effect of conjugative transfer is very similar to electroporation in that it allows plasmids to move between cells and allows recombination between any substrate and the products of previous recombination to occur, merely by propagating the culture.
  • the rate of evolution can also be increased by fusing cells to induce exchange of plasmids or chromosomes. Fusion can be induced by chemical agents, such as PEG, or viral proteins, such as influenza virus hemagglutinin, HSV-1 gB and gD.
  • the rate of evolution can also be increased by use of mutator host cells (e.g., Mut L, S, D, T, H in bacteria and Ataxia telangiectasia human cell lines).
  • the time for which cells are propagated and recombination is allowed to occur varies with the cell type but is generally not critical, because even a small degree of recombination can substantially increase diversity relative to the starting materials.
  • Cells bearing plasmids containing recombined genes are subject to screening or selection for a desired function. For example, if the substrate being evolved contains a drug resistance gene, one would select for drug resistance. Cells surviving screening or selection can be subjected to one or more rounds of screening/selection followed by recombination or can be subjected directly to an additional round of recombination.
  • next round of recombination can be achieved by several different formats independently of the previous round.
  • a further round of recombination can be effected simply by resuming the electroporation or conjugation-mediated intercellular transfer of plasmids described above.
  • a fresh substrate or substrates the same or different from previous substrates, can be transfected into cells surviving selection/screening.
  • the new substrates are included in plasmid vectors bearing a different selective marker and/or from a different incompatibility group than the original plasmids.
  • cells surviving selection/screening can be subdivided into two subpopulations, and plasmid DNA from one subpopulation transfected into the other, where the substrates from the plasmids from the two subpopulations undergo a further round of recombination.
  • the rate of evolution can be increased by employing DNA extraction, electroporation, conjugation or mutator cells, as described above.
  • DNA from cells surviving screening/selection can be extracted and subjected to in vitro recursive sequence recombination.
  • a second round of screening/selection is performed, preferably under conditions of increased stringency. If desired, further rounds of recombination and selection/screening can be performed using the same strategy as for the second round. With successive rounds of recombination and selection/screening, the surviving recombined substrates evolve toward acquisition of a desired phenotype.
  • the final product of recombination that has acquired the desired phenotype differs from starting substrates at 0.1%-25% of positions and has evolved at a rate orders of magnitude in excess (e.g., by at least 10-fold, 100-fold, 1000-fold, or 10,000 fold) of the rate of naturally acquired mutation of about 1 mutation per 10 ⁇ 9 positions per generation (see Anderson et al. Proc. Natl. Acad. Sci. U.S.A. 93:906-907 (1996)).
  • the final products may be transferred to another host more desirable for utilization of the “shuffled” DNA. This is particularly advantageous in situations where the more desirable host is less efficient as a host for the many cycles of mutation/recombination due to the lack of molecular biology or genetic tools available for other organisms such as E. coli.
  • the strategy used for plasmid-plasmid recombination can also be used for virus-plasmid recombination; usually, phage-plasmid recombination.
  • virus-plasmid recombination usually, phage-plasmid recombination.
  • the initial substrates for recombination are cloned into both plasmid and viral vectors. It is usually not critical which substrate(s) are inserted into the viral vector and which into the plasmid, although usually the viral vector should contain different substrate(s) from the plasmid.
  • the plasmid (and the virus) typically contains a selective marker.
  • the plasmid and viral vectors can both be introduced into cells by transfection as described above.
  • Homologous recombination occurs between plasmid and virus generating both recombined plasmids and recombined virus.
  • viruses such as filamentous phage, in which intracellular DNA exists in both double-stranded and single-stranded forms, both can participate in recombination.
  • recombination can be augmented by use of electroporation or conjugation to transfer plasmids between cells.
  • Recombination can also be augmented for some types of virus by allowing the progeny virus from one cell to reinfect other cells.
  • virus infected-cells show resistance to superinfection. However, such resistance can be overcome by infecting at high multiplicity and/or using mutant strains of the virus in which resistance to superinfection is reduced.
  • viruses such as filamentous phage, stably exist with a plasmid in the cell and also extrude progeny phage from the cell.
  • Other viruses such as lambda having a cosmid genome, stably exist in a cell like plasmids without producing progeny virions.
  • Other viruses such as the T-phage and lytic lambda, undergo recombination with the plasmid but ultimately kill the host cell and destroy plasmid DNA.
  • cells containing recombinant plasmids and virus can be screened/selected using the same approach as for plasmid-plasmid recombination. Progeny virus extruded by cells surviving selection/screening can also be collected and used as substrates in subsequent rounds of recombination. For viruses that kill their host cells, recombinant genes resulting from recombination reside only in the progeny virus. If the screening or selective assay requires expression of recombinant genes in a cell, the recombinant genes should be transferred from the progeny virus to another vector, e.g., a plasmid vector, and retransfected into cells before selection/screening is performed.
  • another vector e.g., a plasmid vector
  • filamentous phage the products of recombination are present in both cells surviving recombination and in phage extruded from these cells.
  • the dual source of recombinant products provides some additional options relative to the plasmid-plasmid recombination.
  • DNA can be isolated from phage particles for use in a round of in vitro recombination.
  • the progeny phage can be used to transfect or infect cells surviving a previous round of screening/selection, or fresh cells transfected with fresh substrates for recombination.
  • the principles described for plasmid-plasmid and plasmid-viral recombination can be applied to virus-virus recombination with a few modifications.
  • the initial substrates for recombination are cloned into a viral vector.
  • the same vector is used for all substrates.
  • the virus is one that, naturally or as a result of mutation, does not kill cells.
  • some viral genomes can be packaged in vitro or using a packaging cell line. The packaged viruses are used to infect cells at high multiplicity such that there is a high probability that a cell will receive multiple viruses bearing different substrates.
  • the viruses After the initial round of infection, subsequent steps depend on the nature of infection as discussed in the previous section. For example, if the viruses have phagemid genomes such as lambda cosmids or M13, F1 or Fd phagemids, the phagemids behave as plasmids within the cell and undergo recombination simply by propagating the cells. Recombination is particularly efficient between single-stranded forms of intracellular DNA. Recombination can be augmented by electroporation of cells.
  • phagemid genomes such as lambda cosmids or M13, F1 or Fd phagemids
  • cosmids containing recombinant genes can be recovered from surviving cells, e.g., by heat induction of a cos ⁇ lysogenic host cell, or extraction of DNA by standard procedures, followed by repackaging cosmid DNA in vitro.
  • viruses are filamentous phage
  • recombination of replicating form DNA occurs by propagating the culture of infected cells. Selection/screening identifies colonies of cells containing viral vectors having recombinant genes with improved properties, together with phage extruded from such cells. Subsequent options are essentially the same as for plasmid-viral recombination.
  • This format can be used to especially evolve chromosomal substrates.
  • the format is particularly useful in situations in which many chromosomal genes contribute to a phenotype or one does not know the exact location of the chromosomal gene(s) to be evolved.
  • the initial substrates for recombination are cloned into a plasmid vector. If the chromosomal gene(s) to be evolved are known, the substrates constitute a family of sequences showing a high degree of sequence identity but some divergence from the chromosomal gene. If the chromosomal genes to be evolved have not been located, the initial substrates usually constitute a library of DNA segments of which only a small number show sequence identity to the gene or gene(s) to be evolved. Divergence between plasmid-borne substrate and the chromosomal gene(s) can be induced by mutagenesis or by obtaining the plasmid-borne substrates from a different species than that of the cells bearing the chromosome.
  • the plasmids bearing substrates for recombination are transfected into cells having chromosomal gene(s) to be evolved. Evolution can occur simply by propagating the culture, and can be accelerated by transferring plasmids between cells by conjugation or electroporation. Evolution can be further accelerated by use of mutator host cells or by seeding a culture of nonmutator host cells being evolved with mutator host cells and inducing intercellular transfer of plasmids by electroporation or conjugation.
  • mutator host cells used for seeding contain a negative selectable marker to facilitate isolation of a pure culture of the nonmutator cells being evolved. Selection/screening identifies cells bearing chromosomes and/or plasmids that have evolved toward acquisition of a desired function.
  • Subsequent rounds of recombination and selection/screening proceed in similar fashion to those described for plasmid-plasmid recombination.
  • further recombination can be effected by propagating cells surviving recombination in combination with electroporation or conjugative transfer of plasmids.
  • plasmids bearing additional substrates for recombination can be introduced into the surviving cells.
  • such plasmids are from a different incompatibility group and bear a different selective marker than the original plasmids to allow selection for cells containing at least two different plasmids.
  • plasmid and/or chromosomal DNA can be isolated from a subpopulation of surviving cells and transfected into a second subpopulation. Chromosomal DNA can be cloned into a plasmid vector before transfection.
  • the virus is usually one that does not kill the cells, and is often a phage or phagemid.
  • the procedure is substantially the same as for plasmid-chromosome recombination.
  • Substrates for recombination are cloned into the vector.
  • Vectors including the substrates can then be transfected into cells or in vitro packaged and introduced into cells by infection.
  • Viral genomes recombine with host chromosomes merely by propagating a culture. Evolution can be accelerated by allowing intercellular transfer of viral genomes by electroporation, or reinfection of cells by progeny virions. Screening/selection identifies cells having chromosomes and/or viral genomes that have evolved toward acquisition of a desired function.
  • viral genomes can be transferred between cells surviving selection/recombination by electroporation.
  • viruses extruded from cells surviving selection/screening can be pooled and used to superinfect the cells at high multiplicity.
  • fresh substrates for recombination can be introduced into the cells, either on plasmid or viral vectors.
  • a general method for recursive sequence recombination for the embodiments herein is to begin with a gene encoding an enzyme or enzyme subunit and to evolve that gene either for ability to act on a new substrate, or for enhanced catalytic properties with an old substrate, either alone or in combination with other genes in a multistep pathway.
  • the term “gene” is used herein broadly to refer to any segment or sequence of DNA associated with a biological function. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
  • the ability to use a new substrate can be assayed in some instances by the ability to grow on a substrate as a nutrient source. In other circumstances such ability can be assayed by decreased toxicity of a substrate for a host cell, hence allowing the host to grow in the presence of that substrate.
  • Biosynthesis of new compounds, such as antibiotics can be assayed similarly by growth of an indicator organism in the presence of the host expressing the evolved genes. For example, when an indicator organism used in an overlay of the host expressing the evolved gene(s), wherein the indicator organism is sensitive or expected to be sensitive to the desired antibiotic, growth of the indicator organism would be inhibited in a zone around the host cell or colony expressing the evolved gene(s).
  • Another method of identifying new compounds is the use of standard analytical techniques such as mass spectroscopy, nuclear magnetic resonance, high performance liquid chromatography, etc. Recombinant microorganisms can be pooled and extracts or media supernatants assayed from these pools. Any positive pool can then be subdivided and the procedure repeated until the single positive is identified (“sib-selection”).
  • the starting material for recursive sequence recombination is a discrete gene, cluster of genes, or family of genes known or thought to be associated with metabolism of a particular class of substrates.
  • One of the advantages of the instant invention is that structural information is not required to estimate which parts of a sequence should be mutated to produce a functional hybrid enzyme.
  • an initial screening of enzyme activities in a particular assay can be useful in identifying candidate enzymes as starting materials.
  • high throughput screening can be used to screen enzymes for dioxygenase-type activities using aromatic acids as substrates.
  • Dioxygenases typically transform indole-2-carboxylate and indole-3-carboxylate to colored products, including indigo (Eaton et. al. J. Bacteriol. 177:6983-6988 (1995)).
  • DNA encoding enzymes that give some activity in the initial assay can then be recombined by the recursive techniques of the invention and rescreened.
  • the use of such initial screening for candidate enzymes against a desired target molecule or analog of the target molecule can be especially useful to generate enzymes that catalyze reactions of interest such as catabolism of man-made pollutants.
  • the starting material can also be a segment of such a gene or cluster that is recombined in isolation of its surrounding DNA, but is relinked to its surrounding DNA before screening/selection of recombination products.
  • the starting material for recombination is a larger segment of DNA that includes a coding sequence or other locus associated with metabolism of a particular substrate at an unknown location.
  • the starting material can be a chromosome, episome, YAC, cosmid, or phage P1 clone.
  • the starting material is the whole genome of an organism that is known to have desirable metabolic properties, but for which no information localizing the genes associated with these characteristics is available.
  • Cells of particular interest include many bacterial cell types, both gram-negative and gram-positive, such as Rhodococcus, Streptomycetes, Actinomycetes, Corynebacteria, Penicillium, Bacillus, Escherichia coli , Pseudomonas, Salmonella, and Erwinia.
  • Cells of interest also include eukaryotic cells, particularly mammalian cells (e.g., mouse, hamster, primate, human), both cell lines and primary cultures.
  • Such cells include stem cells, including embryonic stem cells, zygotes, fibroblasts, lymphocytes, Chinese hamster ovary (CHO), mouse fibroblasts (NIH3T3), kidney, liver, muscle, and skin cells.
  • Other eukaryotic cells of interest include plant cells, such as maize, rice, wheat, cotton, soybean, sugarcane, tobacco, and arabidopsis; fish, algae, fungi (Penicillium, Fusarium, Aspergillus, Podospora, Neurospora), insects, yeasts (Picchia and Saccharomyces).
  • the choice of host will depend on a number of factors, depending on the intended use of the engineered host, including pathogenicity, substrate range, environmental hardiness, presence of key intermediates, ease of genetic manipulation, and likelihood of promiscuous transfer of genetic information to other organisms.
  • Particularly advantageous hosts are E. coli , lactobacilli, Streptomycetes, Actinomycetes and filamentous fungi.
  • the breeding procedure starts with at least two substrates, which generally show substantial sequence identity to each other (i.e., at least about 50%, 70%, 80% or 90% sequence identity) but differ from each other at certain positions.
  • the difference can be any type of mutation, for example, substitutions, insertions and deletions.
  • different segments differ from each other in perhaps 5-20 positions.
  • the starting materials must differ from each other in at least two nucleotide positions. That is, if there are only two substrates, there should be at least two divergent positions. If there are three substrates, for example, one substrate can differ from the second as a single position, and the second can differ from the third at a different single position.
  • the starting DNA segments can be natural variants of each other, for example, allelic or species variants.
  • the segments can also be from nonallelic genes showing some degree of structural and usually functional relatedness (e.g., different genes within a superfamily such as the immunoglobulin superfamily).
  • the starting DNA segments can also be induced variants of each other.
  • one DNA segment can be produced by error-prone PCR replication of the other, or by substitution of a mutagenic cassette. Induced mutants can also be prepared by propagating one (or both) of the segments in a mutagenic strain.
  • the second DNA segment is not a single segment but a large family of related segments.
  • the different segments forming the starting materials are often the same length or substantially the same length. However, this need not be the case; for example; one segment can be a subsequence of another.
  • the segments can be present as part of larger molecules, such as vectors, or can be in isolated form.
  • the starting DNA segments are recombined by any of the recursive sequence recombination formats described above to generate a diverse library of recombinant DNA segments.
  • a library can vary widely in size from having fewer than 10 to more than 10 5 , 10 7 , or 10 9 members.
  • the starting segments and the recombinant libraries generated include full-length coding sequences and any essential regulatory sequences, such as a promoter and polyadenylation sequence, required for expression. However, if this is not the case, the recombinant DNA segments in the library can be inserted into a common vector providing the missing sequences before performing screening/selection.
  • the library of recombinant DNA segments generated already exists in a cell which is usually the cell type in which expression of the enzyme with altered substrate specificity is desired. If recursive sequence recombination is performed in vitro, the recombinant library is preferably introduced into the desired cell type before screening/selection. The members of the recombinant library can be linked to an episome or virus before introduction or can be introduced directly. In some embodiments of the invention, the library is amplified in a first host, and is then recovered from that host and introduced to a second host more amenable to expression, selection, or screening, or any other desirable parameter.
  • the manner in which the library is introduced into the cell type depends on the DNA-uptake characteristics of the cell type, e.g., having viral receptors, being capable of conjugation, or being naturally competent. If the cell type is insusceptible to natural and chemical-induced competence, but susceptible to electroporation, one would usually employ electroporation. If the cell type is insusceptible to electroporation as well, one can employ biolistics.
  • the biolistic PDS-1000 Gene Gun uses helium pressure to accelerate DNA-coated gold or tungsten microcarriers toward target cells. The process is applicable to a wide range of tissues, including plants, bacteria, fungi, algae, intact animal tissues, tissue culture cells, and animal embryos.
  • Screening is, in general, a two-step process in which one first determines which cells do and do not express a screening marker and then physically separates the cells having the desired property. Selection is a form of screening in which identification and physical separation are achieved simultaneously, for example, by expression of a selectable marker, which, in some genetic circumstances, allows cells expressing the marker to survive while other cells die (or vice versa). Screening markers include, for example, luciferase, ⁇ -galactosidase, and green fluorescent protein. Screening can also be done by observing such aspects of growth as colony size, halo formation, etc.
  • screening for production of a desired compound can be accomplished by observing binding of cell products to a receptor or ligand, such as on a solid support or on a column. Such screening can additionally be accomplished by binding to antibodies, as in an ELISA.
  • the screening process is preferably automated so as to allow screening of suitable numbers of colonies or cells.
  • automated screening devices include fluorescence activated cell sorting, especially in conjunction with cells immobilized in agarose (see Powell et. al. Bio/Technology 8:333-337 (1990); Weaver et. al. Methods 2:234-247 (1991)), automated ELISA assays, etc.
  • Selectable markers can include, for example, drug, toxin resistance, or nutrient synthesis genes. Selection is also done by such techniques as growth on a toxic substrate to select for hosts having the ability to detoxify a substrate, growth on a new nutrient source to select for hosts having the ability to utilize that nutrient source, competitive growth in culture based on ability to utilize a nutrient source, etc.
  • uncloned but differentially expressed proteins can be screened by differential display (Appleyard et al. Mol. Gen. Gent. 247:338-342 (1995)). Hopwood ( Phil Trans R. Soc. Lond B 324:549-562) provides a review of screens for antibiotic production.
  • Omura Microbio. Rev. 50:259-279 (1986) and Nisbet ( Ann Rep. Med. Chem.
  • Hydrolytic enzymes e.g., proteases, amylases
  • Hydrolytic enzymes can be screened by including the substrate in an agar plate and scoring for a hydrolytic clear zone or by using a calorimetric indicator (Steele et al. Ann. Rev. Microbiol. 45:89-106 (1991)). This can be coupled with the use of stains to detect the effects of enzyme action (such as congo red to detect the extent of degradation of celluloses and hemicelluloses).
  • Tagged substrates can also be used.
  • lipases and esterases can be screened using different lengths of fatty acids linked to umbelliferyl. The action of lipases or esterases removes this tag from the fatty acid, resulting in a quenching of umbelliferyl fluorescence. These enzymes can be screened in microtiter plates by a robotic device.
  • Fluorescence activated cell sorting methods are also a powerful tool for selection/screening.
  • a fluorescent molecule is made within a cell (e.g., green fluorescent protein).
  • the cells producing the protein can simply be sorted by FACS.
  • Gel microdrop technology allows screening of cells encapsulated in agarose microdrops (Weaver et al. Methods 2:234-247 (1991)).
  • products secreted by the cell such as antibodies or antigens
  • Sorting and collection of the drops containing the desired product thus also collects the cells that made the product, and provides a ready source for the cloning of the genes encoding the desired functions.
  • Desired products can be detected by incubating the encapsulated cells with fluorescent antibodies (Powell et al. Bio/Technology 8:333-337 (1990)). FACS sorting can also be used by this technique to assay resistance to toxic compounds and antibiotics by selecting droplets that contain multiple cells (i.e., the product of continued division in the presence of a cytotoxic compound; Goguen et al. Nature 363:189-190 (1995)). This method can select for any enzyme that can change the fluorescence of a substrate that can be immobilized in the agarose droplet.
  • screening can be accomplished by assaying reactivity with a reporter molecule reactive with a desired feature of, for example, a gene product.
  • a reporter molecule reactive with a desired feature of, for example, a gene product e.g., antibodies to those determinants.
  • screening is preferably done with a cell-cell indicator assay.
  • assay format separate library cells (Cell A, the cell being assayed) and reporter cells (Cell B, the assay cell) are used. Only one component of the system, the library cells, is allowed to evolve.
  • the screening is generally carried out in a two-dimensional immobilized format, such as on plates.
  • the products of the metabolic pathways encoded by these genes diffuse out of the library cell to the reporter cell.
  • the product of the library cell may affect the reporter cell in one of a number of ways.
  • the assay system can have a simple readout (e.g., green fluorescent protein, luciferase, ⁇ -galactosidase) which is induced by the library cell product but which does not affect the library cell.
  • a simple readout e.g., green fluorescent protein, luciferase, ⁇ -galactosidase
  • the desired product can be detected by calorimetric changes in the reporter cells adjacent to the library cell.
  • indicator cells can in turn produce something that modifies the growth rate of the library cells via a feedback mechanism.
  • Growth rate feedback can detect and accumulate very small differences. For example, if the library and reporter cells are competing for nutrients, library cells producing compounds to inhibit the growth of the reporter cells will have more available nutrients, and thus will have more opportunity for growth. This is a useful screen for antibiotics or a library of polyketide synthesis gene clusters where each of the library cells is expressing and exporting a different polyketide gene product.
  • the reporter cell for an antibiotic selection can itself secrete a toxin or antibiotic that inhibits growth of the library cell. Production by the library cell of an antibiotic that is able to suppress growth of the reporter cell will thus allow uninhibited growth of the library cell.
  • the library cell may supply nutrients such as amino acids to an auxotrophic reporter, or growth factors to a growth-factor-dependent reporter.
  • the reporter cell in turn should produce a compound that stimulates the growth of the library cell. Interleukins, growth factors, and nutrients are possibilities.
  • markers can be added to DNA constructs used for recursive sequence recombination to make the microorganism dependent on the constructs during the improvement process, even though those markers may be undesirable in the final recombinant microorganism.
  • Evnin et al. Proc. Natl. Acad. Sci. U.S.A. 87:6659-6663 (1990) selected trypsin variants with altered substrate specificity by requiring that variant trypsin generate an essential amino acid for an arginine auxotroph by cleaving arginine ⁇ -naphthylamide. This is thus a selection for arginine-specific trypsin, with the growth rate of the host being proportional to that of the enzyme activity.
  • the pool of cells surviving screening and/or selection is enriched for recombinant genes conferring the desired phenotype (e.g. altered substrate specificity, altered biosynthetic ability, etc.). Further enrichment can be obtained, if desired, by performing a second round of screening and/or selection without generating additional diversity.
  • desired phenotype e.g. altered substrate specificity, altered biosynthetic ability, etc.
  • the recombinant gene or pool of such genes surviving one round of screening/selection forms one or more of the substrates for a second round of recombination.
  • recombination can be performed in vivo or in vitro by any of the recursive sequence recombination formats described above. If recursive sequence recombination is performed in vitro, the recombinant gene or genes to form the substrate for recombination should be extracted from the cells in which screening/selection was performed. Optionally, a subsequence of such gene or genes can be excised for more targeted subsequent recombination. If the recombinant gene(s) are contained within episomes, their isolation presents no difficulties.
  • the recombinant genes are chromosomally integrated, they can be isolated by amplification primed from known sequences flanking the regions in which recombination has occurred.
  • whole genomic DNA can be isolated, optionally amplified, and used as the substrate for recombination.
  • Small samples of genomic DNA can be amplified by whole genome amplification with degenerate primers (Barrett et al. Nucleic Acids Research 23:3488-3492 (1995)). These primers result in a large amount of random 3′ ends, which can undergo homologous recombination when reintroduced into cells.
  • the second round of recombination is to be performed in vivo, as is often the case, it can be performed in the cell surviving screening/selection, or the recombinant genes can be transferred to another cell type (e.g., a cell type having a high frequency of mutation and/or recombination).
  • recombination can be effected by introducing additional DNA segment(s) into cells bearing the recombinant genes.
  • the cells can be induced to exchange genetic information with each other by, for example, electroporation.
  • the second round of recombination is performed by dividing a pool of. cells surviving screening/selection in the first round into two subpopulations.
  • DNA from one subpopulation is isolated and transfected into the other population, where the recombinant gene(s) from the two subpopulations recombine to form a further library of recombinant genes.
  • the second round of recombination is sometimes performed exclusively among the recombinant molecules surviving selection.
  • additional substrates can be introduced.
  • the additional substrates can be of the same form as the substrates used in the first round of recombination, i.e., additional natural or induced mutants of the gene or cluster of genes, forming the substrates for the first round.
  • the additional substrate(s) in the second round of recombination can be exactly the same as the substrate(s) in the first round of replication.
  • recombinant genes conferring the desired phenotype are again selected.
  • the selection process proceeds essentially as before. If a suicide vector bearing a selective marker was used in the first round of selection, the same vector can be used again. Again, a cell or pool of cells surviving selection is selected. If a pool of cells, the cells can be subject to further enrichment.
  • bioremediation is an aspect of pollution control
  • a more useful approach in the long term is one of prevention before industrial waste is pumped into the environment.
  • Exposure of industrial waste streams to recursive sequence recombination-generated microorganisms capable of degrading the pollutants they contain would result in detoxification of mineralization of these pollutants before the waste stream enters the environment. Issues of releasing recombinant organisms can be avoided by containing them within bioreactors fitted to the industrial effluent pipes.
  • This approach would also allow the microbial mixture used to be adjusted to best degrade the particular wastes being produced.
  • this method would avoid the problems of adapting to the outside world and dealing with competition that face many laboratory microorganisms.
  • a recursive sequence recombination approach overcomes a number of limitations in the bioremediation capabilities of naturally occurring microorganisms. Both enzyme activity and specificity can be altered, simultaneously or sequentially, by the methods of the invention. For example, catabolic enzymes can be evolved to increase the rate at which they act on a substrate. Although knowledge of a rate-limiting step in a metabolic pathway is not required to practice the invention, rate-limiting proteins in pathways can be evolved to have increased expression and/or activity, the requirement for inducing substances can be eliminated, and enzymes can be evolved that catalyze novel reactions.
  • Some examples of chemical targets for bioremediation include but are not limited to benzene, xylene, and toluene, camphor, naphthalene, halogenated hydrocarbons, polychlorinated biphenyls (PCBs), trichlorethylene, pesticides such as pentachlorophenyls (PCPs), and herbicides such as atrazine.
  • PCBs polychlorinated biphenyls
  • PCPs pentachlorophenyls
  • herbicides such as atrazine.
  • an enzyme when “evolved” to have a new catalytic function, that function is expressed, either constitutively or in response to the new substrate.
  • Recursive sequence recombination subjects both structural and regulatory elements (including the structure of regulatory proteins) of a protein to recombinogenic mutagenesis simultaneously. Selection of mutants that are efficiently able to use the new substrate as a nutrient source will be sufficient to ensure that both the enzyme and its regulation are optimized, without detailed analysis of either protein structure or operon regulation.
  • aromatic hydrocarbons include but are not limited to benzene, xylene, toluene, biphenyl, and polycyclic aromatic hydrocarbons such as pyrene and naphthalene. These compounds are metabolized via catechol intermediates. Degradation of catechol by Pseudomonas putida requires induction of the catabolic operon by cis, cis-muconate which acts on the CatR regulatory protein. The binding site for the CatR protein is G-N 11 -A, while the optimal sequence for the LysR class of activators (of which CatR is a member) is T-N 11 -A.
  • dioxygenases are required for many pathways in which aromatic compounds are catabolized. Even small differences in dioxygenase sequence can lead to significant differences in substrate specificity (Furukawa et al. J. Bact. 175:5224-5232 (1993); Erickson et al. App. Environ. Micro. 59:3858-3862 (1993)).
  • a hybrid enzyme made using sequences derived from two “parental” enzymes may possess catalytic activities that are intermediate between the parents (Erickson, ibid.), or may actually be better than either parent for a specific reaction (Furukawa et al. J. Bact. 176:2121-2123 (1994)).
  • site directed mutagenesis was used to generate a single polypeptide with hybrid sequence (Erickson, ibid.); in the other, a four subunit enzyme was produced by expressing two subunits from each of two different dioxygenases (Furukawa, ibid.).
  • sequences from one or more genes encoding dioxygenases can be used in the recursive sequence recombination techniques of the instant invention, to generate enzymes with new specificities.
  • other features of the catabolic pathway can also be evolved using these techniques, simultaneously or sequentially, to optimize the metabolic pathway for an activity of interest.
  • halogenated hydrocarbons are produced annually for uses as solvents and biocides. These include, in the United States alone, over 5 million tons of both 1,2-dichloroethane and vinyl chloride used in PVC production in the U.S. alone.
  • the compounds are largely not biodegradable by processes in single organisms, although in principle haloaromatic catabolic pathways can be constructed by combining genes from different microorganisms.
  • Enzymes can be manipulated to change their substrate specificities. Recursive sequence recombination offers the possibility of tailoring enzyme specificity to new substrates without needing detailed structural analysis of the enzymes.
  • Wackett et al. ( Nature 368:627-629 (1994)) recently demonstrated that through classical techniques a recombinant Pseudomonas strain in which seven genes encoding two multi-component oxygenases are combined, generated a single host that can metabolize polyhalogenated compounds by sequential reductive and oxidative techniques to yield non-toxic products.
  • These and/or related materials can be subjected to the techniques discussed above so as to evolve and optimize a biodegradative pathway in a single organism.
  • Trichloroethylene is a significant groundwater contaminant. It is degraded by microorganisms in a cometabolic way (i.e., no energy or nutrients are derived). The enzyme must be induced by a different compound (e.g., Pseudomonas cepacia uses toluene-4-monoxygenase, which requires induction by toluene, to destroy trichloroethylene). Furthermore, the degradation pathway involves formation of highly reactive epoxides that can inactivate the enzyme (Timmis et al. Ann. Rev. Microbiol. 48:525-557 (1994)).
  • the recursive sequence recombination techniques of the invention could be used to mutate the enzyme and its regulatory region such that it is produced constitutively, and is less susceptible to epoxide inactivation.
  • selection of hosts constitutively producing the enzyme and less susceptible to the epoxides can be accomplished by demanding growth in the presence of increasing concentrations of trichloroethylene in the absence of inducing substances.
  • PCBs Polychlorinated Biphenyls
  • PAHs Polycyclic Aromatic Hydrocarbons
  • PCBs and PAHs are families of structurally related compounds that are major pollutants at many Superfund sites. Bacteria transformed with plasmids encoding enzymes with broader substrate specificity have been used commercially. In nature, no known pathways have been generated in a single host that degrade the larger PAHs or more heavily chlorinated PCBs. Indeed, often the collaboration of anaerobic and aerobic bacteria are required for complete metabolism.
  • likely sources for starting material for recursive sequence recombination include identified genes encoding PAH-degrading catabolic pathways on large (20-100 KB) plasmids (Sanseverino et al. Applied Environ. Micro. 59:1931-1937 (1993); Simon et al. Gene 127:31-37 (1993); Zylstra et al. Annals of the NY Acad. Sci. 721:386-398 (1994)); while biphenyl and PCB-metabolizing enzymes are encoded by chromosomal gene clusters, and in a number of cases have been cloned onto plasmids (Hayase et al. J. Bacteriol.
  • Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine] is a moderately persistent herbicide which is frequently detected in ground and surface water at concentrations exceeding the 3 ppb health advisory level set by the EPA. Atrazine can be slowly metabolized by a Pseudomonas species (Mandelbaum et al. Appl. Environ. Micro. 61:1451-1457 (1995)).
  • the enzymes catalyzing the first two steps in atrazine metabolism by Pseudomonas are encoded by genes AtzA and AtzB (de Souza et al. Appl. Environ. Micro. 61:3373-3378 (1995)). These genes have been cloned in a 6.8 kb fragment into pUC18 (AtzAB-pUC). E. coli carrying this plasmid converts atrazine to much more soluble metabolites. It is thus possible to screen for enzyme activity by growing bacteria on plates containing atrazine. The herbicide forms an opaque precipitate in the plates, but cells containing AtzAB-pU18 secrete atrazine degrading enzymes, leading to a clear halo around those cells or colonies.
  • the size of the halo and the rate of its formation can be used to assess the level of activity so that picking colonies with the largest halos allows selection of the more active or highly produced atrazine degrading enzymes.
  • the plasmids carrying these genes can be subjected to the recursive sequence recombination formats described above to optimize the catabolism of atrazine in E. coli or another host of choice, including Pseudomonas. After each round of recombination, screening of host colonies expressing the evolved genes can be done on agar plates containing atrazine to observe halo formation. This is a generally applicable method for screening enzymes that metabolize insoluble compounds to those that are soluble (e.g., polycyclicaromatic hydrocarbons).
  • catabolism of atrazine can provide a source of nitrogen for the cell; if no other nitrogen is available, cell growth will be limited by the rate at which the cells can catabolize nitrogen. Cells able to utilize atrazine as a nitrogen source can thus be selected from a background of non-utilizers or poor-utilizers.
  • Bacteria are used commercially to detoxify arsenate waste generated by the mining of arsenopyrite gold ores. As well as mining effluent, industrial waste water is often contaminated with heavy metals (e.g., those used in the manufacture of electronic components and plastics). Thus, simply to be able to perform other bioremedial functions, microorganisms must be resistant to the levels of heavy metals present, including mercury, arsenate, chromate, cadmium, silver, etc.
  • a strong selective pressure is the ability to metabolize a toxic compound to one less toxic.
  • Heavy metals are toxic largely by virtue of their ability to denature proteins (Ford et al. Bioextraction and Biodeterioration of Metals , p. 1-23).
  • Detoxification of heavy metal contamination can be effected in a number of ways including changing the solubility or bioavailability of the metal, changing its redox state (e.g. toxic mercuric chloride is detoxified by reduction to the much more volatile elemental mercury) and even by bioaccumulation of the metal by immobilized bacteria or plants.
  • the accumulation of metals to a sufficiently high concentration allows metal to be recycled; smelting burns off the organic part of the organism, leaving behind reusable accumulated metal.
  • Resistances to a number of heavy metals are plasmid encoded in a number of species including Staphylococcus and Pseudomonas (Silver et al. Environ. Health Perspect. 102:107-113 (1994); Ji et al. J. Ind. Micro. 14:61-75 (1995)). These genes also confer heavy metal resistance on other species as well (e.g., E. coli).
  • the recursive sequence recombination techniques of the instant invention can be used to increase microbial heavy metal tolerances, as well as to increase the extent to which cells will accumulate heavy metals. For example, the ability of E. coli to detoxify arsenate can be improved at least 100-fold by RSR (see co-pending application Ser. No. 08/621,859, filed Mar. 25, 1996).
  • Cyanide is very efficiently used to extract gold from rock containing as little as 0.2 oz per ton.
  • This cyanide can be microbially neutralized and used as a nitrogen source by fungi or bacteria such as Pseudomonas fluorescens .
  • a problem with microbial cyanide degradation is the presence of toxic heavy metals in the leachate.
  • RSR can be used to increase the resistance of bioremedial microorganisms to toxic heavy metals, so that they will be able to survive the levels present in many industrial and Superfund sites. This will allow them to biodegrade organic pollutants including but not limited to aromatic hydrocarbons, halogenated hydrocarbons, and biocides.
  • Bioleaching is the process by which microbes convert insoluble metal deposits (usually metal sulfides or oxides) into soluble metal sulfates. Bioleaching is commercially important in the mining of arsenopyrite, but has additional potential in the detoxification and recovery of metals and acids from waste dumps.
  • Naturally occurring bacteria capable of bioleaching are reviewed by Rawlings and Silver ( Bio/Technology 13:773-778 (1995)). These bacteria are typically divided into groups by their preferred temperatures for growth. The more important mesophiles are Thiobacillus and Leptospirillum species. Moderate thermophiles include Sulfobacillus species. Extreme thermophiles include Sulfolobus species.
  • the recursive sequence recombination methods described above can be used to optimize the catalytic abilities in native hosts or heterologous hosts for evolved bioleaching genes or pathways, such as the ability to convert metals from insoluble to soluble salts.
  • leach rates of particular ores can be improved as a result of, for example, increased resistance to toxic compounds in the ore concentrate, increased specificity for certain substrates, ability to use different substrates as nutrient sources, and so on.
  • Microbial desulfurization is an appealing bioremediation application.
  • DBT dibenzothiophene
  • U.S. Pat. No. 5,356,801 discloses the cloning of a DNA molecule from Rhodococcus rhodochrous capable of biocatalyzing the desulfurization of oil.
  • Denome et al. ( Gene 175:6890-6901 (1995)) disclose the cloning of a 9.8 kb DNA fragment from Pseudomonas encoding the upper naphthalene catabolizing pathway which also degrades dibenzothiophene.
  • Other genes have been identified that perform similar functions (disclosed in U.S. Pat. No. 5,356,801).
  • the activity of these enzymes is currently too low to be commercially viable, but the pathway could be increased in efficiency using the recursive sequence recombination techniques of the invention.
  • the desired property of the genes of interest is their ability to desulfurize dibenzothiophene.
  • selection is preferably accomplished by coupling this pathway to one providing a nutrient to the bacteria.
  • desulfurization of dibenzothiophene results in formation of hydroxybiphenyl. This is a substrate for the biphenyl-catabolizing pathway which provides carbon and energy.
  • Selection would thus be done by “shuffling” the dibenzothiophene genes and transforming them into a host containing the biphenyl-catabolizing pathway. Increased dibenzothiophene desulfurization will result in increased nutrient availability and increased growth rate. Once the genes have been evolved they are easily separated from the biphenyl degrading genes. The latter are undesirable in the final product since the object is to desulfurize without decreasing the energy content of the oil.
  • Organo-nitro compounds are used as explosives, dyes, drugs, polymers and antimicrobial agents. Biodegradation of these compounds occurs usually by way of reduction of the nitrate group, catalyzed by nitroreductases, a family of broadly-specific enzymes. Partial reduction of organo-nitro compounds often results in the formation of a compound more toxic than the original (Hassan et al. 1979 Arch Bioch Biop. 196:385-395). Recursive sequence recombination of nitroreductases can produce enzymes that are more specific, and able to more completely reduce (and thus detoxify) their target compounds (examples of which include but are not limited to nitrotoluenes and nitrobenzenes).
  • Nitro-reductases can be isolated from bacteria isolated from explosive-contaminated soils, such as Morganella morganii and Enterobacter cloacae (Bryant et. al., 1991 . J. Biol Chem. 266:4126-4130).
  • a preferred selection method is to look for increased resistance to the organo-nitro compound of interest, since that will indicate that the enzyme is also able to reduce any toxic partial reduction products of the original compound.
  • Metabolic engineering can be used to alter microorganisms that produce industrially useful chemicals, so that they will grow using alternate and more abundant sources of nutrients, including human-produced industrial wastes. This typically involves providing both a transport system to get the alternative substrate into the engineered cells and catabolic enzymes from the natural host organisms to the engineered cells.
  • enzymes can be secreted into the medium by engineered cells to degrade the alternate substrate into a form that can more readily be taken up by the engineered cells; in other instances, a batch of engineered cells can be grown on one preferred substrate, then lysed to liberate hydrolytic enzymes for the alternate substrate into the medium, while a second inoculum of the same engineered host or a second host is added to utilize the hydrolyzate.
  • the starting materials for recursive sequence recombination will typically be genes for utilization of a substrate or its transport.
  • nutrient sources of interest include but are not limited to lactose, whey, galactose, mannitol, xylan, cellobiose, cellulose and sucrose, thus allowing cheaper production of compounds including but not limited to ethanol, tryptophan, rhamnolipid surfactants, xanthan gum, and polyhydroxylalkanoate.
  • the recursive sequence recombination methods described above can be used to optimize the ability of native hosts or heterologous hosts to utilize a substrate of interest, to evolve more efficient transport systems, to increase or alter specificity for certain substrates, and so on.
  • Metabolic engineering can be used to alter organisms to optimize the production of practically any metabolic intermediate, including antibiotics, vitamins, amino acids such as phenylalanine and aromatic amino acids, ethanol, butanol, polymers such as xanthan gum and bacterial cellulose, peptides, and lipids.
  • the recursive sequence recombination techniques described above can be used to optimize production of the desired metabolic intermediate, including such features as increasing enzyme substrate specificity and turnover number, altering metabolic fluxes to reduce the concentrations of toxic substrates or intermediates, increasing resistance of the host to such toxic compounds, eliminating, reducing or altering the need for inducers of gene expression/activity, increasing the production of enzymes necessary for metabolism, etc.
  • Enzymes can also be evolved for improved activity in solvents other than water. This is useful because intermediates in chemical syntheses are often protected by blocking groups which dramatically affect the solubility of the compound in aqueous solvents. Many compounds can be produced by a combination of pure chemical and enzymically catalyzed reactions. Performing enzymic reactions on almost insoluble substrates is clearly very inefficient, so the availability of enzymes that are active in other solvents will be of great use.
  • One example of such a scheme is the evolution of a para-nitrobenzyl esterase to remove protecting groups from an intermediate in loracarbef synthesis (Moore, J. C. and Arnold, F. H. Nature Biotechnology 14:458-467 (1996)).
  • the yield of almost any metabolic pathway can be increased, whether consisting entirely of genes endogenous to the host organisms or all or partly heterologous genes. Optimization of the expression levels of the enzymes in a pathway is more complex than simply maximizing expression. In some cases regulation, rather than constitutive expression of an enzyme may be advantageous for cell growth and therefore for product yield, as seen for production of phenylalanine (Backman et al. Ann. NY Acad. Sci. 589:16-24 (1990)) and 2-keto-L-gluconic acid (Anderson et al. U.S. Pat. No. 5,032,514). In addition, it is often advantageous for industrial purposes to express proteins in organisms other than their original hosts.
  • New host strains may be preferable for a variety of reasons, including ease of cloning and transformation, pathogenicity, ability to survive in particular environments and a knowledge of the physiology and genetics of the organisms.
  • proteins expressed in heterologous organisms often show markedly reduced activity for a variety of reasons including inability to fold properly in the new host (Sarthy et al. Appl. Environ. Micro. 53:1996-2000 (1987)).
  • Such difficulties can indeed be overcome by the recursive sequence recombination strategies of the instant invention.
  • the range of natural small molecule antibiotics includes but is not limited to peptides, peptidolactones, thiopeptides, beta-lactams, glycopeptides, lantibiotics, microcins, polyketide-derived antibiotics (anthracyclins, tetracyclins, macrolides, avermectins, polyethers and ansamycins), chloramphenicol, aminoglycosides, aminocyclitols, polyoxins, agrocins and isoprenoids.
  • antibiotic synthesis enzymes can be “evolved” together with transport systems that allow entry of compounds used as antibiotic precursors to improve uptake and incorporation of function-altering artificial side chain precursors.
  • penicillin V is produced by feeding Penicillium the artificial side chain precursor phenoxyacetic acid, and LY146032 by feeding Streptomyces roseosporus decanoic acid (Hopwood, Phil. Trans. R. Soc. Lond. B 324:549-562 (1989)).
  • Poor precursor uptake and poor incorporation by the synthesizing enzyme often lead to inefficient formation of the desired product.
  • Recursive sequence recombination of these two systems can increase the yield of desired product.
  • an enzyme is shuffled for novel catalytic activity/substrate recognition (perhaps by including randomizing oligonucleotides in key positions such as the active site).
  • a number of different substrates for example, analogues of side chains that are normally incorporated into the antibiotic
  • plates are made containing different potential antibiotic precursors (such as the side chain analogues).
  • the microorganisms containing the shuffled library (the library strain) are replicated onto those plates, together with a competing, antibiotic sensitive, microorganism (the indicator strain). Library cells that are able to incorporate the new side chain to produce an effective antibiotic will thus be able to compete with the indicator strain, and will be selected for.
  • the recursive sequence recombination techniques of the instant invention can be used to optimize expression of the foreign genes, to stabilize the enzyme in the new host cell, and to increase the activity of the introduced enzyme against its new substrates in the new host cell.
  • the host genome may also be so optimized.
  • the substrate specificity of an enzyme involved in secondary metabolism can be altered so that it will act on and modify a new compound or so that its activity is changed and it acts at a different subset of positions of its normal substrate.
  • Recursive sequence recombination can be used to alter the substrate specificities of enzymes.
  • recursive sequence recombination of individual enzymes being a strategy to generate novel antibiotics
  • recursive sequence recombination of entire pathways by altering enzyme ratios, will alter metabolite fluxes and may result, not only in increased antibiotic synthesis, but also in the synthesis of different antibiotics.
  • antibiotics can also be produced in vitro by the action of a purified enzyme on a precursor.
  • a purified enzyme for example isopenicillin N synthase catalyses the cyclization of many analogues of its normal substrate (d-(L-a-aminoadipyl)-L-cysteinyl-D-valine) (Hutchinson, Med. Res. Rev. 8:557-567 (1988)).
  • substrate analogues can be tested for incorporation by secondary metabolite synthesizing enzymes without concern for the initial efficiency of the reaction. Recursive sequence recombination can be used subsequently to increase the rate of reaction with a promising new substrate.
  • organisms already producing a desired antibiotic can be evolved with the recursive sequence recombination techniques described above to maximize production of that antibiotic.
  • new antibiotics can be evolved by manipulation of genetic material from the host by the recursive sequence recombination techniques described above.
  • Genes for antibiotic production can be transferred to a preferred host after cycles of recursive sequence recombination or can be evolved in the preferred host as described above.
  • Antibiotic genes are generally clustered and are often positively regulated, making them especially attractive candidates for the recursive sequence recombination techniques of the instant invention.
  • genes of related pathways show cross-hybridization, making them preferred candidates for the generation of new pathways for new antibiotics by the recursive sequence recombination techniques of the invention.
  • increases in secondary metabolite production including enhancement of substrate fluxes (by increasing the rate of a rate limiting enzyme, deregulation of the pathway by suppression of negative control elements or over expression of activators and the relief of feedback controls by mutation of the regulated enzyme to a feedback-insensitive deregulated protein) can be achieved by recursive sequence recombination without exhaustive analysis of the regulatory mechanisms governing expression of the relevant gene clusters.
  • the host chosen for expression of evolved genes is preferably resistant to the antibiotic produced, although in some instances production methods can be designed so as to sacrifice host cells when the amount of antibiotic produced is commercially significant yet lethal to the host. Similarly, bioreactors can be designed so that the growth medium is continually replenished, thereby “drawing off” antibiotic produced and sparing the lives of the producing cells. Preferably, the mechanism of resistance is not the degradation of the antibiotic produced.
  • genes have been cloned from Streptomyces cattleya which direct cephamycin C synthesis in the non-antibiotic producer Streptomyces lividans (Chen et al. Bio/Technology 6:1222-1224 (1988)).
  • 7-aminodeacetooxycephalosporanic acid 7-ADCA
  • 7-ADCA 7-aminodeacetooxycephalosporanic acid
  • 7-ADCA is made by a chemical ring expansion from penicillin V followed by enzymatic deacylation of the phenoxyacetal group.
  • Cephalosporin V could in principle be produced biologically from penicillin V using penicillin N expandase, but penicillin V is not used as a substrate by any known expandase.
  • the recursive sequence recombination techniques of the invention can be used to alter the enzyme so that it will use penicillin V as a substrate.
  • penicillin transacylase could be so modified to accept cephalosporins or cephamycins as substrates.
  • penicillin amidase expressed in E. coli is a key enzyme in the production of penicillin G derivatives.
  • the enzyme is generated from a precursor peptide and tends to accumulate as insoluble aggregates in the periplasm unless non-metabolizable sugars are present in the medium (Scherrer et al. Appl. Microbiol. Biotechnol. 42:85-91 (1994)). Evolution of this enzyme through the methods of the instant invention could be used to generate an enzyme that folds better, leading to a higher level of active enzyme expression.
  • Penicillin G acylase covalently linked to agarose is used in the synthesis of penicillin G derivatives.
  • the enzyme can be stabilized for increased activity, longevity and/or thermal stability by chemical modification (Fernandez-Lafuente et. al. Enzyme Microb. Technol. 14:489-495 (1992). Increased thermal stability is an especially attractive application of the recursive sequence recombination techniques of the instant invention, which can obviate the need for the chemical modification of such enzymes.
  • Selection for thermostability can be performed in vivo in E. coli or in thermophiles at higher temperatures. In general, thermostability is a good first step in enhancing general stabilization of enzymes.
  • Random mutagenesis and selection can also be used to adapt enzymes to function in non-aqueous solvents (Arnold Curr Oin Biotechnol, 4:450-455 (1993); Chen et. al. Proc. Natl. Acad. Sci. U.S.A., 90:5618-5622 (1993)).
  • Recursive sequence recombination represents a more powerful (since recombinogenic) method of generating mutant enzymes that are stable and active in non-aqueous environments. Additional screening can be done on the basis of enzyme stability in solvents.
  • Polyketides include antibiotics such as tetracycline and erythromycin, anti-cancer agents such as daunomycin, immunosuppressants such as FK506 and rapamycin and veterinary products such as monesin and avermectin.
  • PKS's Polyketide syntheses
  • Polyketides are multifunctional enzymes that control the chain length, choice of chain-building units and reductive cycle that generates the huge variation in naturally occurring polyketides.
  • Polyketides are built up by sequential transfers of “extender units” (fatty acyl CoA groups) onto the appropriate starter unit (exacetate, coumarate, propionate and malonamide).
  • the PKS's determine the number of condensation reactions and the type of extender groups added and may also fold and cyclize the polyketide precursor. PKS's reduce specific ⁇ -keto groups and may dehydrate the resultant ⁇ -hydroxyls to form double bonds. Modifications of the nature or number of building blocks used, positions at which ⁇ -keto groups are reduced, the extent of reduction and different positions of possible cyclizations, result in formation of different final products. Polyketide research is currently focused on modification and inhibitor studies, site directed mutagenesis and 3-D structure elucidation to lay the groundwork for rational changes in enzymes that will lead to new polyketide products.
  • the recursive sequence recombination techniques of the instant invention can be used to generate modified enzymes that produce novel polyketides without such detailed analytical effort.
  • the availability of the PKS genes on plasmids and the existence of E. coli -Streptomyces shuttle vectors (Wehmeier Gene 165:149-150 (1995)) makes the process of recursive sequence recombination especially attractive by the techniques described above.
  • Techniques for selection of antibiotic producing organisms can be used as described above; additionally, in some embodiments screening for a particular desired polyketide activity or compound is preferable.
  • Isoprenoids result from cyclization of farnesyl pyrophosphate by sesquiterpene synthases. The diversity of isoprenoids is generated not by the backbone, but by control of cyclization. Cloned examples of isoprenoid synthesis genes include trichodiene synthase from Fusarium sprorotrichioides , pentalene synthase from Streptomyces, aristolochene synthase from Penicillium roquefortii , and epi-aristolochene syrithase from N. tabacum (Cane, D. E. (1995).
  • isoprenoid antibiotics pages 633-655, in “Genetics and Biochemistry of Antibiotic Production” edited by Vining, L. C. & Stuttard, C., published by Butterworth-Heinemann). Recursive sequence recombination of sesquiterpene synthases will be of use both in allowing expression of these enzymes in heterologous hosts (such as plants and industrial microbial strains) and in alteration of enzymes to change the cyclized product made. A large number of isoprenoids are active as antiviral, antibacterial, antifungal, herbicidal, insecticidal or cytostatic agents.
  • Antibacterial and antifungal isoprenoids could thus be preferably screened for using the indicator cell type system described above, with the producing cell competing with bacteria or fungi for nutrients.
  • Antiviral isoprenoids could be screened for preferably by their ability to confer resistance to viral attack on the producing cell.
  • bioactive non-ribosomally synthesized peptides include the antibiotics cyclosporin, pepstatin, actinomycin, gramicidin, depsipeptides, vancomycin, etc. These peptide derivatives are synthesized by complex enzymes rather than ribosomes. Again, increasing the yield of such non-ribosomally synthesized peptide antibiotics has thus far been done by genetic identification of biosynthetic “bottlenecks” and over expression of specific enzymes (See, for example, p. 133-135 in “Genetics and Biochemistry of Antibiotic Production” edited by Vining, L. C. & Stuttard, C., published by Butterworth-Heinemann).
  • peptide synthases are modular and multifunctional enzymes catalyzing condensation reactions between activated building blocks (in this case amino acids) followed by modifications of those building blocks (see Kleinkauf, H. and von Dohren, H. Eur. J. Biochem. 236:335-351 (1996)).
  • recursive sequence recombination can also be used to alter peptide synthases: modifying the specificity of the amino acid recognized by each binding site on the enzyme and altering the activity or substrate specificities of sites that modify these amino acids to produce novel compounds with antibiotic activity.
  • peptide antibiotics are made ribosomally and then post-translationally modified.
  • examples of this type of antibiotics are lantibiotics (produced by gram positive bacteria such Staphylococcus, Streptomyces, Bacillus, and Actinoplanes) and microcins (produced by Enterobacteriaceae). Modifications of the original peptide include (in lantibiotics) dehydration of serine and threonine, condensation of dehydroamino acids with cysteine, or simple N- and C-terminal blocking (microcins).
  • a peptide-encoding sequence and the modifying enzymes may have their expression levels modified by recursive sequence recombination. Again, this will lead to both increased levels of antibiotic synthesis, and by modulation of the levels of the modifying enzymes (and the sequence of the ribosomally synthesized peptide itself) novel antibiotics.
  • Screening can be done as for other antibiotics as described above, including competition with a sensitive (or even initially insensitive) microbial species. Use of competing bacteria that have resistances to the antibiotic being produced will select strongly either for greatly elevated levels of that antibiotic (so that it swamps out the resistance mechanism) or for novel derivatives of that antibiotic that are not neutralized by the resistance mechanism.
  • Examples of starting materials for recursive sequence recombination include but are not limited to genes from bacteria such as Alcallgenes, Zoogloea, Rhizobium, Bacillus, and Azobacter, which produce polyhydroxyalkanoates (PHAS) such as polyhyroxybutyrate (PHB) intracellularly as energy reserve materials in response to stress.
  • PHA polyhydroxyalkanoates
  • PHB polyhyroxybutyrate
  • the recursive sequence recombination techniques of the invention can be used to modify such heterologous genes as well as specific cloned interacting pathways (e.g., mevalonate), and to optimize PHB synthesis in industrial microbial strains, for example to remove the requirement for stresses (such as nitrogen limitation) in growth conditions.
  • specific cloned interacting pathways e.g., mevalonate
  • Carotenoids are a family of over 600 terpenoids produced in the general isoprenoid biosynthetic pathway by bacteria, fungi and plants (for a review, see Armstrong, J. Bact. 176:4795-4802 (1994)). These pigments protect organisms against photooxidative damage as well as functioning as anti-tumor agents, free radical-scavenging anti-oxidants, and enhancers of the immune response. Additionally, they are used commercially in pigmentation of cultured fish and shellfish.
  • carotenoids examples include but are not limited to myxobacton, spheroidene, spheroidenone, lutein, astaxanthin, violaxanthin, 4-ketorulene, myxoxanthrophyll, echinenone, lycopene, zeaxanthin and its mono- and di-glucosides, ⁇ -, ⁇ -, ⁇ - and ⁇ -carotene, ⁇ -cryptoxanthin monoglucoside and neoxanthin.
  • Carotenoid synthesis is catalyzed by relatively small numbers of clustered genes: 11 different genes within 12 kb of DNA from Myxococcus xanthus (Botella et al. Eur. J. Biochem. 233:238-248 (1995)) and 8 genes within 9 kb of DNA from Rhodobacter sphaeroides (Lang et. al. J. Bact. 177:2064-2073 (1995)). In some microorganisms, such as Thermus thermophilus , these genes are plasmid-borne (Tabata et al. FEBS Letts 341:251-255 (1994)). These features make carotenoid synthetic pathways especially attractive candidates for recursive sequence recombination.
  • the recursive sequence recombination techniques of the invention can be used to generate variants in the regulatory and/or structural elements of genes in the carotenoid synthesis pathway, allowing increased expression in heterologous hosts.
  • traditional techniques have been used to increase carotenoid production by increasing expression of a rate limiting enzyme in Thermus thermophilus (Hoshino et al. Appl. Environ. Micro. 59:3150-3153 (1993)).
  • mutation of regulatory genes can cause constitutive expression of carotenoid synthesis in actinomycetes, where carotenoid photoinducibility is otherwise unstable and lost at a relatively high frequency in some species (Kato et al. Mol. Gen. Genet. 247:387-390 (1995)). These are both mutations that can be obtained by recursive sequence recombination.
  • the recursive sequence recombination techniques of the invention as described above can be used to evolve one or more carotenoid synthesis genes in a desired host without the need for analysis of regulatory mechanisms. Since carotenoids are colored, a calorimetric assay in microtiter plates, or even on growth media plates, can be used for screening for increased production.
  • carotenogenic biosynthetic pathways have the potential to produce a wide diversity of carotenoids, as the enzymes involved appear to be specific for the type of reaction they will catalyze, but not for the substrate that they modify.
  • two enzymes from the marine bacterium Agrobacterium aurantiacum (CrtW and CrtZ) synthesize six different ketocarotenoids from ⁇ -carotene (Misawa et al. J. Bact. 177:6576-6584 (1995)).
  • This relaxed substrate specificity means that a diversity of substrates can be transformed into an even greater diversity of products.
  • Another method of identifying new compounds is to use standard analytical techniques such as mass spectroscopy, nuclear magnetic resonance, high performance liquid chromatography, etc. Recombinant microorganisms can be pooled and extracts or media supernatants assayed from these pools. Any positive pool can then be subdivided and the procedure repeated until the single positive is identified (“sib-selection”).
  • indigo is currently produced chemically.
  • nine genes have been combined in E. coli to allow the synthesis of indigo from glucose via the tryptophan/indole pathway (Murdock et al. Bio/Technology 11:381-386 (1993)).
  • a number of manipulations were performed to optimize indigo synthesis: cloning of nine genes, modification of the fermentation medium and directed changes in two operons to increase reaction rates and catalytic activities of several enzymes. Nevertheless, bacterially produced indigo is not currently an economic proposition.
  • the recursive sequence recombination techniques of the instant invention could be used to optimize indigo synthesizing enzyme expression levels and catalytic activities, leading to increased indigo production, thereby making the process commercially viable and reducing the environmental impact of indigo manufacture. Screening for increased indigo production can be done by calorimetric assays of cultures in microtiter plates.
  • Amino acids of particular commercial importance include but are not limited to phenylalanine, monosodium glutamate, glycine, lysine, threonine, tryptophan and methionine.
  • Backman et al. ( Ann. NY Acad. Sci. 589:16-24 (1990)) disclosed the enhanced production of phenylalanine in E. coli via a systematic and downstream strategy covering organism selection, optimization of biosynthetic capacity, and development of fermentation and recovery processes.
  • screening for enhanced production is preferably done in microtiter wells, using chemical tests well known in the art that are specific for the desired amino acid. Screening/selection for amino acid synthesis can also be done by using auxotrophic reporter cells that are themselves unable to synthesize the amino acid in question. If these reporter cells also produce a compound that stimulates the growth of the amino acid producer (this could be a growth factor, or even a different amino acid), then library cells that produce more amino acid will in turn receive more growth stimulant and will therefore grow more rapidly.
  • L-Ascorbic acid is a commercially important vitamin with a world production of over 35,000 tons in 1984.
  • Most vitamin C is currently manufactured chemically by the Reichstein process, although recently bacteria have been engineered that are able to transform glucose to 2,5-keto-gluconic acid, and that product to 2-keto-L-idonic acid, the precursor to L-ascorbic acid (Boudrant, Enzyme Microb. Technol. 12:322-329 (1990)).
  • the genes can be genetically engineered to create one or more operons followed by expression optimization of such a hybrid L-ascorbic acid synthetic pathway to result in commercially viable microbial vitamin C biosynthesis.
  • screening for enhanced L-ascorbic acid production is preferably done in microtiter plates, using assays well known in the art.
  • recursive sequence recombination techniques can be used to improve or alter other aspects of cell properties, from growth rate to ability to secrete certain desired compounds to ability to tolerate increased temperature or other environmental stresses.
  • Some examples of traits engineered by traditional methods include expression of heterologous proteins in bacteria, yeast, and other eukaryotic cells, antibiotic resistance, and phage resistance. Any of these traits is advantageously evolved by the recursive sequence recombination techniques of the instant invention. Examples include replacement of one nutrient uptake system (e.g.
  • heterologous genes encoding these functions all have the potential for further optimization in their new hosts by existing recursive sequence recombination technology. Since these functions increase cell growth rates under the desired growth conditions, optimization of the genes by evolution simply involves recombining the DNA recursively and selecting the recombinants that grow faster with limiting oxygen, higher toxic compound concentration, or whatever is the appropriate growth condition for the parameter being improved.
  • Cultured mammalian cells also require essential amino acids to be present in the growth medium. This requirement could also be circumvented by expression of heterologous metabolic pathways that synthesize these amino acids (Rees et al. Biotechnology 8:629-633 (1990). Recursive sequence recombination would provide a mechanism for optimizing the expression of these genes in mammalian cells. Once again, a preferred selection would be for cells that can grow in the absence of added amino acids.
  • the main function of recursive sequence recombination in this case is in improving the survival of strains that are already known to be better nitrogen fixers. These strains tend to be less good at competing with strains already present in the environment, even though they are better at nitrogen fixation.
  • Targets for recursive sequence recombination such as nodulation and host range determination genes can be modified and selected for by their ability to grow on the new host. Similarly any bacteriocin or energy utilization genes that will improve the competitiveness of the strain will also result in greater growth rates. Selection can simply be performed by subjecting the target genes to recursive sequence recombination and forcing the inoculant to compete with wild type nitrogen fixing bacteria. The better the nitrogen fixing bacteria grow in the new host, the more copies of their recombined genes will be present for the next round of recombination. This growth rate differentiating selection is described above in detail.
  • Bioluminescence or fluorescence genes can be used as reporters by fusing them to specific regulatory genes (Cameron et. al. Appl Biochem Biotechnol, 38:105-140 (1993)).
  • a specific example is one in which the luciferase genes luxCDABE of Vibrio fischeri were fused to the regulatory region of the isopropylbenzene catabolism operon from Pseudomonas putida RE204. Transformation of this fusion construct into E. coli resulted in a strain which produced light in response to a variety of hydrophobic compound such as substituted benzenes, chlorinated solvents and naphthalene (Selifonova et.
  • Recursive sequence recombination can be used in several ways to modify this type of biodetection system. It can be used to increase the amplitude of the response, for example by increasing the fluorescence of the green fluorescent protein. Recursive sequence recombination could also be used to increase induced expression levels or catalytic activities of other signal-generating systems, for example of the luciferase genes.
  • Recursive sequence recombination can also be used to alter the specificity of biosensors.
  • the regulatory region, and transcriptional activators that interact with this region and with the chemicals that induce transcription can also be shuffled. This should generate regulatory systems in which transcription is activated by analogues of the normal inducer, so that biodetectors for different chemicals can be developed. In this case, selection would be for constructs that are activated by the (new) specific chemical to be detected. Screening could be done simply with fluorescence (or light) activated cell sorting, since the desired improvement is in light production.
  • biosensors can be developed that will respond to any chemical for which there are receptors, or for which receptors can be evolved by recursive sequence recombination, such as hormones, growth factors, metals and drugs.
  • receptors may be intracellular and direct activators of transcription, or they may be membrane bound receptors that activate transcription of the signal indirectly, for example by a phosphorylation cascade. They may also not act on transcription at all, but may produce a signal by some post-transcriptional modification of a component of the signal generating pathway.
  • These receptors may also be generated by fusing domains responsible for binding different ligands with different signaling domains.
  • recursive sequence recombination can be used to increase the amplitude of the signal generated to optimize expression and functioning of chimeric receptors, and to alter the specificity of the chemicals detected by the receptor.
  • recursive sequence recombination techniques of the instant invention were used to expand the range of substrates efficiently hydrolyzed by E. coli ⁇ -galactosidase.
  • the goal was to evolve wild type E. coli ⁇ -galactosidase into a fucosidase.
  • the enzyme showed very weak activity with both ⁇ -nitrophenyl- ⁇ -D-fucopyranoside and o-nitrophenyl- ⁇ -D-fucopyranoside (estimated respectively as 80- and 160-fold less efficient than for ⁇ -nitrophenyl- ⁇ -D-galactopyranoside).
  • a lacZ gene (a 3.8 kb Hind III-BamHI fragment from plasmid pCH110, Pharmacia) encoding E. coli ⁇ -galactosidase was subcloned into plasmid pl8SFI-BLA-SFI (Stemmer, Nature, 370:389-391 (1994)). The resulting plasmid, p18-lacZ, was used for recursive sequence recombination and mutant screening.
  • This reaction was diluted 100-fold into a standard PCR reaction using the 40 mer primers p50F 5′-AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC-3′ and pR34 5′-CTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAT-3′. This resulted in amplification of both the desired DNA band (about 4 kb in size) as well as two smaller sized products (about 600bp and 100 bp bands).
  • the PCR products were digested with BamHI and Hind III and the correct size product was cloned into BamHI-HindIII digested p18-lacZ.
  • the resulting plasmid containing a pool of recombined lacZ mutants was plated out on LB plates supplemented with kanamycin and 5-bromo-4-chloro-3-indolyl- ⁇ -D-fucopyranoside (X-fuco). Plates were incubated at 37° C. for 20 hours and screened for colonies with slight blue tint, indicating hydrolysis of the X-fuco. Plasmid DNA was prepared from positive colonies and the procedure was repeated. Thus, six rounds of recursive sequence recombination produced a ten-fold increase in X-fuco hydrolysis activity.
  • the recursive sequence recombination techniques of the invention were used to modify a plasmid encoding resistance to mercury salts.
  • This plasmid as disclosed by Wang et al. ( J. Bact. 171:83-92 (1989)) contains at least 8 genes within 13.5 kb of Bacillus DNA inserted in the cloning vector pUC9.
  • the recursive sequence recombination protocol used for this plasmid was as follows.
  • Plasmid DNA (at 130 ⁇ g/ml) was digested with 0.09 U/ml DNAse in 50 mM Tris-Cl, pH 7.4, 10 mM MnCl 2 , for 10 minutes at 25° C. DNA fragments were not size-selected, but were purified by phenol extraction and ethanol precipitation.
  • the assembly reaction was performed using Tth polymerase (Perkin Elmer) using the manufacturer's supplied buffer, supplemented with the following: 7.5% polyethylene glycol, 8000 MW; 35 mM tetramethylammonium chloride; and 4 U/ml Pwo(Boehringer Mannheim), Pfu(Stratagene), Vent (New England Biolabs), Deep Vent (New England Biolabs), Tfl (Promega) or Tli (Promega) thermostable DNA polymerases. DNA fragments were used at around 10 ⁇ g/ml.
  • the PCR program for assembly was as follows: 94° C. for 20 sec., then 40 cycles of 940C for 15 sec., 40° C. for 30 sec., 72° C. for 30 sec. +2 sec./cycle, and finally 72° C. for 10 min.
  • the recombinant plasmid was then amplified in three fragments by using primers flanking the three relatively evenly spaced AlwNI restriction sites contained in the plasmid.
  • the sequences of these primers were: 1) 5′-CAGGACTTATCGCCACTGGCAGC-3′ 2) 5′-CTCGCTCTGCTAATCCTGTTACC-3′ 3) 5′- GCATATTATGAGCGTTTAGGCTTAATTCC-3′ 4) 5′-CGGTATCCTTTTTCCGTACGTTC-3′ 5) 5′- GTTGAAGAGGTGAAGAAAGTTCTCC-3′ 6) 5′-GTTCGTCGATTTCCACGCTTGGC-3′.
  • nucleotide sequences were recombined between four homologous ⁇ -lactamases from C. freundii, E. cloacae, K. pneumonia , and Y. enterocolitica .
  • the four genes were synthesized from oligonucleotides as described in Stemmer, et al. Gene 164:49-53 (1995). Briefly, the entire coding sequences of the genes were synthesized as overlapping 50-mer oligonucleotides on a commercial oligonucleotide synthesizer.
  • oligonucleotides were then assembled into full length genes by a standard recursive sequence recombination reaction, followed by amplification using primers common to all four genes. oligonucleotides were designed to give optimal E. coli codon usage in the synthetic genes with the goal of increasing the homology to increase the frequency of recombination, and the same 5′ and 3′ terminal sequences. After assembly of the genes and selection for active clones, which is optional, they were DNase treated to produce fragments from 50 to 200 bp in length.
  • the fragments were dissolved at 100 ⁇ g/ml in 15 ⁇ l of Klenow (DNA polymerase I large fragment) buffer (New England Biolabs) and subjected to manual PCR as follows: 15 cycles of 95° C. for 1 min.; freeze on dry ice and ethanol; warm to 25° C. and add 2 Al of Klenow (1 U/ ⁇ l) in Klenow buffer; incubate for 2 min at 25° C.
  • Klenow DNA polymerase I large fragment
  • a 5 ⁇ l aliquot of the manual PCR reaction was then diluted 6-fold into a standard Taq reaction mix (without oligonucleotide primers) and assembled using a standard PCR program consisting of 30 cycles of 940C for 30 sec., 40° C. for 30 sec., and 72° C. for 30 sec.
  • a 4, 8 or 16 ⁇ l aliquot of this second PCR reaction was then diluted into a standard Taq reaction mix containing oligonucleotide primers that prime on sequences contained in all four ⁇ -lactamase genes 5′-AGGGCCTCGTGATACGCCTATT-3′ and 5′-ACGAAAACTCACGTTAAGGGATT-3′.
  • Full-length product was amplified using a standard PCR program consisting of 25 cycles of 94° C. for 30 sec., 45° C. for 30 sec., 72° C. for 45 sec.

Abstract

The present invention is generally directed to the evolution of new metabolic pathways and the enhancement of bioprocessing through a process herein termed recursive sequence recombination. Recursive sequence recombination entails performing iterative cycles of recombination and screening or selection to “evolve” individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes. Such techniques do not require the extensive analysis and computation required by conventional methods for metabolic engineering.

Description

  • This application is a continuation-in-part of U.S. patent application Ser. No. 08/198,431, filed Feb. 17, 1994, Ser. No. PCT/US95/02126, filed, Feb. 17, 1995, Ser. No. 08/537,874, filed Oct. 30, 1995, Ser. No. 08/621,859, filed Mar. 25, 1996, Ser. No. 08/621,430, filed Mar. 25, 1996, and Ser. No. 08/425,684, filed Apr. 18, 1995, the specifications of which are herein incorporated by reference in their entirety for all purposes.[0001]
    • BACKGROUND OF THE INVENTION
  • Metabolic engineering is the manipulation of intermediary metabolism through the use of both classical genetics and genetic engineering techniques. Cellular engineering is generally a more inclusive term referring to the modification of cellular properties. Cameron et al. ([0002] Applied Biochem. Biotech. 38:105-140 (1993)) provide a summary of equivalent terms to describe this type of engineering, including “metabolic engineering”, which is most often used in the context of industrial microbiology and bioprocess engineering, “in vitro evolution” or “directed evolution”, most often used in the context of environmental microbiology, “molecular breeding”, most often used by Japanese researchers, “cellular engineering”, which is used to describe modifications of bacteria, animal, and plant cells, “rational strain development”, and “metabolic pathway evolution”. In this application, the terms “metabolic engineering” and “cellular engineering” are used preferentially for clarity; the term “evolved” genes is used as discussed below.
  • Metabolic engineering can be divided into two basic categories: modification of genes endogenous to the host organism to alter metabolite flux and introduction of foreign genes into an organism. Such introduction can create new metabolic pathways leading to modified cell properties including but not limited to synthesis of known compounds not normally made by the host cell, production of novel compounds (e.g. polymers, antibiotics, etc.) and the ability to utilize new nutrient sources. Specific applications of metabolic engineering can include the production of specialty and novel chemicals, including antibiotics, extension of the range of substrates used for growth and product formation, the production of new catabolic activities in an organism for toxic chemical degradation, and modification of cell properties such as resistance to salt and other environmental factors. [0003]
  • Bailey ([0004] Science 252:1668-1674 (1991)) describes the application of metabolic engineering to the recruitment of heterologous genes for the improvement of a strain, with the caveat that such introduction can result in new compounds that may subsequently undergo further reactions, or that expression of a heterologous protein can result in proteolysis, improper folding, improper modification, or unsuitable intracellular location of the protein, or lack of access to required substrates. Bailey recommends careful configuration of a desired genetic change with minimal perturbation of the host.
  • Liao ([0005] Curr. Opin. Biotech. 4:211-216 (1993)) reviews mathematical modelling and analysis of metabolic pathways, pointing out that in many cases the kinetic parameters of enzymes are unavailable or inaccurate.
  • Stephanopoulos et al. ([0006] Trends. Biotechnol. 11:392-396 (1993)) describe attempts to improve productivity of cellular systems or effect radical alteration of the flux through primary metabolic pathways as having difficulty in that control architectures at key branch points have evolved to resist flux changes. They conclude that identification and characterization of these metabolic nodes is a prerequisite to rational metabolic engineering. Similarly, Stephanopoulos (Curr. Opin. Biotech. 5:196-200 (1994)) concludes that rather than modifying the “rate limiting step” in metabolic engineering, it is necessary to systematically elucidate the control architecture of bioreaction networks.
  • The present invention is generally directed to the evolution of new metabolic pathways and the enhancement of bioprocessing through a process herein termed recursive sequence recombination. Recursive sequence recombination entails performing iterative cycles of recombination and screening or selection to “evolve” individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes (Stemmer, [0007] Bio/Technology 13:549-553 (1995)). Such techniques do not require the extensive analysis and computation required by conventional methods for metabolic engineering. Recursive sequence recombination allows the recombination of large numbers of mutations in a minimum number of selection cycles, in contrast to traditional, pairwise recombination events.
  • Thus, because metabolic and cellular engineering can pose the particular problem of the interaction of many gene products and regulatory mechanisms, recursive sequence recombination (RSR) techniques provide particular advantages in that they provide recombination between mutations in any or all of these, thereby providing a very fast way of exploring the manner in which different combinations of mutations can affect a desired result, whether that result is increased yield of a metabolite, altered catalytic activity or substrate specificity of an enzyme or an entire metabolic pathway, or altered response of a cell to its environment. [0008]
    • SUMMARY OF THE INVENTION
  • One aspect of the invention is a method of evolving a biocatalytic activity of a cell, comprising: [0009]
  • (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; [0010]
  • (b) screening at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; [0011]
  • (c) recombining at least a segment from at least one recombinant gene with a further DNA segment from at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes; [0012]
  • (d) screening at least one further recombinant gene from the further library of recombinant genes that confers enhanced ability to catalyze the reaction of interest in the cell relative to a previous recombinant gene; [0013]
  • (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell. [0014]
  • Another aspect of the invention is a method of evolving a gene to confer ability to catalyze a reaction of interest, the method comprising: [0015]
  • (1) recombining at least first and second DNA segments from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; [0016]
  • (2) screening at least one recombinant gene from the library that confers enhanced ability to catalyze a reaction of interest relative to a wildtype form of the gene; [0017]
  • (3) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes; [0018]
  • (4) screening at least one further recombinant gene from the further library of recombinant genes that confers enhanced ability to catalyze a reaction of interest relative to a previous recombinant gene; [0019]
  • (5) repeating (3) and (4), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze a reaction of interest. [0020]
  • A further aspect of the invention is a method of generating a new biocatalytic activity in a cell, comprising: [0021]
  • (1) recombining at least first and second DNA segments from at least one gene conferring ability to catalyze a first reaction related to a second reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; [0022]
  • (2) screening at least one recombinant gene from the library that confers a new ability to catalyze the second reaction of interest; [0023]
  • (3) recombining at least a segment from at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes; [0024]
  • (4) screening at least one further recombinant gene from the further library of recombinant genes that confers enhanced ability to catalyze the second reaction of interest in the cell relative to a previous recombinant gene; [0025]
  • (5) repeating (3) and (4), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the second reaction of interest in the cell. [0026]
  • Another aspect of the invention is a modified form of a cell, wherein the modification comprises a metabolic pathway evolved by recursive sequence recombination. [0027]
  • A further aspect of the invention is a method of optimizing expression of a gene product, the method comprising: [0028]
  • (1) recombining at least first and second DNA segments from at least one gene conferring ability to produce the gene product, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; [0029]
  • (2) screening at least one recombinant gene from the library that confers optimized expression of the gene product relative to a wildtype form of the gene; [0030]
  • (3) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or-different from the first and second segments, to produce a further library of recombinant genes; [0031]
  • (4) screening at least one further recombinant gene from the further library of recombinant genes that confers optimized ability to produce the gene product relative to a previous recombinant gene; [0032]
  • (5) repeating (3) and (4), as necessary, until the further recombinant gene confers a desired level of optimized ability to express the gene product. [0033]
  • A further aspect of the invention is a method of evolving a biosensor for a compound A of interest, the method comprising: [0034]
  • (1) recombining at least first and second DNA segments from at least one gene conferring ability to detect a related compound B, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; [0035]
  • (2) screening at least one recombinant gene from the library that confers optimized ability to detect compound A relative to a wildtype form of the gene; [0036]
  • (3) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes; [0037]
  • (4) screening at least one further recombinant gene from the further library of recombinant genes that confers optimized ability to detect compound A relative to a previous recombinant gene; [0038]
  • (5) repeating (3) and (4), as necessary, until the further recombinant gene confers a desired level of optimized ability to detect compound A.[0039]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a drawing depicting a scheme for in vitro recursive sequence, recombination.[0040]
    • DESCRIPTION OF THE SPECIFIC EMBODIMENTS
  • The invention provides a number of strategies for evolving metabolic and bioprocessing pathways through the technique of recursive sequence recombination. One strategy entails evolving genes that confer the ability to use a particular substrate of interest as a nutrient source in one species to confer either more efficient use of that substrate in that species, or comparable or more efficient use of that substrate in a second species. Another strategy entails evolving genes that confer the ability to detoxify a compound of interest in one or more species of organisms. Another strategy entails evolving new metabolic pathways by evolving an enzyme or metabolic pathway for biosynthesis or degradation of a compound A related to a compound B for the ability to biosynthesize or degrade compound B, either in the host of origin or a new host. A further strategy entails evolving a gene or metabolic pathway for more efficient or optimized expression of a particular metabolite or gene product. A further strategy entails evolving a host/vector system for expression of a desired heterologous product. These strategies may involve using all the genes in a multi-step pathway, one or several genes, genes from different organisms, or one or more fragments of a gene. [0041]
  • The strategies generally entail evolution of gene(s) or segment(s) thereof to allow retention of function in a heterologous cell or improvement of function in a homologous or heterologous cell. Evolution is effected generally by a process termed recursive sequence recombination. Recursive sequence recombination can be achieved in many different formats and permutations of formats, as described in further detail below. These formats share some common principles. Recursive sequence recombination entails successive cycles of recombination to generate molecular diversity, i.e., the creation of a family of nucleic acid molecules showing substantial sequence identity to each other but differing in the presence of mutations. Each recombination cycle is followed by at least one cycle of screening or selection for molecules having a desired characteristic. The molecule(s) selected in one round form the starting materials for generating diversity in the next round. In any given cycle, recombination can occur in viva or in vitro. Furthermore, diversity resulting from recombination can be augmented in any cycle by applying prior methods of mutagenesis (e.g., error-prone PCR or cassette mutagenesis, passage through bacterial mutator strains, treatment with chemical mutagens) to either the substrates for or products of recombination. [0042]
  • I. Formats for Recursive Sequence Recombination [0043]
  • Some formats and examples for recursive sequence recombination, sometimes referred to as DNA shuffling or molecular breeding, have been described by the present inventors and co-workers in co-pending applications, U.S. patent application Ser. No. 08/621,430, filed Mar. 25, 1996; Ser. No. PCT/US95/02126, filed Feb. 17, 1995; Ser. No. 08/621,859, filed Mar. 25, 1996; Ser. No. 08/198,431, filed Feb. 17, 1994; Stemmer, [0044] Science 270:1510 (1995); Stemmer et al., Gene 164:49-53 (1995); Stemmer, Bio/Technology 13:549-553 (1995); Stemmer, Proc. Natl. Acad. Sci. U.S.A. 91:10747-10751 (1994); Stemmer, Nature 370:389-391 (1994); Crameri et al. Nature Medicine 2(1):1-3 (1996); Crameri et al. Nature Biotechnology 14:315-319 (1996), each of which is incorporated by reference in its entirety for all purposes.
  • (1) In Vitro Formats [0045]
  • One format for recursive sequence recombination in vitro is illustrated in FIG. 1. The initial substrates for recombination are a pool of related sequences. The X's in FIG. 1, panel A, show where the sequences diverge. The sequences can be DNA or RNA and can be of various lengths depending on the size of the gene or DNA fragment to be recombined or reassembled. Preferably the sequences are from 50 bp to 100 kb. [0046]
  • The pool of related substrates can be fragmented, usually at random, into fragments of from about 5 bp to 5 kb or more, as shown in FIG. 1, panel B. Preferably the size of the random fragments is from about 10 bp to 1000 bp, more preferably the size of the DNA fragments is from about 20 bp to 500 bp. The substrates can be digested by a number of different methods, such as DNAseI or RNAse digestion, random shearing or restriction enzyme digestion. The concentration of nucleic acid fragments of a particular length or sequence is often less than 0.1% or 1% by weight of the total nucleic acid. The number of different specific nucleic acid fragments in the mixture is usually at least about 100, 500 or 1000. [0047]
  • The mixed population of nucleic acid fragments are denatured by heating to about 80° C. to 100° C., more preferably from 90° C. to 96° C., to form single-stranded nucleic acid fragments and then reannealed. Single-stranded nucleic acid fragments having regions of sequence identity with other single-stranded nucleic acid fragments can then be reannealed by cooling to 20° C. to 75° C., and preferably from 40° C. to 65° C. Renaturation can be accelerated by the addition of polyethylene glycol (“PEG”) or salt. The salt concentration is preferably from 0 mM to 600 mM, more preferably the salt concentration is from 10 mM to 100 mM. The salt may be such salts as (NH[0048] 4)2SO4, KCl, or NaCl. The concentration of PEG is preferably from 0% to 20%, more preferably from 5% to 10%. The fragments that reanneal can be from different substrates as shown in FIG. 1, panel C.
  • The annealed nucleic acid fragments are incubated in the presence of a nucleic acid polymerase, such as Taq or Klenow, and dNTP's (i.e. DATP, dCTP, dGTP and dTTP). If regions of sequence identity are large, Iaq or other high-temperature polymerase can be used with an annealing temperature of between 45-65° C. If the areas of identity are small, Klenow or other low-temperature polymerases can be used with an annealing temperature of between 20-30° C. The polymerase can be added to the random nucleic acid fragments prior to annealing, simultaneously with annealing or after annealing. [0049]
  • The cycle of denaturation, renaturation and incubation of random nucleic acid fragments in the presence of polymerase is sometimes referred to as “shuffling” of the nucleic acid in vitro. This cycle is repeated for a desired number of times. Preferably the cycle is repeated from 2 to 100 times, more preferably the sequence is repeated from 10 to 40 times. The resulting nucleic acids are a family of double-stranded polynucleotides of from about 50 bp to about 100 kb, preferably from 500 bp to 50 kb, as shown in FIG. 1, panel D. The population represents variants of the starting substrates showing substantial sequence identity thereto but also diverging at several positions. The population has many more members than the starting substrates. The population of fragments resulting from recombination is preferably first amplified by PCR, then cloned into an appropriate vector and the ligation mixture used to transform host cells. [0050]
  • In a variation of in vitro shuffling, subsequences of recombination substrates can be generated by amplifying the full-length sequences under conditions which produce a substantial fraction, typically at least 20 percent or more, of incompletely extended amplification products. The amplification products, including the incompletely extended amplification products are denatured and subjected to at least one additional cycle of reannealing and amplification. This variation, wherein at least one cycle of reannealing and amplification provides a substantial fraction of incompletely extended products, is termed “stuttering.” In the subsequent amplification round, the incompletely extended products anneal to and prime extension on different sequence-related template species. [0051]
  • In a further variation, at least one cycle of amplification can be conducted using a collection of overlapping single-stranded DNA fragments of related sequence, and different lengths. Each fragment can hybridize to and prime polynucleotide chain extension of a second fragment from the collection, thus forming sequence-recombined polynucleotides. In a further variation, single-stranded DNA fragments of variable length can be generated from a single primer by Vent DNA polymerase on a first DNA template. The single stranded DNA fragments are used as primers for a second, Kunkel-type template, consisting of a uracil-containing circular single-stranded DNA. This results in multiple substitutions of the first template into the second (see Levichkin et al. [0052] Mol. Biology 29:572-577 (1995)).
  • Gene clusters such as those involved in polyketide synthesis (or indeed any multi-enzyme pathways catalyzing analogous metabolic reactions) can be recombined by recursive sequence recombination even if they lack DNA sequence homology. Homology can be introduced using synthetic oligonucleotides as PCR primers. In addition to the specific sequences for the gene being amplified, all of the primers used to amplify one type of enzyme (for example the acyl carrier protein in polyketide synthesis) are synthesized to contain an additional sequence of 20-40 bases 5′ to the gene (sequence A) and a different 20-40 base sequence 3′ to the gene (sequence B). The adjacent gene (in this case the keto-synthase) is amplified using a 5′ primer which contains the complementary strand of sequence B (sequence B′), and a 3′ primer containing a different 20-40 base sequence (C). Similarly, primers for the next adjacent gene (keto-reductases) contain sequences C′ (complementary to C) and D. If 5 different polyketide gene clusters are being shuffled, all five acyl carrier proteins are flanked by sequences A and B following their PCR amplification. In this way, small regions of homology are introduced, making the gene clusters into site-specific recombination cassettes. Subsequent to the initial amplification of individual genes, the amplified genes can then be mixed and subjected to primerless PCR. Sequence B at the 3′ end of all of the five acyl carrier protein genes can anneal with and prime DNA synthesis from sequence B′ at the 5′ end of all five keto reductase genes. In this way all possible combinations of genes within the cluster can be obtained. Oligonucleotides allow such recombinants to be obtained in the absence of sufficient sequence homology for recursive sequence recombination described above. Only homology of function is required to produce functional gene clusters. [0053]
  • This method is also useful for exploring permutations of any other multi-subunit enzymes. An example of such enzymes composed of multiple polypeptides that have shown novel functions when the subunits are combined in novel ways are dioxygenases. Directed recombination between the four protein subunits of biphenyl and toluene dioxygenases produced functional dioxygenases with increased activity against trichloroethylene (Furukawa et. al. [0054] J. Bacteriol. 176: 2121-2123 (1994)). This combination of subunits from the two dioxygenases could also have been produced by cassette-shuffling of the dioxygenases as described above, followed by selection for degradation of trichloroethylene.
  • In some polyketide synthases, the separate functions of the acyl carrier protein, keto-synthase, keto-reductase, etc. reside in a single polypeptide. In these cases domains within the single polypeptide may be shuffled, even if sufficient homology does not exist naturally, by introducing regions of homology as described above for entire genes. In this case, it may not be possible to introduce additional flanking sequences to the domains, due to the constraint of maintaining a continuous open reading frame. Instead, groups of oligonucleotides are synthesized that are homologous to the 3′ end of the first domain encoded by one of the genes to be shuffled, and the 5′ ends of the second domains encoded by all of the other genes to be shuffled together. This is repeated with all domains, thus providing sequences that allow recombination between protein domains while maintaining their order. [0055]
  • The cassette-based recombination method can be combined with recursive sequence recombination by including gene fragments (generated by DNase, physical shearing, DNA stuttering, etc.) for one or more of the genes. Thus, in addition to different combinations of entire genes within a cluster (e.g., for polyketide synthesis), individual genes can be shuffled at the same time (e.g., all acyl carrier protein genes can also be provided as fragmented DNA), allowing a more thorough search of sequence space. [0056]
  • (2) In Vivo Formats [0057]
    • (a) Plasmid-plasmid Recombination
  • The initial substrates for recombination are a collection of polynucleotides comprising variant forms of a gene. The variant forms usually show substantial sequence identity to each other sufficient to allow homologous recombination between substrates. The diversity between the polynucleotides can be natural (e.g., allelic or species variants), induced (e.g., error-prone PCR or error-prone recursive sequence recombination), or the result of in vitro recombination. Diversity can also result from resynthesizing genes encoding natural proteins with alternative codon usage. There should be at least sufficient diversity between substrates that recombination can generate more diverse products than there are starting materials. There must be at least two substrates differing in at least two positions. However, commonly a library of substrates of 10[0058] 3-108 members is employed. The degree of diversity depends on the length of the substrate being recombined and the extent of the functional change to be evolved. Diversity at between 0.1-25% of positions is typical. The diverse substrates are incorporated into plasmids. The plasmids are often standard cloning vectors, e.g., bacterial multicopy plasmids. However, in some methods to be described below, the plasmids include mobilization (MOB) functions. The substrates can be incorporated into the same or different plasmids. Often at least two different types of plasmid having different types of selectable markers are used to allow selection for cells containing at least two types of vector. Also, where different types of plasmid are employed, the different plasmids can come from two distinct incompatibility groups to allow stable co-existence of two different plasmids within the cell. Nevertheless, plasmids from the same incompatibility group can still co-exist within the same cell for sufficient time to allow homologous recombination to occur.
  • Plasmids containing diverse substrates are initially introduced into cells by any method (e.g., chemical transformation, natural competence, electroporation, biolistics, packaging into phage or viral systems). Often, the plasmids are present at or near saturating concentration (with respect to maximum transfection capacity) to increase the probability of more than one plasmid entering the same cell. The plasmids containing the various substrates can be transfected simultaneously or in multiple rounds. For example, in the latter approach cells can be transfected with a first aliquot of plasmid, transfectants selected and propagated, and then infected with a second aliquot of plasmid. [0059]
  • Having introduced the plasmids into cells, recombination between substrates to generate recombinant genes occurs within cells containing multiple different plasmids merely by propagating the cells. However, cells that receive only one plasmid are unable to participate in recombination and the potential contribution of substrates on such plasmids to evolution is not fully exploited (although these plasmids may contribute to some extent if they are progagated in mutator cells). The rate of evolution can be increased by allowing all substrates to participate in recombination. Such can be achieved by subjecting transfected cells to electroporation. The conditions for electroporation are the same as those conventionally used for introducing exogenous DNA into cells (e.g., 1,000-2,500 volts, 400 μF and a 1-2 mM gap). Under these conditions, plasmids are exchanged between cells allowing all substrates to participate in recombination. In addition the products of recombination can undergo further rounds of recombination with each other or with the original substrate. The rate of evolution can also be increased by use of conjugative transfer. To exploit conjugative transfer, substrates can be cloned into plasmids having MOB genes, and tra genes are also provided in cis or in trans to the MOB genes. The effect of conjugative transfer is very similar to electroporation in that it allows plasmids to move between cells and allows recombination between any substrate and the products of previous recombination to occur, merely by propagating the culture. The rate of evolution can also be increased by fusing cells to induce exchange of plasmids or chromosomes. Fusion can be induced by chemical agents, such as PEG, or viral proteins, such as influenza virus hemagglutinin, HSV-1 gB and gD. The rate of evolution can also be increased by use of mutator host cells (e.g., Mut L, S, D, T, H in bacteria and Ataxia telangiectasia human cell lines). [0060]
  • The time for which cells are propagated and recombination is allowed to occur, of course, varies with the cell type but is generally not critical, because even a small degree of recombination can substantially increase diversity relative to the starting materials. Cells bearing plasmids containing recombined genes are subject to screening or selection for a desired function. For example, if the substrate being evolved contains a drug resistance gene, one would select for drug resistance. Cells surviving screening or selection can be subjected to one or more rounds of screening/selection followed by recombination or can be subjected directly to an additional round of recombination. [0061]
  • The next round of recombination can be achieved by several different formats independently of the previous round. For example, a further round of recombination can be effected simply by resuming the electroporation or conjugation-mediated intercellular transfer of plasmids described above. Alternatively, a fresh substrate or substrates, the same or different from previous substrates, can be transfected into cells surviving selection/screening. optionally, the new substrates are included in plasmid vectors bearing a different selective marker and/or from a different incompatibility group than the original plasmids. As a further alternative, cells surviving selection/screening can be subdivided into two subpopulations, and plasmid DNA from one subpopulation transfected into the other, where the substrates from the plasmids from the two subpopulations undergo a further round of recombination. In either of the latter two options, the rate of evolution can be increased by employing DNA extraction, electroporation, conjugation or mutator cells, as described above. In a still further variation, DNA from cells surviving screening/selection can be extracted and subjected to in vitro recursive sequence recombination. [0062]
  • After the second round of recombination, a second round of screening/selection is performed, preferably under conditions of increased stringency. If desired, further rounds of recombination and selection/screening can be performed using the same strategy as for the second round. With successive rounds of recombination and selection/screening, the surviving recombined substrates evolve toward acquisition of a desired phenotype. Typically, in this and other methods of recursive recombination, the final product of recombination that has acquired the desired phenotype differs from starting substrates at 0.1%-25% of positions and has evolved at a rate orders of magnitude in excess (e.g., by at least 10-fold, 100-fold, 1000-fold, or 10,000 fold) of the rate of naturally acquired mutation of about 1 mutation per 10[0063] −9 positions per generation (see Anderson et al. Proc. Natl. Acad. Sci. U.S.A. 93:906-907 (1996)). The final products may be transferred to another host more desirable for utilization of the “shuffled” DNA. This is particularly advantageous in situations where the more desirable host is less efficient as a host for the many cycles of mutation/recombination due to the lack of molecular biology or genetic tools available for other organisms such as E. coli.
    • (b) Virus-plasmid Recombination
  • The strategy used for plasmid-plasmid recombination can also be used for virus-plasmid recombination; usually, phage-plasmid recombination. However, some additional comments particular to the use of viruses are appropriate. The initial substrates for recombination are cloned into both plasmid and viral vectors. It is usually not critical which substrate(s) are inserted into the viral vector and which into the plasmid, although usually the viral vector should contain different substrate(s) from the plasmid. As before, the plasmid (and the virus) typically contains a selective marker. The plasmid and viral vectors can both be introduced into cells by transfection as described above. However, a more efficient procedure is to transfect the cells with plasmid, select transfectants and infect the transfectants with virus. Because the efficiency of infection of many viruses approaches 100% of cells, most cells transfected and infected by this route contain both a plasmid and virus bearing different substrates. [0064]
  • Homologous recombination occurs between plasmid and virus generating both recombined plasmids and recombined virus. For some viruses, such as filamentous phage, in which intracellular DNA exists in both double-stranded and single-stranded forms, both can participate in recombination. Provided that the virus is not one that rapidly kills cells, recombination can be augmented by use of electroporation or conjugation to transfer plasmids between cells. Recombination can also be augmented for some types of virus by allowing the progeny virus from one cell to reinfect other cells. For some types of virus, virus infected-cells show resistance to superinfection. However, such resistance can be overcome by infecting at high multiplicity and/or using mutant strains of the virus in which resistance to superinfection is reduced. [0065]
  • The result of infecting plasmid-containing cells with virus depends on the nature of the virus. Some viruses, such as filamentous phage, stably exist with a plasmid in the cell and also extrude progeny phage from the cell. Other viruses, such as lambda having a cosmid genome, stably exist in a cell like plasmids without producing progeny virions. Other viruses, such as the T-phage and lytic lambda, undergo recombination with the plasmid but ultimately kill the host cell and destroy plasmid DNA. For viruses that infect cells without killing the host, cells containing recombinant plasmids and virus can be screened/selected using the same approach as for plasmid-plasmid recombination. Progeny virus extruded by cells surviving selection/screening can also be collected and used as substrates in subsequent rounds of recombination. For viruses that kill their host cells, recombinant genes resulting from recombination reside only in the progeny virus. If the screening or selective assay requires expression of recombinant genes in a cell, the recombinant genes should be transferred from the progeny virus to another vector, e.g., a plasmid vector, and retransfected into cells before selection/screening is performed. [0066]
  • For filamentous phage, the products of recombination are present in both cells surviving recombination and in phage extruded from these cells. The dual source of recombinant products provides some additional options relative to the plasmid-plasmid recombination. For example, DNA can be isolated from phage particles for use in a round of in vitro recombination. Alternatively, the progeny phage can be used to transfect or infect cells surviving a previous round of screening/selection, or fresh cells transfected with fresh substrates for recombination. [0067]
    • (c) Virus-virus Recombination
  • The principles described for plasmid-plasmid and plasmid-viral recombination can be applied to virus-virus recombination with a few modifications. The initial substrates for recombination are cloned into a viral vector. Usually, the same vector is used for all substrates. Preferably, the virus is one that, naturally or as a result of mutation, does not kill cells. After insertion, some viral genomes can be packaged in vitro or using a packaging cell line. The packaged viruses are used to infect cells at high multiplicity such that there is a high probability that a cell will receive multiple viruses bearing different substrates. [0068]
  • After the initial round of infection, subsequent steps depend on the nature of infection as discussed in the previous section. For example, if the viruses have phagemid genomes such as lambda cosmids or M13, F1 or Fd phagemids, the phagemids behave as plasmids within the cell and undergo recombination simply by propagating the cells. Recombination is particularly efficient between single-stranded forms of intracellular DNA. Recombination can be augmented by electroporation of cells. [0069]
  • Following selection/screening, cosmids containing recombinant genes can be recovered from surviving cells, e.g., by heat induction of a cos[0070] − lysogenic host cell, or extraction of DNA by standard procedures, followed by repackaging cosmid DNA in vitro.
  • If the viruses are filamentous phage, recombination of replicating form DNA occurs by propagating the culture of infected cells. Selection/screening identifies colonies of cells containing viral vectors having recombinant genes with improved properties, together with phage extruded from such cells. Subsequent options are essentially the same as for plasmid-viral recombination. [0071]
    • (d) Chromosome Recombination
  • This format can be used to especially evolve chromosomal substrates. The format is particularly useful in situations in which many chromosomal genes contribute to a phenotype or one does not know the exact location of the chromosomal gene(s) to be evolved. The initial substrates for recombination are cloned into a plasmid vector. If the chromosomal gene(s) to be evolved are known, the substrates constitute a family of sequences showing a high degree of sequence identity but some divergence from the chromosomal gene. If the chromosomal genes to be evolved have not been located, the initial substrates usually constitute a library of DNA segments of which only a small number show sequence identity to the gene or gene(s) to be evolved. Divergence between plasmid-borne substrate and the chromosomal gene(s) can be induced by mutagenesis or by obtaining the plasmid-borne substrates from a different species than that of the cells bearing the chromosome. [0072]
  • The plasmids bearing substrates for recombination are transfected into cells having chromosomal gene(s) to be evolved. Evolution can occur simply by propagating the culture, and can be accelerated by transferring plasmids between cells by conjugation or electroporation. Evolution can be further accelerated by use of mutator host cells or by seeding a culture of nonmutator host cells being evolved with mutator host cells and inducing intercellular transfer of plasmids by electroporation or conjugation. Preferably, mutator host cells used for seeding contain a negative selectable marker to facilitate isolation of a pure culture of the nonmutator cells being evolved. Selection/screening identifies cells bearing chromosomes and/or plasmids that have evolved toward acquisition of a desired function. [0073]
  • Subsequent rounds of recombination and selection/screening proceed in similar fashion to those described for plasmid-plasmid recombination. For example, further recombination can be effected by propagating cells surviving recombination in combination with electroporation or conjugative transfer of plasmids. Alternatively, plasmids bearing additional substrates for recombination can be introduced into the surviving cells. Preferably, such plasmids are from a different incompatibility group and bear a different selective marker than the original plasmids to allow selection for cells containing at least two different plasmids. As a further alternative, plasmid and/or chromosomal DNA can be isolated from a subpopulation of surviving cells and transfected into a second subpopulation. Chromosomal DNA can be cloned into a plasmid vector before transfection. [0074]
    • (e) Virus-chromosome Recombination
  • As in the other methods described above, the virus is usually one that does not kill the cells, and is often a phage or phagemid. The procedure is substantially the same as for plasmid-chromosome recombination. Substrates for recombination are cloned into the vector. Vectors including the substrates can then be transfected into cells or in vitro packaged and introduced into cells by infection. Viral genomes recombine with host chromosomes merely by propagating a culture. Evolution can be accelerated by allowing intercellular transfer of viral genomes by electroporation, or reinfection of cells by progeny virions. Screening/selection identifies cells having chromosomes and/or viral genomes that have evolved toward acquisition of a desired function. [0075]
  • There are several options for subsequent rounds of recombination. For example, viral genomes can be transferred between cells surviving selection/recombination by electroporation. Alternatively, viruses extruded from cells surviving selection/screening can be pooled and used to superinfect the cells at high multiplicity. Alternatively, fresh substrates for recombination can be introduced into the cells, either on plasmid or viral vectors. [0076]
  • II. Recursive Sequence Recombination Techniques for Metabolic and Cellular Engineering [0077]
  • A. Starting Materials [0078]
  • Thus, a general method for recursive sequence recombination for the embodiments herein is to begin with a gene encoding an enzyme or enzyme subunit and to evolve that gene either for ability to act on a new substrate, or for enhanced catalytic properties with an old substrate, either alone or in combination with other genes in a multistep pathway. The term “gene” is used herein broadly to refer to any segment or sequence of DNA associated with a biological function. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters. The ability to use a new substrate can be assayed in some instances by the ability to grow on a substrate as a nutrient source. In other circumstances such ability can be assayed by decreased toxicity of a substrate for a host cell, hence allowing the host to grow in the presence of that substrate. Biosynthesis of new compounds, such as antibiotics, can be assayed similarly by growth of an indicator organism in the presence of the host expressing the evolved genes. For example, when an indicator organism used in an overlay of the host expressing the evolved gene(s), wherein the indicator organism is sensitive or expected to be sensitive to the desired antibiotic, growth of the indicator organism would be inhibited in a zone around the host cell or colony expressing the evolved gene(s). [0079]
  • Another method of identifying new compounds is the use of standard analytical techniques such as mass spectroscopy, nuclear magnetic resonance, high performance liquid chromatography, etc. Recombinant microorganisms can be pooled and extracts or media supernatants assayed from these pools. Any positive pool can then be subdivided and the procedure repeated until the single positive is identified (“sib-selection”). [0080]
  • In some instances, the starting material for recursive sequence recombination is a discrete gene, cluster of genes, or family of genes known or thought to be associated with metabolism of a particular class of substrates. [0081]
  • One of the advantages of the instant invention is that structural information is not required to estimate which parts of a sequence should be mutated to produce a functional hybrid enzyme. [0082]
  • In some embodiments of the invention, an initial screening of enzyme activities in a particular assay can be useful in identifying candidate enzymes as starting materials. For example, high throughput screening can be used to screen enzymes for dioxygenase-type activities using aromatic acids as substrates. Dioxygenases typically transform indole-2-carboxylate and indole-3-carboxylate to colored products, including indigo (Eaton et. al. [0083] J. Bacteriol. 177:6983-6988 (1995)). DNA encoding enzymes that give some activity in the initial assay can then be recombined by the recursive techniques of the invention and rescreened. The use of such initial screening for candidate enzymes against a desired target molecule or analog of the target molecule can be especially useful to generate enzymes that catalyze reactions of interest such as catabolism of man-made pollutants.
  • The starting material can also be a segment of such a gene or cluster that is recombined in isolation of its surrounding DNA, but is relinked to its surrounding DNA before screening/selection of recombination products. In other instances, the starting material for recombination is a larger segment of DNA that includes a coding sequence or other locus associated with metabolism of a particular substrate at an unknown location. For example, the starting material can be a chromosome, episome, YAC, cosmid, or phage P1 clone. In still other instances, the starting material is the whole genome of an organism that is known to have desirable metabolic properties, but for which no information localizing the genes associated with these characteristics is available. [0084]
  • In general any type of cells can be used as a recipient of evolved genes. Cells of particular interest include many bacterial cell types, both gram-negative and gram-positive, such as Rhodococcus, Streptomycetes, Actinomycetes, Corynebacteria, Penicillium, Bacillus, [0085] Escherichia coli, Pseudomonas, Salmonella, and Erwinia. Cells of interest also include eukaryotic cells, particularly mammalian cells (e.g., mouse, hamster, primate, human), both cell lines and primary cultures. Such cells include stem cells, including embryonic stem cells, zygotes, fibroblasts, lymphocytes, Chinese hamster ovary (CHO), mouse fibroblasts (NIH3T3), kidney, liver, muscle, and skin cells. Other eukaryotic cells of interest include plant cells, such as maize, rice, wheat, cotton, soybean, sugarcane, tobacco, and arabidopsis; fish, algae, fungi (Penicillium, Fusarium, Aspergillus, Podospora, Neurospora), insects, yeasts (Picchia and Saccharomyces).
  • The choice of host will depend on a number of factors, depending on the intended use of the engineered host, including pathogenicity, substrate range, environmental hardiness, presence of key intermediates, ease of genetic manipulation, and likelihood of promiscuous transfer of genetic information to other organisms. Particularly advantageous hosts are [0086] E. coli, lactobacilli, Streptomycetes, Actinomycetes and filamentous fungi.
  • The breeding procedure starts with at least two substrates, which generally show substantial sequence identity to each other (i.e., at least about 50%, 70%, 80% or 90% sequence identity) but differ from each other at certain positions. The difference can be any type of mutation, for example, substitutions, insertions and deletions. Often, different segments differ from each other in perhaps 5-20 positions. For recombination to generate increased diversity relative to the starting materials, the starting materials must differ from each other in at least two nucleotide positions. That is, if there are only two substrates, there should be at least two divergent positions. If there are three substrates, for example, one substrate can differ from the second as a single position, and the second can differ from the third at a different single position. The starting DNA segments can be natural variants of each other, for example, allelic or species variants. The segments can also be from nonallelic genes showing some degree of structural and usually functional relatedness (e.g., different genes within a superfamily such as the immunoglobulin superfamily). The starting DNA segments can also be induced variants of each other. For example, one DNA segment can be produced by error-prone PCR replication of the other, or by substitution of a mutagenic cassette. Induced mutants can also be prepared by propagating one (or both) of the segments in a mutagenic strain. In these situations, strictly speaking, the second DNA segment is not a single segment but a large family of related segments. The different segments forming the starting materials are often the same length or substantially the same length. However, this need not be the case; for example; one segment can be a subsequence of another. The segments can be present as part of larger molecules, such as vectors, or can be in isolated form. [0087]
  • The starting DNA segments are recombined by any of the recursive sequence recombination formats described above to generate a diverse library of recombinant DNA segments. Such a library can vary widely in size from having fewer than 10 to more than 10[0088] 5, 107, or 109 members. In general, the starting segments and the recombinant libraries generated include full-length coding sequences and any essential regulatory sequences, such as a promoter and polyadenylation sequence, required for expression. However, if this is not the case, the recombinant DNA segments in the library can be inserted into a common vector providing the missing sequences before performing screening/selection.
  • If the recursive sequence recombination format employed is an in vivo format, the library of recombinant DNA segments generated already exists in a cell, which is usually the cell type in which expression of the enzyme with altered substrate specificity is desired. If recursive sequence recombination is performed in vitro, the recombinant library is preferably introduced into the desired cell type before screening/selection. The members of the recombinant library can be linked to an episome or virus before introduction or can be introduced directly. In some embodiments of the invention, the library is amplified in a first host, and is then recovered from that host and introduced to a second host more amenable to expression, selection, or screening, or any other desirable parameter. The manner in which the library is introduced into the cell type depends on the DNA-uptake characteristics of the cell type, e.g., having viral receptors, being capable of conjugation, or being naturally competent. If the cell type is insusceptible to natural and chemical-induced competence, but susceptible to electroporation, one would usually employ electroporation. If the cell type is insusceptible to electroporation as well, one can employ biolistics. The biolistic PDS-1000 Gene Gun (Biorad, Hercules, Calif.) uses helium pressure to accelerate DNA-coated gold or tungsten microcarriers toward target cells. The process is applicable to a wide range of tissues, including plants, bacteria, fungi, algae, intact animal tissues, tissue culture cells, and animal embryos. One can employ electronic pulse delivery, which is essentially a mild electroporation format for live tissues in animals and patients. Zhao, [0089] Advanced Drug Delivery Reviews 17:257-262 (1995). Novel methods for making cells competent are described in co-pending application U.S. patent application Ser. No. 08/621,430, filed Mar. 25, 1996. After introduction of the library of recombinant DNA genes, the cells are optionally propagated to allow expression of genes to occur.
  • B. Selection and Screening [0090]
  • Screening is, in general, a two-step process in which one first determines which cells do and do not express a screening marker and then physically separates the cells having the desired property. Selection is a form of screening in which identification and physical separation are achieved simultaneously, for example, by expression of a selectable marker, which, in some genetic circumstances, allows cells expressing the marker to survive while other cells die (or vice versa). Screening markers include, for example, luciferase, β-galactosidase, and green fluorescent protein. Screening can also be done by observing such aspects of growth as colony size, halo formation, etc. Additionally, screening for production of a desired compound, such as a therapeutic drug or “designer chemical” can be accomplished by observing binding of cell products to a receptor or ligand, such as on a solid support or on a column. Such screening can additionally be accomplished by binding to antibodies, as in an ELISA. In some instances the screening process is preferably automated so as to allow screening of suitable numbers of colonies or cells. Some examples of automated screening devices include fluorescence activated cell sorting, especially in conjunction with cells immobilized in agarose (see Powell et. al. [0091] Bio/Technology 8:333-337 (1990); Weaver et. al. Methods 2:234-247 (1991)), automated ELISA assays, etc. Selectable markers can include, for example, drug, toxin resistance, or nutrient synthesis genes. Selection is also done by such techniques as growth on a toxic substrate to select for hosts having the ability to detoxify a substrate, growth on a new nutrient source to select for hosts having the ability to utilize that nutrient source, competitive growth in culture based on ability to utilize a nutrient source, etc.
  • In particular, uncloned but differentially expressed proteins (e.g., those induced in response to new compounds, such as biodegradable pollutants in the medium) can be screened by differential display (Appleyard et al. [0092] Mol. Gen. Gent. 247:338-342 (1995)). Hopwood (Phil Trans R. Soc. Lond B 324:549-562) provides a review of screens for antibiotic production. Omura (Microbio. Rev. 50:259-279 (1986) and Nisbet (Ann Rep. Med. Chem. 21:149-157 (1986)) disclose screens for antimicrobial agents, including supersensitive bacteria, detection of β-lactamase and D,D-carboxypeptidase inhibition, β-lactamase induction, chromogenic substrates and monoclonal antibody screens. Antibiotic targets can also be used as screening targets in high throughput screening. Antifungals are typically screened by inhibition of fungal growth. Pharmacological agents can be identified as enzyme inhibitors using plates containing the enzyme and a chromogenic substrate, or by automated receptor assays. Hydrolytic enzymes (e.g., proteases, amylases) can be screened by including the substrate in an agar plate and scoring for a hydrolytic clear zone or by using a calorimetric indicator (Steele et al. Ann. Rev. Microbiol. 45:89-106 (1991)). This can be coupled with the use of stains to detect the effects of enzyme action (such as congo red to detect the extent of degradation of celluloses and hemicelluloses). Tagged substrates can also be used. For example, lipases and esterases can be screened using different lengths of fatty acids linked to umbelliferyl. The action of lipases or esterases removes this tag from the fatty acid, resulting in a quenching of umbelliferyl fluorescence. These enzymes can be screened in microtiter plates by a robotic device.
  • Fluorescence activated cell sorting (FACS) methods are also a powerful tool for selection/screening. In some instances a fluorescent molecule is made within a cell (e.g., green fluorescent protein). The cells producing the protein can simply be sorted by FACS. Gel microdrop technology allows screening of cells encapsulated in agarose microdrops (Weaver et al. [0093] Methods 2:234-247 (1991)). In this technique products secreted by the cell (such as antibodies or antigens) are immobilized with the cell that generated them. Sorting and collection of the drops containing the desired product thus also collects the cells that made the product, and provides a ready source for the cloning of the genes encoding the desired functions. Desired products can be detected by incubating the encapsulated cells with fluorescent antibodies (Powell et al. Bio/Technology 8:333-337 (1990)). FACS sorting can also be used by this technique to assay resistance to toxic compounds and antibiotics by selecting droplets that contain multiple cells (i.e., the product of continued division in the presence of a cytotoxic compound; Goguen et al. Nature 363:189-190 (1995)). This method can select for any enzyme that can change the fluorescence of a substrate that can be immobilized in the agarose droplet.
  • In some embodiments of the invention, screening can be accomplished by assaying reactivity with a reporter molecule reactive with a desired feature of, for example, a gene product. Thus, specific functionalities such as antigenic domains can be screened with antibodies specific for those determinants. [0094]
  • In other embodiments of the invention, screening is preferably done with a cell-cell indicator assay. In this assay format, separate library cells (Cell A, the cell being assayed) and reporter cells (Cell B, the assay cell) are used. Only one component of the system, the library cells, is allowed to evolve. The screening is generally carried out in a two-dimensional immobilized format, such as on plates. The products of the metabolic pathways encoded by these genes (in this case, usually secondary metabolites such as antibiotics, polyketides, carotenoids, etc.) diffuse out of the library cell to the reporter cell. The product of the library cell may affect the reporter cell in one of a number of ways. [0095]
  • The assay system (indicator cell) can have a simple readout (e.g., green fluorescent protein, luciferase, β-galactosidase) which is induced by the library cell product but which does not affect the library cell. In these examples the desired product can be detected by calorimetric changes in the reporter cells adjacent to the library cell. [0096]
  • In other embodiments, indicator cells can in turn produce something that modifies the growth rate of the library cells via a feedback mechanism. Growth rate feedback can detect and accumulate very small differences. For example, if the library and reporter cells are competing for nutrients, library cells producing compounds to inhibit the growth of the reporter cells will have more available nutrients, and thus will have more opportunity for growth. This is a useful screen for antibiotics or a library of polyketide synthesis gene clusters where each of the library cells is expressing and exporting a different polyketide gene product. [0097]
  • Another variation of this theme is that the reporter cell for an antibiotic selection can itself secrete a toxin or antibiotic that inhibits growth of the library cell. Production by the library cell of an antibiotic that is able to suppress growth of the reporter cell will thus allow uninhibited growth of the library cell. [0098]
  • Conversely, if the library is being screened for production of a compound that stimulates the growth of the reporter cell (for example, in improving chemical syntheses, the library cell may supply nutrients such as amino acids to an auxotrophic reporter, or growth factors to a growth-factor-dependent reporter. The reporter cell in turn should produce a compound that stimulates the growth of the library cell. Interleukins, growth factors, and nutrients are possibilities. [0099]
  • Further possibilities include competition based on ability to kill surrounding cells, positive feedback loops in which the desired product made by the evolved cell stimulates the indicator cell to produce a positive growth factor for cell A, thus indirectly selecting for increased product formation. [0100]
  • In some embodiments of the invention it can be advantageous to use a different organism (or genetic background) for screening than the one that will be used in the final product. For example, markers can be added to DNA constructs used for recursive sequence recombination to make the microorganism dependent on the constructs during the improvement process, even though those markers may be undesirable in the final recombinant microorganism. [0101]
  • Likewise, in some embodiments it is advantageous to use a different substrate for screening an evolved enzyme than the one that will be used in the final product. For example, Evnin et al. ([0102] Proc. Natl. Acad. Sci. U.S.A. 87:6659-6663 (1990)) selected trypsin variants with altered substrate specificity by requiring that variant trypsin generate an essential amino acid for an arginine auxotroph by cleaving arginine β-naphthylamide. This is thus a selection for arginine-specific trypsin, with the growth rate of the host being proportional to that of the enzyme activity.
  • The pool of cells surviving screening and/or selection is enriched for recombinant genes conferring the desired phenotype (e.g. altered substrate specificity, altered biosynthetic ability, etc.). Further enrichment can be obtained, if desired, by performing a second round of screening and/or selection without generating additional diversity. [0103]
  • The recombinant gene or pool of such genes surviving one round of screening/selection forms one or more of the substrates for a second round of recombination. Again, recombination can be performed in vivo or in vitro by any of the recursive sequence recombination formats described above. If recursive sequence recombination is performed in vitro, the recombinant gene or genes to form the substrate for recombination should be extracted from the cells in which screening/selection was performed. Optionally, a subsequence of such gene or genes can be excised for more targeted subsequent recombination. If the recombinant gene(s) are contained within episomes, their isolation presents no difficulties. If the recombinant genes are chromosomally integrated, they can be isolated by amplification primed from known sequences flanking the regions in which recombination has occurred. Alternatively, whole genomic DNA can be isolated, optionally amplified, and used as the substrate for recombination. Small samples of genomic DNA can be amplified by whole genome amplification with degenerate primers (Barrett et al. [0104] Nucleic Acids Research 23:3488-3492 (1995)). These primers result in a large amount of random 3′ ends, which can undergo homologous recombination when reintroduced into cells.
  • If the second round of recombination is to be performed in vivo, as is often the case, it can be performed in the cell surviving screening/selection, or the recombinant genes can be transferred to another cell type (e.g., a cell type having a high frequency of mutation and/or recombination). In this situation, recombination can be effected by introducing additional DNA segment(s) into cells bearing the recombinant genes. In other methods, the cells can be induced to exchange genetic information with each other by, for example, electroporation. In some methods, the second round of recombination is performed by dividing a pool of. cells surviving screening/selection in the first round into two subpopulations. DNA from one subpopulation is isolated and transfected into the other population, where the recombinant gene(s) from the two subpopulations recombine to form a further library of recombinant genes. In these methods, it is not necessary to isolate particular genes from the first subpopulation or to take steps to avoid random shearing of DNA during extraction. Rather, the whole genome of DNA sheared or otherwise cleaved into manageable sized fragments is transfected into the second subpopulation. This approach is particularly useful when several genes are being evolved simultaneously and/or the location and identity of such genes within chromosome are not known. [0105]
  • The second round of recombination is sometimes performed exclusively among the recombinant molecules surviving selection. However, in other embodiments, additional substrates can be introduced. The additional substrates can be of the same form as the substrates used in the first round of recombination, i.e., additional natural or induced mutants of the gene or cluster of genes, forming the substrates for the first round. Alternatively, the additional substrate(s) in the second round of recombination can be exactly the same as the substrate(s) in the first round of replication. [0106]
  • After the second round of recombination, recombinant genes conferring the desired phenotype are again selected. The selection process proceeds essentially as before. If a suicide vector bearing a selective marker was used in the first round of selection, the same vector can be used again. Again, a cell or pool of cells surviving selection is selected. If a pool of cells, the cells can be subject to further enrichment. [0107]
  • III. Recursive Sequence Recombination of Genes For Bioremediation [0108]
  • Modern industry generates many pollutants for which the environment can no longer be considered an infinite sink. Naturally occurring microorganisms are able to metabolize thousands of organic compounds, including many not found in nature (e.g xenobiotics). Bioremediation, the deliberate use of microorganisms for the biodegradation of man-made wastes, is an emerging technology that offers cost and practicality advantages over traditional methods of disposal. The success of bioremediation depends on the availability of organisms that are able to detoxify or mineralize pollutants. Microorganisms capable of degrading specific pollutants can be generated by genetic engineering and recursive sequence recombination. [0109]
  • Although bioremediation is an aspect of pollution control, a more useful approach in the long term is one of prevention before industrial waste is pumped into the environment. Exposure of industrial waste streams to recursive sequence recombination-generated microorganisms capable of degrading the pollutants they contain would result in detoxification of mineralization of these pollutants before the waste stream enters the environment. Issues of releasing recombinant organisms can be avoided by containing them within bioreactors fitted to the industrial effluent pipes. This approach would also allow the microbial mixture used to be adjusted to best degrade the particular wastes being produced. Finally, this method would avoid the problems of adapting to the outside world and dealing with competition that face many laboratory microorganisms. [0110]
  • In the wild, microorganisms have evolved new catabolic activities enabling them to exploit pollutants as nutrient sources for which there is no competition. However, pollutants that are present at low concentrations in the environment may not provide a sufficient advantage to stimulate the evolution of catabolic enzymes. For a review of such naturally occurring evolution of biodegradative pathways and the manipulation of some of microorganisms by classical techniques, see Ramos et al., [0111] Bio/Technology 12:1349-1355 (1994).
  • Generation of new catabolic enzymes or pathways for bioremediation has thus relied upon deliberate transfer of specific genes between organisms (Wackett et al., supra), forced matings between bacteria with specific catabolic capabilities (Brenner et al. [0112] Biodegradation 5:359-377 (1994)), or prolonged selection in a chemostat. Some researchers have attempted to facilitate evolution via naturally occurring genetic mechanisms in their chemostat selections by including microorganisms with a variety of catabolic pathways (Kellogg et. al. Science 214:1133-1135 (1981); Chakrabarty American Society of Micro. Biol. News 62:130-137 (1996)). For a review of efforts in this area, see Cameron et al. Applied Biochem. Biotech. 38:105-140 (1993).
  • Current efforts in improving organisms for bioremediation take a labor-intensive approach in which many parameters are optimized independently, including transcription efficiency from native and heterologous promoters, regulatory circuits and translational efficiency as well as improvement of protein stability and activity (Timmis et al. [0113] Ann. Rev. Microbiol. 48:525-527 (1994)).
  • A recursive sequence recombination approach overcomes a number of limitations in the bioremediation capabilities of naturally occurring microorganisms. Both enzyme activity and specificity can be altered, simultaneously or sequentially, by the methods of the invention. For example, catabolic enzymes can be evolved to increase the rate at which they act on a substrate. Although knowledge of a rate-limiting step in a metabolic pathway is not required to practice the invention, rate-limiting proteins in pathways can be evolved to have increased expression and/or activity, the requirement for inducing substances can be eliminated, and enzymes can be evolved that catalyze novel reactions. [0114]
  • Some examples of chemical targets for bioremediation include but are not limited to benzene, xylene, and toluene, camphor, naphthalene, halogenated hydrocarbons, polychlorinated biphenyls (PCBs), trichlorethylene, pesticides such as pentachlorophenyls (PCPs), and herbicides such as atrazine. [0115]
  • A. Aromatic Hydrocarbons [0116]
  • Preferably, when an enzyme is “evolved” to have a new catalytic function, that function is expressed, either constitutively or in response to the new substrate. Recursive sequence recombination subjects both structural and regulatory elements (including the structure of regulatory proteins) of a protein to recombinogenic mutagenesis simultaneously. Selection of mutants that are efficiently able to use the new substrate as a nutrient source will be sufficient to ensure that both the enzyme and its regulation are optimized, without detailed analysis of either protein structure or operon regulation. [0117]
  • Examples of aromatic hydrocarbons include but are not limited to benzene, xylene, toluene, biphenyl, and polycyclic aromatic hydrocarbons such as pyrene and naphthalene. These compounds are metabolized via catechol intermediates. Degradation of catechol by [0118] Pseudomonas putida requires induction of the catabolic operon by cis, cis-muconate which acts on the CatR regulatory protein. The binding site for the CatR protein is G-N11-A, while the optimal sequence for the LysR class of activators (of which CatR is a member) is T-N11-A. Mutation of the G to a T in the CatR binding site enhances the expression of catechol metabolizing genes (Chakrabarty, American Society of Microbiology News 62:130-137 (1996)). This demonstrates that the control of existing catabolic pathways is not optimized for the metabolism of specific xenobiotics. It is also an example of a type of mutant that would be expected from recursive sequence recombination of the operon followed by selection of bacteria that are better able to degrade the target compound.
  • As an example of starting materials, dioxygenases are required for many pathways in which aromatic compounds are catabolized. Even small differences in dioxygenase sequence can lead to significant differences in substrate specificity (Furukawa et al. [0119] J. Bact. 175:5224-5232 (1993); Erickson et al. App. Environ. Micro. 59:3858-3862 (1993)). A hybrid enzyme made using sequences derived from two “parental” enzymes may possess catalytic activities that are intermediate between the parents (Erickson, ibid.), or may actually be better than either parent for a specific reaction (Furukawa et al. J. Bact. 176:2121-2123 (1994)). In one of these cases site directed mutagenesis was used to generate a single polypeptide with hybrid sequence (Erickson, ibid.); in the other, a four subunit enzyme was produced by expressing two subunits from each of two different dioxygenases (Furukawa, ibid.). Thus, sequences from one or more genes encoding dioxygenases can be used in the recursive sequence recombination techniques of the instant invention, to generate enzymes with new specificities. In addition, other features of the catabolic pathway can also be evolved using these techniques, simultaneously or sequentially, to optimize the metabolic pathway for an activity of interest.
  • B. Halogenated Hydrocarbons [0120]
  • Large quantities of halogenated hydrocarbons are produced annually for uses as solvents and biocides. These include, in the United States alone, over 5 million tons of both 1,2-dichloroethane and vinyl chloride used in PVC production in the U.S. alone. The compounds are largely not biodegradable by processes in single organisms, although in principle haloaromatic catabolic pathways can be constructed by combining genes from different microorganisms. Enzymes can be manipulated to change their substrate specificities. Recursive sequence recombination offers the possibility of tailoring enzyme specificity to new substrates without needing detailed structural analysis of the enzymes. [0121]
  • As an example of possible starting materials for the methods of the instant invention, Wackett et al. ([0122] Nature 368:627-629 (1994)) recently demonstrated that through classical techniques a recombinant Pseudomonas strain in which seven genes encoding two multi-component oxygenases are combined, generated a single host that can metabolize polyhalogenated compounds by sequential reductive and oxidative techniques to yield non-toxic products. These and/or related materials can be subjected to the techniques discussed above so as to evolve and optimize a biodegradative pathway in a single organism.
  • Trichloroethylene is a significant groundwater contaminant. It is degraded by microorganisms in a cometabolic way (i.e., no energy or nutrients are derived). The enzyme must be induced by a different compound (e.g., [0123] Pseudomonas cepacia uses toluene-4-monoxygenase, which requires induction by toluene, to destroy trichloroethylene). Furthermore, the degradation pathway involves formation of highly reactive epoxides that can inactivate the enzyme (Timmis et al. Ann. Rev. Microbiol. 48:525-557 (1994)). The recursive sequence recombination techniques of the invention could be used to mutate the enzyme and its regulatory region such that it is produced constitutively, and is less susceptible to epoxide inactivation. In some embodiments of the invention, selection of hosts constitutively producing the enzyme and less susceptible to the epoxides can be accomplished by demanding growth in the presence of increasing concentrations of trichloroethylene in the absence of inducing substances.
  • C. Polychlorinated Biphenyls (PCBs) and Polycyclic Aromatic Hydrocarbons (PAHs) [0124]
  • PCBs and PAHs are families of structurally related compounds that are major pollutants at many Superfund sites. Bacteria transformed with plasmids encoding enzymes with broader substrate specificity have been used commercially. In nature, no known pathways have been generated in a single host that degrade the larger PAHs or more heavily chlorinated PCBs. Indeed, often the collaboration of anaerobic and aerobic bacteria are required for complete metabolism. [0125]
  • Thus, likely sources for starting material for recursive sequence recombination include identified genes encoding PAH-degrading catabolic pathways on large (20-100 KB) plasmids (Sanseverino et al. [0126] Applied Environ. Micro. 59:1931-1937 (1993); Simon et al. Gene 127:31-37 (1993); Zylstra et al. Annals of the NY Acad. Sci. 721:386-398 (1994)); while biphenyl and PCB-metabolizing enzymes are encoded by chromosomal gene clusters, and in a number of cases have been cloned onto plasmids (Hayase et al. J. Bacteriol. 172:1160-1164 (1990); Furukawa et al. Gene 98:21-28 (1992); Hofer et al. Gene 144:9-16 (1994)). The materials can be subjected to the techniques discussed above so as to evolve a biodegradative pathway in a single organism.
  • Substrate specificity in the PCB pathway largely results from enzymes involved in initial dioxygenation reactions, and can be significantly altered by mutations in those enzymes (Erickson et al. [0127] Applied Environ. Micro. 59:3858-38662 (1993); Furukawa et al. J. Bact. 175:5224-5232 (1993). Mineralization of PAHs and PCBs requires that the downstream pathway is able to metabolize the products of the initial reaction (Brenner et al. Biodegradation 5:359-377 (1994)). In this case, recursive sequence recombination of the entire pathway with selection for bacteria able to use the PCB or PAH as the sole carbon source will allow production of novel PCB and PAH degrading bacteria.
  • D. Herbicides [0128]
  • A general method for evolving genes for the catabolism of insoluble herbicides is exemplified as follows for atrazine. Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine] is a moderately persistent herbicide which is frequently detected in ground and surface water at concentrations exceeding the 3 ppb health advisory level set by the EPA. Atrazine can be slowly metabolized by a Pseudomonas species (Mandelbaum et al. [0129] Appl. Environ. Micro. 61:1451-1457 (1995)). The enzymes catalyzing the first two steps in atrazine metabolism by Pseudomonas are encoded by genes AtzA and AtzB (de Souza et al. Appl. Environ. Micro. 61:3373-3378 (1995)). These genes have been cloned in a 6.8 kb fragment into pUC18 (AtzAB-pUC). E. coli carrying this plasmid converts atrazine to much more soluble metabolites. It is thus possible to screen for enzyme activity by growing bacteria on plates containing atrazine. The herbicide forms an opaque precipitate in the plates, but cells containing AtzAB-pU18 secrete atrazine degrading enzymes, leading to a clear halo around those cells or colonies. Typically, the size of the halo and the rate of its formation can be used to assess the level of activity so that picking colonies with the largest halos allows selection of the more active or highly produced atrazine degrading enzymes. Thus, the plasmids carrying these genes can be subjected to the recursive sequence recombination formats described above to optimize the catabolism of atrazine in E. coli or another host of choice, including Pseudomonas. After each round of recombination, screening of host colonies expressing the evolved genes can be done on agar plates containing atrazine to observe halo formation. This is a generally applicable method for screening enzymes that metabolize insoluble compounds to those that are soluble (e.g., polycyclicaromatic hydrocarbons). Additionally, catabolism of atrazine can provide a source of nitrogen for the cell; if no other nitrogen is available, cell growth will be limited by the rate at which the cells can catabolize nitrogen. Cells able to utilize atrazine as a nitrogen source can thus be selected from a background of non-utilizers or poor-utilizers.
  • E. Heavy Metal Detoxification [0130]
  • Bacteria are used commercially to detoxify arsenate waste generated by the mining of arsenopyrite gold ores. As well as mining effluent, industrial waste water is often contaminated with heavy metals (e.g., those used in the manufacture of electronic components and plastics). Thus, simply to be able to perform other bioremedial functions, microorganisms must be resistant to the levels of heavy metals present, including mercury, arsenate, chromate, cadmium, silver, etc. [0131]
  • A strong selective pressure is the ability to metabolize a toxic compound to one less toxic. Heavy metals are toxic largely by virtue of their ability to denature proteins (Ford et al. [0132] Bioextraction and Biodeterioration of Metals, p. 1-23). Detoxification of heavy metal contamination can be effected in a number of ways including changing the solubility or bioavailability of the metal, changing its redox state (e.g. toxic mercuric chloride is detoxified by reduction to the much more volatile elemental mercury) and even by bioaccumulation of the metal by immobilized bacteria or plants. The accumulation of metals to a sufficiently high concentration allows metal to be recycled; smelting burns off the organic part of the organism, leaving behind reusable accumulated metal. Resistances to a number of heavy metals (arsenate, cadmium, cobalt, chromium, copper, mercury, nickel, lead, silver, and zinc) are plasmid encoded in a number of species including Staphylococcus and Pseudomonas (Silver et al. Environ. Health Perspect. 102:107-113 (1994); Ji et al. J. Ind. Micro. 14:61-75 (1995)). These genes also confer heavy metal resistance on other species as well (e.g., E. coli). The recursive sequence recombination techniques of the instant invention (RSR) can be used to increase microbial heavy metal tolerances, as well as to increase the extent to which cells will accumulate heavy metals. For example, the ability of E. coli to detoxify arsenate can be improved at least 100-fold by RSR (see co-pending application Ser. No. 08/621,859, filed Mar. 25, 1996).
  • Cyanide is very efficiently used to extract gold from rock containing as little as 0.2 oz per ton. This cyanide can be microbially neutralized and used as a nitrogen source by fungi or bacteria such as [0133] Pseudomonas fluorescens. A problem with microbial cyanide degradation is the presence of toxic heavy metals in the leachate. RSR can be used to increase the resistance of bioremedial microorganisms to toxic heavy metals, so that they will be able to survive the levels present in many industrial and Superfund sites. This will allow them to biodegrade organic pollutants including but not limited to aromatic hydrocarbons, halogenated hydrocarbons, and biocides.
  • F. Microbial Mining [0134]
  • “Bioleaching” is the process by which microbes convert insoluble metal deposits (usually metal sulfides or oxides) into soluble metal sulfates. Bioleaching is commercially important in the mining of arsenopyrite, but has additional potential in the detoxification and recovery of metals and acids from waste dumps. Naturally occurring bacteria capable of bioleaching are reviewed by Rawlings and Silver ([0135] Bio/Technology 13:773-778 (1995)). These bacteria are typically divided into groups by their preferred temperatures for growth. The more important mesophiles are Thiobacillus and Leptospirillum species. Moderate thermophiles include Sulfobacillus species. Extreme thermophiles include Sulfolobus species. Many of these organisms are difficult to grow in commercial industrial settings, making their catabolic abilities attractive candidates for transfer to and optimization in other organisms such as Pseudomonas, Rhodococcus, T. ferrooxidans or E. coli. Genetic systems are available for at least one strain of T. ferrooxidans, allowing the manipulation of its genetic material on plasmids.
  • The recursive sequence recombination methods described above can be used to optimize the catalytic abilities in native hosts or heterologous hosts for evolved bioleaching genes or pathways, such as the ability to convert metals from insoluble to soluble salts. In addition, leach rates of particular ores can be improved as a result of, for example, increased resistance to toxic compounds in the ore concentrate, increased specificity for certain substrates, ability to use different substrates as nutrient sources, and so on. [0136]
  • G. Oil Desulfurization [0137]
  • The presence of sulfur in fossil fuels has been correlated with corrosion of pipelines, pumping, and refining equipment, and with the premature breakdown of combustion engines. Sulfur also poisons many catalysts used in the refining of fossil fuels. The atmospheric emission of sulfur combustion products is known as acid rain. [0138]
  • Microbial desulfurization is an appealing bioremediation application. Several bacteria have been reported that are capable of catabolizing dibenzothiophene (DBT), which is the representative compound of the class of sulfur compounds found in fossil fuels. U.S. Pat. No. 5,356,801 discloses the cloning of a DNA molecule from [0139] Rhodococcus rhodochrous capable of biocatalyzing the desulfurization of oil. Denome et al. (Gene 175:6890-6901 (1995)) disclose the cloning of a 9.8 kb DNA fragment from Pseudomonas encoding the upper naphthalene catabolizing pathway which also degrades dibenzothiophene. Other genes have been identified that perform similar functions (disclosed in U.S. Pat. No. 5,356,801).
  • The activity of these enzymes is currently too low to be commercially viable, but the pathway could be increased in efficiency using the recursive sequence recombination techniques of the invention. The desired property of the genes of interest is their ability to desulfurize dibenzothiophene. In some embodiments of the invention, selection is preferably accomplished by coupling this pathway to one providing a nutrient to the bacteria. Thus, for example, desulfurization of dibenzothiophene results in formation of hydroxybiphenyl. This is a substrate for the biphenyl-catabolizing pathway which provides carbon and energy. Selection would thus be done by “shuffling” the dibenzothiophene genes and transforming them into a host containing the biphenyl-catabolizing pathway. Increased dibenzothiophene desulfurization will result in increased nutrient availability and increased growth rate. Once the genes have been evolved they are easily separated from the biphenyl degrading genes. The latter are undesirable in the final product since the object is to desulfurize without decreasing the energy content of the oil. [0140]
  • H. Organo-nitro Compounds [0141]
  • Organo-nitro compounds are used as explosives, dyes, drugs, polymers and antimicrobial agents. Biodegradation of these compounds occurs usually by way of reduction of the nitrate group, catalyzed by nitroreductases, a family of broadly-specific enzymes. Partial reduction of organo-nitro compounds often results in the formation of a compound more toxic than the original (Hassan et al. 1979 [0142] Arch Bioch Biop. 196:385-395). Recursive sequence recombination of nitroreductases can produce enzymes that are more specific, and able to more completely reduce (and thus detoxify) their target compounds (examples of which include but are not limited to nitrotoluenes and nitrobenzenes). Nitro-reductases can be isolated from bacteria isolated from explosive-contaminated soils, such as Morganella morganii and Enterobacter cloacae (Bryant et. al., 1991. J. Biol Chem. 266:4126-4130). A preferred selection method is to look for increased resistance to the organo-nitro compound of interest, since that will indicate that the enzyme is also able to reduce any toxic partial reduction products of the original compound.
  • IV. Use of Alternative Substrates for Chemical Synthesis [0143]
  • Metabolic engineering can be used to alter microorganisms that produce industrially useful chemicals, so that they will grow using alternate and more abundant sources of nutrients, including human-produced industrial wastes. This typically involves providing both a transport system to get the alternative substrate into the engineered cells and catabolic enzymes from the natural host organisms to the engineered cells. In some instances, enzymes can be secreted into the medium by engineered cells to degrade the alternate substrate into a form that can more readily be taken up by the engineered cells; in other instances, a batch of engineered cells can be grown on one preferred substrate, then lysed to liberate hydrolytic enzymes for the alternate substrate into the medium, while a second inoculum of the same engineered host or a second host is added to utilize the hydrolyzate. [0144]
  • The starting materials for recursive sequence recombination will typically be genes for utilization of a substrate or its transport. Examples of nutrient sources of interest include but are not limited to lactose, whey, galactose, mannitol, xylan, cellobiose, cellulose and sucrose, thus allowing cheaper production of compounds including but not limited to ethanol, tryptophan, rhamnolipid surfactants, xanthan gum, and polyhydroxylalkanoate. For a review of such substrates as desired target substances, see Cameron et al. ([0145] Appl. Biochem. Biotechnol. 38:105-140 (1993)).
  • The recursive sequence recombination methods described above can be used to optimize the ability of native hosts or heterologous hosts to utilize a substrate of interest, to evolve more efficient transport systems, to increase or alter specificity for certain substrates, and so on. [0146]
  • V. Biosynthesis [0147]
  • Metabolic engineering can be used to alter organisms to optimize the production of practically any metabolic intermediate, including antibiotics, vitamins, amino acids such as phenylalanine and aromatic amino acids, ethanol, butanol, polymers such as xanthan gum and bacterial cellulose, peptides, and lipids. When such compounds are already produced by a host, the recursive sequence recombination techniques described above can be used to optimize production of the desired metabolic intermediate, including such features as increasing enzyme substrate specificity and turnover number, altering metabolic fluxes to reduce the concentrations of toxic substrates or intermediates, increasing resistance of the host to such toxic compounds, eliminating, reducing or altering the need for inducers of gene expression/activity, increasing the production of enzymes necessary for metabolism, etc. [0148]
  • Enzymes can also be evolved for improved activity in solvents other than water. This is useful because intermediates in chemical syntheses are often protected by blocking groups which dramatically affect the solubility of the compound in aqueous solvents. Many compounds can be produced by a combination of pure chemical and enzymically catalyzed reactions. Performing enzymic reactions on almost insoluble substrates is clearly very inefficient, so the availability of enzymes that are active in other solvents will be of great use. One example of such a scheme is the evolution of a para-nitrobenzyl esterase to remove protecting groups from an intermediate in loracarbef synthesis (Moore, J. C. and Arnold, F. H. [0149] Nature Biotechnology 14:458-467 (1996)). In this case alternating rounds of error-prone PCR and colony screening for production of a fluorescent reporter from a substrate analogue were used to generate a mutant esterase that was 16-fold more active than the parent molecule in 30% dimethylformamide. No individual mutation was found to contribute more than a 2-fold increase in activity, but it was the combination of a number of mutations which led to the overall increase. Structural analysis of the mutant protein showed that the amino acid changes were distributed throughout the length of the protein in a manner that could not have been rationally predicted. Sequential rounds of error-prone PCR have the problem that after each round all but one mutant is discarded, with a concomitant loss of information contained in all the other beneficial mutations. Recursive sequence recombination avoids this problem, and would thus be ideally suited to evolving enzymes for catalysis in other solvents, as well as in conditions where salt concentrations or pH were different from the original enzyme optimas.
  • In addition, the yield of almost any metabolic pathway can be increased, whether consisting entirely of genes endogenous to the host organisms or all or partly heterologous genes. Optimization of the expression levels of the enzymes in a pathway is more complex than simply maximizing expression. In some cases regulation, rather than constitutive expression of an enzyme may be advantageous for cell growth and therefore for product yield, as seen for production of phenylalanine (Backman et al. [0150] Ann. NY Acad. Sci. 589:16-24 (1990)) and 2-keto-L-gluconic acid (Anderson et al. U.S. Pat. No. 5,032,514). In addition, it is often advantageous for industrial purposes to express proteins in organisms other than their original hosts. New host strains may be preferable for a variety of reasons, including ease of cloning and transformation, pathogenicity, ability to survive in particular environments and a knowledge of the physiology and genetics of the organisms. However, proteins expressed in heterologous organisms often show markedly reduced activity for a variety of reasons including inability to fold properly in the new host (Sarthy et al. Appl. Environ. Micro. 53:1996-2000 (1987)). Such difficulties can indeed be overcome by the recursive sequence recombination strategies of the instant invention.
  • A. Antibiotics [0151]
  • The range of natural small molecule antibiotics includes but is not limited to peptides, peptidolactones, thiopeptides, beta-lactams, glycopeptides, lantibiotics, microcins, polyketide-derived antibiotics (anthracyclins, tetracyclins, macrolides, avermectins, polyethers and ansamycins), chloramphenicol, aminoglycosides, aminocyclitols, polyoxins, agrocins and isoprenoids. [0152]
  • There are at least three ways in which recursive sequence recombination techniques of the instant invention can be used to facilitate novel drug synthesis, or to improve biosynthesis of existing antibiotics. [0153]
  • First, antibiotic synthesis enzymes can be “evolved” together with transport systems that allow entry of compounds used as antibiotic precursors to improve uptake and incorporation of function-altering artificial side chain precursors. For example, penicillin V is produced by feeding Penicillium the artificial side chain precursor phenoxyacetic acid, and LY146032 by feeding Streptomyces roseosporus decanoic acid (Hopwood, [0154] Phil. Trans. R. Soc. Lond. B 324:549-562 (1989)). Poor precursor uptake and poor incorporation by the synthesizing enzyme often lead to inefficient formation of the desired product. Recursive sequence recombination of these two systems can increase the yield of desired product.
  • Furthermore, a combinatorial approach can be taken in which an enzyme is shuffled for novel catalytic activity/substrate recognition (perhaps by including randomizing oligonucleotides in key positions such as the active site). A number of different substrates (for example, analogues of side chains that are normally incorporated into the antibiotic) can then be tested in combination with all the different enzymes and tested for biological activity. In this embodiment, plates are made containing different potential antibiotic precursors (such as the side chain analogues). The microorganisms containing the shuffled library (the library strain) are replicated onto those plates, together with a competing, antibiotic sensitive, microorganism (the indicator strain). Library cells that are able to incorporate the new side chain to produce an effective antibiotic will thus be able to compete with the indicator strain, and will be selected for. [0155]
  • Second, the expression of heterologous genes transferred from one antibiotic synthesizing organism to another can be optimized. The newly introduced enzyme(s) act on secondary metabolites in the host cell, transforming them into new compounds with novel properties. Using traditional methods, introduction of foreign genes into antibiotic synthesizing hosts has already resulted in the production of novel hybrid antibiotics. Examples include mederrhodin, dihydrogranatirhodin, 6-deoxyerythromycin A, isovalerylspiramycin and other hybrid macrolides (Cameron et. al. [0156] Appl. Biochem. Biotechnol. 38:105-140 (1993)). The recursive sequence recombination techniques of the instant invention can be used to optimize expression of the foreign genes, to stabilize the enzyme in the new host cell, and to increase the activity of the introduced enzyme against its new substrates in the new host cell. In some embodiments of the invention, the host genome may also be so optimized.
  • Third, the substrate specificity of an enzyme involved in secondary metabolism can be altered so that it will act on and modify a new compound or so that its activity is changed and it acts at a different subset of positions of its normal substrate. Recursive sequence recombination can be used to alter the substrate specificities of enzymes. Furthermore, in addition to recursive sequence recombination of individual enzymes being a strategy to generate novel antibiotics, recursive sequence recombination of entire pathways, by altering enzyme ratios, will alter metabolite fluxes and may result, not only in increased antibiotic synthesis, but also in the synthesis of different antibiotics. This can be deduced from the observation that expression of different genes from the same cluster in a foreign host leads to different products being formed (see p. 80 in Hutchinson et. al., (1991) [0157] Ann NY Acad Sci, 646:78-93). Recursive sequence recombination of the introduced gene clusters may result in a variety of expression levels of different proteins within the cluster (because it produces different combinations of, in this case regulatory, mutations). This in turn may lead to a variety of different end products. Thus, “evolution” of an existing antibiotic synthesizing pathway could be used to generate novel antibiotics either by modifying the rates or substrate specificities of enzymes in that pathway.
  • Additionally, antibiotics can also be produced in vitro by the action of a purified enzyme on a precursor. For example isopenicillin N synthase catalyses the cyclization of many analogues of its normal substrate (d-(L-a-aminoadipyl)-L-cysteinyl-D-valine) (Hutchinson, [0158] Med. Res. Rev. 8:557-567 (1988)). Many of these products are active as antibiotics. A wide variety of substrate analogues can be tested for incorporation by secondary metabolite synthesizing enzymes without concern for the initial efficiency of the reaction. Recursive sequence recombination can be used subsequently to increase the rate of reaction with a promising new substrate.
  • Thus, organisms already producing a desired antibiotic can be evolved with the recursive sequence recombination techniques described above to maximize production of that antibiotic. Additionally, new antibiotics can be evolved by manipulation of genetic material from the host by the recursive sequence recombination techniques described above. Genes for antibiotic production can be transferred to a preferred host after cycles of recursive sequence recombination or can be evolved in the preferred host as described above. Antibiotic genes are generally clustered and are often positively regulated, making them especially attractive candidates for the recursive sequence recombination techniques of the instant invention. Additionally, some genes of related pathways show cross-hybridization, making them preferred candidates for the generation of new pathways for new antibiotics by the recursive sequence recombination techniques of the invention. Furthermore, increases in secondary metabolite production including enhancement of substrate fluxes (by increasing the rate of a rate limiting enzyme, deregulation of the pathway by suppression of negative control elements or over expression of activators and the relief of feedback controls by mutation of the regulated enzyme to a feedback-insensitive deregulated protein) can be achieved by recursive sequence recombination without exhaustive analysis of the regulatory mechanisms governing expression of the relevant gene clusters. [0159]
  • The host chosen for expression of evolved genes is preferably resistant to the antibiotic produced, although in some instances production methods can be designed so as to sacrifice host cells when the amount of antibiotic produced is commercially significant yet lethal to the host. Similarly, bioreactors can be designed so that the growth medium is continually replenished, thereby “drawing off” antibiotic produced and sparing the lives of the producing cells. Preferably, the mechanism of resistance is not the degradation of the antibiotic produced. [0160]
  • Numerous screening methods for increased antibiotic expression are known in the art, as discussed above, including screening for organisms that are more resistant to the antibiotic that they produce. This may result from linkage between expression of the antibiotic synthesis and antibiotic resistance genes (Chater, [0161] Bio/Technology 8:115-121 (1990)). Another screening method is to fuse a reporter gene (e.g. xylE from the Pseudomonas TOL plasmid) to the antibiotic production genes. Antibiotic synthesis gene expression can then be measured by looking for expression of the reporter (e.g. xylE encodes a catechol dioxygenase which produces yellow muconic semialdehyde when colonies are sprayed with catechol (Zukowski et al. Proc. Natl. Acad. Sci. U.S.A. 80:1101-1105 (1983)).
  • The wide variety of cloned antibiotic genes provides a wealth of starting materials for the recursive sequence recombination techniques of the instant invention. For example, genes have been cloned from Streptomyces cattleya which direct cephamycin C synthesis in the non-antibiotic producer Streptomyces lividans (Chen et al. [0162] Bio/Technology 6:1222-1224 (1988)). Clustered genes for penicillin biosynthesis (δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase; isopenicillin N synthetase and acyl coenzyme A:6-aminopenicillanic acid acyltransferase) have been cloned from Penicillium chrysogenum. Transfer of these genes into Neurospora crassa and Aspergillus niger result in the synthesis of active penicillin V (Smith et al. Bio/Technology 8:39-41 (1990)). For a review of cloned genes involved in Cephalosporin C, Penicillins G and V and Cephamycin C biosynthesis, see Piepersberg, Crit. Rev. Biotechnol. 14:251-285 (1994). For a review of cloned clusters of antibiotic-producing genes, see Chater Bio/Technoloqv 8:115-121 (1990). Other examples of antibiotic synthesis genes transferred to industrial producing strains, or over expression of genes, include tylosin, cephamycin C, cephalosporin C, LL-E33288 complex (an antitumor and antibacterial agent), doxorubicin, spiramycin and other macrolide antibiotics, reviewed in Cameron et al. Appl. Biochem. Biotechnol. 38:105-140 (1993).
  • B. Biosynthesis to Replace Chemical Synthesis of Antibiotics [0163]
  • Some antibiotics are currently made by chemical modifications of biologically produced starting compounds. Complete biosynthesis of the desired molecules may currently be impractical because of the lack of an enzyme with the required enzymatic activity and substrate specificity. For example, 7-aminodeacetooxycephalosporanic acid (7-ADCA) is a precursor for semi-synthetically produced cephalosporins. 7-ADCA is made by a chemical ring expansion from penicillin V followed by enzymatic deacylation of the phenoxyacetal group. Cephalosporin V could in principle be produced biologically from penicillin V using penicillin N expandase, but penicillin V is not used as a substrate by any known expandase. The recursive sequence recombination techniques of the invention can be used to alter the enzyme so that it will use penicillin V as a substrate. Similarly, penicillin transacylase could be so modified to accept cephalosporins or cephamycins as substrates. [0164]
  • In yet another example, penicillin amidase expressed in [0165] E. coli is a key enzyme in the production of penicillin G derivatives. The enzyme is generated from a precursor peptide and tends to accumulate as insoluble aggregates in the periplasm unless non-metabolizable sugars are present in the medium (Scherrer et al. Appl. Microbiol. Biotechnol. 42:85-91 (1994)). Evolution of this enzyme through the methods of the instant invention could be used to generate an enzyme that folds better, leading to a higher level of active enzyme expression.
  • In yet another example, Penicillin G acylase covalently linked to agarose is used in the synthesis of penicillin G derivatives. The enzyme can be stabilized for increased activity, longevity and/or thermal stability by chemical modification (Fernandez-Lafuente et. al. [0166] Enzyme Microb. Technol. 14:489-495 (1992). Increased thermal stability is an especially attractive application of the recursive sequence recombination techniques of the instant invention, which can obviate the need for the chemical modification of such enzymes. Selection for thermostability can be performed in vivo in E. coli or in thermophiles at higher temperatures. In general, thermostability is a good first step in enhancing general stabilization of enzymes. Random mutagenesis and selection can also be used to adapt enzymes to function in non-aqueous solvents (Arnold Curr Oin Biotechnol, 4:450-455 (1993); Chen et. al. Proc. Natl. Acad. Sci. U.S.A., 90:5618-5622 (1993)). Recursive sequence recombination represents a more powerful (since recombinogenic) method of generating mutant enzymes that are stable and active in non-aqueous environments. Additional screening can be done on the basis of enzyme stability in solvents.
  • C. Polyketides [0167]
  • Polyketides include antibiotics such as tetracycline and erythromycin, anti-cancer agents such as daunomycin, immunosuppressants such as FK506 and rapamycin and veterinary products such as monesin and avermectin. Polyketide syntheses (PKS's) are multifunctional enzymes that control the chain length, choice of chain-building units and reductive cycle that generates the huge variation in naturally occurring polyketides. Polyketides are built up by sequential transfers of “extender units” (fatty acyl CoA groups) onto the appropriate starter unit (examples are acetate, coumarate, propionate and malonamide). The PKS's determine the number of condensation reactions and the type of extender groups added and may also fold and cyclize the polyketide precursor. PKS's reduce specific β-keto groups and may dehydrate the resultant β-hydroxyls to form double bonds. Modifications of the nature or number of building blocks used, positions at which β-keto groups are reduced, the extent of reduction and different positions of possible cyclizations, result in formation of different final products. Polyketide research is currently focused on modification and inhibitor studies, site directed mutagenesis and 3-D structure elucidation to lay the groundwork for rational changes in enzymes that will lead to new polyketide products. [0168]
  • Recently, McDaniel et al. ([0169] Science 262:1546-1550 (1995)) have developed a Streptomyces host-vector system for efficient construction and expression of recombinant PKSs. Hutchinson (Bio/Technology 12:375-308 (1994)) reviewed targeted mutation of specific biosynthetic genes and suggested that microbial isolates can be screened by DNA hybridization for genes associated with known pharmacologically active agents so as to provide new metabolites and large amounts of old ones. In particular, that review focuses on polyketide synthase and pathways to aminoglycoside and oligopeptide antibiotics.
  • The recursive sequence recombination techniques of the instant invention can be used to generate modified enzymes that produce novel polyketides without such detailed analytical effort. The availability of the PKS genes on plasmids and the existence of [0170] E. coli-Streptomyces shuttle vectors (Wehmeier Gene 165:149-150 (1995)) makes the process of recursive sequence recombination especially attractive by the techniques described above. Techniques for selection of antibiotic producing organisms can be used as described above; additionally, in some embodiments screening for a particular desired polyketide activity or compound is preferable.
  • D. Isoprenoids [0171]
  • Isoprenoids result from cyclization of farnesyl pyrophosphate by sesquiterpene synthases. The diversity of isoprenoids is generated not by the backbone, but by control of cyclization. Cloned examples of isoprenoid synthesis genes include trichodiene synthase from [0172] Fusarium sprorotrichioides, pentalene synthase from Streptomyces, aristolochene synthase from Penicillium roquefortii, and epi-aristolochene syrithase from N. tabacum (Cane, D. E. (1995). Isoprenoid antibiotics, pages 633-655, in “Genetics and Biochemistry of Antibiotic Production” edited by Vining, L. C. & Stuttard, C., published by Butterworth-Heinemann). Recursive sequence recombination of sesquiterpene synthases will be of use both in allowing expression of these enzymes in heterologous hosts (such as plants and industrial microbial strains) and in alteration of enzymes to change the cyclized product made. A large number of isoprenoids are active as antiviral, antibacterial, antifungal, herbicidal, insecticidal or cytostatic agents. Antibacterial and antifungal isoprenoids could thus be preferably screened for using the indicator cell type system described above, with the producing cell competing with bacteria or fungi for nutrients. Antiviral isoprenoids could be screened for preferably by their ability to confer resistance to viral attack on the producing cell.
  • E. Bioactive Peptide Derivatives [0173]
  • Examples of bioactive non-ribosomally synthesized peptides include the antibiotics cyclosporin, pepstatin, actinomycin, gramicidin, depsipeptides, vancomycin, etc. These peptide derivatives are synthesized by complex enzymes rather than ribosomes. Again, increasing the yield of such non-ribosomally synthesized peptide antibiotics has thus far been done by genetic identification of biosynthetic “bottlenecks” and over expression of specific enzymes (See, for example, p. 133-135 in “Genetics and Biochemistry of Antibiotic Production” edited by Vining, L. C. & Stuttard, C., published by Butterworth-Heinemann). Recursive sequence recombination of the enzyme clusters can be used to improve the yields of existing bioactive non-ribosomally made peptides in both natural and heterologous hosts. Like polyketide synthases, peptide synthases are modular and multifunctional enzymes catalyzing condensation reactions between activated building blocks (in this case amino acids) followed by modifications of those building blocks (see Kleinkauf, H. and von Dohren, H. [0174] Eur. J. Biochem. 236:335-351 (1996)). Thus, as for polyketide synthases, recursive sequence recombination can also be used to alter peptide synthases: modifying the specificity of the amino acid recognized by each binding site on the enzyme and altering the activity or substrate specificities of sites that modify these amino acids to produce novel compounds with antibiotic activity.
  • Other peptide antibiotics are made ribosomally and then post-translationally modified. Examples of this type of antibiotics are lantibiotics (produced by gram positive bacteria such Staphylococcus, Streptomyces, Bacillus, and Actinoplanes) and microcins (produced by Enterobacteriaceae). Modifications of the original peptide include (in lantibiotics) dehydration of serine and threonine, condensation of dehydroamino acids with cysteine, or simple N- and C-terminal blocking (microcins). For ribosomally made antibiotics both the peptide-encoding sequence and the modifying enzymes may have their expression levels modified by recursive sequence recombination. Again, this will lead to both increased levels of antibiotic synthesis, and by modulation of the levels of the modifying enzymes (and the sequence of the ribosomally synthesized peptide itself) novel antibiotics. [0175]
  • Screening can be done as for other antibiotics as described above, including competition with a sensitive (or even initially insensitive) microbial species. Use of competing bacteria that have resistances to the antibiotic being produced will select strongly either for greatly elevated levels of that antibiotic (so that it swamps out the resistance mechanism) or for novel derivatives of that antibiotic that are not neutralized by the resistance mechanism. [0176]
  • F. Polymers [0177]
  • Several examples of metabolic engineering to produce biopolymers have been reported, including the production of the biodegradable plastic polyhydroxybutarate (PHB), and the polysaccharide xanthan gum. For a review, see Cameron et al. [0178] Applied Biochem. Biotech. 38:105-140 (1993). Genes for these pathways have been cloned, making them excellent candidates for the recursive sequence recombination techniques described above. Expression of such evolved genes in a commercially viable host such as E. coli is an especially attractive application of this technology.
  • Examples of starting materials for recursive sequence recombination include but are not limited to genes from bacteria such as Alcallgenes, Zoogloea, Rhizobium, Bacillus, and Azobacter, which produce polyhydroxyalkanoates (PHAS) such as polyhyroxybutyrate (PHB) intracellularly as energy reserve materials in response to stress. Genes from [0179] Alcaligenes eutrophus that encode enzymes catalyzing the conversion of acetoacetyl CoA to PHB have been transferred both to E. coli and to the plant Arabidopsis thaliana (Poirier et al. Science 256:520-523 (1992)). Two of these genes (phbB and phbc, encoding acetoacetyl-CoA reductase and PHB synthase respectively) allow production of PHB in Arabidopsis. The plants producing the plastic are stunted, probably because of adverse interactions between the new metabolic pathway and the plants' original metabolism (i.e., depletion of substrate from the mevalonate pathway). Improved production of PHB in plants has been attempted by localization of the pathway enzymes to organelles such as plastids. Other strategies such as regulation of tissue specificity, expression timing and cellular localization have been suggested to solve the deleterious effects of PHB expression in plants. The recursive sequence recombination techniques of the invention can be used to modify such heterologous genes as well as specific cloned interacting pathways (e.g., mevalonate), and to optimize PHB synthesis in industrial microbial strains, for example to remove the requirement for stresses (such as nitrogen limitation) in growth conditions.
  • Additionally, other microbial polyesters are made by different bacteria in which additional monomers are incorporated into the polymer (Peoples et al. in Novel Biodegradable Microbial Polymers, E A Dawes, ed., pp191-202 (1990)). Recursive sequence recombination of these genes or pathways singly or in combination into a heterologous host will allow the production of a variety of polymers with differing properties, including variation of the monomer subunit ratios in the polymer. Another polymer whose synthesis may be manipulated by recursive sequence recombination is cellulose. The genes for cellulose biosynthesis have been cloned from [0180] Agrobacterium tumefaciens (Matthysse, A. G. et. al. J. Bacterial. 177:1069-1075 (1995)). Recursive sequence recombination of this biosynthetic pathway could be used either to increase synthesis of cellulose, or to produce mutants in which alternative sugars are incorporated into the polymer.
  • G. Carotenoids [0181]
  • Carotenoids are a family of over 600 terpenoids produced in the general isoprenoid biosynthetic pathway by bacteria, fungi and plants (for a review, see Armstrong, [0182] J. Bact. 176:4795-4802 (1994)). These pigments protect organisms against photooxidative damage as well as functioning as anti-tumor agents, free radical-scavenging anti-oxidants, and enhancers of the immune response. Additionally, they are used commercially in pigmentation of cultured fish and shellfish. Examples of carotenoids include but are not limited to myxobacton, spheroidene, spheroidenone, lutein, astaxanthin, violaxanthin, 4-ketorulene, myxoxanthrophyll, echinenone, lycopene, zeaxanthin and its mono- and di-glucosides, α-, β-, γ- and δ-carotene, β-cryptoxanthin monoglucoside and neoxanthin.
  • Carotenoid synthesis is catalyzed by relatively small numbers of clustered genes: 11 different genes within 12 kb of DNA from [0183] Myxococcus xanthus (Botella et al. Eur. J. Biochem. 233:238-248 (1995)) and 8 genes within 9 kb of DNA from Rhodobacter sphaeroides (Lang et. al. J. Bact. 177:2064-2073 (1995)). In some microorganisms, such as Thermus thermophilus, these genes are plasmid-borne (Tabata et al. FEBS Letts 341:251-255 (1994)). These features make carotenoid synthetic pathways especially attractive candidates for recursive sequence recombination.
  • Transfer of some carotenoid genes into heterologous organisms results in expression. For example, genes from [0184] Erwina uredovora and Haematococcus pluvialis. will function together in E. coli (Kajiwara et al. Plant Mol. Biol. 29:343-352 (1995)). E. herbicola genes will function in R. sphaeroides (Hunter et al. J. Bact. 176:3692-3697 (1994)). However, some other genes do not; for example, R. capsulatus genes do not direct carotenoid synthesis in E. coli (Marrs, J. Bact. 146:1003-1012 (1981)).
  • In an embodiment of the invention, the recursive sequence recombination techniques of the invention can be used to generate variants in the regulatory and/or structural elements of genes in the carotenoid synthesis pathway, allowing increased expression in heterologous hosts. Indeed, traditional techniques have been used to increase carotenoid production by increasing expression of a rate limiting enzyme in [0185] Thermus thermophilus (Hoshino et al. Appl. Environ. Micro. 59:3150-3153 (1993)). Furthermore, mutation of regulatory genes can cause constitutive expression of carotenoid synthesis in actinomycetes, where carotenoid photoinducibility is otherwise unstable and lost at a relatively high frequency in some species (Kato et al. Mol. Gen. Genet. 247:387-390 (1995)). These are both mutations that can be obtained by recursive sequence recombination.
  • The recursive sequence recombination techniques of the invention as described above can be used to evolve one or more carotenoid synthesis genes in a desired host without the need for analysis of regulatory mechanisms. Since carotenoids are colored, a calorimetric assay in microtiter plates, or even on growth media plates, can be used for screening for increased production. [0186]
  • In addition to increasing expression of carotenoids, carotenogenic biosynthetic pathways have the potential to produce a wide diversity of carotenoids, as the enzymes involved appear to be specific for the type of reaction they will catalyze, but not for the substrate that they modify. For example, two enzymes from the marine bacterium [0187] Agrobacterium aurantiacum (CrtW and CrtZ) synthesize six different ketocarotenoids from β-carotene (Misawa et al. J. Bact. 177:6576-6584 (1995)). This relaxed substrate specificity means that a diversity of substrates can be transformed into an even greater diversity of products. Introduction of foreign carotenoid genes into a cell can lead to novel and functional carotenoid-protein complexes, for example in photosynthetic complexes (Hunter et al. J. Bact. 176:3692-3697 (1994)). Thus, the deliberate recombination of enzymes through the recursive sequence recombination techniques of the invention is likely to generate novel compounds. Screening for such compounds can be accomplished, for example, by the cell competition/survival techniques discussed above and by a calorimetric assay for pigmented compounds.
  • Another method of identifying new compounds is to use standard analytical techniques such as mass spectroscopy, nuclear magnetic resonance, high performance liquid chromatography, etc. Recombinant microorganisms can be pooled and extracts or media supernatants assayed from these pools. Any positive pool can then be subdivided and the procedure repeated until the single positive is identified (“sib-selection”). [0188]
  • H. Indigo Biosynthesis [0189]
  • Many dyes, i.e. agents for imparting color, are specialty chemicals with significant markets. As an example, indigo is currently produced chemically. However, nine genes have been combined in [0190] E. coli to allow the synthesis of indigo from glucose via the tryptophan/indole pathway (Murdock et al. Bio/Technology 11:381-386 (1993)). A number of manipulations were performed to optimize indigo synthesis: cloning of nine genes, modification of the fermentation medium and directed changes in two operons to increase reaction rates and catalytic activities of several enzymes. Nevertheless, bacterially produced indigo is not currently an economic proposition. The recursive sequence recombination techniques of the instant invention could be used to optimize indigo synthesizing enzyme expression levels and catalytic activities, leading to increased indigo production, thereby making the process commercially viable and reducing the environmental impact of indigo manufacture. Screening for increased indigo production can be done by calorimetric assays of cultures in microtiter plates.
  • I. Amino Acids [0191]
  • Amino acids of particular commercial importance include but are not limited to phenylalanine, monosodium glutamate, glycine, lysine, threonine, tryptophan and methionine. Backman et al. ([0192] Ann. NY Acad. Sci. 589:16-24 (1990)) disclosed the enhanced production of phenylalanine in E. coli via a systematic and downstream strategy covering organism selection, optimization of biosynthetic capacity, and development of fermentation and recovery processes.
  • As described in Simpson et al. ([0193] Biochem Soc Trans, 23:381-387 (1995)), current work in the field of amino acid production is focused on understanding the regulation of these pathways in great molecular detail. The recursive sequence recombination techniques of the instant invention would obviate the need for this analysis to obtain bacterial strains with higher secreted amino acid yields. Amino acid production could be optimized for expression using recursive sequence recombination of the amino acid synthesis and secretion genes as well as enzymes at the regulatory phosphoenolpyruvate branchpoint, from such organisms as Serratia marcescens, Bacillus, and the Corynebacterium-Brevibacterium group. In some embodiments of the invention, screening for enhanced production is preferably done in microtiter wells, using chemical tests well known in the art that are specific for the desired amino acid. Screening/selection for amino acid synthesis can also be done by using auxotrophic reporter cells that are themselves unable to synthesize the amino acid in question. If these reporter cells also produce a compound that stimulates the growth of the amino acid producer (this could be a growth factor, or even a different amino acid), then library cells that produce more amino acid will in turn receive more growth stimulant and will therefore grow more rapidly.
  • J. Vitamin C Synthesis [0194]
  • L-Ascorbic acid (vitamin C) is a commercially important vitamin with a world production of over 35,000 tons in 1984. Most vitamin C is currently manufactured chemically by the Reichstein process, although recently bacteria have been engineered that are able to transform glucose to 2,5-keto-gluconic acid, and that product to 2-keto-L-idonic acid, the precursor to L-ascorbic acid (Boudrant, [0195] Enzyme Microb. Technol. 12:322-329 (1990)).
  • The efficiencies of these enzymatic steps in bacteria are currently low. Using the recursive sequence recombination techniques of the instant invention, the genes can be genetically engineered to create one or more operons followed by expression optimization of such a hybrid L-ascorbic acid synthetic pathway to result in commercially viable microbial vitamin C biosynthesis. In some embodiments, screening for enhanced L-ascorbic acid production is preferably done in microtiter plates, using assays well known in the art. [0196]
  • VI. Modification of Cell Properties. [0197]
  • Although not strictly examples of manipulation of intermediary metabolism, recursive sequence recombination techniques can be used to improve or alter other aspects of cell properties, from growth rate to ability to secrete certain desired compounds to ability to tolerate increased temperature or other environmental stresses. Some examples of traits engineered by traditional methods include expression of heterologous proteins in bacteria, yeast, and other eukaryotic cells, antibiotic resistance, and phage resistance. Any of these traits is advantageously evolved by the recursive sequence recombination techniques of the instant invention. Examples include replacement of one nutrient uptake system (e.g. ammonia in [0198] Methylophilus methylotrophus) with another that is more energy efficient; expression of haemoglobin to improve growth under conditions of limiting oxygen; redirection of toxic metabolic end products to less toxic compounds; expression of genes conferring tolerance to salt, drought and toxic compounds and resistance to pathogens, antibiotics and bacteriophage, reviewed in Cameron et. al. Appl Biochem Biotechnol, 38:105-140 (1993).
  • The heterologous genes encoding these functions all have the potential for further optimization in their new hosts by existing recursive sequence recombination technology. Since these functions increase cell growth rates under the desired growth conditions, optimization of the genes by evolution simply involves recombining the DNA recursively and selecting the recombinants that grow faster with limiting oxygen, higher toxic compound concentration, or whatever is the appropriate growth condition for the parameter being improved. [0199]
  • Since these functions increase cell growth rates under the desired growth conditions, optimization of the genes by “evolution” can simply involve “shuffling” the DNA and selecting the recombinants that grow faster with limiting oxygen, higher toxic compound concentration or whatever restrictive condition is being overcome. [0200]
  • Cultured mammalian cells also require essential amino acids to be present in the growth medium. This requirement could also be circumvented by expression of heterologous metabolic pathways that synthesize these amino acids (Rees et al. [0201] Biotechnology 8:629-633 (1990). Recursive sequence recombination would provide a mechanism for optimizing the expression of these genes in mammalian cells. Once again, a preferred selection would be for cells that can grow in the absence of added amino acids.
  • Yet another candidate for improvement through the techniques of the invention is symbiotic nitrogen fixation. Genes involved in nodulation (nod, ndv), nitrogen reduction (nif, fix), host range determination (nod, hsp), bacteriocin production (tfx), surface polysaccharide synthesis (exo) and energy utilization (dct, hup) which have been identified (Paau, [0202] Biotech. Adv. 9:173-184 (1991)).
  • The main function of recursive sequence recombination in this case is in improving the survival of strains that are already known to be better nitrogen fixers. These strains tend to be less good at competing with strains already present in the environment, even though they are better at nitrogen fixation. Targets for recursive sequence recombination such as nodulation and host range determination genes can be modified and selected for by their ability to grow on the new host. Similarly any bacteriocin or energy utilization genes that will improve the competitiveness of the strain will also result in greater growth rates. Selection can simply be performed by subjecting the target genes to recursive sequence recombination and forcing the inoculant to compete with wild type nitrogen fixing bacteria. The better the nitrogen fixing bacteria grow in the new host, the more copies of their recombined genes will be present for the next round of recombination. This growth rate differentiating selection is described above in detail. [0203]
  • VI. Biodetectors/Biosensors [0204]
  • Bioluminescence or fluorescence genes can be used as reporters by fusing them to specific regulatory genes (Cameron et. al. [0205] Appl Biochem Biotechnol, 38:105-140 (1993)). A specific example is one in which the luciferase genes luxCDABE of Vibrio fischeri were fused to the regulatory region of the isopropylbenzene catabolism operon from Pseudomonas putida RE204. Transformation of this fusion construct into E. coli resulted in a strain which produced light in response to a variety of hydrophobic compound such as substituted benzenes, chlorinated solvents and naphthalene (Selifonova et. al., Appl Environ Microbiol 62:778-783 (1996)). This type of construct is useful for the detection of pollutant levels, and has the added benefit of only measuring those pollutants that are bioavailable (and therefore potentially toxic). Other signal molecules such as jellyfish green fluorescent protein could also be fused to genetic regulatory regions that respond to chemicals in the environment. This should allow a variety of molecules to be detected by their ability to induce expression of a protein or proteins which result in light, fluorescence or some other easily detected signal.
  • Recursive sequence recombination can be used in several ways to modify this type of biodetection system. It can be used to increase the amplitude of the response, for example by increasing the fluorescence of the green fluorescent protein. Recursive sequence recombination could also be used to increase induced expression levels or catalytic activities of other signal-generating systems, for example of the luciferase genes. [0206]
  • Recursive sequence recombination can also be used to alter the specificity of biosensors. The regulatory region, and transcriptional activators that interact with this region and with the chemicals that induce transcription can also be shuffled. This should generate regulatory systems in which transcription is activated by analogues of the normal inducer, so that biodetectors for different chemicals can be developed. In this case, selection would be for constructs that are activated by the (new) specific chemical to be detected. Screening could be done simply with fluorescence (or light) activated cell sorting, since the desired improvement is in light production. [0207]
  • In addition to detection of environmental pollutants, biosensors can be developed that will respond to any chemical for which there are receptors, or for which receptors can be evolved by recursive sequence recombination, such as hormones, growth factors, metals and drugs. These receptors may be intracellular and direct activators of transcription, or they may be membrane bound receptors that activate transcription of the signal indirectly, for example by a phosphorylation cascade. They may also not act on transcription at all, but may produce a signal by some post-transcriptional modification of a component of the signal generating pathway. These receptors may also be generated by fusing domains responsible for binding different ligands with different signaling domains. Again, recursive sequence recombination can be used to increase the amplitude of the signal generated to optimize expression and functioning of chimeric receptors, and to alter the specificity of the chemicals detected by the receptor. [0208]
  • The following examples are offered by way of illustration, not by way of limitation. [0209]
    • EXAMPLES
  • I. Alteration of Enzyme Activity and Specificity. [0210]
  • In this example, recursive sequence recombination techniques of the instant invention were used to expand the range of substrates efficiently hydrolyzed by [0211] E. coli β-galactosidase. The goal was to evolve wild type E. coli β-galactosidase into a fucosidase. The enzyme showed very weak activity with both ρ-nitrophenyl-β-D-fucopyranoside and o-nitrophenyl-β-D-fucopyranoside (estimated respectively as 80- and 160-fold less efficient than for ρ-nitrophenyl-β-D-galactopyranoside).
  • To increase the activity of [0212] E. coli β-galactosidase against these fucopyranoside derivatives, a lacZ gene (a 3.8 kb Hind III-BamHI fragment from plasmid pCH110, Pharmacia) encoding E. coli β-galactosidase was subcloned into plasmid pl8SFI-BLA-SFI (Stemmer, Nature, 370:389-391 (1994)). The resulting plasmid, p18-lacZ, was used for recursive sequence recombination and mutant screening.
  • Purified plasmid p18-lacZ (4-5 μg) was used directly for DNase I fragmentation. Fragments with sizes between 50 and 200 bp were purified from a 2% agarose gel and used for reassembly PCR (Stemmer, [0213] Nature 370:389-391 (1994)). Assembly reactions used Tth polymerase (Perkin Elmer) in the manufacturer's supplied buffer. The PCR program for assembly was as follows: 94° C., 2 min., then 40 cycles of 94° C. for 30 sec.; 55° C. for 3 sec.; 72° C. for 1 min. +5 sec. per cycle; then finally 72° C. for S min.
  • This reaction was diluted 100-fold into a standard PCR reaction using the 40 mer primers p50F 5′-AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC-3′ and pR34 5′-CTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAT-3′. This resulted in amplification of both the desired DNA band (about 4 kb in size) as well as two smaller sized products (about 600bp and 100 bp bands). The PCR products were digested with BamHI and Hind III and the correct size product was cloned into BamHI-HindIII digested p18-lacZ. The resulting plasmid containing a pool of recombined lacZ mutants was plated out on LB plates supplemented with kanamycin and 5-bromo-4-chloro-3-indolyl-β-D-fucopyranoside (X-fuco). Plates were incubated at 37° C. for 20 hours and screened for colonies with slight blue tint, indicating hydrolysis of the X-fuco. Plasmid DNA was prepared from positive colonies and the procedure was repeated. Thus, six rounds of recursive sequence recombination produced a ten-fold increase in X-fuco hydrolysis activity. [0214]
  • II. Evolution of an Entire Metabolic Pathway [0215]
  • As an example of evolution of an entire metabolic pathway, the recursive sequence recombination techniques of the invention were used to modify a plasmid encoding resistance to mercury salts. This plasmid, as disclosed by Wang et al. ([0216] J. Bact. 171:83-92 (1989)) contains at least 8 genes within 13.5 kb of Bacillus DNA inserted in the cloning vector pUC9. The recursive sequence recombination protocol used for this plasmid was as follows.
  • Plasmid DNA (at 130 μg/ml) was digested with 0.09 U/ml DNAse in 50 mM Tris-Cl, pH 7.4, 10 mM MnCl[0217] 2, for 10 minutes at 25° C. DNA fragments were not size-selected, but were purified by phenol extraction and ethanol precipitation. The assembly reaction was performed using Tth polymerase (Perkin Elmer) using the manufacturer's supplied buffer, supplemented with the following: 7.5% polyethylene glycol, 8000 MW; 35 mM tetramethylammonium chloride; and 4 U/ml Pwo(Boehringer Mannheim), Pfu(Stratagene), Vent (New England Biolabs), Deep Vent (New England Biolabs), Tfl (Promega) or Tli (Promega) thermostable DNA polymerases. DNA fragments were used at around 10 μg/ml.
  • The PCR program for assembly was as follows: 94° C. for 20 sec., then 40 cycles of 940C for 15 sec., 40° C. for 30 sec., 72° C. for 30 sec. +2 sec./cycle, and finally 72° C. for 10 min. [0218]
  • The recombinant plasmid was then amplified in three fragments by using primers flanking the three relatively evenly spaced AlwNI restriction sites contained in the plasmid. The sequences of these primers were: [0219]
    1) 5′-CAGGACTTATCGCCACTGGCAGC-3′
    2) 5′-CTCGCTCTGCTAATCCTGTTACC-3′
    3) 5′-  GCATATTATGAGCGTTTAGGCTTAATTCC-3′
    4) 5′-CGGTATCCTTTTTCCGTACGTTC-3′
    5) 5′-  GTTGAAGAGGTGAAGAAAGTTCTCC-3′
    6) 5′-GTTCGTCGATTTCCACGCTTGGC-3′.
  • Three fragments were amplified using primers 1+4 (6 kb fragment), 2+5 (4 kb fragment) and 3+6 (6 kb fragment). These were then digested with AlwNI, gel purified and ligated together. As AlwNI is a non-palindromic cutter, the plasmid could only reassemble in the correct (original) order. The resultant plasmids were transformed into [0220] E. coli strain DHlOB (Gibco BRL) and selected on nutrient agar containing ampicillin 50 μg/ml and increasing concentrations of mercuric chloride (100 μM to 1000 μM) or phenylmercuric acetate (50 μM to 400 μM) Thus, in 2 rounds of recursive sequence recombination the tolerance of E. coli to these compounds increased by a factor of 10 (from about 100 to about 1,000 μM).
  • III. Recursive Sequence Recombination of a Family of Related Enzymes [0221]
  • In this example nucleotide sequences were recombined between four homologous β-lactamases from [0222] C. freundii, E. cloacae, K. pneumonia, and Y. enterocolitica. The four genes were synthesized from oligonucleotides as described in Stemmer, et al. Gene 164:49-53 (1995). Briefly, the entire coding sequences of the genes were synthesized as overlapping 50-mer oligonucleotides on a commercial oligonucleotide synthesizer. The oligonucleotides were then assembled into full length genes by a standard recursive sequence recombination reaction, followed by amplification using primers common to all four genes. oligonucleotides were designed to give optimal E. coli codon usage in the synthetic genes with the goal of increasing the homology to increase the frequency of recombination, and the same 5′ and 3′ terminal sequences. After assembly of the genes and selection for active clones, which is optional, they were DNase treated to produce fragments from 50 to 200 bp in length. The fragments were dissolved at 100 μg/ml in 15 μl of Klenow (DNA polymerase I large fragment) buffer (New England Biolabs) and subjected to manual PCR as follows: 15 cycles of 95° C. for 1 min.; freeze on dry ice and ethanol; warm to 25° C. and add 2 Al of Klenow (1 U/μl) in Klenow buffer; incubate for 2 min at 25° C.
  • A 5 μl aliquot of the manual PCR reaction was then diluted 6-fold into a standard Taq reaction mix (without oligonucleotide primers) and assembled using a standard PCR program consisting of 30 cycles of 940C for 30 sec., 40° C. for 30 sec., and 72° C. for 30 sec. [0223]
  • A 4, 8 or 16 μl aliquot of this second PCR reaction was then diluted into a standard Taq reaction mix containing oligonucleotide primers that prime on sequences contained in all four β-lactamase genes 5′-AGGGCCTCGTGATACGCCTATT-3′ and 5′-ACGAAAACTCACGTTAAGGGATT-3′. Full-length product was amplified using a standard PCR program consisting of 25 cycles of 94° C. for 30 sec., 45° C. for 30 sec., 72° C. for 45 sec. [0224]
  • This procedure produced hybrid β-lactamase genes whose activities can be tested against antibiotics including but not limited to ampicillin, carbenicillin, cefotaxime, cefoxitine, cloxacillin, ceftazidime, cephaloridine and moxalactam, to determine the specificities of the hybrid enzymes so created. Moxalactam was chosen as the test antibiotic for hybrid genes. The best of the original β-lactamase genes used in this study conferred resistance to 0.125 μg/ml of moxalactam. After the first round of recursive sequence recombination hybrid genes were isolated that conferred resistance to 0.5 μg/ml moxalactam, yielding a 4-fold increase. [0225]
  • Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. [0226]
  • All references cited herein are expressly incorporated in their entirety for all purposes. [0227]

Claims (30)

What is claimed is:
1. A method of evolving a biocatalytic activity of a cell, comprising:
(a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes;
(b) screening at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene;
(c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes;
(d) screening at least one further recombinant gene from the further library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene;
(e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell.
2. The method of claim 1, wherein the reaction of interest is the ability to utilize a substrate as a nutrient source.
3. The method of claim 1, wherein the reaction of interest is the ability to catabolize a compound.
4. The method of claim 1, wherein the reaction of interest is the ability to detoxify a compound.
5. The method of claim 1, wherein the reaction of interest is the ability to synthesize a compound of interest.
6. The method of claim 4, wherein the compound is an antibiotic.
7. The method of claim 4, wherein the compound is an amino acid.
8. The method of claim 4, wherein the compound is a polymer.
9. The method of claim 4, wherein the compound is a carotenoid.
10. The method of claim 4, wherein the compound is vitamin C.
11. The method of claim 4, wherein the compound is indigo.
12. The method of claim 1, wherein at least one recombining step is performed in vitro, and the resulting library of recombinants is introduced into the cell whose biocatalytic activity is to be enhanced generating a library of cells containing different recombinants.
13. The method of claim 12, wherein the in vitro recombining step comprises:
cleaving the first and second segments into fragments;
mixing and denaturing the fragments; and
incubating the denatured fragments with a polymerase under conditions which result in annealing of the denatured fragments and formation of the library of recombinant genes.
14. The method of claim 1, wherein at least one recombining step is performed in vivo.
15. The method of claim 1, wherein the recombining step is performed in the cell whose biocatalytic activity is to be enhanced.
16. The method of claim 1, wherein at least one DNA segment comprises a cluster of genes collectively conferring ability to catalyze a reaction of interest.
17. A method of evolving a gene to confer ability to catalyze a reaction of interest, the method comprising:
(1) recombining at least first and second DNA segments from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes;
(2) screening at least one recombinant gene from the library that confers enhanced ability to catalyze a reaction of interest relative to a wildtype form of the gene;
(3) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes;
(4) screening at least one further recombinant gene from the further library of recombinant genes that confers enhanced ability to catalyze a reaction of interest relative to a previous recombinant gene;
(5) repeating (3) and (4), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze a reaction of interest.
18. A method of generating a new biocatalytic activity in a cell, comprising:
(1) recombining at least first and second DNA segments from at least one gene conferring ability to catalyze a first reaction related to a second reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes;
(2) screening at least one recombinant gene from the library that confers a new ability to catalyze the second reaction of interest;
(3) recombining at least a segment from at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes;
(4) screening at least one further recombinant gene from the further library of recombinant genes that confers enhanced ability to catalyze the second reaction of interest in the cell relative to a previous recombinant gene;
(5) repeating (3) and (4), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the second reaction of interest in the cell.
19. A modified form of a cell, wherein the modification comprises a metabolic pathway evolved by recursive sequence recombination.
20. A method of optimizing expression of a gene product, the method comprising:
(1) recombining at least first and second DNA segments from at least one gene conferring ability to produce the gene product, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes;
(2) screening at least one recombinant gene from the library that confers optimized expression of the gene product relative to a wildtype form of the gene;
(3) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes;
(4) screening at least one further recombinant gene from the further library of recombinant genes that confers optimized ability to produce the gene product relative to a previous recombinant gene;
(5) repeating (3) and (4), as necessary, until the further recombinant gene confers a desired level of optimized ability to express the gene product.
21. The method of claim 20, wherein the at least one gene encodes the gene product.
22. The method of claim 20, wherein the at least one gene is a vector comprising a gene encoding the gene product.
23. The method of claim 20, wherein at least one recombining step is performed in vivo.
24. The method of claim 23, wherein the recombining step is performed in a host cell wherein the gene product is expressed.
25. The method of claim 20, wherein the at least one gene is a host cell gene and wherein the host cell gene does not encode the gene product.
26. The method of claim 20, wherein optimization results in increased expression of the gene product.
27. A method of evolving a biosensor for a compound A of interest, the method comprising:
(1) recombining at least first and second DNA segments from at least one gene conferring ability to detect a related compound B, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes;
(2) screening at least one recombinant gene from the library that confers optimized ability to detect compound A relative to a wildtype form of the gene;
(3) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes;
(4) screening at least one further recombinant gene from the further library of recombinant genes that confers optimized ability to detect compound A relative to a previous recombinant gene;
(5) repeating (3) and (4), as necessary, until the further recombinant gene confers Et desired level of optimized ability to detect compound A.
28. The method of claim 27, wherein optimization results in increased amplitude of response by the biosensor.
29. The method of claim 27, wherein compound A and compound B are different.
30. The method of claim 27, wherein compound A and compound B are identical.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040110174A1 (en) * 2001-01-25 2004-06-10 Niel Goldsmith Concatemers of differentially expressed multiple genes
US20040241672A1 (en) * 2001-01-25 2004-12-02 Neil Goldsmith Library of a collection of cells
US20050164162A1 (en) * 2002-01-25 2005-07-28 Evolva Ltd., C/O Dr. Iur. Martin Eisenring Methods for multiple parameters screening and evolution of cells to produce small molecules with multiple functionalities
US20070048793A1 (en) * 2005-07-12 2007-03-01 Baynes Brian M Compositions and methods for biocatalytic engineering
US11728008B2 (en) * 2020-09-03 2023-08-15 Melonfrost, Inc. Machine learning and control systems and methods for learning and steering evolutionary dynamics

Families Citing this family (1093)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6759226B1 (en) * 2000-05-24 2004-07-06 Third Wave Technologies, Inc. Enzymes for the detection of specific nucleic acid sequences
US7045289B2 (en) * 1991-09-09 2006-05-16 Third Wave Technologies, Inc. Detection of RNA Sequences
US7150982B2 (en) * 1991-09-09 2006-12-19 Third Wave Technologies, Inc. RNA detection assays
US6268140B1 (en) * 1994-02-03 2001-07-31 Neugenesis Combinatorial metabolic libraries
US6165793A (en) * 1996-03-25 2000-12-26 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6406855B1 (en) 1994-02-17 2002-06-18 Maxygen, Inc. Methods and compositions for polypeptide engineering
US5837458A (en) 1994-02-17 1998-11-17 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
US6995017B1 (en) 1994-02-17 2006-02-07 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6117679A (en) * 1994-02-17 2000-09-12 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US5605793A (en) * 1994-02-17 1997-02-25 Affymax Technologies N.V. Methods for in vitro recombination
US6395547B1 (en) 1994-02-17 2002-05-28 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US20060257890A1 (en) * 1996-05-20 2006-11-16 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
US6309883B1 (en) 1994-02-17 2001-10-30 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
US6335160B1 (en) 1995-02-17 2002-01-01 Maxygen, Inc. Methods and compositions for polypeptide engineering
US7597886B2 (en) * 1994-11-07 2009-10-06 Human Genome Sciences, Inc. Tumor necrosis factor-gamma
US7820798B2 (en) * 1994-11-07 2010-10-26 Human Genome Sciences, Inc. Tumor necrosis factor-gamma
US6143557A (en) * 1995-06-07 2000-11-07 Life Technologies, Inc. Recombination cloning using engineered recombination sites
US6720140B1 (en) 1995-06-07 2004-04-13 Invitrogen Corporation Recombinational cloning using engineered recombination sites
US6964861B1 (en) 1998-11-13 2005-11-15 Invitrogen Corporation Enhanced in vitro recombinational cloning of using ribosomal proteins
US6455254B1 (en) 1995-07-18 2002-09-24 Diversa Corporation Sequence based screening
US5958672A (en) 1995-07-18 1999-09-28 Diversa Corporation Protein activity screening of clones having DNA from uncultivated microorganisms
US6004788A (en) * 1995-07-18 1999-12-21 Diversa Corporation Enzyme kits and libraries
US20020028443A1 (en) * 1999-09-27 2002-03-07 Jay M. Short Method of dna shuffling with polynucleotides produced by blocking or interrupting a synthesis or amplification process
US20030219752A1 (en) * 1995-12-07 2003-11-27 Diversa Corporation Novel antigen binding molecules for therapeutic, diagnostic, prophylactic, enzymatic, industrial, and agricultural applications, and methods for generating and screening thereof
US6939689B2 (en) 1995-12-07 2005-09-06 Diversa Corporation Exonuclease-mediated nucleic acid reassembly in directed evolution
US5939250A (en) * 1995-12-07 1999-08-17 Diversa Corporation Production of enzymes having desired activities by mutagenesis
US7018793B1 (en) 1995-12-07 2006-03-28 Diversa Corporation Combinatorial screening of mixed populations of organisms
US6344328B1 (en) 1995-12-07 2002-02-05 Diversa Corporation Method for screening for enzyme activity
US6489145B1 (en) 1996-07-09 2002-12-03 Diversa Corporation Method of DNA shuffling
US6740506B2 (en) 1995-12-07 2004-05-25 Diversa Corporation End selection in directed evolution
US5965408A (en) * 1996-07-09 1999-10-12 Diversa Corporation Method of DNA reassembly by interrupting synthesis
US6358709B1 (en) 1995-12-07 2002-03-19 Diversa Corporation End selection in directed evolution
US6537776B1 (en) 1999-06-14 2003-03-25 Diversa Corporation Synthetic ligation reassembly in directed evolution
US20020164580A1 (en) * 1995-12-07 2002-11-07 Diversa Corporation Combinatorial screening of mixed populations of organisms
US6713279B1 (en) 1995-12-07 2004-03-30 Diversa Corporation Non-stochastic generation of genetic vaccines and enzymes
US6764835B2 (en) 1995-12-07 2004-07-20 Diversa Corporation Saturation mutageneis in directed evolution
US6361974B1 (en) 1995-12-07 2002-03-26 Diversa Corporation Exonuclease-mediated nucleic acid reassembly in directed evolution
US20020031771A1 (en) * 1995-12-07 2002-03-14 Short Jay M. Sequence based screening
US5830696A (en) 1996-12-05 1998-11-03 Diversa Corporation Directed evolution of thermophilic enzymes
US7888466B2 (en) 1996-01-11 2011-02-15 Human Genome Sciences, Inc. Human G-protein chemokine receptor HSATU68
US6096548A (en) 1996-03-25 2000-08-01 Maxygen, Inc. Method for directing evolution of a virus
US6506602B1 (en) 1996-03-25 2003-01-14 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6242211B1 (en) 1996-04-24 2001-06-05 Terragen Discovery, Inc. Methods for generating and screening novel metabolic pathways
AU4113797A (en) 1996-07-10 1998-02-02 Lonza A.G. Method of preparing (s) - or (r) -3,3,3-trifluoro-2-hydroxy-2- methylpropionic acid
US20070009930A1 (en) * 1996-12-18 2007-01-11 Maxygen, Inc. Methods and compositions for polypeptide engineering
WO1998028416A1 (en) * 1996-12-20 1998-07-02 Novo Nordisk A/S In vivo recombination
US6326204B1 (en) 1997-01-17 2001-12-04 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
KR100570935B1 (en) * 1997-01-17 2006-04-13 맥시겐, 인크. Evolution of whole cells and organisms by recursive sequence recombination
US7148054B2 (en) * 1997-01-17 2006-12-12 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
GB9701425D0 (en) 1997-01-24 1997-03-12 Bioinvent Int Ab A method for in vitro molecular evolution of protein function
US6159687A (en) 1997-03-18 2000-12-12 Novo Nordisk A/S Methods for generating recombined polynucleotides
DE69840382D1 (en) * 1997-03-18 2009-02-05 Novozymes As METHOD FOR THE PRODUCTION OF A LIBRARY THROUGH DNA SHUFFLING
US6291165B1 (en) * 1997-03-18 2001-09-18 Novo Nordisk A/S Shuffling of heterologous DNA sequences
US6159688A (en) * 1997-03-18 2000-12-12 Novo Nordisk A/S Methods of producing polynucleotide variants
US6153410A (en) * 1997-03-25 2000-11-28 California Institute Of Technology Recombination of polynucleotide sequences using random or defined primers
US6969584B2 (en) * 1997-06-12 2005-11-29 Rigel Pharmaceuticals, Inc. Combinatorial enzymatic complexes
US20050070005A1 (en) * 1997-06-16 2005-03-31 Martin Keller High throughput or capillary-based screening for a bioactivity or biomolecule
US20040241759A1 (en) * 1997-06-16 2004-12-02 Eileen Tozer High throughput screening of libraries
US6225074B1 (en) * 1997-08-18 2001-05-01 Dennis Wright Direct chloramphenicol acetyl transferase assay
CN101125873A (en) * 1997-10-24 2008-02-20 茵维特罗根公司 Recombinational cloning using nucleic acids having recombination sites
US7351578B2 (en) * 1999-12-10 2008-04-01 Invitrogen Corp. Use of multiple recombination sites with unique specificity in recombinational cloning
US20030124555A1 (en) * 2001-05-21 2003-07-03 Invitrogen Corporation Compositions and methods for use in isolation of nucleic acid molecules
WO1999021977A1 (en) 1997-10-24 1999-05-06 Life Technologies, Inc. Recombinational cloning using nucleic acids having recombination sites
WO1999021979A1 (en) 1997-10-28 1999-05-06 Maxygen, Inc. Human papillomavirus vectors
EP1030861A4 (en) 1997-10-31 2001-09-05 Maxygen Inc Modification of virus tropism and host range by viral genome shuffling
KR20010032861A (en) 1997-12-08 2001-04-25 캘리포니아 인스티튜트 오브 테크놀로지 Method for creating polynucleotide and polypeptide sequences
US20030166169A1 (en) * 1998-01-16 2003-09-04 Padgett Hal S. Method for constructing viral nucleic acids in a cell-free manner
US6468745B1 (en) 1998-01-16 2002-10-22 Large Scale Biology Corporation Method for expressing a library of nucleic acid sequence variants and selecting desired traits
US6426185B1 (en) 1998-01-16 2002-07-30 Large Scale Biology Corporation Method of compiling a functional gene profile in a plant by transfecting a nucleic acid sequence of a donor plant into a different host plant in an anti-sense orientation
US6303848B1 (en) * 1998-01-16 2001-10-16 Large Scale Biology Corporation Method for conferring herbicide, pest, or disease resistance in plant hosts
US20020164585A1 (en) * 1998-01-16 2002-11-07 Sean Chapman Method for enhancing RNA or protein production using non-native 5' untranslated sequences in recombinant viral nucleic acids
US6841347B1 (en) * 1998-02-05 2005-01-11 The General Hospital Corporation In vivo construction of DNA libraries
US6541011B2 (en) 1998-02-11 2003-04-01 Maxygen, Inc. Antigen library immunization
US7390619B1 (en) * 1998-02-11 2008-06-24 Maxygen, Inc. Optimization of immunomodulatory properties of genetic vaccines
US20020147143A1 (en) 1998-03-18 2002-10-10 Corixa Corporation Compositions and methods for the therapy and diagnosis of lung cancer
WO1999057128A1 (en) 1998-05-01 1999-11-11 Maxygen, Inc. Optimization of pest resistance genes using dna shuffling
US6902918B1 (en) 1998-05-21 2005-06-07 California Institute Of Technology Oxygenase enzymes and screening method
US20030207345A1 (en) * 1998-05-21 2003-11-06 California Institute Of Technology Oxygenase enzymes and screening method
US7153655B2 (en) 1998-06-16 2006-12-26 Alligator Bioscience Ab Method for in vitro molecular evolution of protein function involving the use of exonuclease enzyme and two populations of parent polynucleotide sequence
JP2002517995A (en) * 1998-06-17 2002-06-25 マキシジェン， インコーポレイテッド Method for producing a polynucleotide having desired properties
US6365408B1 (en) 1998-06-19 2002-04-02 Maxygen, Inc. Methods of evolving a polynucleotides by mutagenesis and recombination
US6846655B1 (en) 1998-06-29 2005-01-25 Phylos, Inc. Methods for generating highly diverse libraries
US20030153042A1 (en) * 1998-07-28 2003-08-14 California Institute Of Technology Expression of functional eukaryotic proteins
US20030092023A1 (en) * 1998-08-12 2003-05-15 Daniel Dupret Method of shuffling polynucleotides using templates
US20030104417A1 (en) * 1998-08-12 2003-06-05 Proteus S.A. Template-mediated, ligation-oriented method of nonrandomly shuffling polynucleotides
US20060242731A1 (en) * 1998-08-12 2006-10-26 Venkiteswaran Subramanian DNA shuffling to produce herbicide selective crops
US6991922B2 (en) * 1998-08-12 2006-01-31 Proteus S.A. Process for in vitro creation of recombinant polynucleotide sequences by oriented ligation
US6951719B1 (en) * 1999-08-11 2005-10-04 Proteus S.A. Process for obtaining recombined nucleotide sequences in vitro, libraries of sequences and sequences thus obtained
JP2002522072A (en) 1998-08-12 2002-07-23 マキシジェン， インコーポレイテッド DNA shuffling of monooxygenase gene for production of industrial chemicals.
US20020058249A1 (en) * 1998-08-12 2002-05-16 Venkiteswaran Subramanian Dna shuffling to produce herbicide selective crops
WO2000015809A2 (en) * 1998-09-15 2000-03-23 Syngenta Participations Ag Uracil permease from arabidopsis as herbicidal target gene
US20070178475A1 (en) * 1998-09-17 2007-08-02 Nehls Michael C Novel human polynucleotides and polypeptides encoded thereby
KR20010085850A (en) * 1998-09-29 2001-09-07 추후제출 Shuffling of codon altered genes
AU6510799A (en) * 1998-10-07 2000-04-26 Maxygen, Inc. Dna shuffling to produce nucleic acids for mycotoxin detoxification
WO2000028018A1 (en) 1998-11-10 2000-05-18 Maxygen, Inc. Modified adp-glucose pyrophosphorylase for improvement and optimization of plant phenotypes
AU2023700A (en) * 1998-11-10 2000-05-29 Maxygen, Inc. Modified ribulose 1,5-bisphosphate carboxylase/oxygenase
US6438561B1 (en) * 1998-11-19 2002-08-20 Navigation Technologies Corp. Method and system for using real-time traffic broadcasts with navigation systems
ATE338120T2 (en) * 1998-11-27 2006-09-15 Ucb Sa COMPOSITIONS AND METHODS FOR INCREASE BONE MINERALIZATION
US20040009535A1 (en) 1998-11-27 2004-01-15 Celltech R&D, Inc. Compositions and methods for increasing bone mineralization
US6570003B1 (en) * 2001-01-09 2003-05-27 Lexion Genetics Incorporated Human 7TM proteins and polynucleotides encoding the same
US20020031802A1 (en) * 2000-05-12 2002-03-14 Yi Hu Novel seven transmembrane proteins and polynucleotides encoding the same
DE60042730D1 (en) 1999-01-05 2009-09-24 Univ Boston IMPROVED CLONING PROCESS
US6358712B1 (en) 1999-01-05 2002-03-19 Trustee Of Boston University Ordered gene assembly
US20040005673A1 (en) * 2001-06-29 2004-01-08 Kevin Jarrell System for manipulating nucleic acids
EP1151409A1 (en) * 1999-01-18 2001-11-07 Maxygen, Inc. Methods of populating data stuctures for use in evolutionary simulations
US6917882B2 (en) * 1999-01-19 2005-07-12 Maxygen, Inc. Methods for making character strings, polynucleotides and polypeptides having desired characteristics
US6368861B1 (en) * 1999-01-19 2002-04-09 Maxygen, Inc. Oligonucleotide mediated nucleic acid recombination
US6376246B1 (en) 1999-02-05 2002-04-23 Maxygen, Inc. Oligonucleotide mediated nucleic acid recombination
US20030054390A1 (en) * 1999-01-19 2003-03-20 Maxygen, Inc. Oligonucleotide mediated nucleic acid recombination
US6436675B1 (en) 1999-09-28 2002-08-20 Maxygen, Inc. Use of codon-varied oligonucleotide synthesis for synthetic shuffling
US7024312B1 (en) 1999-01-19 2006-04-04 Maxygen, Inc. Methods for making character strings, polynucleotides and polypeptides having desired characteristics
AU777909B2 (en) 1999-01-19 2004-11-04 Lexicon Genetics Incorporated Mammalian cortexin-like proteins and polynucleotides encoding the same
US20070065838A1 (en) * 1999-01-19 2007-03-22 Maxygen, Inc. Oligonucleotide mediated nucleic acid recombination
EP2253704B1 (en) * 1999-01-19 2015-08-19 Codexis Mayflower Holdings, LLC Oligonucleotide mediated nucleic acid recombination
US6961664B2 (en) 1999-01-19 2005-11-01 Maxygen Methods of populating data structures for use in evolutionary simulations
US20090130718A1 (en) * 1999-02-04 2009-05-21 Diversa Corporation Gene site saturation mutagenesis
EP1153037A1 (en) * 1999-02-09 2001-11-14 Lexicon Genetics Incorporated Human uncoupling proteins and polynucleotides encoding the same
IL144657A0 (en) 1999-02-11 2002-06-30 Maxygen Inc High throughput mass spectrometry
AU3501700A (en) 1999-02-26 2000-09-14 Human Genome Sciences, Inc. Human endokine alpha and methods of use
NZ525134A (en) 1999-03-02 2004-09-24 Invitrogen Corp Compositions and methods for use in recombinational cloning of nucleic acids
JP3399518B2 (en) * 1999-03-03 2003-04-21 インターナショナル・ビジネス・マシーンズ・コーポレーション Semiconductor structure and method of manufacturing the same
EP1165775A2 (en) 1999-03-05 2002-01-02 Maxygen, Inc. Recombination of insertion modified nucleic acids
US6531316B1 (en) 1999-03-05 2003-03-11 Maxyag, Inc. Encryption of traits using split gene sequences and engineered genetic elements
US20030186226A1 (en) * 1999-03-08 2003-10-02 Brennan Thomas M. Methods and compositions for economically synthesizing and assembling long DNA sequences
US6773918B2 (en) * 1999-03-09 2004-08-10 The Regents Of The University Of California Detection of phenols using engineered bacteria
AU4211600A (en) * 1999-04-10 2000-11-14 Maxygen, Inc. Modified lipid production
AU4456200A (en) * 1999-04-13 2000-11-14 Maxygen, Inc. Modified starch metabolism enzymes and encoding genes for improvement and optimization of plant phenotypes
AU4469700A (en) * 1999-04-19 2000-11-02 Bios Group Lp A system and method for molecule selection using extended target shape
US20020110809A1 (en) * 1999-04-30 2002-08-15 Nehls Michael C. Novel human polynucleotides and polypeptides encoded thereby
US20020095031A1 (en) * 1999-05-04 2002-07-18 Nehls Michael C. Novel human polynucleotides and polypeptides encoded thereby
WO2001004287A1 (en) * 1999-07-07 2001-01-18 Maxygen Aps A method for preparing modified polypeptides
WO2001010903A2 (en) * 1999-08-09 2001-02-15 Incyte Genomics, Inc. Proteases and protease inhibitors
IL147384A0 (en) * 1999-08-12 2002-08-14 Maxygen Inc Dna shuffling of dioxygenase genes for production of industrial chemicals
AU778837B2 (en) * 1999-08-12 2004-12-23 Zoetis Services Llc Streptomyces avermitilis gene directing the ratio of B2:B1 avermectins
AU784533B2 (en) * 1999-08-24 2006-04-27 Lexicon Genetics Incorporated Sequence derived from a human mammary gland CDNA library
US20030166902A1 (en) * 2001-02-20 2003-09-04 Yi Hu Novel human protease and polynucleotides encoding the same
US6433153B1 (en) 1999-09-02 2002-08-13 Lexicon Genetics Incorporated Human calcium dependent proteases and polynucleotides encoding the same
US20030050464A1 (en) * 2000-07-28 2003-03-13 Yi Hu Novel human proteases and polynucleotides encoding the same
US6448388B1 (en) 2000-08-16 2002-09-10 Lexicon Genetics Incorporated Human proteases and polynucleotides encoding the same
US6716614B1 (en) 1999-09-02 2004-04-06 Lexicon Genetics Incorporated Human calcium dependent proteases, polynucleotides encoding the same, and uses thereof
US20050153323A1 (en) * 2000-07-28 2005-07-14 Yi Hu Novel human proteases and polynucleotides encoding the same
US20080003673A1 (en) * 1999-09-02 2008-01-03 Alejandro Abuin Novel human proteases and polynucleotides encoding the same
WO2001018042A2 (en) * 1999-09-10 2001-03-15 Incyte Genomics, Inc. Apoptosis proteins
US20020107382A1 (en) * 2000-09-27 2002-08-08 Friddle Carl Johan Novel human protease inhibitor proteins and polynucleotides encoding the same
AU780770B2 (en) 1999-09-24 2005-04-14 Lexicon Pharmaceuticals, Inc. Novel human endothelin converting enzyme-like proteins and polynucleotides encoding the same
US6790660B1 (en) * 2001-09-18 2004-09-14 Lexicon Genetics Incorporated Human kielin-like proteins and polynucleotides encoding the same
WO2001021651A2 (en) * 1999-09-24 2001-03-29 Lexicon Genetics Incorporated Novel human protease inhibitor-like proteins and polynucleotides encoding the same
US6867291B1 (en) * 2000-09-15 2005-03-15 Lexicon Genetics Incorporated Human hemicentin proteins and polynucleotides encoding the same
US6841377B1 (en) * 2001-06-13 2005-01-11 Lexicon Genetics Incorporated Human kinase and polynucleotides encoding the same
US20080050809A1 (en) * 1999-09-28 2008-02-28 Alejandro Abuin Novel human kinases and polynucleotides encoding the same
US6541252B1 (en) 2000-05-19 2003-04-01 Lexicon Genetics Incorporated Human kinases and polynucleotides encoding the same
US6720173B1 (en) 1999-11-08 2004-04-13 Lexicon Genetics Incorporated Human kinase protein and polynucleotides encoding the same
US6797510B1 (en) 2001-05-24 2004-09-28 Lexicon Genetics Incorporated Human kinases and polynucleotides encoding the same
US6864079B2 (en) 2001-04-06 2005-03-08 Lexicon Genetics Incorporated Human kinase and polynucleotides encoding the same
US6716616B1 (en) * 1999-09-28 2004-04-06 Lexicon Genetics Incorporated Human kinase proteins and polynucleotides encoding the same
US6734010B2 (en) * 2001-05-09 2004-05-11 Lexicon Genetics Incorporated Human kinases and polynucleotides encoding the same
US6511840B1 (en) 2000-06-15 2003-01-28 Lexicon Genetics Incorporated Human kinase proteins and polynucleotides encoding the same
US6734009B2 (en) 2000-12-27 2004-05-11 Lexicon Genetics Incorporated Human kinases and polynucleotides encoding the same
US6759527B2 (en) 2001-03-20 2004-07-06 Lexicon Genetics Incorporated Human kinase and polynucleotides encoding the same
US6586230B1 (en) * 2000-10-27 2003-07-01 Lexicon Genetics Incorporated Human kinase and polynucleotides encoding the same
JP2003512027A (en) 1999-09-29 2003-04-02 レキシコン・ジェネティクス・インコーポレーテッド Novel human carboxypeptidase and polynucleotide encoding the same
US20040002474A1 (en) * 1999-10-07 2004-01-01 Maxygen Inc. IFN-alpha homologues
WO2001027274A1 (en) 1999-10-12 2001-04-19 Lexicon Genetics Incorporated Human ldl receptor family proteins and polynucleotides encoding the same
US7430477B2 (en) * 1999-10-12 2008-09-30 Maxygen, Inc. Methods of populating data structures for use in evolutionary simulations
AU1198701A (en) * 1999-10-13 2001-04-23 Lexicon Genetics Incorporated Novel human membrane proteins
US6743907B1 (en) * 1999-10-19 2004-06-01 Lexicon Genetics Incorporated Human proteins and polynucleotides encoding the same
US20080213878A1 (en) * 1999-10-19 2008-09-04 Gregory Donoho Novel human membrane proteins and polynucleotides encoding the same
EP1244698A2 (en) 1999-11-02 2002-10-02 Lexicon Genetics Incorporated Novel human transporter proteins and polynucleotides encoding the same
AU783485B2 (en) 1999-11-10 2005-11-03 Lexicon Pharmaceuticals, Inc. Human atpase proteins and polynucleotides encoding the same
AU783948B2 (en) * 1999-11-12 2006-01-05 Lexicon Genetics Incorporated Novel human proteases and polynucleotides encoding the same
AU1920601A (en) * 1999-11-19 2001-05-30 Lexicon Genetics Incorporated Novel human secreted proteins and polynucleotides encoding the same
US6686515B1 (en) 1999-11-23 2004-02-03 Maxygen, Inc. Homologous recombination in plants
US7115712B1 (en) * 1999-12-02 2006-10-03 Maxygen, Inc. Cytokine polypeptides
AU2070701A (en) * 1999-12-07 2001-06-18 Lexicon Genetics Incorporated Novel human membrane proteins and polynucleotides encoding the same
AU785082B2 (en) * 1999-12-07 2006-09-14 Lexicon Pharmaceuticals, Inc. Novel human kinase proteins and polynucleotides encoding the same
EP1238068A1 (en) * 1999-12-08 2002-09-11 California Institute Of Technology Directed evolution of biosynthetic and biodegration pathways
JP2004500060A (en) * 1999-12-09 2004-01-08 レキシコン・ジェネティクス・インコーポレーテッド Human ADAM-TS protease and polynucleotide encoding the same
NZ539430A (en) 1999-12-10 2006-09-29 Invitrogen Corp Use of multiple recombination sites with unique specificity in recombinational cloning
EP1263966A1 (en) * 1999-12-13 2002-12-11 Lexicon Genetics Incorporated Novel human transferase proteins and polynucleotides encoding the same
EP1242832A1 (en) * 1999-12-17 2002-09-25 Maxygen, Inc. Methods for parallel detection of compositions having desired characteristics by means of mri spectroscopy
AU783901B2 (en) * 1999-12-22 2005-12-22 Lexicon Pharmaceuticals, Inc. Human membrane proteins and polynucleotides encoding the same having Homology to CD20 Proteins and IGE Receptors
EP1244795A1 (en) * 2000-01-06 2002-10-02 Lexicon Genetics Incorporated Novel human proteases and polynucleotides encoding the same
EP2133098A1 (en) 2000-01-10 2009-12-16 Maxygen Holdings Ltd G-CSF conjugates
SG121902A1 (en) * 2000-01-11 2006-05-26 Maxygen Inc Integrated systems for diversity generation and screening
AU2001229318A1 (en) * 2000-01-12 2001-07-24 Lexicon Genetics Incorporated Human olfactory receptor and polynucleotides encoding the same
AU784540B2 (en) * 2000-01-18 2006-04-27 Lexicon Pharmaceuticals, Inc. Novel human kinase protein and polynucleotides encoding the same
DE60118703T2 (en) * 2000-01-26 2006-12-14 Lexicon Genetics Inc., The Woodlands HUMAN NEUREXIN-SIMILAR PROTEINS AND POLYNUCLEOTIDES THAT CODE
US20020115837A1 (en) * 2000-01-28 2002-08-22 Turner C. Alexander Novel human membrane proteins and polynucleotides encoding the same
AU9256001A (en) * 2000-01-28 2001-12-17 Lexicon Genetics Incorporated Novel human enzymes and polynucleotides encoding the same
WO2001057086A2 (en) * 2000-02-04 2001-08-09 Lexicon Genetics Incorporated Novel human g protein coupled receptor proteins and polynucleotides encoding the same
RU2278123C2 (en) 2000-02-11 2006-06-20 Максиджен Холдингз Лтд. Molecules similar to factor vii or viia
AU2001241939A1 (en) * 2000-02-28 2001-09-12 Maxygen, Inc. Single-stranded nucleic acid template-mediated recombination and nucleic acid fragment isolation
JP2003531582A (en) * 2000-02-29 2003-10-28 レキシコン・ジェネティクス・インコーポレーテッド Human transporter protein and polynucleotide encoding the same
WO2001064903A2 (en) * 2000-02-29 2001-09-07 Lexicon Genetics Incorporated Novel human transferase proteins and polynucleotides encoding the same
CA2401454A1 (en) * 2000-03-03 2001-09-13 Incyte Genomics, Inc. G-protein coupled receptors
AU2001247283A1 (en) * 2000-03-06 2001-09-17 Lexicon Genetics Incorporated Human transporter proteins and polynucleotides encoding the same
CA2402227A1 (en) 2000-03-10 2001-09-20 Lexicon Genetics Incorporated Human g-coupled protein receptor kinases and polynucleotides encoding the same
EP1317551B1 (en) * 2000-03-13 2007-02-28 Lexicon Genetics Incorporated Human phospholipases and polynucleotides encoding the same
EP1268538A2 (en) * 2000-03-20 2003-01-02 Lexicon Genetics Incorporated Human secreted proteins and polynucleotides encoding the same
AU2001287273A1 (en) * 2000-03-24 2001-10-08 Maxygen, Inc. Methods for modulating cellular and organismal phenotypes
WO2001075108A1 (en) * 2000-04-03 2001-10-11 Lexicon Genetics Incorporated Human ion channel protein and polynucleotides encoding the same
EP1832599A3 (en) 2000-04-12 2007-11-21 Human Genome Sciences, Inc. Albumin fusion proteins
CA2405729A1 (en) * 2000-04-12 2001-10-25 Lexicon Genetics Incorporated Human metalloprotease and polynucleotides encoding the same
EP1276873A2 (en) 2000-04-25 2003-01-22 Lexicon Genetics Incorporated Human kinase proteins and polynucleotides encoding the same
AU2001259156A1 (en) * 2000-04-27 2001-11-07 Lexicon Genetics Incorporated Novel membrane proteins and polynucleotides encoding the same
JP2003533205A (en) * 2000-05-12 2003-11-11 レキシコン・ジェネティクス・インコーポレーテッド Novel human lipocalin homologs and polynucleotides encoding the same
US7115403B1 (en) 2000-05-16 2006-10-03 The California Institute Of Technology Directed evolution of galactose oxidase enzymes
WO2001088133A2 (en) 2000-05-18 2001-11-22 Lexicon Genetics Incorporated Human semaphorin homologs and polynucleotides encoding the same
US7244560B2 (en) * 2000-05-21 2007-07-17 Invitrogen Corporation Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites
WO2001090179A2 (en) * 2000-05-23 2001-11-29 Lexicon Genetics Incorporated Novel human thrombospondin-like proteins and polynucleotides encoding the same
US6790667B1 (en) * 2000-05-30 2004-09-14 Lexicon Genetics Incorporated Human mitochondrial proteins and polynucleotides encoding the same
US20020111500A1 (en) * 2000-05-31 2002-08-15 Selifonov Sergey A. Preparation of 4-hydroxy-3[2H]-furanones
US20030031675A1 (en) 2000-06-06 2003-02-13 Mikesell Glen E. B7-related nucleic acids and polypeptides useful for immunomodulation
AU2001275337A1 (en) * 2000-06-07 2001-12-17 Lexicon Genetics Incorporated Novel human transporter proteins and polynucleotides encoding the same
US6465632B1 (en) * 2000-06-09 2002-10-15 Lexicon Genetics Incorporated Human phosphatases and polynucleotides encoding the same
AU2001266844A1 (en) * 2000-06-09 2001-12-17 Lexicon Genetics Incorporated Novel human seven transmembrane proteins and polynucleotides encoding the same
CA2413160A1 (en) 2000-06-15 2001-12-20 Human Genome Sciences, Inc. Human tumor necrosis factor delta and epsilon
EP2093293A3 (en) 2000-06-16 2010-07-07 Incyte Corporation G-Protein coupled receptors
PT2281843T (en) 2000-06-16 2017-01-02 Human Genome Sciences Inc Antibodies that immunospecifically bind to blys
WO2002000897A2 (en) * 2000-06-23 2002-01-03 Maxygen, Inc. Novel chimeric promoters
AU2001272978A1 (en) * 2000-06-23 2002-01-08 Maxygen, Inc. Novel co-stimulatory molecules
US7074591B2 (en) * 2000-06-27 2006-07-11 Lexicon Genetics Incorporated Polynucleotides encoding human GABA receptors
EP1961819A3 (en) 2000-06-28 2008-11-12 Corixa Corporation Composition and methods for the therapy and diagnosis of lung cancer
WO2002004629A2 (en) * 2000-07-07 2002-01-17 Maxygen, Inc. Molecular breeding of transposable elements
US6858422B2 (en) * 2000-07-13 2005-02-22 Codexis, Inc. Lipase genes
EP1315800A2 (en) * 2000-07-21 2003-06-04 Incyte Genomics, Inc. Human proteases
US6878861B2 (en) * 2000-07-21 2005-04-12 Washington State University Research Foundation Acyl coenzyme A thioesterases
US6887685B1 (en) 2000-07-25 2005-05-03 Lexicon Genetics Incorporated Human thymosin protein and polynucleotides encoding the same
EP1354031A2 (en) * 2000-07-31 2003-10-22 Maxygen, Inc. Nucleotide incorporating enzymes
AU2001280968A1 (en) * 2000-07-31 2002-02-13 Menzel, Rolf Compositions and methods for directed gene assembly
US20020076780A1 (en) * 2000-08-16 2002-06-20 Turner C. Alexander Novel human ion channel proteins and polynucleotides encoding the same
US7198924B2 (en) 2000-12-11 2007-04-03 Invitrogen Corporation Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites
JP2004531201A (en) * 2000-08-22 2004-10-14 レキシコン・ジェネティクス・インコーポレーテッド Novel human protease and polynucleotide encoding the protease
EP1311676A2 (en) * 2000-08-22 2003-05-21 Lexicon Genetics Incorporated Human 7tm proteins and polynucleotides encoding the same
WO2002016583A2 (en) * 2000-08-24 2002-02-28 Maxygen, Inc. Constructs and their use in metabolic pathway engineering
EP2157183A1 (en) 2000-08-25 2010-02-24 BASF Plant Science GmbH Plant polynucleotides encoding prenyl proteases
WO2002018555A2 (en) * 2000-08-31 2002-03-07 Lexicon Genetics Incorporated Human kinase proteins and polynucleotides encoding the same
US6660507B2 (en) 2000-09-01 2003-12-09 E. I. Du Pont De Nemours And Company Genes involved in isoprenoid compound production
WO2002020753A2 (en) * 2000-09-01 2002-03-14 Lexicon Genetics Incorporated Human gaba transporter protein and polynucleotides encoding the same
AU2001293019B2 (en) * 2000-09-27 2007-01-18 Lexicon Pharmaceuticals, Inc. Human ion-exchanger proteins and polynucleotides encoding the same
US6777232B1 (en) * 2000-10-02 2004-08-17 Lexicon Genetics Incorporated Human membrane proteins and polynucleotides encoding the same
WO2002033089A2 (en) * 2000-10-05 2002-04-25 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Ixodes scapularis tissue factor pathway inhibitor
WO2002031129A2 (en) 2000-10-12 2002-04-18 Lexicon Genetics Incorporated Human kinases and polynucleotides encoding the same
US7001763B1 (en) 2000-10-17 2006-02-21 Lexicon Genetics Incorporated Human semaphorin proteins and polynucleotides encoding the same
US7605304B2 (en) * 2000-10-24 2009-10-20 E.I. Du Pont De Nemours And Company Genes encoding novel bacillus thuringiensis proteins with pesticidal activity against coleopterans
US7060442B2 (en) * 2000-10-30 2006-06-13 Regents Of The University Of Michigan Modulators on Nod2 signaling
CA2427701A1 (en) * 2000-10-30 2002-05-10 Lexicon Genetics Incorporated Human 7tm proteins and polynucleotides encoding the same
AU2004260931B9 (en) 2003-04-29 2012-01-19 E.I. Du Pont De Nemours And Company Novel glyphosate-N-acetyltransferase (GAT) genes
US7202070B2 (en) * 2000-10-31 2007-04-10 Biocatalytics, Inc. Method for reductive amination of a ketone using a mutated enzyme
ATE336571T1 (en) * 2000-11-01 2006-09-15 Lexicon Genetics Inc POLYNUCLEOTIDES THAT CODE FOR HUMAN MELTRIN BETA (ADAM19) METALLOENDOPEPTIDASES
US7608704B2 (en) 2000-11-08 2009-10-27 Incyte Corporation Secreted proteins
WO2002050278A2 (en) * 2000-11-15 2002-06-27 Lexicon Genetics Incorporated Novel human secreted proteins and polynucleotides encoding the same
AU2002228633A1 (en) * 2000-11-20 2002-06-03 Lexicon Genetics Incorporated Human kinases and polynucleotides encoding the same
TWI327599B (en) 2000-11-28 2010-07-21 Medimmune Llc Methods of administering/dosing anti-rsv antibodies for prophylaxis and treatment
JP2004517622A (en) 2000-12-11 2004-06-17 レキシコン・ジェネティクス・インコーポレーテッド Novel human kinase and polynucleotide encoding the kinase
JP2004530417A (en) * 2000-12-12 2004-10-07 レキシコン・ジェネティクス・インコーポレーテッド Novel human kinase and polynucleotide encoding it
US6958213B2 (en) 2000-12-12 2005-10-25 Alligator Bioscience Ab Method for in vitro molecular evolution of protein function
DE10062086A1 (en) * 2000-12-13 2002-07-04 Wella Ag Agent and method of dyeing keratin fibers
US6583269B1 (en) 2000-12-18 2003-06-24 Lexicon Genetics Incorporated Human protease inhibitor and polynucleotides encoding the same
US7367155B2 (en) 2000-12-20 2008-05-06 Monsanto Technology Llc Apparatus and methods for analyzing and improving agricultural products
CA2432737A1 (en) * 2000-12-20 2002-06-27 Lexicon Genetics Incorporated Human ion channel protein and polynucleotides encoding the same
US6852844B1 (en) * 2000-12-20 2005-02-08 Lexicon Genetics Incorporated Human protocadherin proteins and polynucleotides encoding the same
US20020086292A1 (en) 2000-12-22 2002-07-04 Shigeaki Harayama Synthesis of hybrid polynucleotide molecules using single-stranded polynucleotide molecules
AU2001297533A1 (en) * 2000-12-28 2002-09-12 Lexicon Genetics Incorporated Novel human ion channel-related proteins and polynucleotides encoding the same
WO2002053753A2 (en) * 2001-01-05 2002-07-11 Lexicon Genetics Incorporated Novel human lipase and polynucleotides encoding the same
JP2004524835A (en) * 2001-01-23 2004-08-19 レキシコン・ジェネティクス・インコーポレーテッド Novel human kinase and polynucleotide encoding it
CA2434897A1 (en) * 2001-01-24 2002-08-01 Lexicon Genetics Incorporated Human lipase and polynucleotides encoding the same
EP1683865A3 (en) 2001-02-02 2006-10-25 Eli Lilly & Company Mammalian proteins and in particular CD200
WO2002063002A2 (en) * 2001-02-02 2002-08-15 Lexicon Genetics Incorporated Novel human transporter protein and polynucleotides encoding the same
EP1366058B1 (en) 2001-02-09 2011-01-26 Human Genome Sciences, Inc. Human g-protein chemokine receptor (ccr5) hdgnr10
US6849449B2 (en) * 2001-03-01 2005-02-01 Regents Of The University Of Michigan Orphanin FQ receptor nucleic acids
JP3929250B2 (en) * 2001-03-08 2007-06-13 株式会社ルネサステクノロジ Semiconductor device
US20020164627A1 (en) * 2001-03-12 2002-11-07 Wilganowski Nathaniel L. Novel human transporter proteins and polynucleotides encoding the same
AU2002306696B2 (en) * 2001-03-12 2007-12-20 Lexicon Pharmaceuticals, Inc. Novel human EGF-family proteins and polynucleotides encoding the same
US7252990B2 (en) * 2001-03-12 2007-08-07 Lexicon Genetics Incorporated Human dectin proteins and polynucleotides encoding the same
US6994995B1 (en) * 2001-03-16 2006-02-07 Lexicon Genetics Incorporated Human synaptotagmin and polynucleotides encoding the same
KR20030088472A (en) * 2001-03-30 2003-11-19 더 내셔날 푸드 리서치 인스티튜트 Method for increasing productivity of secondary metabolite by conferring drug-resistant mutations
AR035799A1 (en) 2001-03-30 2004-07-14 Syngenta Participations Ag INSECTICIDE TOXINS ISOLATED FROM BACILLUS THURINGIENSIS AND ITS USES.
EP1383879A4 (en) * 2001-04-06 2005-05-11 Lexicon Genetics Inc Novel human kinases and polynucleotides encoding the same
US6644173B2 (en) * 2001-04-11 2003-11-11 Keuring, Incorporated Beverage filter cartridge holder
EP2228389B1 (en) 2001-04-13 2015-07-08 Human Genome Sciences, Inc. Antibodies against vascular endothelial growth factor 2
EP1470219A4 (en) * 2001-04-16 2005-10-05 California Inst Of Techn Peroxide-driven cytochrome p450 oxygenase variants
US6607895B2 (en) * 2001-04-16 2003-08-19 Lexicon Genetics Incorporated Human adenylsuccinate synthetase and polynucleotides encoding the same
US20030077804A1 (en) * 2001-04-19 2003-04-24 Invitrogen Corporation Compositions and methods for recombinational cloning of nucleic acid molecules
WO2002088313A2 (en) * 2001-04-30 2002-11-07 Lexicon Genetics Incorporated Novel human nuclear transporters and polynucleotides encoding the same
EP1383887A4 (en) * 2001-05-03 2004-07-07 Rensselaer Polytech Inst Novel methods of directed evolution
JP2005504513A (en) 2001-05-09 2005-02-17 コリクサ コーポレイション Compositions and methods for treatment and diagnosis of prostate cancer
KR100430534B1 (en) * 2001-05-23 2004-05-10 주식회사 마이크로아이디 Method for constructing a chimeric dna library using a single strand specific dnase
US20020193585A1 (en) * 2001-05-25 2002-12-19 Walke D. Wade Novel human transporter proteins and polynucleotides encoding the same
US7064189B2 (en) 2001-05-25 2006-06-20 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to trail receptors
EP1461425B1 (en) 2001-05-29 2008-02-13 Lexicon Pharmaceuticals, Inc. Novel human hydroxylases and polynucleotides encoding the same
GB0113435D0 (en) * 2001-06-04 2001-07-25 Amersham Pharm Biotech Uk Ltd Acridone derivatives as labels for fluorescence detection of target materials
JP2004531268A (en) * 2001-06-14 2004-10-14 レキシコン・ジェネティクス・インコーポレーテッド Novel human transporter protein and polynucleotide encoding the same
MXPA03011890A (en) 2001-06-22 2004-06-03 Du Pont Defensin polynucleotides and methods of use.
AU2002318186A1 (en) * 2001-07-03 2003-01-21 Lexicon Genetics Incorporated Novel human kielin-like proteins and polynucleotides encoding the same
US7226768B2 (en) 2001-07-20 2007-06-05 The California Institute Of Technology Cytochrome P450 oxygenases
US6867189B2 (en) * 2001-07-26 2005-03-15 Genset S.A. Use of adipsin/complement factor D in the treatment of metabolic related disorders
JP2005500060A (en) * 2001-08-14 2005-01-06 レキシコン・ジェネティクス・インコーポレーテッド Novel human collagen protein and polynucleotide encoding the same
ATE557092T1 (en) 2001-08-29 2012-05-15 Genentech Inc BV8 NUCLEIC ACIDS AND POLYPEPTIDES WITH MITOGENIC ACTIVITY
WO2003020936A1 (en) 2001-08-31 2003-03-13 The Dow Chemical Company Nucleic acid compositions conferring altered metabolic characteristics
US20070020622A1 (en) * 2001-09-14 2007-01-25 Invitrogen Corporation DNA Polymerases and mutants thereof
US7108975B2 (en) * 2001-09-21 2006-09-19 Regents Of The University Of Michigan Atlastin
US7582425B2 (en) * 2001-09-21 2009-09-01 The Regents Of The University Of Michigan Atlastin
US7157277B2 (en) 2001-11-28 2007-01-02 Neose Technologies, Inc. Factor VIII remodeling and glycoconjugation of Factor VIII
US8008252B2 (en) 2001-10-10 2011-08-30 Novo Nordisk A/S Factor VII: remodeling and glycoconjugation of Factor VII
US7173003B2 (en) 2001-10-10 2007-02-06 Neose Technologies, Inc. Granulocyte colony stimulating factor: remodeling and glycoconjugation of G-CSF
AU2004236174B2 (en) 2001-10-10 2011-06-02 Novo Nordisk A/S Glycopegylation methods and proteins/peptides produced by the methods
EP2305311A3 (en) 2001-10-10 2011-07-20 BioGeneriX AG Glycoconjugation of peptides
ES2538342T3 (en) 2001-10-10 2015-06-19 Ratiopharm Gmbh Remodeling and glycoconjugation of follicle stimulating hormone (FSH)
US7214660B2 (en) 2001-10-10 2007-05-08 Neose Technologies, Inc. Erythropoietin: remodeling and glycoconjugation of erythropoietin
WO2003035842A2 (en) * 2001-10-24 2003-05-01 Dyax Corporation Hybridization control of sequence variation
US6768476B2 (en) * 2001-12-05 2004-07-27 Etenna Corporation Capacitively-loaded bent-wire monopole on an artificial magnetic conductor
ES2405790T3 (en) 2001-12-17 2013-06-03 Corixa Corporation Compositions and methods for therapy and diagnosis of inflammatory bowel disease
TWI262083B (en) * 2001-12-28 2006-09-21 Syngenta Participations Ag Microbially-expressed thermotolerant phytase for animal feed
US20070166704A1 (en) * 2002-01-18 2007-07-19 Fei Huang Identification of polynucleotides and polypeptide for predicting activity of compounds that interact with protein tyrosine kinases and/or protein tyrosine kinase pathways
US7255986B2 (en) * 2002-01-31 2007-08-14 The Board Of Trustees Operating Michigan State University Compositions for the diagnosis and treatment of epizootic catarrhal enteritis in ferrets
BRPI0307620B8 (en) * 2002-02-12 2016-09-13 Pah Usa 15 Llc polynucleotide molecules encoding avec variants, methods for producing a recombinant streptomyces avermitilis strain, as well as said recombinant cells and avermectin composition
AU2003222225A1 (en) * 2002-02-20 2003-09-09 Incyte Corporation Receptors and membrane-associated proteins
US20030166010A1 (en) * 2002-02-25 2003-09-04 Affholter Joseph A. Custom ligand design for biomolecular filtration and purification for bioseperation
US7879582B2 (en) * 2002-02-26 2011-02-01 E. I. Du Pont De Nemours And Company Method for the recombination of genetic elements
WO2003075129A2 (en) 2002-03-01 2003-09-12 Maxygen, Inc. Methods, systems, and software for identifying functional bio-molecules
US20040132101A1 (en) 2002-09-27 2004-07-08 Xencor Optimized Fc variants and methods for their generation
EP1488335A4 (en) 2002-03-09 2006-11-15 Maxygen Inc Optimization of crossover points for directed evolution
AU2003218456A1 (en) * 2002-04-01 2003-10-20 Human Genome Sciences, Inc. Antibodies that specifically bind to gmad
AU2003221841A1 (en) 2002-04-03 2003-10-27 Celltech R And D, Inc. Association of polymorphisms in the sost gene region with bone mineral density
BR0309017B1 (en) * 2002-04-04 2015-02-18 Monsanto Technology Llc AUTOMATIC MACHINE FOR HANDLING AND HANDLING INDIVIDUAL PARTS OF PRIVATE MATERIAL
JP2005535572A (en) 2002-04-12 2005-11-24 メディミューン，インコーポレーテッド Recombinant anti-interleukin-9 antibody
CN102174546A (en) 2002-04-19 2011-09-07 维莱尼姆公司 Phospholipases, nucleic acids encoding them and methods for making and using them
US7226771B2 (en) 2002-04-19 2007-06-05 Diversa Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
JP4372679B2 (en) * 2002-05-17 2009-11-25 アリゲーター・バイオサイエンス・アーベー In vitro molecular evolution of protein function
US7132100B2 (en) 2002-06-14 2006-11-07 Medimmune, Inc. Stabilized liquid anti-RSV antibody formulations
US7425618B2 (en) 2002-06-14 2008-09-16 Medimmune, Inc. Stabilized anti-respiratory syncytial virus (RSV) antibody formulations
AU2003247582A1 (en) * 2002-06-20 2004-01-06 Board Of Trustees Operating Michigan State University Plastid division and related genes and proteins, and methods of use
DE60336555D1 (en) 2002-06-21 2011-05-12 Novo Nordisk Healthcare Ag PEGYLATED GLYCO FORMS OF FACTOR VII
US9321832B2 (en) 2002-06-28 2016-04-26 Domantis Limited Ligand
CA2492407C (en) 2002-07-18 2014-09-30 Monsanto Technology Llc Methods for using artificial polynucleotides and compositions thereof to reduce transgene silencing
EP1529110B1 (en) * 2002-08-01 2011-10-05 Evolva Ltd. Methods of mixing large numbers of heterologous genes
EP1546349A1 (en) * 2002-08-02 2005-06-29 Aromagen Corporation Methods for making (-) -menthol and oxygenated menthane compounds
BR0313281A (en) 2002-08-06 2007-07-24 Verdia Inc ap1 amine oxidase variants
AU2003265431A1 (en) * 2002-08-13 2004-02-25 Incyte Corporation Cell adhesion and extracellular matrix proteins
ES2381617T5 (en) 2002-08-14 2016-02-24 Macrogenics, Inc. Specific antibodies against FcgammaRIIB and its procedures for use
US20070219353A1 (en) * 2002-09-03 2007-09-20 Incyte Corporation Immune Response Associated Proteins
EP2298805A3 (en) 2002-09-27 2011-04-13 Xencor, Inc. Optimized Fc variants and methods for their generation
AU2003279829A1 (en) * 2002-10-04 2004-05-04 Incyte Corp Protein modification and maintenance molecules
US6863731B2 (en) * 2002-10-18 2005-03-08 Controls Corporation Of America System for deposition of inert barrier coating to increase corrosion resistance
US20050164275A1 (en) * 2002-10-18 2005-07-28 Incyte Corporation Phosphodiesterases
JP3447009B1 (en) * 2002-10-29 2003-09-16 實 平垣 Construct structure and method for producing the same
US20070009886A1 (en) * 2002-11-12 2007-01-11 Incyte Corporation Carbohydrate-associated proteins
WO2004044165A2 (en) * 2002-11-13 2004-05-27 Incyte Corporation Lipid-associated proteins
WO2004048550A2 (en) * 2002-11-26 2004-06-10 Incyte Corporation Immune response associated proteins
US7553930B2 (en) * 2003-01-06 2009-06-30 Xencor, Inc. BAFF variants and methods thereof
US20050221443A1 (en) * 2003-01-06 2005-10-06 Xencor, Inc. Tumor necrosis factor super family agonists
US20060014248A1 (en) * 2003-01-06 2006-01-19 Xencor, Inc. TNF super family members with altered immunogenicity
US20050130892A1 (en) * 2003-03-07 2005-06-16 Xencor, Inc. BAFF variants and methods thereof
CA2512729C (en) 2003-01-09 2014-09-16 Macrogenics, Inc. Identification and engineering of antibodies with variant fc regions and methods of using same
WO2004065551A2 (en) * 2003-01-21 2004-08-05 Bristol-Myers Squibb Company Polynucleotide encoding a novel acyl coenzyme a, monoacylglycerol acyltransferase-3 (mgat3), and uses thereof
AU2004213873C1 (en) 2003-02-20 2009-08-13 Athenix Corporation Delta-endotoxin genes and methods for their use
EP3023498B1 (en) 2003-03-06 2018-11-28 BASF Enzymes LLC Amylases, nucleic acids encoding them and methods for making and using them
DK2853593T3 (en) 2003-03-07 2018-01-08 Dsm Ip Assets Bv Hydrolases, nucleic acids encoding them, and processes for their preparation and use
JP4912144B2 (en) 2003-03-12 2012-04-11 ジェネンテック， インコーポレイテッド Use of BV8 and / or EG-VEGF to promote hematopoiesis
BRPI0408358A (en) 2003-03-14 2006-03-21 Neose Technologies Inc branched water-soluble polymers and their conjugates
US7323303B2 (en) * 2003-03-31 2008-01-29 Hong Kong Polytechnic University Modified β-lactamases and uses thereof
DK1613733T3 (en) 2003-04-04 2015-08-31 Basf Enzymes Llc PEKTATLYASER, nucleic acids encoding them, and methods for making and using the
US8791070B2 (en) 2003-04-09 2014-07-29 Novo Nordisk A/S Glycopegylated factor IX
CA2521826C (en) 2003-04-11 2013-08-06 Jennifer L. Reed Recombinant il-9 antibodies and uses thereof
US7348420B2 (en) 2003-04-25 2008-03-25 North Carolina State University Lactobacillus acidophilus nucleic acid sequences encoding cell surface protein homologues and uses therefore
US7834147B2 (en) * 2003-04-28 2010-11-16 Childrens Hospital Medical Center Saposin C-DOPS: a novel anti-tumor agent
US7829682B1 (en) 2003-04-30 2010-11-09 Incyte Corporation Human β-adrenergic receptor kinase nucleic acid molecule
EP1624847B1 (en) 2003-05-09 2012-01-04 BioGeneriX AG Compositions and methods for the preparation of human growth hormone glycosylation mutants
US7173010B2 (en) * 2003-05-19 2007-02-06 Regents Of The University Of Michigan Orphanin FQ receptor
CA2529623A1 (en) 2003-06-16 2005-02-17 Celltech R & D, Inc. Antibodies specific for sclerostin and methods for increasing bone mineralization
EP1639091B1 (en) 2003-06-17 2012-12-05 California University Of Technology Regio- and enantioselective alkane hydroxylation with modified cytochrome p450
EP2319863A1 (en) 2003-06-23 2011-05-11 North Carolina State University Lactobacillus acidophilus nucleic acids encoding fructo-oligosaccharide utilization compounds and uses thereof
BRPI0412279A (en) 2003-07-02 2006-09-19 Diversa Corp glucanases, nucleic acids encoding the same and methods for preparing and applying them
WO2005012484A2 (en) 2003-07-25 2005-02-10 Neose Technologies, Inc. Antibody-toxin conjugates
EP1660646B1 (en) * 2003-08-11 2014-12-31 California Institute Of Technology Thermostable peroxide-driven cytochrome p450 oxygenase variants and methods of use
EP1668113B1 (en) 2003-08-11 2013-06-19 Verenium Corporation Laccases, nucleic acids encoding them and methods for making and using them
CA2536238C (en) 2003-08-18 2015-04-07 Medimmune, Inc. Humanization of antibodies
WO2005021586A2 (en) * 2003-08-21 2005-03-10 Wisconsin Alumni Research Foundation Metabolic engineering of viomycin biosynthesis
CA2536635C (en) 2003-08-25 2015-05-12 Funzyme Biotechnologies Sa Novel fungal proteins and nucleic acids encoding same
EP1671134A2 (en) * 2003-09-23 2006-06-21 Monsanto Technology LLC High throughput automated seed analysis system
AU2004279895A1 (en) 2003-10-10 2005-04-21 Xencor, Inc Protein based TNF-alpha variants for the treatment of TNF-alpha related disorders
AU2004290052B2 (en) 2003-11-12 2008-12-04 Corteva Agriscience Llc Delta-15 desaturases suitable for altering levels of polyunsaturated fatty acids in oleaginous plants and yeast
CA2546698A1 (en) * 2003-11-20 2005-06-02 Agency For Science, Technology And Research Method
US8633157B2 (en) 2003-11-24 2014-01-21 Novo Nordisk A/S Glycopegylated erythropoietin
US20080305992A1 (en) 2003-11-24 2008-12-11 Neose Technologies, Inc. Glycopegylated erythropoietin
JP2007512838A (en) 2003-12-01 2007-05-24 インヴィトロジェン コーポレーション Nucleic acid molecules containing recombination sites and methods of use thereof
US7956032B2 (en) 2003-12-03 2011-06-07 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
US20060040856A1 (en) * 2003-12-03 2006-02-23 Neose Technologies, Inc. Glycopegylated factor IX
ATE352616T1 (en) * 2003-12-04 2007-02-15 Hoffmann La Roche METHOD FOR PRODUCING CIRCULAR MUTATED AND/OR CHIMERIC POLYNUCLEOTIDES
US20070169227A1 (en) 2003-12-16 2007-07-19 Pioneer Hi-Bred International Inc. Dominant Gene Suppression Transgenes and Methods of Using Same
JP5743368B2 (en) 2004-01-08 2015-07-01 ラショファーム ゲーエムベーハー O-linked glycosylation of peptides
US7432057B2 (en) * 2004-01-30 2008-10-07 Michigan State University Genetic test for PSE-susceptible turkeys
WO2005082077A2 (en) 2004-02-25 2005-09-09 Pioneer Hi-Bred International, Inc. Novel bacillus thuringiensis crystal polypeptides, polynucleotides, and compositions thereof
US7459289B2 (en) 2004-03-08 2008-12-02 North Carolina State University Lactobacillus acidophilus nucleic acid sequences encoding carbohydrate utilization-related proteins and uses therefor
FR2868081A1 (en) * 2004-03-23 2005-09-30 Libragen Sa METHOD OF IDENTIFYING METABOLIC PATHWAY FAMILY BY POSITIVE SELECTION
US7973139B2 (en) * 2004-03-26 2011-07-05 Human Genome Sciences, Inc. Antibodies against nogo receptor
JP4702284B2 (en) * 2004-03-30 2011-06-15 日本電気株式会社 Protein analysis method
JP2008505853A (en) 2004-04-13 2008-02-28 クインテセンス バイオサイエンシーズ インコーポレーティッド Non-natural ribonuclease complex as a cytotoxic agent
AR049214A1 (en) 2004-06-09 2006-07-05 Pioneer Hi Bred Int PEPTIDES OF TRANSIT TO PLASTIDES
EA013993B1 (en) 2004-06-16 2010-08-30 Верениум Корпорейшн Compositions and methods for enzymatic de-colorization of chlorophyll
EP1781795B1 (en) 2004-06-30 2011-01-19 Pioneer-Hi-Bred International, Inc. Methods of protecting plants from pathogenic fungi
EP2264053B1 (en) 2004-07-02 2014-02-26 Pioneer-Hi-Bred International, Inc. Antifungal polypeptides
US20080300173A1 (en) 2004-07-13 2008-12-04 Defrees Shawn Branched Peg Remodeling and Glycosylation of Glucagon-Like Peptides-1 [Glp-1]
US7148051B2 (en) * 2004-08-16 2006-12-12 E. I. Du Pont De Nemours And Company Production of 3-hydroxycarboxylic acid using nitrilase
US7703238B2 (en) 2004-08-26 2010-04-27 Monsanto Technology Llc Methods of seed breeding using high throughput nondestructive seed sampling
ES2439898T3 (en) * 2004-08-26 2014-01-27 Monsanto Technology, Llc Automated Seed Testing
US7832143B2 (en) 2004-08-26 2010-11-16 Monsanto Technology Llc High throughput methods for sampling seeds
FI20050753A (en) 2004-09-03 2006-03-04 Licentia Oy New peptides
US8268967B2 (en) 2004-09-10 2012-09-18 Novo Nordisk A/S Glycopegylated interferon α
US7700720B2 (en) 2004-09-21 2010-04-20 Medimmune, Llc Antibodies against and methods for producing vaccines for respiratory syncytial virus
US7666616B2 (en) 2004-09-30 2010-02-23 Trustees Of Dartmouth College Nucleic acid sequences encoding luciferase for expression in filamentous fungi
EP2166098B1 (en) 2004-10-05 2013-11-06 SunGene GmbH Constitutive expression cassettes for regulation of plant expression
JP2008523786A (en) * 2004-10-18 2008-07-10 コドン デバイシズ インコーポレイテッド Method for assembling high fidelity synthetic polynucleotides
JP2008518023A (en) 2004-10-27 2008-05-29 メディミューン，インコーポレーテッド Regulation of antibody specificity by altering affinity for cognate antigens
EP3061461A1 (en) 2004-10-29 2016-08-31 ratiopharm GmbH Remodeling and glycopegylation of fibroblast growth factor (fgf)
EP1655364A3 (en) 2004-11-05 2006-08-02 BASF Plant Science GmbH Expression cassettes for seed-preferential expression in plants
EP2163631B1 (en) 2004-11-25 2013-05-22 SunGene GmbH Expression cassettes for guard cell-preferential expression in plants
EP1666599A3 (en) 2004-12-04 2006-07-12 SunGene GmbH Expression cassettes for mesophyll- and/or epidermis-preferential expression in plants
EP2163634A1 (en) 2004-12-08 2010-03-17 SunGene GmbH Expression cassettes for vascular tissue-preferential expression in plants
EP1669456A3 (en) 2004-12-11 2006-07-12 SunGene GmbH Expression cassettes for meristem-preferential expression in plants
US7198927B2 (en) 2004-12-22 2007-04-03 E. I. Du Pont De Nemours And Company Enzymatic production of glycolic acid
EP2003205B1 (en) 2004-12-28 2013-05-01 Pioneer Hi-Bred International, Inc. Improved grain quality through altered expression of seed proteins
JP4951527B2 (en) 2005-01-10 2012-06-13 バイオジェネリックス アーゲー GlycoPEGylated granulocyte colony stimulating factor
MX2007008486A (en) 2005-01-13 2008-02-15 Pioneer Hi Bred Int Maize cyclo1 gene and promoter.
US7402730B1 (en) 2005-02-03 2008-07-22 Lexicon Pharmaceuticals, Inc. Knockout animals manifesting hyperlipidemia
CA2597924C (en) 2005-02-15 2018-10-02 Duke University Anti-cd19 antibodies and uses in oncology
EP1856264B1 (en) 2005-02-26 2014-07-23 BASF Plant Science GmbH Expression cassettes for seed-preferential expression in plants
MX2007010552A (en) 2005-03-02 2009-02-19 Inst Nac De Tecnologia Agropec Herbicide-resistant rice plants, polynucleotides encoding herbicide-resistant acetohydroxyacid synthase large subunit proteins, and methods of use.
CA2614769A1 (en) 2005-03-10 2006-09-21 Verenium Corporation Lyase enzymes, nucleic acids encoding them and methods for making and using them
CA2611859C (en) 2005-03-15 2015-03-31 Verenium Corporation Cellulases, nucleic acids encoding them and methods for making and using them
WO2006102095A2 (en) 2005-03-18 2006-09-28 Medimmune, Inc. Framework-shuffling of antibodies
US11214817B2 (en) 2005-03-28 2022-01-04 California Institute Of Technology Alkane oxidation by modified hydroxylases
US8715988B2 (en) 2005-03-28 2014-05-06 California Institute Of Technology Alkane oxidation by modified hydroxylases
WO2006107761A2 (en) 2005-04-01 2006-10-12 Athenix Corporation Axmi-027, axmi-036 and axmi-038, a family of delta-endotoxin genes and methods for their use
WO2006121569A2 (en) 2005-04-08 2006-11-16 Neose Technologies, Inc. Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
CN101198689A (en) 2005-04-15 2008-06-11 北卡罗来纳州大学 Methods and compositions to modulate adhesion and stress tolerance in bacteria
EP1868650B1 (en) 2005-04-15 2018-10-03 MacroGenics, Inc. Covalent diabodies and uses thereof
CA2606220A1 (en) 2005-04-19 2006-12-21 Basf Plant Science Gmbh Starchy-endosperm and/or germinating embryo-specific expression in mono-cotyledonous plants
US8003108B2 (en) 2005-05-03 2011-08-23 Amgen Inc. Sclerostin epitopes
US7592429B2 (en) 2005-05-03 2009-09-22 Ucb Sa Sclerostin-binding antibody
AU2006244445B2 (en) 2005-05-05 2013-04-18 Duke University Anti-CD19 antibody therapy for autoimmune disease
EP1888098A2 (en) 2005-05-25 2008-02-20 Neose Technologies, Inc. Glycopegylated erythropoietin formulations
JP5216580B2 (en) * 2005-05-25 2013-06-19 ノヴォ ノルディスク アー／エス Glycopegylated factor IX
KR20080025174A (en) 2005-06-23 2008-03-19 메디뮨 인코포레이티드 Antibody formulations having optimized aggregation and fragmentation profiles
CA3006126C (en) 2005-07-01 2022-04-19 Basf Se Herbicide-resistant sunflower plants, polynucleotides encoding herbicide-resistant acetohydroxy acid synthase large subunit proteins, and methods of use
US7494788B2 (en) * 2005-07-11 2009-02-24 Molecular Kinetics, Inc. Entropic bristle domain sequences and their use in recombinant protein production
EP1907553B1 (en) 2005-07-18 2012-08-22 Pioneer Hi-Bred International Inc. Modified frt recombination sites and methods of use
KR20080052570A (en) 2005-07-29 2008-06-11 타게티드 그로스 인코퍼레이티드 Dominant negative mutant krp protein protection of active cyclin-cdk complex inhibition by wild-type krp
CA2618681C (en) 2005-08-10 2015-10-27 Macrogenics, Inc. Identification and engineering of antibodies with variant fc regions and methods of using same
US20070105755A1 (en) 2005-10-26 2007-05-10 Neose Technologies, Inc. One pot desialylation and glycopegylation of therapeutic peptides
AU2006292224B2 (en) 2005-09-19 2013-08-01 Histogenics Corporation Cell-support matrix and a method for preparation thereof
US7312079B1 (en) 2005-10-06 2007-12-25 Lexicon Pharmaceuticals, Inc. Variants of FAM3C
US7470532B2 (en) 2005-10-19 2008-12-30 E.I. Du Pont De Nemours And Company Mortierella alpina C16/18 fatty acid elongase
US20090048440A1 (en) 2005-11-03 2009-02-19 Neose Technologies, Inc. Nucleotide Sugar Purification Using Membranes
US20070118920A1 (en) 2005-11-09 2007-05-24 Basf Agrochemical Products B.V. Herbicide-resistant sunflower plants, polynucleotides encoding herbicide-resistant acetohydroxyacid synthase large subunit proteins, and methods of use
AU2006315788A1 (en) 2005-11-10 2007-05-24 Pioneer Hi-Bred International, Inc. Dof (DNA binding with one finger) sequences and methods of use
US20070199096A1 (en) 2005-11-14 2007-08-23 E.I. Du Pont De Nemours And Company Compositions and Methods for Altering Alpha- and Beta-Tocotrienol Content
JP2009519224A (en) * 2005-11-18 2009-05-14 グロスター ファーマシューティカルズ， インコーポレイテッド Metabolite derivatives of HDAC inhibitor FK228
GB2432366B (en) * 2005-11-19 2007-11-21 Alligator Bioscience Ab A method for in vitro molecular evolution of protein function
US7674958B2 (en) 2005-12-01 2010-03-09 Athenix Corporation GRG23 and GRG51 genes conferring herbicide resistance
US20080313769A9 (en) 2006-01-12 2008-12-18 Athenix Corporation EPSP synthase domains conferring glyphosate resistance
US20090324574A1 (en) 2006-02-02 2009-12-31 Verenium Corporation Esterases and Related Nucleic Acids and Methods
MY160772A (en) 2006-02-10 2017-03-15 Verenium Corp Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them
PL1989302T3 (en) 2006-02-14 2019-03-29 Bp Corp North America Inc Xylanases, nucleic acids encoding them and methods for making and using them
US7998669B2 (en) 2006-03-02 2011-08-16 Monsanto Technology Llc Automated contamination-free seed sampler and methods of sampling, testing and bulking seeds
AU2007223364B2 (en) 2006-03-02 2014-02-13 Athenix Corporation Methods and compositions for improved enzyme activity in transgenic plant
US8028469B2 (en) 2006-03-02 2011-10-04 Monsanto Technology Llc Automated high-throughput seed sampler and methods of sampling, testing and bulking seeds
EP2650306A1 (en) 2006-03-06 2013-10-16 Aeres Biomedical Limited Humanized Anti-CD22 antibodies and their use in treatment of oncology, transplantation and autoimmune disease
EP3153580A3 (en) 2006-03-07 2017-04-19 BASF Enzymes LLC Aldolases, nucleic acids encoding them and methods for making and using them
EP2112227B1 (en) 2006-03-07 2013-05-15 Cargill, Incorporated Aldolases, nucleic acids encoding them and methods for making and using them
US20070214515A1 (en) 2006-03-09 2007-09-13 E.I.Du Pont De Nemours And Company Polynucleotide encoding a maize herbicide resistance gene and methods for use
EP2007799A2 (en) 2006-04-19 2008-12-31 Pioneer Hi-Bred International, Inc. Isolated polynucleotide molecules corresponding to mutant and wild-type alleles of the maize d9 gene and methods of use
WO2008013589A2 (en) * 2006-04-24 2008-01-31 Gloucester Pharmaceuticals Treatment of ras-expressing tumors
US7598346B1 (en) 2006-05-16 2009-10-06 Pioneer Hi-Bred International, Inc. Antifungal polypeptides
CN103215304A (en) 2006-05-17 2013-07-24 先锋高级育种国际公司 Artificial plant minichromosomes
WO2007146730A2 (en) * 2006-06-08 2007-12-21 Gloucester Pharmaceuticals Deacetylase inhibitor therapy
EP3800198A1 (en) 2006-06-15 2021-04-07 Basf Agricultural Solutions Seed Us Llc A family of pesticidal proteins and methods for their use
WO2007149594A2 (en) * 2006-06-23 2007-12-27 Quintessence Biosciences, Inc. Modified ribonucleases
HUE030269T2 (en) 2006-06-26 2017-04-28 Macrogenics Inc Fc riib-specific antibodies and methods of use thereof
EP3162454B1 (en) 2006-06-28 2019-04-24 Monsanto Technology LLC Small object sorting method
US7572618B2 (en) 2006-06-30 2009-08-11 Bristol-Myers Squibb Company Polynucleotides encoding novel PCSK9 variants
US20080004410A1 (en) * 2006-06-30 2008-01-03 Yu-Chin Lai Hydrophilic macromonomers having alpha,beta-conjugated carboxylic terminal group and medical devices incorporating same
US7977535B2 (en) 2006-07-12 2011-07-12 Board Of Trustees Of Michigan State University DNA encoding ring zinc-finger protein and the use of the DNA in vectors and bacteria and in plants
EP2049151A4 (en) 2006-07-17 2010-03-24 Quintessence Biosciences Inc Methods and compositions for the treatment of cancer
JP2009544327A (en) 2006-07-21 2009-12-17 ノヴォ ノルディスク アー／エス Glycosylation of peptides with O-linked glycosylation sequences
US8252559B2 (en) * 2006-08-04 2012-08-28 The California Institute Of Technology Methods and systems for selective fluorination of organic molecules
AU2007356171B8 (en) 2006-08-04 2014-01-16 Bp Corporation North America Inc. Glucanases, nucleic acids encoding them, and methods for making and using them
EP2069513A4 (en) * 2006-08-04 2012-03-21 California Inst Of Techn Methods and systems for selective fluorination of organic molecules
SG170032A1 (en) 2006-08-28 2011-04-29 Kyowa Hakko Kirin Co Ltd Antagonistic human light-specific human monoclonal antibodies
WO2008030735A2 (en) 2006-09-06 2008-03-13 Janssen Pharmaceutical N.V. Biomarkers for assessing response to c-met treatment
ATE551071T1 (en) 2006-09-08 2012-04-15 Medimmune Llc HUMANIZED ANTI-CD19 ANTIBODIES AND THEIR USE FOR THE TREATMENT OF CANCER, TRANSPLANTATION AND AUTOIMMUNE DISEASES
CN101558154B (en) 2006-09-21 2016-02-24 帝斯曼知识产权资产管理有限公司 Phospholipid hydrolase, encode their nucleic acid and methods for making and using same thereof
MX2009003032A (en) 2006-09-21 2009-11-18 Verenium Corp Phytases, nucleic acids encoding them and methods for making and using them.
JP2010505874A (en) 2006-10-03 2010-02-25 ノヴォ ノルディスク アー／エス Purification method for polypeptide conjugates
CA2665480C (en) 2006-10-04 2019-11-12 Shawn Defrees Glycerol linked pegylated sugars and glycopeptides
US20080124755A1 (en) * 2006-10-12 2008-05-29 Michael Tai-Man Louie Biosynthesis of beta-cryptoxanthin in microbial hosts using an Arabidopsis thaliana beta-carotene hydroxylase gene
US20100162433A1 (en) 2006-10-27 2010-06-24 Mclaren James Plants with improved nitrogen utilization and stress tolerance
CA2566906A1 (en) * 2006-10-30 2008-04-30 Nathalie Ravet Carbon-coated lifepo4 storage and handling
EP2423226A3 (en) * 2006-11-10 2012-05-30 Amgen Inc. Antibody-based diagnostics and therapeutics
EP2094731A2 (en) * 2006-11-10 2009-09-02 UCB Pharma S.A. Anti human sclerostin antibodies
PL2479267T3 (en) 2006-12-21 2017-06-30 Basf Enzymes Llc Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them
EP2102230A2 (en) * 2006-12-29 2009-09-23 Gloucester Pharmaceuticals, Inc. Purifiction of romidepsin
CN103497238A (en) * 2006-12-29 2014-01-08 细胞基因公司 Romidepsin preparation
BRPI0807132A2 (en) 2007-01-30 2018-12-04 Syngenta Participations Ag enzymes for the treatment of lignocellulosics, nucleic acids encoding them, and methods and use thereof
US20080248545A1 (en) * 2007-02-02 2008-10-09 The California Institute Of Technology Methods for Generating Novel Stabilized Proteins
EP2115130B1 (en) 2007-02-08 2011-08-03 Codexis, Inc. Ketoreductases and uses thereof
US20080268517A1 (en) * 2007-02-08 2008-10-30 The California Institute Of Technology Stable, functional chimeric cytochrome p450 holoenzymes
BRPI0808008B1 (en) 2007-02-16 2016-08-09 Basf Plant Science Gmbh isolated nucleic acid molecule, expression cassette, expression vector, methods for excising target sequences from a plant to produce a plant with increased yield, and / or increased stress tolerance, and / or increased nutritional quality, and / or increased or modified oil content of a seed or sprout for the plant, and use of the nucleic acid molecule
CA2682292A1 (en) 2007-03-30 2008-10-09 Medimmune, Llc Aqueous formulation comprising an anti-human interferon alpha antibody
ES2406267T3 (en) 2007-04-03 2013-06-06 Biogenerix Ag Treatment methods using glycopegylated G-CSF
KR20100016165A (en) 2007-04-04 2010-02-12 바스프 에스이 Herbicide-resistant brassica plants and methods of use
AU2008246442B2 (en) 2007-05-04 2014-07-03 Technophage, Investigacao E Desenvolvimento Em Biotecnologia, Sa Engineered rabbit antibody variable domains and uses thereof
WO2008137915A2 (en) 2007-05-07 2008-11-13 Medimmune, Llc Anti-icos antibodies and their use in treatment of oncology, transplantation and autoimmune disease
WO2008137993A1 (en) * 2007-05-08 2008-11-13 Duke University Compositions and methods for characterizing and regulating olfactory sensation
WO2008141190A1 (en) * 2007-05-11 2008-11-20 Wisconsin Alumni Research Foundation Heterologous production of capreomycin and generation of new capreomycin derivatives through metabolic engineering
HUE036885T2 (en) 2007-05-14 2018-08-28 Astrazeneca Ab Methods of reducing basophil levels
CN101679493A (en) 2007-05-25 2010-03-24 克罗普迪塞恩股份有限公司 In plant, strengthen by the output of regulating corn ALFIN
WO2008150903A1 (en) 2007-05-31 2008-12-11 Monsanto Technology Llc Seed sorter
CN101778859B (en) 2007-06-12 2014-03-26 诺和诺德公司 Improved process for the production of nucleotide sugars
US8802401B2 (en) * 2007-06-18 2014-08-12 The California Institute Of Technology Methods and compositions for preparation of selectively protected carbohydrates
ES2702087T3 (en) 2007-06-21 2019-02-27 Macrogenics Inc Covalent diabodies and their uses
CN101784669B (en) 2007-08-24 2015-02-18 科德克希思公司 Improved ketoreductase polypeptides for the stereoselective production of (R)-3-hydroxythiolane
US8207112B2 (en) 2007-08-29 2012-06-26 Biogenerix Ag Liquid formulation of G-CSF conjugate
CL2008002775A1 (en) 2007-09-17 2008-11-07 Amgen Inc Use of a sclerostin binding agent to inhibit bone resorption.
NZ601191A (en) 2007-10-03 2014-01-31 Verenium Corp Xylanases, nucleic acids encoding them and methods for making and using them
EP2205271B1 (en) * 2007-10-08 2014-05-21 Quintessence Biosciences, Inc. Compositions and methods for ribonuclease-based therapies
EP2198030B1 (en) 2007-10-10 2016-09-14 Athenix Corporation Synthetic genes encoding cry1ac
EA201070371A1 (en) 2007-10-16 2011-02-28 Атеникс Корпорейшн AXMI-066 AND AXMI-076: DELTA-ENDOTOXIN PROTEINS AND METHODS OF THEIR APPLICATION
US7741088B2 (en) * 2007-10-31 2010-06-22 E.I. Dupont De Nemours And Company Immobilized microbial nitrilase for production of glycolic acid
US7871802B2 (en) * 2007-10-31 2011-01-18 E.I. Du Pont De Nemours And Company Process for enzymatically converting glycolonitrile to glycolic acid
WO2009076196A1 (en) * 2007-12-07 2009-06-18 Dow Global Technologies Inc. High copy number self-replicating plasmids in pseudomonas
BRPI0819688A2 (en) * 2007-12-14 2015-06-16 Amgen Inc Process for treating bone fracture with anti-sclerostin antibodies.
EP2865750A3 (en) 2008-01-03 2015-08-05 BASF Enzymes LLC Transferases and oxidoreductases, nucleic acids encoding them and methods for making and using them
US8541220B2 (en) 2008-01-03 2013-09-24 Verenium Corporation Isomerases, nucleic acids encoding them and methods for making and using them
AU2009205995B2 (en) 2008-01-18 2014-04-03 Medimmune, Llc Cysteine engineered antibodies for site-specific conjugation
MX2010008578A (en) 2008-02-08 2010-11-10 Medimmune Llc Anti-ifnar1 antibodies with reduced fc ligand affinity.
WO2009102901A1 (en) * 2008-02-12 2009-08-20 Codexis, Inc. Method of generating an optimized, diverse population of variants
ES2476690T3 (en) 2008-02-27 2014-07-15 Novo Nordisk A/S Factor VIII conjugated molecules
WO2009118300A1 (en) 2008-03-25 2009-10-01 Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Treating cancer by down-regulating frizzled-4 and/or frizzled-1
EP2285965A1 (en) * 2008-03-31 2011-02-23 Pfenex Inc A design for rapidly cloning one or more polypeptide chains into an expression system
CA2723235A1 (en) 2008-05-02 2009-11-05 Epicentre Technologies Corporation Rna polyphosphatase compositions, kits, and uses thereof
EP3115459A3 (en) 2008-05-23 2017-04-26 E. I. du Pont de Nemours and Company Novel dgat genes for increased seed storage lipid production and altered fatty acid profiles in oilseed plants
US20090312196A1 (en) 2008-06-13 2009-12-17 Codexis, Inc. Method of synthesizing polynucleotide variants
EP2285958B1 (en) 2008-06-13 2016-03-09 Codexis, Inc. Method of synthesizing polynucleotide variants
US8178333B2 (en) 2008-06-24 2012-05-15 Codexis, Inc. Biocatalytic processes for the preparation of substantially stereomerically pure fused bicyclic proline compounds
ES2911327T3 (en) 2008-06-25 2022-05-18 BASF Agricultural Solutions Seed US LLC Toxin genes and procedures for their use
NZ590039A (en) 2008-07-02 2012-11-30 Athenix Corp Axmi-il 5, axmi-113, axmi-005, axmi-163 and axmi-184 : vip3a insecticidal proteins from bacillus thuringiensis and methods for their use
NZ600622A (en) 2008-08-05 2013-12-20 Novartis Ag Compositions and methods for antibodies targeting complement protein c5
US8426178B2 (en) 2008-08-27 2013-04-23 Codexis, Inc. Ketoreductase polypeptides for the production of a 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine
WO2010025287A2 (en) 2008-08-27 2010-03-04 Codexis, Inc. Ketoreductase polypeptides for the production of 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine
US8288131B2 (en) 2008-08-27 2012-10-16 Codexis, Inc. Ketoreductase polypeptides and uses thereof
US8198062B2 (en) 2008-08-29 2012-06-12 Dsm Ip Assets B.V. Hydrolases, nucleic acids encoding them and methods for making and using them
SI2329014T1 (en) 2008-08-29 2015-01-30 Codexis, Inc. Ketoreductase polypeptides for the stereoselective production of (4s)-3š(5s)-5(4-fluorophenyl)-5-hydroxypentanoylć-4-phenyl-1,3-oxazolidin-2-one
US8357503B2 (en) 2008-08-29 2013-01-22 Bunge Oils, Inc. Hydrolases, nucleic acids encoding them and methods for making and using them
US8153391B2 (en) 2008-08-29 2012-04-10 Bunge Oils, Inc. Hydrolases, nucleic acids encoding them and methods for making and using them
CN102216453B (en) 2008-09-26 2014-02-05 巴斯夫农化产品有限公司 Herbicide-resistant AHAS-mutants and methods of use
KR102025502B1 (en) 2008-09-26 2019-09-25 토카겐 인크. Gene therapy vectors and cytosine deaminases
CN102227443B (en) 2008-10-01 2014-05-14 昆特森斯生物科学公司 Therapeutic ribonucleases
JP5701762B2 (en) 2008-10-03 2015-04-15 イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニーＥ．Ｉ．Ｄｕ Ｐｏｎｔ Ｄｅ Ｎｅｍｏｕｒｓ Ａｎｄ Ｃｏｍｐａｎｙ Enzymatic peracid generating formulation
ES2719496T3 (en) 2008-11-12 2019-07-10 Medimmune Llc Antibody formulation
CA2743707A1 (en) 2008-12-04 2010-06-10 Pioneer Hi-Bred International, Inc. Methods and compositions for enhanced yield by targeted expression of knotted1
US8404934B2 (en) 2008-12-15 2013-03-26 Athenix Corporation Genes encoding nematode toxins
PL2786762T3 (en) 2008-12-19 2019-09-30 Macrogenics, Inc. Covalent diabodies and uses thereof
EP2671951A3 (en) 2008-12-22 2014-06-04 Athenix Corporation Pesticidal genes from Brevibacillus and methods for their use
US8084416B2 (en) 2008-12-23 2011-12-27 Athenix Corp. AXMI-150 delta-endotoxin gene and methods for its use
EP3354727B1 (en) 2009-01-08 2020-10-07 Codexis, Inc. Transaminase polypeptides
EP2206723A1 (en) 2009-01-12 2010-07-14 Bonas, Ulla Modular DNA-binding domains
US20110239315A1 (en) 2009-01-12 2011-09-29 Ulla Bonas Modular dna-binding domains and methods of use
US8852608B2 (en) 2009-02-02 2014-10-07 Medimmune, Llc Antibodies against and methods for producing vaccines for respiratory syncytial virus
BRPI1007915B1 (en) 2009-02-05 2019-05-14 Athenix Corporation AXMI-R1 VARIANT GENES DELTA-ENDOTOXIN, VECTOR, MICROBIAN HOST CELL, RECOMBINANT POLYPEPTIDE AND ITS METHOD OF PRODUCTION, COMPOSITION, AND METHODS FOR CONTROLING AND KILLING A CLETETETE CLETETE PANTOTTETE POPULATION
MY189620A (en) * 2009-02-20 2022-02-21 Malaysian Palm Oil Board A constitutive promoter from oil palm
CA2752818A1 (en) 2009-02-26 2010-09-02 Codexis, Inc. Beta-glucosidase variant enzymes and related polynucleotides
EP2957638B1 (en) 2009-02-27 2018-12-12 Athenix Corporation Pesticidal proteins and methods for their use
EP2405920A1 (en) 2009-03-06 2012-01-18 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Novel therapy for anxiety
CN102395679A (en) 2009-03-06 2012-03-28 阿森尼克斯公司 Methods and compositions for controlling plant pests
AR075818A1 (en) 2009-03-11 2011-04-27 Athenix Corp AXMI-001, AXMI- 002, AXMI-030 AND AXMI -035 AND AXMI -045: TOXIN GENES AND METHODS FOR USE
BRPI1013886A2 (en) 2009-03-17 2016-10-11 Codexis Inc endoglucanase variants, polynucleotides and related uses
WO2010109053A1 (en) 2009-03-27 2010-09-30 Proyeto De Biomedicina Cima, S.L. Methods and compositions for the treatment of cirrhosis and liver fibrosis
EP2414506B1 (en) 2009-03-31 2017-05-03 Codexis, Inc. Improved endoglucanases
EP2241323A1 (en) 2009-04-14 2010-10-20 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Tenascin-W and brain cancers
KR101815716B1 (en) 2009-04-22 2018-01-05 인디애나 유니버시티 리서치 앤드 테크놀로지 코퍼레이션 Compositions for use in the treatment of chronic obstructive pulmonary diseases and asthma
CA2760700A1 (en) 2009-05-04 2010-11-11 Pioneer Hi-Bred International, Inc. Yield enhancement in plants by modulation of ap2 transcription factor
MY186558A (en) * 2009-05-13 2021-07-27 Malaysian Palm Oil Board A constitutive promoter from oil palm type 2
JP2012527247A (en) 2009-05-21 2012-11-08 ヴェレニウム コーポレイション Phytase, nucleic acid encoding phytase and method for producing and using the same
WO2010138709A1 (en) 2009-05-28 2010-12-02 Government Of The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Anti-tnf induced apoptosis (atia) diagnostic markers and therapies
EP2440663A1 (en) 2009-06-09 2012-04-18 Pioneer Hi-Bred International Inc. Early endosperm promoter and methods of use
WO2010148128A1 (en) 2009-06-16 2010-12-23 Codexis, Inc. Beta-glucosidase variant enzymes and related polynucleotides
BRPI1015234A2 (en) 2009-06-22 2018-02-20 Medimmune Llc fc regions designed for site specific conjugation.
WO2011005527A2 (en) 2009-06-22 2011-01-13 Codexis, Inc. Ketoreductase-mediated stereoselective route to alpha chloroalcohols
UA105046C2 (en) 2009-07-02 2014-04-10 Атенікс Корп. Pesticide gene axmi-205 and its using for plants protection againts insect pests
WO2011005823A1 (en) 2009-07-07 2011-01-13 Castle Linda A Crystal structure of glyphosate acetyltransferase (glyat) and methods of use
WO2011011630A2 (en) 2009-07-23 2011-01-27 Codexis, Inc. Nitrilase biocatalysts
CN102656185B (en) 2009-07-31 2018-04-24 阿森尼克斯公司 The AXMI-192 families of killing gene and use their method
US9133500B2 (en) 2009-08-10 2015-09-15 MorphoSys A6 Screening strategies for the identification of binders
EP3381937A3 (en) 2009-08-13 2018-10-31 The Johns Hopkins University Methods of modulating immune function
JP5762408B2 (en) * 2009-08-13 2015-08-12 クルセル ホランド ベー ヴェー Antibodies against human respiratory syncytial virus (RSV) and methods of use
EP2467473B1 (en) 2009-08-19 2016-03-23 Codexis, Inc. Ketoreductase polypeptides for the preparation of phenylephrine
WO2011022597A1 (en) 2009-08-20 2011-02-24 Pioneer Hi-Bred International, Inc. Functional expression of shuffled yeast nitrate transporter (ynti) in maize to improve nitrate uptake under low nitrate environment
EP2292266A1 (en) 2009-08-27 2011-03-09 Novartis Forschungsstiftung, Zweigniederlassung Treating cancer by modulating copine III
EP2473604B1 (en) 2009-09-04 2017-01-11 Codexis, Inc. Variant cbh2 cellulases and related polynucleotides
EP2464735A1 (en) 2009-09-09 2012-06-20 Braskem S.A. Microorganisms and process for producing n-propanol
DK3301208T3 (en) 2009-09-18 2020-12-14 Codexis Inc REDUCED CODON MUTAGENESIS
US20120244170A1 (en) 2009-09-22 2012-09-27 Rafal Ciosk Treating cancer by modulating mex-3
US8222012B2 (en) * 2009-10-01 2012-07-17 E. I. Du Pont De Nemours And Company Perhydrolase for enzymatic peracid production
JP5898082B2 (en) 2009-10-07 2016-04-06 マクロジェニクス，インコーポレーテッド Fc region-containing polypeptide exhibiting improved effector function by changing the degree of fucosylation and use thereof
WO2011045352A2 (en) 2009-10-15 2011-04-21 Novartis Forschungsstiftung Spleen tyrosine kinase and brain cancers
UA109884C2 (en) 2009-10-16 2015-10-26 A POLYPEPTIDE THAT HAS THE ACTIVITY OF THE PHOSPHATIDYLINOSYTOL-SPECIFIC PHOSPHOLIPASE C, NUCLEIC ACID, AND METHOD OF METHOD
UA111708C2 (en) 2009-10-16 2016-06-10 Бандж Ойлз, Інк. METHOD OF OIL REFINING
WO2011056544A1 (en) 2009-10-26 2011-05-12 Pioneer Hi-Bred International, Inc. Somatic ovule specific promoter and methods of use
US20120213801A1 (en) 2009-10-30 2012-08-23 Ekaterina Gresko Phosphorylated Twist1 and cancer
WO2011063198A2 (en) 2009-11-20 2011-05-26 St. Jude Children's Research Hospital Methods and compositions for modulating the activity of the interleukin-35 receptor complex
EP2504441B1 (en) 2009-11-23 2020-07-22 E. I. du Pont de Nemours and Company Sucrose transporter genes for increasing plant seed lipids
PL2504017T3 (en) 2009-11-25 2014-06-30 Codexis Inc Recombinant beta-glucosidase variants for production of soluble sugars from cellulosic biomass
US8652813B2 (en) 2009-11-25 2014-02-18 Codexis, Inc. Recombinant Thermoascus aurantiacus β-glucosidase variants
BR112012012588B1 (en) 2009-11-27 2019-03-26 Basf Plant Science Company Gmbh ENDONUCLEASE, METHOD FOR HOMOLOGICAL RECOMBINATION OF POLINUCLEOTIDES AND METHOD FOR DIRECTED POLINUCLEOTIDE MUTATION
JP2013511979A (en) 2009-11-27 2013-04-11 ビーエーエスエフ プラント サイエンス カンパニー ゲーエムベーハー Chimeric endonuclease and use thereof
CA2781693C (en) 2009-11-27 2018-12-18 Basf Plant Science Company Gmbh Chimeric endonucleases and uses thereof
WO2011067712A1 (en) 2009-12-03 2011-06-09 Basf Plant Science Company Gmbh Expression cassettes for embryo-specific expression in plants
US8895271B2 (en) 2009-12-08 2014-11-25 Codexis, Inc. Synthesis of prazole compounds
BR112012016290A2 (en) 2009-12-31 2015-09-01 Pioneer Hi Bred Int Isolated or recombinant nucleic acid, expression cassette, non-human host cell, transgenic plant and seed, isolated or recombinant oxox polypeptide variant, oxalate oxidase (oxox) protein level modulation method in a plant or plant cell, method for enhancing plant resistance to a pathogen, pathogen resistant plant, method to identify oxox variants with sustained or increased oxox activity, method to generate a plant that has increased resistance to a pathogen
CA2789629A1 (en) 2010-02-10 2011-08-18 Immunogen, Inc. Cd20 antibodies and uses thereof
US9080192B2 (en) 2010-02-10 2015-07-14 Codexis, Inc. Processes using amino acid dehydrogenases and ketoreductase-based cofactor regenerating system
AR080200A1 (en) 2010-02-18 2012-03-21 Athenix Corp DELTA-ENDOTOXINIC GENES AXMI221Z, AXMI222Z, AXMI223Z, AXMI224Z, AND AXMI225Z AND METHODS FOR USE
CA2790023A1 (en) 2010-02-18 2011-08-25 Athenix Corp. Axmi218, axmi219, axmi220, axmi226, axmi227, axmi228, axmi229, axmi230, and axmi231 delta-endotoxin genes and methods for their use
US20130004519A1 (en) 2010-03-05 2013-01-03 Ruth Chiquet-Ehrismann Smoci, tenascin-c and brain cancers
WO2011116396A2 (en) 2010-03-19 2011-09-22 The Board Of Trustees Of The Leland Stanford Junior University Hepatocyte growth factor fragments that function as potent met receptor agonists and antagonists
WO2011123576A2 (en) 2010-03-31 2011-10-06 Codexis, Inc. Production of monoterpenes
US8383793B2 (en) 2010-04-15 2013-02-26 St. Jude Children's Research Hospital Methods and compositions for the diagnosis and treatment of cancer resistant to anaplastic lymphoma kinase (ALK) kinase inhibitors
US20130034543A1 (en) 2010-04-19 2013-02-07 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Resear Modulating xrn1
WO2011140219A1 (en) 2010-05-04 2011-11-10 Codexis, Inc. Biocatalysts for ezetimibe synthesis
WO2011139431A1 (en) 2010-05-06 2011-11-10 Pioneer Hi-Bred International, Inc. Maize acc synthase 3 gene and protein and uses thereof
PE20130206A1 (en) 2010-05-06 2013-02-28 Novartis Ag MULTIVALENT ANTIBODIES ANTAGONISTS OF LRP6 (LOW DENSITY LIPOPROTEIN-RELATED PROTEIN 6) AND COMPOSITIONS
EP4234698A3 (en) 2010-05-06 2023-11-08 Novartis AG Compositions and methods of use for therapeutic low density lipoprotein-related protein 6 (lrp6) antibodies
WO2011143274A1 (en) 2010-05-10 2011-11-17 Perseid Therapeutics Polypeptide inhibitors of vla4
AU2011250920B2 (en) 2010-05-14 2015-05-21 Amgen Inc. High concentration antibody formulations
BR112012029881A2 (en) 2010-05-25 2019-09-24 Inserm (Institut National De La Santé De La Recherche Médicale) combination of anti-envelope and anti-receptor antibodies for the treatment and presence of hcv infection
WO2011150313A1 (en) 2010-05-28 2011-12-01 Codexis, Inc. Pentose fermentation by a recombinant microorganism
EP2580239A1 (en) 2010-06-10 2013-04-17 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Treating cancer by modulating mammalian sterile 20-like kinase 3
HUE038434T2 (en) 2010-06-17 2018-10-29 Codexis Inc Biocatalysts and methods for the synthesis of (s)-3-(1-aminoethyl)-phenol
CN102958349B (en) 2010-06-25 2015-12-02 纳幕尔杜邦公司 For strengthening composition and the method for corn northern leaf blight resistance in corn
US8574878B2 (en) 2010-06-28 2013-11-05 Behnaz Behrouzian Fatty alcohol forming acyl reductases (FARs) and methods of use thereof
AU2011272878B2 (en) 2010-06-30 2015-04-23 Codexis, Inc. Highly stable beta-class carbonic anhydrases useful in carbon capture systems
EA027835B1 (en) 2010-07-09 2017-09-29 Круселл Холланд Б.В. Anti-human respiratory syncytial virus (rsv) antibodies and methods of use thereof
NZ706052A (en) 2010-07-12 2016-07-29 Celgene Corp Romidepsin solid forms and uses thereof
JP5964300B2 (en) 2010-08-02 2016-08-03 マクロジェニクス，インコーポレーテッド Covalently bonded diabody and its use
WO2012021797A1 (en) 2010-08-13 2012-02-16 Pioneer Hi-Bred International, Inc. Methods and compositions for targeting sequences of interest to the chloroplast
CA2808098C (en) 2010-08-16 2020-03-10 Merck Sharp & Dohme Corp. Process for preparing aminocyclohexyl ether compounds
WO2012022734A2 (en) 2010-08-16 2012-02-23 Medimmune Limited Anti-icam-1 antibodies and methods of use
US9322027B2 (en) 2010-08-20 2016-04-26 Shell Oil Company Expression constructs comprising fungal promoters
CA2807702C (en) 2010-08-20 2018-07-24 Codexis, Inc. Use of glycoside hydrolase 61 family proteins in processing of cellulose
MY162825A (en) 2010-08-20 2017-07-31 Novartis Ag Antibodies for epidermal growth factor receptor 3 (her3)
BR112013003223A2 (en) 2010-08-23 2016-06-07 Pioneer Hi Bred Int "isolated polynucleotide, expression cassette, host cell, microorganism, plant or plant part, method of obtaining a transformed plant, antipathogenic composition, method of protecting a plant against a pathogen or use of an isolated polynucleotide"
JP5963749B2 (en) 2010-08-26 2016-08-03 イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニーＥ．Ｉ．Ｄｕ Ｐｏｎｔ Ｄｅ Ｎｅｍｏｕｒｓ Ａｎｄ Ｃｏｍｐａｎｙ Mutant Δ9 elongases and their use in the production of polyunsaturated fatty acids
EP2609202A1 (en) 2010-08-26 2013-07-03 E.I. Du Pont De Nemours And Company Mutant hpgg motif and hdash motif delta-5 desaturases and their use in making polyunsaturated fatty acids
EP2614080A1 (en) 2010-09-10 2013-07-17 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Phosphorylated twist1 and metastasis
RU2013115927A (en) 2010-09-10 2014-10-20 Апексиджен, Инк. ANTIBODIES AGAINST IL-1β AND METHODS OF APPLICATION
US8859502B2 (en) 2010-09-13 2014-10-14 Celgene Corporation Therapy for MLL-rearranged leukemia
CN103298341B (en) 2010-09-22 2016-06-08 拜耳知识产权有限责任公司 Active component is used for preventing and treating the purposes of nematicide in nematicide crop
EP2635607B1 (en) 2010-11-05 2019-09-04 Zymeworks Inc. Stable heterodimeric antibody design with mutations in the fc domain
EP2640738A1 (en) 2010-11-15 2013-09-25 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Anti-fungal agents
WO2012069466A1 (en) 2010-11-24 2012-05-31 Novartis Ag Multispecific molecules
US9267159B2 (en) 2010-12-08 2016-02-23 Codexis, Inc. Biocatalysts and methods for the synthesis of armodafinil
BR112013014988A2 (en) 2010-12-22 2017-06-27 Du Pont isolated nucleic acid molecule, dna construct, vector, plant cell, plant, transgenic seed from plant, method for expression of a nucleotide sequence in a plant, method for expression of a nucleotide sequence in a plant cell, method for selectively express a nucleotide sequence in green corn plant tissues
MX2013007087A (en) 2010-12-22 2013-07-29 Du Pont Viral promoter, truncations thereof, and methods of use.
WO2012092106A1 (en) 2010-12-28 2012-07-05 Pioneer Hi-Bred International, Inc. Novel bacillus thuringiensis gene with lepidopteran activity
BR112013017955A2 (en) 2011-01-14 2019-09-24 Florida Res Foundation Inc methods for preparing a citrus-resistant citrus-resistant plant and for producing citrus fruit, nucleic acid molecule, expression cassette or vector, plant or plant cell, nucleic acid molecule, and host cell
WO2012103165A2 (en) 2011-01-26 2012-08-02 Kolltan Pharmaceuticals, Inc. Anti-kit antibodies and uses thereof
WO2012106579A1 (en) 2011-02-03 2012-08-09 Bristol-Myers Squibb Company Amino acid dehydrogenase and its use in preparing amino acids from keto acids
WO2012112411A1 (en) 2011-02-15 2012-08-23 Pioneer Hi-Bred International, Inc. Root-preferred promoter and methods of use
AU2012223358A1 (en) 2011-03-01 2013-09-05 Amgen Inc. Sclerostin and DKK-1 bispecific binding agents
RU2013144392A (en) 2011-03-03 2015-04-10 Апексиджен, Инк. ANTIBODIES TO IL-6 RECEPTOR AND METHODS OF APPLICATION
AU2012225246B2 (en) 2011-03-10 2016-01-21 Omeros Corporation Generation of anti-FN14 monoclonal antibodies by ex-vivo accelerated antibody evolution
US8878007B2 (en) 2011-03-10 2014-11-04 Pioneer Hi Bred International Inc Bacillus thuringiensis gene with lepidopteran activity
MX338078B (en) 2011-03-25 2016-04-01 Amgen Inc Anti - sclerostin antibody crystals and formulations thereof.
US9321814B2 (en) 2011-03-30 2016-04-26 Athenix Corp. AXMI238 toxin gene and methods for its use
EP2691517A4 (en) 2011-03-30 2015-04-22 Codexis Inc Pentose fermentation by a recombinant microorganism
US9695440B2 (en) 2011-03-30 2017-07-04 Athenix Corp. AXMI232, AXMI233, and AXMI249 toxin genes and methods for their use
BR112013025665B8 (en) 2011-04-05 2022-07-05 Athenix Corp RECOMBINANT NUCLEIC ACID MOLECULE, VECTOR, TRANSGENIC MICRO-ORGANISM, RECOMBINANT POLYPEPTIDE AND ITS PRODUCTION METHOD, COMPOSITION, METHODS TO CONTROL OR EXTERMINATE A PEST POPULATION, TO PROTECT A PLANT FROM A PEST, AND TO INCREASE YIELD IN A PLANT
US9102963B2 (en) 2011-04-13 2015-08-11 Codexis, Inc. Biocatalytic process for preparing eslicarbazepine and analogs thereof
WO2012142233A1 (en) 2011-04-14 2012-10-18 St. Jude Children's Research Hospital Methods and compositions for detecting and modulating a novel mtor complex
CN106928362B (en) 2011-04-29 2021-10-26 埃派斯进有限公司 anti-CD 40 antibodies and methods of use thereof
EP2710042A2 (en) 2011-05-16 2014-03-26 Fabion Pharmaceuticals, Inc. Multi-specific fab fusion proteins and methods of use
EA201391753A1 (en) 2011-05-21 2014-08-29 Макродженикс, Инк. DOMAINS CONNECTING WITH DEIMMUNIZED SERUM AND THEIR APPLICATION TO INCREASE THE TIME OF HALF-DURATION
US9150625B2 (en) 2011-05-23 2015-10-06 E I Du Pont De Nemours And Company Chloroplast transit peptides and methods of their use
EP2714738B1 (en) 2011-05-24 2018-10-10 Zyngenia, Inc. Multivalent and monovalent multispecific complexes and their uses
EP2717911A1 (en) 2011-06-06 2014-04-16 Novartis Forschungsstiftung, Zweigniederlassung Protein tyrosine phosphatase, non-receptor type 11 (ptpn11) and triple-negative breast cancer
WO2012170742A2 (en) 2011-06-07 2012-12-13 University Of Hawaii Treatment and prevention of cancer with hmgb1 antagonists
WO2012170740A2 (en) 2011-06-07 2012-12-13 University Of Hawaii Biomarker of asbestos exposure and mesothelioma
WO2012172495A1 (en) 2011-06-14 2012-12-20 Novartis Ag Compositions and methods for antibodies targeting tem8
WO2012174139A2 (en) 2011-06-14 2012-12-20 Synthon Biopharmaceuticals B.V. Compositions and methods for making and b ioc ont aining auxotrophic transgenic plants
MX2013015174A (en) 2011-06-21 2014-09-22 Pioneer Hi Bred Int Methods and compositions for producing male sterile plants.
BR112013033614A2 (en) 2011-06-30 2018-05-29 Codexis Inc pentose fermentation by recombinant microorganism
WO2013009935A2 (en) 2011-07-12 2013-01-17 Two Blades Foundation Late blight resistance genes
US9322007B2 (en) 2011-07-22 2016-04-26 The California Institute Of Technology Stable fungal Cel6 enzyme variants
US9120858B2 (en) 2011-07-22 2015-09-01 The Research Foundation Of State University Of New York Antibodies to the B12-transcobalamin receptor
WO2013015821A1 (en) 2011-07-22 2013-01-31 The Research Foundation Of State University Of New York Antibodies to the b12-transcobalamin receptor
AR087367A1 (en) 2011-07-28 2014-03-19 Athenix Corp AXMI 270 TOXIN GEN AND ITS METHODS OF USE
HUE037346T2 (en) 2011-07-28 2018-08-28 Athenix Corp Axmi205 variant proteins and methods for their use
MX2014001070A (en) 2011-07-29 2014-04-14 Athenix Corp Axmi279 pesticidal gene and methods for its use.
CA2844101A1 (en) 2011-08-03 2013-02-07 E. I. Du Pont De Nemours And Company Methods and compositions for targeted integration in a plant
MX355816B (en) 2011-08-04 2018-05-02 Amgen Inc Method for treating bone gap defects.
CA2844289C (en) 2011-08-12 2020-01-14 Omeros Corporation Anti-fzd10 monoclonal antibodies and methods for their use
WO2013024157A2 (en) 2011-08-17 2013-02-21 INSERM (Institut National de la Santé et de la Recherche Médicale) Combinations of host targeting agents for the treatment and the prevention of hcv infection
WO2013024156A2 (en) 2011-08-17 2013-02-21 INSERM (Institut National de la Santé et de la Recherche Médicale) Combinations of anti-hcv-entry factor antibodies and interferons for the treatment and the prevention of hcv infection
WO2013024155A1 (en) 2011-08-17 2013-02-21 Inserm (Institut National De La Sante Et De La Recherche Medicale) Combinations of anti-hcv-entry factor antibodies and direct acting antivirals for the treatment and the prevention of hcv infection
EP2748317B1 (en) 2011-08-22 2017-04-19 Codexis, Inc. Gh61 glycoside hydrolase protein variants and cofactors that enhance gh61 activity
US11377663B1 (en) 2011-08-30 2022-07-05 Monsanto Technology Llc Genetic regulatory elements
PT2751279T (en) 2011-08-31 2017-12-29 St Jude Children`S Res Hospital Methods and compositions to detect the level of lysosomal exocytosis activity and methods of use
WO2013036861A1 (en) 2011-09-08 2013-03-14 Codexis, Inc Biocatalysts and methods for the synthesis of substituted lactams
US20130084608A1 (en) 2011-09-30 2013-04-04 Codexis, Inc. Fungal proteases
US20130111634A1 (en) 2011-10-28 2013-05-02 E I Du Pont De Nemours And Company And Pioneer Hi-Bred International Methods and compositions for silencing genes using artificial micrornas
EP2773373B1 (en) 2011-11-01 2018-08-22 Bionomics, Inc. Methods of blocking cancer stem cell growth
US9221907B2 (en) 2011-11-01 2015-12-29 Bionomics Inc. Anti-GPR49 monoclonal antibodies
WO2013067057A1 (en) 2011-11-01 2013-05-10 Bionomics, Inc. Anti-gpr49 antibodies
CN104053671A (en) 2011-11-01 2014-09-17 生态学有限公司 Antibodies and methods of treating cancer
WO2013067098A1 (en) 2011-11-02 2013-05-10 Apexigen, Inc. Anti-kdr antibodies and methods of use
MX358862B (en) 2011-11-04 2018-09-06 Zymeworks Inc Stable heterodimeric antibody design with mutations in the fc domain.
EP2776838A1 (en) 2011-11-08 2014-09-17 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Early diagnostic of neurodegenerative diseases
EP2776022A1 (en) 2011-11-08 2014-09-17 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research New treatment for neurodegenerative diseases
EP2780448B1 (en) 2011-11-18 2019-09-11 Codexis, Inc. Biocatalysts for the preparation of hydroxy substituted carbamates
KR102151383B1 (en) 2011-12-05 2020-09-03 노파르티스 아게 Antibodies for epidermal growth factor receptor 3 (her3)
EA201491107A1 (en) 2011-12-05 2014-11-28 Новартис Аг ANTIBODIES TO THE RECEPTOR EPIDERMAL GROWTH FACTOR 3 (HER3), DIRECTED TO DOMAIN II HER3
BR112014015030A2 (en) 2011-12-20 2017-06-13 Codexis Inc endoflucanase variants 1b (eg1b)
EP2794656B1 (en) 2011-12-21 2019-02-27 Novartis AG Compositions comprising antibodies targeting factor p and c5
CN104114707A (en) 2011-12-22 2014-10-22 纳幕尔杜邦公司 Use of the soybean sucrose synthase promoter to increase plant seed lipid content
WO2013093809A1 (en) 2011-12-23 2013-06-27 Pfizer Inc. Engineered antibody constant regions for site-specific conjugation and methods and uses therefor
IN2014CN04634A (en) 2011-12-28 2015-09-18 Amgen Inc
EP2800583A1 (en) 2012-01-02 2014-11-12 Novartis AG Cdcp1 and breast cancer
US20130180008A1 (en) 2012-01-06 2013-07-11 Pioneer Hi Bred International Inc Ovule Specific Promoter and Methods of Use
WO2013103365A1 (en) 2012-01-06 2013-07-11 Pioneer Hi-Bred International, Inc. Pollen preferred promoters and methods of use
EP2800814A1 (en) 2012-01-06 2014-11-12 Pioneer Hi-Bred International Inc. A method to screen plants for genetic elements inducing parthenogenesis in plants
EP2800760A1 (en) 2012-01-06 2014-11-12 Pioneer Hi-Bred International Inc. Compositions and methods for the expression of a sequence in a reproductive tissue of a plant
CN102568437B (en) 2012-01-20 2014-07-30 华为终端有限公司 Image displaying method and equipment
BR112014018294B1 (en) 2012-01-26 2022-01-11 Norfolk Plant Sciences, Ltd METHOD FOR PRODUCING A PLANT, EXPRESSION CASSETTE, AND BACTERIAL CELL
AR089793A1 (en) 2012-01-27 2014-09-17 Du Pont METHODS AND COMPOSITIONS TO GENERATE COMPOSITE TRANSGENIC RISK LOCUS
US9790258B2 (en) 2012-03-08 2017-10-17 Athenix Corp. AXMI335 toxin gene and methods for its use
UA115236C2 (en) 2012-03-08 2017-10-10 Атенікс Корп. Axmi345 delta-endotoxin gene and methods for its use
CN104508126B (en) 2012-03-23 2017-06-30 科德克希思公司 Biocatalyst and method for synthesizing the derivative of tryptamines and tryptamine analogues
US20150266961A1 (en) 2012-03-29 2015-09-24 Novartis Forschungsstiftung, Zweigniederlassung, Fridrich Miescher Institute Inhibition of interleukin-8 and/or its receptor cxcr1 in the treatment of her2/her3-overexpressing breast cancer
WO2013166113A1 (en) 2012-05-04 2013-11-07 E. I. Du Pont De Nemours And Company Compositions and methods comprising sequences having meganuclease activity
CN104428412B (en) 2012-05-08 2017-09-08 科德克希思公司 Hydroxylated biocatalyst and method for compound
US20130336973A1 (en) 2012-05-10 2013-12-19 Zymeworks Inc. Heteromultimer Constructs of Immunoglobulin Heavy Chains with Mutations in the Fc Domain
CN107904216B (en) 2012-05-11 2021-06-29 科德克希思公司 Engineered imine reductases and methods for reductive amination of ketone and amine compounds
ES2703540T3 (en) 2012-06-04 2019-03-11 Novartis Ag Site-specific marking methods and molecules produced in this way
US8956844B2 (en) 2012-06-11 2015-02-17 Codexis, Inc. Fungal xylanases and xylosidases
BR112014031260A2 (en) 2012-06-15 2019-08-20 Du Pont methods and compositions involving native substrate-preferable als variants
US20130337442A1 (en) 2012-06-15 2013-12-19 Pioneer Hi-Bred International, Inc. Genetic loci associated with soybean cyst nematode resistance and methods of use
US20150337336A1 (en) 2012-06-22 2015-11-26 Phytogene, Inc. Enzymes and methods for styrene synthesis
CA2840683C (en) 2012-06-29 2020-03-10 Athenix Corp. Axmi277 nematode toxin and methods for its use
EP2866831A1 (en) 2012-06-29 2015-05-06 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Treating diseases by modulating a specific isoform of mkl1
EP3626267A1 (en) 2012-07-05 2020-03-25 UCB Pharma, S.A. Treatment for bone diseases
WO2014006114A1 (en) 2012-07-05 2014-01-09 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research New treatment for neurodegenerative diseases
CA2880007C (en) 2012-07-25 2021-12-28 Kolltan Pharmaceuticals, Inc. Anti-kit antibodies and uses thereof
EP2880159A1 (en) 2012-07-30 2015-06-10 Allylix, Inc. Sclareol and labdenediol diphosphate synthase polypeptides encoding nucleic acid molecules and uses thereof
WO2014036238A1 (en) 2012-08-30 2014-03-06 Athenix Corp. Axmi-234 and axmi-235 toxin genes and methods for their use
WO2014033266A1 (en) 2012-08-31 2014-03-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Anti-sr-bi antibodies for the inhibition of hepatitis c virus infection
AU2013202506B2 (en) 2012-09-07 2015-06-18 Celgene Corporation Resistance biomarkers for hdac inhibitors
EP2895610B1 (en) 2012-09-13 2019-11-06 Indiana University Research and Technology Corporation Compositions and systems for conferring disease resistance in plants and methods of use thereof
EP2906598A1 (en) 2012-10-09 2015-08-19 Igenica Biotherapeutics, Inc. Anti-c16orf54 antibodies and methods of use thereof
US20140109259A1 (en) 2012-10-11 2014-04-17 Pioneer Hi Bred International Inc Guard Cell Promoters and Uses Thereof
BR112015008504A2 (en) 2012-10-15 2017-08-22 Pioneer Hi Bred Int METHOD FOR ENHANCED PESTICIDAL ACTIVITY, POLYPEPTIDE, NUCLEIC ACID MOLECULE, EXPRESSION CASSETTE, VECTOR, METHOD FOR OBTAINING A PLANT, METHOD FOR OBTAINING A PLANT CELL, PESTICIDE COMPOSITION, METHOD FOR PROTECTING A PLANT, METHOD FOR PROTECTING A PLANT PLANT, METHOD TO INCREASE THE RESISTANCE OF A PLANT
WO2014066497A2 (en) 2012-10-23 2014-05-01 Montana State University Production of high quality durum wheat having increased amylose content
WO2014070673A1 (en) 2012-10-29 2014-05-08 University Of Rochester Artemisinin derivatives, methods for their preparation and their use as antimalarial agents
CN109265552A (en) 2012-10-30 2019-01-25 埃派斯进有限公司 Anti-CD 40 antibodies and its application method
BR112015009774A2 (en) 2012-11-01 2017-12-05 Univ British Columbia cytochrome p450 and cytochrome p450 reductase polypeptides encoding nucleic acid molecules and their uses
AU2013202507B9 (en) 2012-11-14 2015-08-13 Celgene Corporation Inhibition of drug resistant cancer cells
WO2014081605A1 (en) 2012-11-20 2014-05-30 Codexis, Inc. Pentose fermentation by a recombinant microorganism
UY35148A (en) 2012-11-21 2014-05-30 Amgen Inc HETERODIMERIC IMMUNOGLOBULINS
WO2014084859A1 (en) 2012-11-30 2014-06-05 Novartis Ag Molecules and methods for modulating tmem16a activities
HUE053669T2 (en) 2012-12-05 2021-07-28 Novartis Ag Compositions and methods for antibodies targeting epo
WO2014088984A1 (en) 2012-12-07 2014-06-12 Merck Sharp & Dohme Corp. Biocatalytic transamination process
AR093909A1 (en) 2012-12-12 2015-06-24 Bayer Cropscience Ag USE OF ACTIVE INGREDIENTS TO CONTROL NEMATODES IN CULTURES RESISTANT TO NEMATODES
MX2015007931A (en) 2012-12-18 2015-10-05 Novartis Ag Compositions and methods that utilize a peptide tag that binds to hyaluronan.
JP6618180B2 (en) 2012-12-21 2019-12-11 コデクシス， インコーポレイテッド Engineered biocatalysts and methods for the synthesis of chiral amines
WO2014113521A1 (en) 2013-01-18 2014-07-24 Codexis, Inc. Engineered biocatalysts useful for carbapenem synthesis
US9684771B2 (en) 2013-01-31 2017-06-20 Codexis, Inc. Methods, systems, and software for identifying bio-molecules using models of multiplicative form
WO2014124258A2 (en) 2013-02-08 2014-08-14 Irm Llc Specific sites for modifying antibodies to make immunoconjugates
SI2953976T1 (en) 2013-02-08 2021-08-31 Novartis Ag Specific sites for modifying antibodies to make immunoconjugates
HUE039836T2 (en) 2013-02-13 2019-02-28 Athenix Corp Use of axmi184 for the control of rootworm insects
US9617573B2 (en) 2013-02-28 2017-04-11 Codexis, Inc. Engineered transaminase polypeptides for industrial biocatalysis
WO2014138339A2 (en) 2013-03-07 2014-09-12 Athenix Corp. Toxin genes and methods for their use
WO2014164775A1 (en) 2013-03-11 2014-10-09 Pioneer Hi-Bred International, Inc. Methods and compositions to improve the spread of chemical signals in plants
US20160326540A1 (en) 2013-03-11 2016-11-10 Pioneer Hi-Bred International, Inc. Methods and Compositions Employing a Sulfonylurea-Dependent Stabilization Domain
US9803214B2 (en) 2013-03-12 2017-10-31 Pioneer Hi-Bred International, Inc. Breeding pair of wheat plants comprising an MS45 promoter inverted repeat that confers male sterility and a construct that restores fertility
EP2970496B1 (en) 2013-03-12 2018-07-18 Institute of Arthritis Research LLC Immunologically active polypeptide
US9273322B2 (en) 2013-03-12 2016-03-01 Pioneer Hi Bred International Inc Root-preferred promoter and methods of use
US9243258B2 (en) 2013-03-12 2016-01-26 Pioneer Hi Bred International Inc Root-preferred promoter and methods of use
US9498532B2 (en) 2013-03-13 2016-11-22 Novartis Ag Antibody drug conjugates
EP2970479B1 (en) 2013-03-14 2019-04-24 Novartis AG Antibodies against notch 3
ES2647828T3 (en) 2013-03-14 2017-12-26 Evolva, Inc. Valencene synthase polypeptides, nucleic acid molecules that encode them, and uses thereof
EP2970935A1 (en) 2013-03-14 2016-01-20 Pioneer Hi-Bred International, Inc. Compositions having dicamba decarboxylase activity and methods of use
CA2905595A1 (en) 2013-03-14 2014-09-25 Pioneer Hi-Bred International, Inc. Compositions having dicamba decarboxylase activity and methods of use
WO2014150937A1 (en) 2013-03-15 2014-09-25 Novartis Ag Antibody drug conjugates
CA2901316A1 (en) 2013-03-15 2014-09-25 Pioneer Hi-Bred International, Inc. Phi-4 polypeptides and methods for their use
WO2014144600A2 (en) 2013-03-15 2014-09-18 Viktor Roschke Multivalent and monovalent multispecific complexes and their uses
EP2970434B1 (en) 2013-03-15 2018-12-05 The Regents of The University of California Dual reactivity potent kunitz inhibitor of fibrinolysis
PE20151775A1 (en) 2013-04-18 2015-12-02 Codexis Inc POLYPEPTIDES OF PHENYLALANINE AMMONIA LIASA MODIFIED BY GENETIC ENGINEERING
NZ714765A (en) 2013-06-06 2021-12-24 Pf Medicament Anti-c10orf54 antibodies and uses thereof
AR096601A1 (en) 2013-06-21 2016-01-20 Novartis Ag ANTIBODIES OF LEXINED OXIDATED LDL RECEIVER 1 AND METHODS OF USE
US9562101B2 (en) 2013-06-21 2017-02-07 Novartis Ag Lectin-like oxidized LDL receptor 1 antibodies and methods of use
CA2917103C (en) 2013-07-09 2021-01-12 Board Of Trustees Of Michigan State University Transgenic plants produced with a k-domain, and methods and expression cassettes related thereto
US11459579B2 (en) 2013-07-09 2022-10-04 Board Of Trustees Of Michigan State University Transgenic plants produced with a K-domain, and methods and expression cassettes related thereto
EP3030902B1 (en) 2013-08-07 2019-09-25 Friedrich Miescher Institute for Biomedical Research New screening method for the treatment friedreich's ataxia
US10287329B2 (en) 2013-08-08 2019-05-14 Pioneer Hi-Bred International, Inc. Insecticidal polypeptides having broad spectrum activity and uses thereof
UY35696A (en) 2013-08-09 2015-03-27 Athenix Corp ? RECOMBINANT DNA MOLECULE THAT INCLUDES AXMI440 TOXIN GENE, VECTOR, GUEST CELL, PLANTS, COMPOSITIONS AND RELATED METHODS ?.
WO2015038262A2 (en) 2013-08-09 2015-03-19 Athenix Corp. Axmi281 toxin gene and methods for its use
CN106232820A (en) 2013-08-16 2016-12-14 先锋国际良种公司 Insecticidal protein and using method thereof
BR112016005541A2 (en) 2013-09-11 2018-01-30 Du Pont recombinant nucleic acid molecule, dna construct, transgenic plant, transgenic plant seed
BR122020001770B1 (en) 2013-09-13 2022-11-29 Pioneer Hi-Bred International, Inc DNA CONSTRUCTION, METHOD FOR OBTAINING A TRANSGENIC PLANT, FUSION PROTEIN, METHOD FOR CONTROLLING AN INSECT PEST POPULATION, METHOD FOR INHIBITING THE GROWTH OR KILLING AN INSECT PEST
US10696964B2 (en) 2013-09-27 2020-06-30 Codexis, Inc. Automated screening of enzyme variants
WO2015050959A1 (en) 2013-10-01 2015-04-09 Yale University Anti-kit antibodies and methods of use thereof
EP3068793B1 (en) 2013-11-13 2021-02-17 Codexis, Inc. Engineered imine reductases and methods for the reductive amination of ketone and amine compounds
WO2015077525A1 (en) 2013-11-25 2015-05-28 Bayer Cropscience Lp Use of axmi-011 for the control of hemipteran insects
EA033842B1 (en) 2013-12-09 2019-12-02 Атеникс Корп. Axmi477, Axmi482, Axmi486 AND Axmi525 TOXIN GENES AND METHODS FOR THEIR USE
US10544191B2 (en) 2013-12-23 2020-01-28 University Of Rochester Methods and compositions for ribosomal synthesis of macrocyclic peptides
NZ630311A (en) 2013-12-27 2016-03-31 Celgene Corp Romidepsin formulations and uses thereof
SI3097122T1 (en) 2014-01-24 2020-07-31 Ngm Biopharmaceuticals, Inc. Antibodies binding beta klotho domain 2 and methods of use thereof
US20170166920A1 (en) 2014-01-30 2017-06-15 Two Blades Foundation Plants with enhanced resistance to phytophthora
CA2938979C (en) 2014-02-07 2024-02-20 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
UA120608C2 (en) 2014-02-07 2020-01-10 Піонір Хай-Бред Інтернешнл, Інк. Insecticidal proteins and methods for their use
GB201403775D0 (en) 2014-03-04 2014-04-16 Kymab Ltd Antibodies, uses & methods
WO2015138615A2 (en) 2014-03-12 2015-09-17 Irm Llc Specific sites for modifying antibodies to make immunoconjugates
KR102352573B1 (en) 2014-04-04 2022-01-18 바이오노믹스 인코포레이티드 Humanized antibodies that bind lgr5
EP3145536B1 (en) 2014-04-16 2021-10-27 Codexis, Inc. Engineered tyrosine ammonia lyase
US10053702B2 (en) 2014-04-22 2018-08-21 E I Du Pont De Nemours And Company Plastidic carbonic anhydrase genes for oil augmentation in seeds with increased DGAT expression
WO2015171603A1 (en) 2014-05-06 2015-11-12 Two Blades Foundation Methods for producing plants with enhanced resistance to oomycete pathogens
EP3753573A1 (en) 2014-05-09 2020-12-23 Board of Supervisors of Louisiana State University and Agricultural and Mechanical College Vaccines against genital herpes simplex infections
US20170137824A1 (en) 2014-06-13 2017-05-18 Indranil BANERJEE New treatment against influenza virus
ES2694296T3 (en) 2014-06-17 2018-12-19 Centre National De La Recherche Scientifique (Cnrs) Anti-pVHL monoclonal antibodies and uses thereof
EP3157945A4 (en) 2014-06-20 2017-11-22 F. Hoffmann-La Roche AG Chagasin-based scaffold compositions, methods, and uses
EP3160990A2 (en) 2014-06-25 2017-05-03 Novartis AG Compositions and methods for long acting proteins
EP3160991A2 (en) 2014-06-25 2017-05-03 Novartis AG Compositions and methods for long acting proteins
WO2016007623A1 (en) 2014-07-09 2016-01-14 Codexis, Inc. Novel p450-bm3 variants with improved activity
WO2016020791A1 (en) 2014-08-05 2016-02-11 Novartis Ag Ckit antibody drug conjugates
WO2016020882A2 (en) 2014-08-07 2016-02-11 Novartis Ag Angiopoetin-like 4 (angptl4) antibodies and methods of use
CU24453B1 (en) 2014-08-07 2019-11-04 Novartis Ag ANTI-PROTEIN ANTIBODIES SIMILAR TO ANGIOPOYETIN 4
US20170218384A1 (en) 2014-08-08 2017-08-03 Pioneer Hi-Bred International, Inc. Ubiquitin promoters and introns and methods of use
US9982045B2 (en) 2014-08-12 2018-05-29 Novartis Ag Anti-CDH6 antibody drug conjugates
MX2017002930A (en) 2014-09-12 2017-06-06 Du Pont Generation of site-specific-integration sites for complex trait loci in corn and soybean, and methods of use.
EP3197557A1 (en) 2014-09-24 2017-08-02 Friedrich Miescher Institute for Biomedical Research Lats and breast cancer
MA41044A (en) 2014-10-08 2017-08-15 Novartis Ag COMPOSITIONS AND METHODS OF USE FOR INCREASED IMMUNE RESPONSE AND CANCER TREATMENT
EP3207126A4 (en) 2014-10-16 2018-04-04 Pioneer Hi-Bred International, Inc. Insecticidal polypeptides having broad spectrum activity and uses thereof
CN113372421A (en) 2014-10-16 2021-09-10 先锋国际良种公司 Insecticidal proteins and methods of use thereof
MX2017004814A (en) 2014-10-16 2017-08-02 Pioneer Hi Bred Int Insecticidal polypeptides having improved activity spectrum and uses thereof.
GB201418713D0 (en) 2014-10-21 2014-12-03 Kymab Ltd Bindings Proteins
SG11201703464UA (en) 2014-11-14 2017-05-30 Novartis Ag Antibody drug conjugates
WO2016085916A1 (en) 2014-11-25 2016-06-02 Codexis, Inc. Engineered imine reductases and methods for the reductive amination of ketone and amine compounds
US10077420B2 (en) 2014-12-02 2018-09-18 Histogenics Corporation Cell and tissue culture container
CN108064167A (en) 2014-12-11 2018-05-22 皮埃尔法布雷医药公司 Anti- C10ORF54 antibody and application thereof
MA41142A (en) 2014-12-12 2017-10-17 Amgen Inc ANTI-SCLEROSTINE ANTIBODIES AND THE USE OF THEM TO TREAT BONE CONDITIONS AS PART OF THE TREATMENT PROTOCOL
RU2741833C2 (en) 2014-12-12 2021-01-29 Зингента Партисипейшнс Аг Compositions and methods for controlling plant pests
US20170369902A1 (en) 2014-12-16 2017-12-28 Pioneer Hi-Bred International, Inc. Restoration of male fertility in wheat
UY36449A (en) 2014-12-19 2016-07-29 Novartis Ag COMPOSITIONS AND METHODS FOR ANTIBODIES DIRECTED TO BMP6
PL3237621T3 (en) 2014-12-22 2023-10-30 Codexis, Inc. Human alpha-galactosidase variants
CA2976045A1 (en) 2015-02-09 2016-08-18 Bioconsortia, Inc. Agriculturally beneficial microbes, microbial compositions, and consortia
ES2830725T3 (en) 2015-02-10 2021-06-04 Codexis Inc Ketoreductase polypeptides for the synthesis of chiral compounds
ES2937020T3 (en) 2015-03-03 2023-03-23 Kymab Ltd Antibodies, uses and methods
CA2977026A1 (en) 2015-03-11 2016-09-15 E.I. Du Pont De Nemours And Company Insecticidal combinations of pip-72 and methods of use
CA2975279A1 (en) 2015-03-19 2016-09-22 Pioneer Hi-Bred International, Inc. Methods and compositions for accelerated trait introgression
EP3070103A1 (en) 2015-03-19 2016-09-21 Institut Hospitalier Universitaire De Strasbourg Anti-Claudin 1 monoclonal antibodies for the prevention and treatment of hepatocellular carcinoma
BR112017022383A2 (en) 2015-04-17 2018-07-17 Agbiome Inc recombinant polypeptide, composition, recombinant nucleic acid molecule, recombinant nucleic acid, host cell, dna construct, vector, method for controlling a pest population, method for producing a polypeptide, plant, transgenic plant seed, method for protecting a plant , method to increase yield in a plant
WO2016171999A2 (en) 2015-04-22 2016-10-27 AgBiome, Inc. Pesticidal genes and methods of use
WO2016179522A1 (en) 2015-05-06 2016-11-10 Pioneer Hi-Bred International, Inc. Methods and compositions for the production of unreduced, non-recombined gametes and clonal offspring
MX2017014213A (en) 2015-05-07 2018-03-28 Codexis Inc Penicillin-g acylases.
EP3294892B1 (en) 2015-05-09 2020-11-25 Two Blades Foundation Late blight resistance gene from solanum americanum and methods of use
CN108064233B (en) 2015-05-19 2022-07-15 先锋国际良种公司 Insecticidal proteins and methods of use thereof
CN108271390A (en) 2015-06-03 2018-07-10 农业生物群落股份有限公司 Killing gene and application method
SG11201707815RA (en) 2015-06-05 2017-12-28 Novartis Ag Antibodies targeting bone morphogenetic protein 9 (bmp9) and methods therefor
CN107771218B (en) 2015-06-17 2021-12-31 先锋国际良种公司 Plant regulatory elements and methods of use thereof
WO2016203432A1 (en) 2015-06-17 2016-12-22 Novartis Ag Antibody drug conjugates
CN107849097A (en) 2015-06-22 2018-03-27 农业生物群落股份有限公司 Killing gene and application method
CN107849597B (en) 2015-06-25 2022-07-26 原生微生物股份有限公司 Method for forming a population of active microbial strains based on analysis of heterogeneous microbial communities
WO2017120495A1 (en) 2016-01-07 2017-07-13 Ascus Biosciences, Inc. Methods for improving milk production by administration of microbial consortia
WO2018126026A1 (en) 2016-12-28 2018-07-05 Ascus Biosciences, Inc. Methods, apparatuses, and systems for analyzing complete microorganism strains in complex heterogeneous communities, determining functional relationships and interactions thereof, and identifying and synthesizing bioreactive modificators based thereon
US10632157B2 (en) 2016-04-15 2020-04-28 Ascus Biosciences, Inc. Microbial compositions and methods of use for improving fowl production
US9938558B2 (en) 2015-06-25 2018-04-10 Ascus Biosciences, Inc. Methods, apparatuses, and systems for analyzing microorganism strains from complex heterogeneous communities, predicting and identifying functional relationships and interactions thereof, and selecting and synthesizing microbial ensembles based thereon
US10851399B2 (en) 2015-06-25 2020-12-01 Native Microbials, Inc. Methods, apparatuses, and systems for microorganism strain analysis of complex heterogeneous communities, predicting and identifying functional relationships and interactions thereof, and selecting and synthesizing microbial ensembles based thereon
JOP20200312A1 (en) 2015-06-26 2017-06-16 Novartis Ag Factor xi antibodies and methods of use
DK3319987T3 (en) 2015-07-07 2021-06-21 Codexis Inc NEW P450-BM3 VARIANTS WITH IMPROVED ACTIVITY
EP3325604B1 (en) 2015-07-25 2022-02-09 BioConsortia, Inc. Agriculturally beneficial microbes, microbial compositions, and consortia
CA2994516A1 (en) 2015-08-03 2017-02-09 Novartis Ag Methods of treating fgf21-associated disorders
WO2017023486A1 (en) 2015-08-06 2017-02-09 Pioneer Hi-Bred International, Inc. Plant derived insecticidal proteins and methods for their use
WO2017042701A1 (en) 2015-09-09 2017-03-16 Novartis Ag Thymic stromal lymphopoietin (tslp)-binding antibodies and methods of using the antibodies
LT3347377T (en) 2015-09-09 2021-05-25 Novartis Ag Thymic stromal lymphopoietin (tslp)-binding antibodies and methods of using the antibodies
TWI799366B (en) 2015-09-15 2023-04-21 美商建南德克公司 Cystine knot scaffold platform
CN108289953B (en) 2015-09-29 2022-03-11 细胞基因公司 PD-1 binding proteins and methods of use thereof
WO2017062790A1 (en) 2015-10-09 2017-04-13 Two Blades Foundation Cold shock protein receptors and methods of use
WO2017072669A1 (en) 2015-10-28 2017-05-04 Friedrich Miescher Institute For Biomedical Research Tenascin-w and biliary tract cancers
EP3370509A1 (en) 2015-11-03 2018-09-12 Two Blades Foundation Wheat stripe rust resistance genes and methods of use
US20180258438A1 (en) 2015-11-06 2018-09-13 Pioneer Hi-Bred International, Inc. Generation of complex trait loci in soybean and methods of use
CA3004913A1 (en) 2015-11-30 2017-06-08 Pioneer Hi-Bred International, Inc. Plant regulatory elements and methods of use thereof
ES2878014T3 (en) 2015-12-07 2021-11-18 Zymergen Inc Improvement of microbial strains using an HTP genomic engineering platform
US11208649B2 (en) 2015-12-07 2021-12-28 Zymergen Inc. HTP genomic engineering platform
US9988624B2 (en) 2015-12-07 2018-06-05 Zymergen Inc. Microbial strain improvement by a HTP genomic engineering platform
CA3007635A1 (en) 2015-12-07 2017-06-15 Zymergen Inc. Promoters from corynebacterium glutamicum
KR20180089510A (en) 2015-12-18 2018-08-08 노파르티스 아게 Antibodies targeting CD32b and methods of using the same
WO2017105987A1 (en) 2015-12-18 2017-06-22 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2017112006A1 (en) 2015-12-22 2017-06-29 Pioneer Hi-Bred International, Inc. Tissue-preferred promoters and methods of use
BR112018012893A2 (en) 2015-12-22 2018-12-04 AgBiome, Inc. pesticide genes and methods of use
US20210198368A1 (en) 2016-01-21 2021-07-01 Novartis Ag Multispecific molecules targeting cll-1
GB201604124D0 (en) 2016-03-10 2016-04-27 Ucb Biopharma Sprl Pharmaceutical formulation
WO2017157305A1 (en) 2016-03-15 2017-09-21 Generon (Shanghai) Corporation Ltd. Multispecific fab fusion proteins and use thereof
CN108697799A (en) 2016-03-22 2018-10-23 生态学有限公司 The application of anti-LGR5 monoclonal antibodies
US9988641B2 (en) 2016-04-05 2018-06-05 Corn Products Development, Inc. Compositions and methods for producing starch with novel functionality
CN117003840A (en) 2016-04-14 2023-11-07 先锋国际良种公司 Insecticidal polypeptides having improved activity profile and uses thereof
CN109874294B (en) 2016-04-15 2023-06-20 原生微生物股份有限公司 Method for improving agricultural production of poultry by administering a microbial consortium or purified strain thereof
EP3445861B1 (en) 2016-04-19 2021-12-08 Pioneer Hi-Bred International, Inc. Insecticidal combinations of polypeptides having improved activity spectrum and uses thereof
WO2017189724A1 (en) 2016-04-27 2017-11-02 Novartis Ag Antibodies against growth differentiation factor 15 and uses thereof
BR112018072417B1 (en) 2016-05-04 2023-03-14 E. I. Du Pont De Nemours And Company RECOMBINANT INSECTICIDAL POLYPEPTIDE, CHIMERIC POLYPEPTIDE, COMPOSITION, RECOMBINANT POLYNUCLEOTIDE, DNA CONSTRUCTS, METHODS FOR OBTAINING A TRANSGENIC PLANT, METHODS FOR INHIBITING THE GROWTH OR EXTERMINATION OF AN INSECT PEST OR PEST POPULATION, METHOD FOR OBTAINING A TRANSFORMED PROKARYOTIC CELL TRANSFORMED AND METHOD TO GENETICALLY MODIFY THE INSECTICIDAL POLYPEPTIDE
CN109715793A (en) 2016-05-05 2019-05-03 科德克希思公司 Penicillin-G acylase
TW201802121A (en) 2016-05-25 2018-01-16 諾華公司 Reversal binding agents for anti-factor XI/XIa antibodies and uses thereof
EP3469088B1 (en) 2016-06-09 2023-09-06 Codexis, Inc. Biocatalysts and methods for hydroxylation of chemical compounds
US11299723B2 (en) 2016-06-15 2022-04-12 Codexis, Inc. Engineered beta-glucosidases and glucosylation methods
JP7231411B2 (en) 2016-06-15 2023-03-01 ノバルティス アーゲー Methods of treating diseases using inhibitors of bone morphogenetic protein 6 (BMP6)
CA3026653A1 (en) 2016-06-24 2017-12-28 Pioneer Hi-Bred International, Inc. Plant regulatory elements and methods of use thereof
EP3478845A4 (en) 2016-06-30 2019-07-31 Zymergen, Inc. Methods for generating a glucose permease library and uses thereof
WO2018005655A2 (en) 2016-06-30 2018-01-04 Zymergen Inc. Methods for generating a bacterial hemoglobin library and uses thereof
RU2019102714A (en) 2016-07-01 2020-08-03 Пайонир Хай-Бред Интернэшнл, Инк. INSECTICIDE PROTEINS FROM PLANTS AND METHODS OF THEIR APPLICATION
EP3494143B1 (en) 2016-08-03 2022-10-05 Cipla USA, Inc. Plazomicin antibodies and methods of use
SG10202111589RA (en) 2016-08-26 2021-11-29 Codexis Inc Engineered imine reductases and methods for the reductive amination of ketone and amine compounds
CN110214189A (en) 2016-09-06 2019-09-06 农业生物群落股份有限公司 Killing gene and application method
EP3509616A1 (en) 2016-09-09 2019-07-17 H. Hoffnabb-La Roche Ag Selective peptide inhibitors of frizzled
MX2019002934A (en) 2016-09-16 2019-06-17 Dupont Nutrition Biosci Aps Acetolactate decarboxylase variants having improved specific activity.
EP3515943A4 (en) 2016-09-19 2020-05-06 Celgene Corporation Methods of treating vitiligo using pd-1 binding proteins
JP2019531284A (en) 2016-09-19 2019-10-31 セルジーン コーポレイション Methods of treating immune disorders using PD-1 binding proteins
EP3535285B1 (en) 2016-11-01 2022-04-06 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
US11779604B2 (en) 2016-11-03 2023-10-10 Kymab Limited Antibodies, combinations comprising antibodies, biomarkers, uses and methods
JP7155136B2 (en) 2016-11-11 2022-10-18 バイオ－ラド ラボラトリーズ，インコーポレイティド Methods of processing nucleic acid samples
MX2019005835A (en) 2016-11-23 2019-10-30 BASF Agricultural Solutions Seed US LLC Axmi669 and axmi991 toxin genes and methods for their use.
CN110088123B (en) 2016-12-14 2023-10-20 先锋国际良种公司 Insecticidal proteins and methods of use thereof
US10669537B2 (en) 2016-12-14 2020-06-02 Biological Research Centre Mutagenizing intracellular nucleic acids
WO2018112356A1 (en) 2016-12-16 2018-06-21 Two Blades Foundation Late blight resistance genes and methods of use
RU2745517C2 (en) 2016-12-20 2021-03-25 Колгейт-Палмолив Компани Oral cavity care compositions and methods for increasing their stability
BR112019012099B1 (en) 2016-12-20 2023-02-14 Colgate-Palmolive Company ORAL CARE COMPOSITION FOR TEETH WHITENING
CN110087625B (en) 2016-12-20 2022-04-26 高露洁-棕榄公司 Oral care compositions and methods for whitening teeth
KR20190095411A (en) 2016-12-22 2019-08-14 바스프 아그리컬쳐럴 솔루션즈 시드 유에스 엘엘씨 Use of CR14 for the control of nematode pests
WO2018118811A1 (en) 2016-12-22 2018-06-28 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
JP7110199B2 (en) 2016-12-23 2022-08-01 ノバルティス アーゲー Methods of treatment with anti-factor XI/XIa antibodies
CN110325550B (en) 2016-12-23 2024-03-08 诺华股份有限公司 Factor XI antibodies and methods of use
TW201825515A (en) 2017-01-04 2018-07-16 美商伊繆諾金公司 Met antibodies and immunoconjugates and uses thereof
WO2018129130A1 (en) 2017-01-05 2018-07-12 Codexis, Inc. Penicillin-g acylases
AR110757A1 (en) 2017-01-18 2019-05-02 Bayer Cropscience Lp BP005 TOXIN GEN AND PROCEDURES FOR USE
WO2018136611A1 (en) 2017-01-18 2018-07-26 Bayer Cropscience Lp Use of bp005 for the control of plant pathogens
WO2018140214A1 (en) 2017-01-24 2018-08-02 Pioneer Hi-Bred International, Inc. Nematicidal protein from pseudomonas
WO2018140859A2 (en) 2017-01-30 2018-08-02 AgBiome, Inc. Pesticidal genes and methods of use
JOP20190187A1 (en) 2017-02-03 2019-08-01 Novartis Ag Anti-ccr7 antibody drug conjugates
EP3577229A4 (en) 2017-02-03 2020-12-23 Codexis, Inc. Engineered glycosyltransferases and steviol glycoside glucosylation methods
US20190390219A1 (en) 2017-02-08 2019-12-26 Pioneer Hi-Bred International, Inc. Insecticidal combinations of plant derived insecticidal proteins and methods for their use
JP6991246B2 (en) 2017-02-08 2022-02-03 ノバルティス アーゲー FGF21 mimetic antibody and its use
SG11201906880VA (en) 2017-02-13 2019-08-27 Codexis Inc Engineered phenylalanine ammonia lyase polypeptides
WO2018185618A1 (en) 2017-04-03 2018-10-11 Novartis Ag Anti-cdh6 antibody drug conjugates and anti-gitr antibody combinations and methods of treatment
CA3058757A1 (en) 2017-04-11 2018-10-18 AgBiome, Inc. Pesticidal genes and methods of use
AU2018258274C1 (en) 2017-04-24 2021-12-02 Tesaro, Inc. Methods of manufacturing of niraparib
WO2018197520A1 (en) 2017-04-24 2018-11-01 Dupont Nutrition Biosciences Aps Methods and compositions of anti-crispr proteins for use in plants
WO2018200214A2 (en) 2017-04-27 2018-11-01 Codexis, Inc. Ketoreductase polypeptides and polynucleotides
UY37708A (en) 2017-04-28 2018-10-31 Ascus Biosciences Inc METHODS TO COMPLEMENT DIETS WITH HIGH CONSUMPTION OF GRAINS AND / OR HIGH ENERGY VALUE IN RUMINANTS, THROUGH THE ADMINISTRATION OF A SYNTHETIC BIOENSAMBLE OF MICROBIES, OR PURIFIED VINTAGES OF THE SAME
EP3635101B1 (en) 2017-05-08 2021-08-18 Codexis, Inc. Engineered ligase variants
US11555203B2 (en) 2017-05-11 2023-01-17 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2018213796A1 (en) 2017-05-19 2018-11-22 Zymergen Inc. Genomic engineering of biosynthetic pathways leading to increased nadph
JOP20190271A1 (en) 2017-05-24 2019-11-21 Novartis Ag Antibody-cytokine engrafted proteins and methods of use for immune related disorders
WO2018215937A1 (en) 2017-05-24 2018-11-29 Novartis Ag Interleukin-7 antibody cytokine engrafted proteins and methods of use in the treatment of cancer
EP3630813A1 (en) 2017-05-24 2020-04-08 Novartis AG Antibody-cytokine engrafted proteins and methods of use in the treatment of cancer
CN111107868A (en) 2017-05-24 2020-05-05 诺华股份有限公司 Antibody cytokine transplantation proteins and methods of use
EA201992824A1 (en) 2017-05-26 2020-05-06 Пайонир Хай-Бред Интернэшнл, Инк. INSECTICIDAL POLYPEPTIDES POSSESSING AN IMPROVED ACTIVITY SPECTRUM, AND WAYS OF THEIR APPLICATION
CA3064612A1 (en) 2017-06-06 2018-12-13 Zymergen Inc. A htp genomic engineering platform for improving escherichia coli
WO2018226810A1 (en) 2017-06-06 2018-12-13 Zymergen Inc. High throughput transposon mutagenesis
EP3635112A2 (en) 2017-06-06 2020-04-15 Zymergen, Inc. A htp genomic engineering platform for improving fungal strains
WO2018226893A2 (en) 2017-06-06 2018-12-13 Zymergen Inc. A high-throughput (htp) genomic engineering platform for improving saccharopolyspora spinosa
US11359183B2 (en) 2017-06-14 2022-06-14 Codexis, Inc. Engineered transaminase polypeptides for industrial biocatalysis
WO2018229715A1 (en) 2017-06-16 2018-12-20 Novartis Ag Compositions comprising anti-cd32b antibodies and methods of use thereof
CN111051506B (en) 2017-06-27 2023-11-24 科德克希思公司 Penicillin G acylase
EP3645712A4 (en) 2017-06-30 2021-07-07 Codexis, Inc. T7 rna polymerase variants
US10793841B2 (en) 2017-06-30 2020-10-06 Codexis, Inc. T7 RNA polymerase variants
WO2019016772A2 (en) 2017-07-21 2019-01-24 Novartis Ag Compositions and methods to treat cancer
WO2019023587A1 (en) 2017-07-28 2019-01-31 Two Blades Foundation Potyvirus resistance genes and methods of use
EP4036105A3 (en) 2017-08-03 2022-11-02 AgBiome, Inc. Pesticidal genes and methods of use
US11634400B2 (en) 2017-08-19 2023-04-25 University Of Rochester Micheliolide derivatives, methods for their preparation and their use as anticancer and antiinflammatory agents
JP2020536515A (en) 2017-09-25 2020-12-17 パイオニア ハイ−ブレッド インターナショナル， インコーポレイテッド Tissue-preferred promoters and how to use them
US20200165626A1 (en) 2017-10-13 2020-05-28 Pioneer Hi-Bred International, Inc. Virus-induced gene silencing technology for insect control in maize
AR113371A1 (en) 2017-10-18 2020-04-22 Ascus Biosciences Inc INCREASE IN POULTRY PRODUCTION THROUGH THE ADMINISTRATION OF A SYNTHETIC BIOASSEMBLY OF MICROBES OR PURIFIED STRAINS OF THEM
EP3700933A1 (en) 2017-10-25 2020-09-02 Novartis AG Antibodies targeting cd32b and methods of use thereof
EP3706780A4 (en) 2017-11-07 2021-12-22 Codexis, Inc. Transglutaminase variants
WO2019108619A1 (en) 2017-11-28 2019-06-06 Two Blades Foundation Methods and compositions for enhancing the disease resistance of plants
SG11202004185PA (en) 2017-12-13 2020-06-29 Codexis Inc Carboxyesterase polypeptides for amide coupling
US11535833B2 (en) 2017-12-13 2022-12-27 Glaxosmithkline Intellectual Property Development Limited Carboxyesterase biocatalysts
WO2019125651A1 (en) 2017-12-19 2019-06-27 Pioneer Hi-Bred International, Inc. Insecticidal polypeptides and uses thereof
CA3085363A1 (en) 2017-12-22 2019-06-27 AgBiome, Inc. Pesticidal genes and methods of use
RU2020122331A (en) 2018-01-10 2022-02-10 Брайтсид, Инк. METABOLISM MODULATION METHOD
EP3737226A1 (en) 2018-01-12 2020-11-18 Two Blades Foundation Stem rust resistance genes and methods of use
WO2019157522A1 (en) 2018-02-12 2019-08-15 Curators Of The University Of Missouri Small auxin upregulated (saur) gene for the improvement of plant root system architecture, waterlogging tolerance, drought resistance and yield
KR20200132870A (en) 2018-03-20 2020-11-25 지머젠 인코포레이티드 HTP platform for genetic manipulation of Chinese hamster ovary cells
MX2020009975A (en) 2018-03-28 2020-10-12 Bristol Myers Squibb Co Interleukin-2/interleukin-2 receptor alpha fusion proteins and methods of use.
SG11202008206TA (en) 2018-03-30 2020-09-29 Amgen Inc C-terminal antibody variants
BR112020020285A2 (en) 2018-04-06 2021-01-19 Braskem S.A. NADH-DEPENDENT ENZYME MUTANTS TO CONVERT ACETONE IN ISOPROPANOL
CN112218952A (en) 2018-04-20 2021-01-12 农业生物群落股份有限公司 Insecticidal genes and methods of use
CA3096516A1 (en) 2018-05-22 2019-11-28 Pioneer Hi-Bred International, Inc. Plant regulatory elements and methods of use thereof
UY38247A (en) 2018-05-30 2019-12-31 Novartis Ag ANTIBODIES AGAINST ENTPD2, COMBINATION THERAPIES AND METHODS OF USE OF ANTIBODIES AND COMBINATION THERAPIES
US11440967B2 (en) 2018-05-31 2022-09-13 Glyconex Inc. Therapeutic antibodies
PE20210320A1 (en) 2018-06-01 2021-02-16 Novartis Ag BINDING MOLECULES AGAINST BCMA AND THE USES OF THEM
MX2020013124A (en) 2018-06-05 2021-07-15 Lifeedit Therapeutics Inc Rna-guided nucleases and active fragments and variants thereof and methods of use.
US11028401B2 (en) 2018-06-06 2021-06-08 Zymergen Inc. Manipulation of genes involved in signal transduction to control fungal morphology during fermentation and production
CA3102968A1 (en) 2018-06-12 2019-12-19 Codexis, Inc. Engineered tyrosine ammonia lyase
US20210277409A1 (en) 2018-06-28 2021-09-09 Pioneer Hi-Bred International, Inc. Methods for selecting transformed plants
AU2019300836A1 (en) 2018-07-09 2021-01-07 Codexis, Inc. Engineered deoxyribose-phosphate aldolases
WO2020014050A1 (en) 2018-07-09 2020-01-16 Codexis, Inc. Engineered pantothenate kinase variant enzymes
AU2019302422B2 (en) 2018-07-09 2022-08-04 Codexis, Inc. Engineered phosphopentomutase variant enzymes
JP7461658B2 (en) 2018-07-09 2024-04-04 コデクシス， インコーポレイテッド Engineered Purine Nucleoside Phosphorylase Variant Enzymes
EP3821000A4 (en) 2018-07-09 2022-07-20 Codexis, Inc. Engineered galactose oxidase variant enzymes
WO2020014306A1 (en) 2018-07-10 2020-01-16 Immunogen, Inc. Met antibodies and immunoconjugates and uses thereof
US11198861B2 (en) 2018-07-12 2021-12-14 Codexis, Inc. Engineered phenylalanine ammonia lyase polypeptides
AU2019306165A1 (en) 2018-07-20 2021-02-25 Pierre Fabre Medicament Receptor for vista
CN112805295A (en) 2018-07-30 2021-05-14 科德克希思公司 Engineering glycosyltransferases and methods of glycosylation of steviol glycosides
US20210315984A1 (en) 2018-08-01 2021-10-14 Konstantin G. Kousoulas Compositions comprising herpes simplex virus-i for use in methods of treating and preventing cancer
BR112021003797A2 (en) 2018-08-29 2021-05-25 Pioneer Hi-Bred International, Inc. insecticidal proteins and methods for their use
UY38407A (en) 2018-10-15 2020-05-29 Novartis Ag TREM2 STABILIZING ANTIBODIES
US11060075B2 (en) 2018-10-29 2021-07-13 Codexis, Inc. Engineered DNA polymerase variants
EP3873532A1 (en) 2018-10-31 2021-09-08 Novartis AG Dc-sign antibody drug conjugates
US11066663B2 (en) 2018-10-31 2021-07-20 Zymergen Inc. Multiplexed deterministic assembly of DNA libraries
CA3111090A1 (en) 2018-10-31 2020-05-07 Pioneer Hi-Bred International, Inc. Compositions and methods for ochrobactrum-mediated plant transformation
US11473077B2 (en) 2018-12-14 2022-10-18 Codexis, Inc. Engineered tyrosine ammonia lyase
JP2022515742A (en) 2018-12-20 2022-02-22 コデクシス， インコーポレイテッド Alpha-galactosidase variant
CN113195541A (en) 2018-12-21 2021-07-30 诺华股份有限公司 Antibodies against PMEL17 and conjugates thereof
EP3883537A1 (en) 2018-12-27 2021-09-29 Colgate-Palmolive Company Oral care compositions
EP4339286A2 (en) 2018-12-27 2024-03-20 LifeEDIT Therapeutics, Inc. Polypeptides useful for gene editing and methods of use
US11053515B2 (en) 2019-03-08 2021-07-06 Zymergen Inc. Pooled genome editing in microbes
JP2022524043A (en) 2019-03-08 2022-04-27 ザイマージェン インコーポレイテッド Repeated genome editing of microorganisms
CN113412333A (en) 2019-03-11 2021-09-17 先锋国际良种公司 Method for clonal plant production
US20220170033A1 (en) 2019-03-27 2022-06-02 Pioneer Hi-Bred International, Inc. Plant explant transformation
EP3947696A1 (en) 2019-03-28 2022-02-09 Pioneer Hi-Bred International, Inc. Modified agrobacterium strains and use thereof for plant transformation
JP2022524215A (en) 2019-03-28 2022-04-28 ダニスコ・ユーエス・インク Modified antibody
AU2020279974A1 (en) 2019-05-21 2021-11-18 Novartis Ag CD19 binding molecules and uses thereof
US20230071196A1 (en) 2019-05-21 2023-03-09 Novartis Ag Variant cd58 domains and uses thereof
WO2020236795A2 (en) 2019-05-21 2020-11-26 Novartis Ag Trispecific binding molecules against bcma and uses thereof
AR122276A1 (en) 2019-06-27 2022-08-31 Two Blades Found ENGINEERED ATRLP23 PATTERN RECOGNITION RECEIVERS AND METHODS OF USE
CN114929033A (en) 2019-07-29 2022-08-19 布莱特西德公司 Methods for improving digestive health
EP4013864A1 (en) 2019-08-12 2022-06-22 Lifeedit Therapeutics, Inc. Rna-guided nucleases and active fragments and variants thereof and methods of use
TW202124446A (en) 2019-09-18 2021-07-01 瑞士商諾華公司 Combination therapies with entpd2 antibodies
JP2022548881A (en) 2019-09-18 2022-11-22 ノバルティス アーゲー ENTPD2 Antibodies, Combination Therapy and Methods of Using Antibodies and Combination Therapy
AU2020358714A1 (en) 2019-10-02 2022-05-12 Abbott Diabetes Care Inc. Detection of analytes by protein switches
AU2020383340A1 (en) 2019-11-11 2022-06-09 Brightseed, Inc Extract, consumable product and method for enriching bioactive metabolite in an extract
EP3821946A1 (en) 2019-11-12 2021-05-19 Université de Strasbourg Anti-claudin-1 monoclonal antibodies for the prevention and treatment of fibrotic diseases
CN115190912A (en) 2019-12-30 2022-10-14 生命编辑制药股份有限公司 RNA-guided nucleases, active fragments and variants thereof, and methods of use
WO2021202473A2 (en) 2020-03-30 2021-10-07 Danisco Us Inc Engineered antibodies
WO2021202463A1 (en) 2020-03-30 2021-10-07 Danisco Us Inc Anti-rsv antibodies
US11788071B2 (en) 2020-04-10 2023-10-17 Codexis, Inc. Engineered transaminase polypeptides
TW202208626A (en) 2020-04-24 2022-03-01 美商生命編輯公司 Rna-guided nucleases and active fragments and variants thereof and methods of use
US20230181756A1 (en) 2020-04-30 2023-06-15 Novartis Ag Ccr7 antibody drug conjugates for treating cancer
CN115461363A (en) 2020-05-01 2022-12-09 诺华股份有限公司 Immunoglobulin variants
US20230242647A1 (en) 2020-05-01 2023-08-03 Novartis Ag Engineered immunoglobulins
AU2021267953A1 (en) 2020-05-04 2022-11-17 Immunorizon Ltd. Precursor tri-specific antibody constructs and methods of use thereof
WO2021231437A1 (en) 2020-05-11 2021-11-18 LifeEDIT Therapeutics, Inc. Rna-guided nucleic acid binding proteins and active fragments and variants thereof and methods of use
BR112022027035A2 (en) 2020-07-14 2023-04-11 Pioneer Hi Bred Int INSECTICIDAL PROTEINS AND METHODS FOR THE USE OF THEM
CA3173949A1 (en) 2020-07-15 2022-01-20 LifeEDIT Therapeutics, Inc. Uracil stabilizing proteins and active fragments and variants thereof and methods of use
US11479779B2 (en) 2020-07-31 2022-10-25 Zymergen Inc. Systems and methods for high-throughput automated strain generation for non-sporulating fungi
WO2022035649A1 (en) 2020-08-10 2022-02-17 Pioneer Hi-Bred International, Inc. Plant regulatory elements and methods of use thereof
BR112023002602A2 (en) 2020-08-10 2023-04-04 Du Pont COMPOSITIONS AND METHODS TO INCREASE RESISTANCE TO HELMINTOSPORIOSIS IN CORN
JP2023539634A (en) 2020-08-28 2023-09-15 コデクシス， インコーポレイテッド Engineered amylase variants
JP2023541400A (en) 2020-09-11 2023-10-02 ライフエディット セラピューティクス，インコーポレイティド DNA modification enzymes, active fragments and variants thereof, and methods for using the same
CN116249780A (en) 2020-09-30 2023-06-09 先锋国际良种公司 Rapid transformation of monocot leaf explants
IL302412A (en) 2020-11-06 2023-06-01 Novartis Ag Anti-cd19 agent and b cell targeting agent combination therapy for treating b cell malignancies
WO2022097060A1 (en) 2020-11-06 2022-05-12 Novartis Ag Cd19 binding molecules and uses thereof
CA3200858A1 (en) 2020-11-24 2022-06-02 Novartis Ag Anti-cd48 antibodies, antibody drug conjugates, and uses thereof
EP4251755A2 (en) 2020-11-24 2023-10-04 AgBiome, Inc. Pesticidal genes and methods of use
EP4259772A1 (en) 2020-12-11 2023-10-18 Willow Biosciences, Inc. Recombinant acyl activating enzyme (aae) genes for enhanced biosynthesis of cannabinoids and cannabinoid precursors
US11898174B2 (en) 2020-12-18 2024-02-13 Codexis, Inc. Engineered uridine phosphorylase variant enzymes
JP2024502832A (en) 2020-12-31 2024-01-23 アラマー バイオサイエンシーズ， インコーポレイテッド Binding agent molecules with high affinity and/or specificity and methods for their production and use
AU2022242754A1 (en) 2021-03-22 2023-11-02 LifeEDIT Therapeutics, Inc. Dna modifyng enzymes and active fragments and variants thereof and methods of use
US20220325285A1 (en) 2021-04-02 2022-10-13 Codexis, Inc. ENGINEERED CYCLIC GMP-AMP SYNTHASE (cGAS) VARIANT ENZYMES
CN117222735A (en) 2021-04-02 2023-12-12 科德克希思公司 Engineered acetate kinase variant enzymes
US20220325284A1 (en) 2021-04-02 2022-10-13 Codexis, Inc. Engineered guanylate kinase variant enzymes
IL305919A (en) 2021-04-02 2023-11-01 Codexis Inc Engineered adenylate kinase variant enzymes
CA3216880A1 (en) 2021-04-16 2022-10-20 Novartis Ag Antibody drug conjugates and methods for making thereof
AR125794A1 (en) 2021-05-06 2023-08-16 Agbiome Inc PESTICIDE GENES AND METHODS OF USE
WO2022240931A1 (en) 2021-05-11 2022-11-17 Two Blades Foundation Methods for preparing a library of plant disease resistance genes for functional testing for disease resistance
WO2022261394A1 (en) 2021-06-11 2022-12-15 LifeEDIT Therapeutics, Inc. Rna polymerase iii promoters and methods of use
WO2023010083A2 (en) 2021-07-30 2023-02-02 Willow Biosciences, Inc. Recombinant prenyltransferase polypeptides engineered for enhanced biosynthesis of cannabinoids
CA3227236A1 (en) 2021-08-19 2023-02-23 Trish Choudhary Recombinant olivetolic acid cyclase polypeptides engineered for enhanced biosynthesis of cannabinoids
WO2023056269A1 (en) 2021-09-30 2023-04-06 Two Blades Foundation Plant disease resistance genes against stem rust and methods of use
WO2023069921A1 (en) 2021-10-19 2023-04-27 Epimeron Usa, Inc. Recombinant thca synthase polypeptides engineered for enhanced biosynthesis of cannabinoids
WO2023107943A1 (en) 2021-12-07 2023-06-15 AgBiome, Inc. Pesticidal genes and methods of use
US20230227545A1 (en) 2022-01-07 2023-07-20 Johnson & Johnson Enterprise Innovation Inc. Materials and methods of il-1beta binding proteins
WO2023139557A1 (en) 2022-01-24 2023-07-27 LifeEDIT Therapeutics, Inc. Rna-guided nucleases and active fragments and variants thereof and methods of use
WO2023173084A1 (en) 2022-03-11 2023-09-14 University Of Rochester Cyclopeptibodies and uses thereof
US20230357381A1 (en) 2022-04-26 2023-11-09 Novartis Ag Multispecific antibodies targeting il-13 and il-18
WO2024013727A1 (en) 2022-07-15 2024-01-18 Janssen Biotech, Inc. Material and methods for improved bioengineered pairing of antigen-binding variable regions
WO2024015953A1 (en) 2022-07-15 2024-01-18 Danisco Us Inc. Methods for producing monoclonal antibodies
EP4311430A1 (en) 2022-07-28 2024-01-31 Limagrain Europe Chlorotoluron tolerance gene and methods of use thereof
WO2024026406A2 (en) 2022-07-29 2024-02-01 Vestaron Corporation Next Generation ACTX Peptides
WO2024033901A1 (en) 2022-08-12 2024-02-15 LifeEDIT Therapeutics, Inc. Rna-guided nucleases and active fragments and variants thereof and methods of use
WO2024040020A1 (en) 2022-08-15 2024-02-22 Absci Corporation Quantitative affinity activity specific cell enrichment
WO2024044596A1 (en) 2022-08-23 2024-02-29 AgBiome, Inc. Pesticidal genes and methods of use
WO2024042489A1 (en) 2022-08-25 2024-02-29 LifeEDIT Therapeutics, Inc. Chemical modification of guide rnas with locked nucleic acid for rna guided nuclease-mediated gene editing

Family Cites Families (87)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4994379A (en) 1983-03-09 1991-02-19 Cetus Corporation Modified signal peptides
US5422266A (en) 1984-12-31 1995-06-06 University Of Georgia Research Foundation, Inc. Recombinant DNA vectors capable of expressing apoaequorin
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US5176995A (en) 1985-03-28 1993-01-05 Hoffmann-La Roche Inc. Detection of viruses by amplification and hybridization
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
EP0590689B2 (en) 1985-03-30 2006-08-16 KAUFFMAN, Stuart A. Method for obtaining DNA, RNA, peptides, polypeptides or proteins by means of a DNA-recombinant technique
EP0208491B1 (en) * 1985-07-03 1993-08-25 Genencor International, Inc. Hybrid polypeptides and process for their preparation
US4800159A (en) 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
DK311186D0 (en) * 1986-06-30 1986-06-30 Novo Industri As ENZYMES
US5824469A (en) 1986-07-17 1998-10-20 University Of Washington Method for producing novel DNA sequences with biological activity
US5763192A (en) 1986-11-20 1998-06-09 Ixsys, Incorporated Process for obtaining DNA, RNA, peptides, polypeptides, or protein, by recombinant DNA technique
US5192507A (en) 1987-06-05 1993-03-09 Arthur D. Little, Inc. Receptor-based biosensors
EP0321546A1 (en) 1987-06-09 1989-06-28 PECK, Dana P. Immunoassay for aromatic ring containing compounds
US4994368A (en) * 1987-07-23 1991-02-19 Syntex (U.S.A.) Inc. Amplification method for polynucleotide assays
DK6488D0 (en) * 1988-01-07 1988-01-07 Novo Industri As ENZYMES
US5032514A (en) * 1988-08-08 1991-07-16 Genentech, Inc. Metabolic pathway engineering to increase production of ascorbic acid intermediates
FR2641793B1 (en) * 1988-12-26 1993-10-01 Setratech METHOD OF IN VIVO RECOMBINATION OF DNA SEQUENCES HAVING BASIC MATCHING
US5043272A (en) * 1989-04-27 1991-08-27 Life Technologies, Incorporated Amplification of nucleic acid sequences using oligonucleotides of random sequence as primers
US5556750A (en) 1989-05-12 1996-09-17 Duke University Methods and kits for fractionating a population of DNA molecules based on the presence or absence of a base-pair mismatch utilizing mismatch repair systems
CA2016842A1 (en) 1989-05-16 1990-11-16 Richard A. Lerner Method for tapping the immunological repertoire
IT1231157B (en) * 1989-07-20 1991-11-19 Crc Ricerca Chim NEW FUNCTIONAL HYBRID GENES OF BACILLUS THURINGIENSIS OBTAINED THROUGH IN VIVO RECOMBINATION.
US5023171A (en) 1989-08-10 1991-06-11 Mayo Foundation For Medical Education And Research Method for gene splicing by overlap extension using the polymerase chain reaction
WO1991006570A1 (en) 1989-10-25 1991-05-16 The University Of Melbourne HYBRID Fc RECEPTOR MOLECULES
WO1991007506A1 (en) 1989-11-08 1991-05-30 The United States Of America, Represented By The Secretary, United States Department Of Commerce A method of synthesizing double-stranded dna molecules
EP0527809B1 (en) 1990-04-05 1995-08-16 CREA, Roberto Walk-through mutagenesis
AU7791991A (en) 1990-04-24 1991-11-11 Stratagene Methods for phenotype creation from multiple gene populations
US5851813A (en) 1990-07-12 1998-12-22 President And Fellows Of Harvard College Primate lentivirus antigenic compositions
JP3392863B2 (en) 1990-07-24 2003-03-31 エフ．ホフマン ― ラ ロシュ アーゲー Reduction of non-specific amplification in in vitro nucleic acid amplification using modified nucleobases
US5858725A (en) 1990-10-10 1999-01-12 Glaxo Wellcome Inc. Preparation of chimaeric antibodies using the recombinant PCR strategy
US5234824A (en) 1990-11-13 1993-08-10 Specialty Laboratories, Inc. Rapid purification of DNA
US5187083A (en) 1990-11-13 1993-02-16 Specialty Laboratories, Inc. Rapid purification of DNA
US5489523A (en) * 1990-12-03 1996-02-06 Stratagene Exonuclease-deficient thermostable Pyrococcus furiosus DNA polymerase I
DE69129154T2 (en) * 1990-12-03 1998-08-20 Genentech Inc METHOD FOR ENRICHING PROTEIN VARIANTS WITH CHANGED BINDING PROPERTIES
US5795747A (en) 1991-04-16 1998-08-18 Evotec Biosystems Gmbh Process for manufacturing new biopolymers
DE4112440C1 (en) 1991-04-16 1992-10-22 Diagen Institut Fuer Molekularbiologische Diagnostik Gmbh, 4000 Duesseldorf, De
US5512463A (en) 1991-04-26 1996-04-30 Eli Lilly And Company Enzymatic inverse polymerase chain reaction library mutagenesis
WO1993000103A1 (en) 1991-06-21 1993-01-07 The Wistar Institute Of Anatomy And Biology Chimeric envelope proteins for viral targeting
US5223408A (en) * 1991-07-11 1993-06-29 Genentech, Inc. Method for making variant secreted proteins with altered properties
GB9115364D0 (en) 1991-07-16 1991-08-28 Wellcome Found Antibody
US5279952A (en) * 1991-08-09 1994-01-18 Board Of Regents, The University Of Texas System PCR-based strategy of constructing chimeric DNA molecules
ATE181571T1 (en) 1991-09-23 1999-07-15 Medical Res Council METHODS FOR PRODUCING HUMANIZED ANTIBODIES
GB9125979D0 (en) 1991-12-06 1992-02-05 Wellcome Found Antibody
EP0625205A1 (en) 1992-01-30 1994-11-23 Genzyme Limited Chiral synthesis with modified enzymes
US5773267A (en) 1992-02-07 1998-06-30 Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University D29 shuttle phasmids and uses thereof
GB9202796D0 (en) 1992-02-11 1992-03-25 Wellcome Found Antiviral antibody
WO1993018141A1 (en) 1992-03-02 1993-09-16 Biogen, Inc. Thrombin receptor antagonists
US5316935A (en) 1992-04-06 1994-05-31 California Institute Of Technology Subtilisin variants suitable for hydrolysis and synthesis in organic media
EP0606430A1 (en) 1992-06-15 1994-07-20 City Of Hope Chimeric anti-cea antibody
CA2139876A1 (en) * 1992-07-10 1994-01-20 John Rambosek Recombinant dna encoding a desulfurization biocatalyst
US6107062A (en) 1992-07-30 2000-08-22 Inpax, Inc. Antisense viruses and antisense-ribozyme viruses
DE69232604T2 (en) 1992-11-04 2002-11-07 City Of Hope Duarte ANTIBODY CONSTRUCTS
US5360728A (en) * 1992-12-01 1994-11-01 Woods Hole Oceanographic Institution (W.H.O.I.) Modified apoaequorin having increased bioluminescent activity
DK0672142T3 (en) 1992-12-04 2001-06-18 Medical Res Council Multivalent and multi-specific binding proteins as well as their preparation and use
IT1264712B1 (en) * 1993-07-13 1996-10-04 Eniricerche Spa BIOLOGICAL SYNTHESIS METHOD OF PEPTIDES
US6150141A (en) 1993-09-10 2000-11-21 Trustees Of Boston University Intron-mediated recombinant techniques and reagents
US5498531A (en) 1993-09-10 1996-03-12 President And Fellows Of Harvard College Intron-mediated recombinant techniques and reagents
IL107268A0 (en) * 1993-10-12 1994-01-25 Yeda Res & Dev Molecules influencing the shedding of the tnf receptor, their preparation and their use
US5556772A (en) 1993-12-08 1996-09-17 Stratagene Polymerase compositions and uses thereof
DE4343591A1 (en) * 1993-12-21 1995-06-22 Evotec Biosystems Gmbh Process for the evolutionary design and synthesis of functional polymers based on shape elements and shape codes
US5834252A (en) 1995-04-18 1998-11-10 Glaxo Group Limited End-complementary polymerase reaction
US5928905A (en) 1995-04-18 1999-07-27 Glaxo Group Limited End-complementary polymerase reaction
US6335160B1 (en) 1995-02-17 2002-01-01 Maxygen, Inc. Methods and compositions for polypeptide engineering
US5837458A (en) 1994-02-17 1998-11-17 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
US5605793A (en) 1994-02-17 1997-02-25 Affymax Technologies N.V. Methods for in vitro recombination
US6117679A (en) * 1994-02-17 2000-09-12 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US5521077A (en) * 1994-04-28 1996-05-28 The Leland Stanford Junior University Method of generating multiple protein variants and populations of protein variants prepared thereby
US5514588A (en) 1994-12-13 1996-05-07 Exxon Research And Engineering Company Surfactant-nutrients for bioremediation of hydrocarbon contaminated soils and water
US5629179A (en) 1995-03-17 1997-05-13 Novagen, Inc. Method and kit for making CDNA library
HUP9801871A3 (en) 1995-04-24 1999-12-28 Chromaxome Corp San Diego Methods for generating and screening novel metabolic pathways
US5958672A (en) 1995-07-18 1999-09-28 Diversa Corporation Protein activity screening of clones having DNA from uncultivated microorganisms
WO1997007206A1 (en) * 1995-08-11 1997-02-27 Okkels, Jens, Sigurd Method for preparing polypeptide variants
US5962258A (en) 1995-08-23 1999-10-05 Diversa Corporation Carboxymethyl cellulase fromthermotoga maritima
US5756316A (en) 1995-11-02 1998-05-26 Genencor International, Inc. Molecular cloning by multimerization of plasmids
US5962283A (en) 1995-12-07 1999-10-05 Diversa Corporation Transminases and amnotransferases
US5939250A (en) 1995-12-07 1999-08-17 Diversa Corporation Production of enzymes having desired activities by mutagenesis
US5814473A (en) 1996-02-09 1998-09-29 Diversa Corporation Transaminases and aminotransferases
US5965408A (en) 1996-07-09 1999-10-12 Diversa Corporation Method of DNA reassembly by interrupting synthesis
US5830696A (en) 1996-12-05 1998-11-03 Diversa Corporation Directed evolution of thermophilic enzymes
AU1367897A (en) 1996-01-10 1997-08-01 Novo Nordisk A/S A method for in vivo production of a mutant library in cells
US5942430A (en) 1996-02-16 1999-08-24 Diversa Corporation Esterases
US5958751A (en) 1996-03-08 1999-09-28 Diversa Corporation α-galactosidase
US6096548A (en) * 1996-03-25 2000-08-01 Maxygen, Inc. Method for directing evolution of a virus
US5783431A (en) 1996-04-24 1998-07-21 Chromaxome Corporation Methods for generating and screening novel metabolic pathways
US5789228A (en) 1996-05-22 1998-08-04 Diversa Corporation Endoglucanases
US5939300A (en) 1996-07-03 1999-08-17 Diversa Corporation Catalases
US5948666A (en) 1997-08-06 1999-09-07 Diversa Corporation Isolation and identification of polymerases
US5876997A (en) 1997-08-13 1999-03-02 Diversa Corporation Phytase

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040110174A1 (en) * 2001-01-25 2004-06-10 Niel Goldsmith Concatemers of differentially expressed multiple genes
US20040241672A1 (en) * 2001-01-25 2004-12-02 Neil Goldsmith Library of a collection of cells
US7838287B2 (en) 2001-01-25 2010-11-23 Evolva Sa Library of a collection of cells
US8008459B2 (en) 2001-01-25 2011-08-30 Evolva Sa Concatemers of differentially expressed multiple genes
US20050164162A1 (en) * 2002-01-25 2005-07-28 Evolva Ltd., C/O Dr. Iur. Martin Eisenring Methods for multiple parameters screening and evolution of cells to produce small molecules with multiple functionalities
US20070048793A1 (en) * 2005-07-12 2007-03-01 Baynes Brian M Compositions and methods for biocatalytic engineering
US11728008B2 (en) * 2020-09-03 2023-08-15 Melonfrost, Inc. Machine learning and control systems and methods for learning and steering evolutionary dynamics

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