WO2000031247A2 - Recombinant methods and materials for producing epothilone and epothilone derivatives - Google Patents
Recombinant methods and materials for producing epothilone and epothilone derivatives Download PDFInfo
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- WO2000031247A2 WO2000031247A2 PCT/US1999/027438 US9927438W WO0031247A2 WO 2000031247 A2 WO2000031247 A2 WO 2000031247A2 US 9927438 W US9927438 W US 9927438W WO 0031247 A2 WO0031247 A2 WO 0031247A2
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Definitions
- the present invention provides recombinant methods and materials for producing epothilone and epothilone derivatives.
- the invention relates to the fields of agriculture, chemistry, medicinal chemistry, medicine, molecular biology, and pharmacology.
- the epothilones were first identified by Gerhard Hofle and colleagues at the National Biotechnology Research Institute as an antifungal activity extracted from the myxobacterium Sorangium cellulosum (see K. Gerth et al., 1996, J. Antibiotics 49: 560- 563 and Germany Patent No. DE 41 38 042). The epothilones were later found to have activity in a tubulin polymerization assay (see D. Bollag et al., 1995, Cancer Res.
- epothilones E and F Two other naturally occurring epothilones have been described. These are epothilones E and F, in which the methyl side chain of the thiazole moiety of epothilones A and B has been hydroxylated to yield epothilones E and F, respectively.
- the present invention provides recombinant DNA compounds that encode the proteins required to produce epothilones A, B, C, and D.
- the present invention also provides recombinant DNA compounds that encode portions of these proteins.
- the present invention also provides recombinant DNA compounds that encode a hybrid protein, which hybrid protein includes all or a portion of a protein involved in epothilone biosynthesis and all or a portion of a protein involved in the biosynthesis of another polyketide or non-ribosomal-derived peptide.
- the recombinant DNA compounds of the invention are recombinant DNA cloning vectors that facilitate manipulation of the coding sequences or recombinant DNA expression vectors that code for the expression of one or more of the proteins of the invention in recombinant host cells.
- the present invention provides recombinant host cells that produce a desired epothilone or epothilone derivative.
- the invention provides host cells that produce one or more of the epothilones or epothilone derivatives at higher levels than produced in the naturally occurring organisms that produce epothilones.
- the invention provides host cells that produce mixtures of epothilones that are less complex than the mixtures produced by naturally occurring host cells.
- the present invention provides non-Sorangium recombinant host cells that produce an epothilone or epothilone derivative.
- the host cells of the invention produce less complex mixtures of epothilones than do naturally occurring cells that produce epothilones.
- Naturally occurring cells that produce epothilones typically produce a mixture of epothilones A, B, C, D, E, and F.
- the table below summarizes the epothilones produced in different illustrative host cells of the invention.
- cell types may be constructed which produce only the newly discovered epothilones G and H, further discussed below, and one or the other of G and H or both in combination with the downstream epothilones.
- the biosynthetic pathway which relates the naturally occurring epothilones is, respectively, G ⁇ C ⁇ A ⁇ E and H ⁇ D ⁇ B ⁇ F.
- Appropriate enzymes may also convert members of each pathway to the corresponding member of the other.
- the recombinant host cells of the invention also include host cells that produce only one desired epothilone or epothilone derivative.
- the invention provides Sorangium host cells that have been modified genetically to produce epothilones either at levels greater than those observed in naturally occurring host cells or as less complex mixtures of epothilones than produced by naturally occurring host cells, or produce an epothilone derivative that is not produced in nature.
- the host cell produces the epothilones at equal to or greater than 20 mg/L.
- the recombinant host cells of the invention are host cells other than Sorangium cellulosum that have been modified genetically to produce an epothilone or an epothilone derivative.
- the host cell produces the epothilones at equal to or greater than 20 mg/L.
- the recombinant host cells are Myxococcus, Pseudomonas, or Streptomyces host cells that produce the epothilones or an epothilone derivative at equal to or greater than 20 mg/L.
- the present invention provides novel compounds useful in agriculture, veterinary practice, and medicine.
- the compounds are useful as fungicides.
- the compounds are useful in cancer chemotherapy.
- the compound is an epothilone derivative that is at least as potent against tumor cells as epothilone B or D.
- the compounds are useful as immunosuppressants.
- the compounds are useful in the manufacture of another compound.
- the compounds are formulated in a mixture or solution for administration to a human or animal.
- Figure 1 shows a restriction site map of the insert Sorangium cellulosum genomic
- DNA in four overlapping cosmid clones (designated 8A3, 1 A2, 4, and 85 and corresponding to pKOS35-70.8A3, pKOS35-70.1A2, pKOS35-70.4, and pKOS35-79.85, respectively) spanning the epothilone gene cluster.
- a functional map of the epothilone gene cluster is also shown.
- the loading domain (Loading, epoA), the non-ribosomal peptide synthase (NRPS, Module 1, epoB) module, and each module (Modules 2 through 9, epoC, epoD, epoE, and epoF) of the remaining eight modules of the epothilone synthase gene are shown, as is the location of the epoK gene that encodes a cytochrome P450-like epoxidation enzyme.
- Figure 2 shows a number of precursor compounds to N-acylcysteamine thioester derivatives that can be supplied to an epothilone PKS of the invention in which the NRPS- like module 1 or module 2 KS domain has been inactivated to produce a novel epothilone derivative. A general synthetic procedure for making such compounds is also shown.
- Figure 3 shows restriction site and function maps of plasmids pKOS35-82.1 and pKOS35-82.2.
- Figure 4 shows restriction site and function maps of plasmids pKOS35-154 and pKOS90-22.
- Figure 5 shows a schematic of a protocol for introducing the epothilone PKS and modification enzyme genes into the chromosome of a Myxococcus xanthus host cell as described in Example 3.
- Figure 6 shows restriction site and function maps of plasmids pKOS039-124 and pKOS039-124R.
- Figure 7 shows a restriction site and function map of plasmid pKOS039-126R.
- Figure 8 shows a restriction site and function map of plasmid pKOS039-141.
- Figure 9 shows a restriction site and function map of plasmid pKOS045-12.
- the present invention provides the genes and proteins that synthesize the epothilones in Sorangium cellulosum in recombinant and isolated form.
- the term recombinant refers to a compound or composition produced by human intervention, typically by specific and directed manipulation of a gene or portion thereof.
- isolated refers to a compound or composition in a preparation that is substantially free of contaminating or undesired materials or, with respect to a compound or composition found in nature, substantially free of the materials with which that compound or composition is associated in its natural state.
- the epothilones (epothilone A, B, C, D, E, and F) and compounds structurally related thereto (epothilone derivatives) are potent cytotoxic agents specific for eukaryotic cells. These compounds have application as anti-fungals, cancer chemotherapeutics, and immunosuppressants.
- the epothilones are produced at very low levels in the naturally occurring Sorangium cellulosum cells in which they have been identified. Moreover, S. cellulosum is very slow growing, and fermentation of S. cellulosum strains is difficult and time-consuming.
- One important benefit conferred by the present invention is the ability simply to produce an epothilone or epothilone derivative in a non-S.
- Another advantage of the present invention is the ability to produce the epothilones at higher levels and in greater amounts in the recombinant host cells provided by the invention than possible in the naturally occurring epothilone producer cells. Yet another advantage is the ability to produce an epothilone derivative in a recombinant host cell.
- the isolation of recombinant DNA encoding the epothilone biosynthetic genes resulted from the probing of a genomic library of Sorangium cellulosum SMP44 DNA.
- the library was prepared by partially digesting S. cellulosum genomic DNA with restriction enzyme SauIIIAl and inserting the DNA fragments generated into BamHI-digested SupercosTM cosmid DNA (Stratagene). Cosmid clones containing epothilone gene sequences were identified by probing with DNA probes specific for sequences from PKS genes and reprobing with secondary probes comprising nucleotide sequences identified with the primary probes.
- cosmids Four overlapping cosmid clones were identified by this effort. These four cosmids were deposited with the American Type Culture Collection (ATCC), Manassas, VA, USA, under the terms of the Budapest Treaty, and assigned ATCC accession numbers. The clones (and accession numbers) were designated as cosmids pKOS35-70.1 A2 (ATCC 203782), pKOS35-70.4 (ATCC 203781), pKOS35-70.8A3 (ATCC 203783), and pKOS35- 79.85 (ATCC 203780). The cosmids contain insert DNA that completely spans the epothilone gene cluster. A restriction site map of these cosmids is shown in Figure 1.
- Figure 1 also provides a function map of the epothilone gene cluster, showing the location of the six epothilone PKS genes and the epoKV 50 epoxidase gene.
- the epothilone PKS genes like other PKS genes, are composed of coding sequences organized to encode a loading domain, a number of modules, and a thioesterase domain. As described more fully below, each of these domains and modules corresponds to a polypeptide with one or more specific functions.
- the loading domain is responsible for binding the first building block used to synthesize the polyketide and transferring it to the first module.
- the building blocks used to form complex polyketides are typically acylthioesters, most commonly acetyl, propionyl, malonyl, methylmalonyl, and ethylmalonyl CoA. Other building blocks include amino acid-like acylthioesters.
- PKSs catalyze the biosynthesis of polyketides through repeated, decarboxylative Claisen condensations between the acylthioester building blocks.
- Each module is responsible for binding a building block, performing one or more functions on that building block, and transferring the resulting compound to the next module.
- the next module is responsible for attaching the next building block and transferring the growing compound to the next module until synthesis is complete.
- an enzymatic thioesterase (TE) activity cleaves the polyketide from the PKS.
- Such modular organization is characteristic of the class of PKS enzymes that synthesize complex polyketides and is well known in the art.
- Recombinant methods for manipulating modular PKS genes are described in U.S. Patent Nos. 5,672,491 ; 5,712,146; 5,830,750; and 5,843,718; and in PCT patent publication Nos. 98/49315 and 97/02358, each of which is inco ⁇ orated herein by reference.
- the polyketide known as 6- deoxyerythronolide B (6-dEB) is synthesized by a PKS that is a prototypical modular PKS enzyme.
- the loading domain of the DEBS PKS consists of an acyltransferase (AT) and an acyl carrier protein (ACP).
- AT acyltransferase
- ACP acyl carrier protein
- the AT of the DEBS loading domain recognizes propionyl CoA (other loading domain ATs can recognize other acyl-CoAs, such as acetyl, malonyl, methylmalonyl, or butyryl CoA) and transfers it as a thioester to the ACP of the loading domain.
- the AT on each of the six extender modules recognizes a methylmalonyl CoA (other extender module ATs can recognize other CoAs, such as malonyl or alpha-substituted malonyl CoAs, i.e., malonyl, ethylmalonyl, and 2- hydroxymalonyl CoA) and transfers it to the ACP of that module to form a thioester.
- other extender module ATs can recognize other CoAs, such as malonyl or alpha-substituted malonyl CoAs, i.e., malonyl, ethylmalonyl, and 2- hydroxymalonyl CoA
- the acyl group of the loading domain migrates to form a thioester (trans-esterification) at the KS of the first module; at this stage, module one possesses an acyl-KS adjacent to a methylmalonyl ACP.
- the acyl group derived from the DEBS loading domain is then covalently attached to the alpha- carbon of the extender group to form a carbon-carbon bond, driven by concomitant decarboxylation, and generating a new acyl-ACP that has a backbone two carbons longer than the loading unit (elongation or extension).
- the growing polyketide chain is transferred from the ACP to the KS of the next module of DEBS, and the process continues.
- the polyketide chain, growing by two carbons for each module of DEBS is sequentially passed as a covalently bound thioester from module to module, in an assembly line-like process.
- the carbon chain produced by this process alone would possess a ketone at every other carbon atom, producing a polyketone, from which the name polyketide arises.
- additional enzymatic activities modify the beta keto group of each two carbon unit just after it has been added to the growing polyketide chain but before it is transferred to the next module.
- modules may contain a ketoreductase (KR) that reduces the keto group to an alcohol.
- Modules may also contain a KR plus a dehydratase (DH) that dehydrates the alcohol to a double bond.
- Modules may also contain a KR, a DH, and an enoylreductase (ER) that converts the double bond to a saturated single bond using the beta carbon as a methylene function.
- the DEBS modules include those with only a KR domain, only an inactive KR domain, and with all three KR, DH, and ER domains.
- a polyketide chain traverses the final module of a PKS, it encounters the releasing domain or thioesterase found at the carboxyl end of most PKSs.
- the polyketide is cleaved from the enzyme and, for most but not all polyketides, cyclized.
- the polyketide can be modified further by tailoring or modification enzymes; these enzymes add carbohydrate groups or methyl groups, or make other modifications, i.e., oxidation or reduction, on the polyketide core molecule.
- 6-dEB is hydroxylated, methylated, and glycosylated (glycosidated) to yield the well known antibiotic erythromycin A in the Saccharopolyspora erythraea cells in which it is produced naturally.
- PKS enzymes comprise loading domains that, unlike the loading domain of DEBS, comprise an "inactive" KS domain that functions as a decarboxylase.
- KS This inactive KS is in most instances called KS , where the superscript is the single-letter abbreviation for the amino acid (glutamine) that is present instead of the active site cysteine required for ketosynthase activity.
- the epothilone PKS loading domain contains a KS Y domain not present in other PKS enzymes for which amino acid sequence is currently available in which the amino acid tyrosine has replaced the cysteine.
- the present invention provides recombinant DNA coding sequences for this novel KS domain.
- Another important variation in PKS enzymes relates to the type of building block inco ⁇ orated. Some polyketides, including epothilone, inco ⁇ orate an amino acid derived building block.
- NRPS non-ribosomal peptide synthetase
- the epothilone PKS contains an NRPS module.
- Another example of a variation relates to additional activities in a module.
- one module of the epothilone PKS contains a methyltransferase (MT) domain, a heretofore unknown domain of PKS enzymes that make modular polyketides.
- MT methyltransferase
- ORFs open reading frames
- a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity and, in some instances, even an improvement of a desired activity.
- the present invention includes such polypeptides with alternate amino acid sequences, and the amino acid sequences shown merely illustrate preferred embodiments of the invention.
- T7he present invention provides recombinant genes for the production of epothilones.
- the invention is exemplified by the cloning, characterization, and manipulation of the epothilone PKS and modification enzymes of Sorangium cellulosum SMP44.
- the description of the invention and the recombinant vectors deposited in connection with that description enable the identification, cloning, and manipulation of epothilone PKS and modification enzymes from any naturally occurring host cell that produces an epothilone.
- host cells include other S. cellulosum strains, such as So ce 90, other Sorangium species, and non-Sorangium cells.
- Such identification, cloning, and characterization can be conducted by those of ordinary skill in accordance with the present invention using standard methodology for identifying homologous DNA sequences and for identifying genes that encode a protein of function similar to a known protein.
- the present invention provides recombinant epothilone PKS and modification enzyme genes that are synthesized de novo or are assembled from non-epothilone PKS genes to provide an ordered array of domains and modules in one or more proteins that assemble to form a PKS that produces epothilone or an epothilone derivative.
- the recombinant nucleic acids, proteins, and peptides of the invention are many and diverse.
- the following discussion describes various regions of the epothilone PKS and corresponding coding sequences. This discussion begins with a general discussion of the genes that encode the PKS, the location of the various domains and modules in those genes, and the location of the various domains in those modules. Then, a more detailed discussion follows, focusing first on the loading domain, followed by the NRPS module, and then the remaining eight modules of the epothilone PKS.
- the epoA gene encodes the 149 kDa loading domain (which can also be referred to as a loading module).
- the epoB gene encodes module 1, the 158 kDa NRPS module.
- the epoC gene encodes the 193 kDa module 2.
- the epoD gene encodes a 765 kDa protein that comprises modules 3 through 6, inclusive.
- the epoE gene encodes a 405 kDa protein that comprises modules 7 and 8.
- the epoF gene encodes a 257 kDa protein that comprises module 9 and the thioesterase domain.
- the P450 epoxidase gene which encodes a 47 kDa protein, followed immediately by the epoL gene, which may encode a 24 kDa dehydratase.
- the epoL gene is followed by a number of ORFs that include genes believed to encode proteins involved in transport and regulation.
- Example 1 The sequences of these genes are shown in Example 1 in one contiguous sequence or contig of 71,989 nucleotides.
- This contig also contains two genes that appear to originate from a transposon and are identified below as ORF A and ORF B. These two genes are believed not to be involved in epothilone biosynthesis but could possibly contain sequences that function as a promoter or enhancer.
- the contig also contains more than 12 additional ORFs, only 12 of which, designated ORF2 through ORF 12 and ORF2 complement, are identified below.
- ORF2 actually is two ORFs, because the complement of the strand shown also comprises an ORF. The function of the corresponding gene product, if any, of these ORFs has not yet been established.
- the Table below provides the location of various open reading frames, module-coding sequences, and domain encoding sequences within the contig sequence shown in Example 1. Those of skill in the art will recognize, upon consideration of the sequence shown in Example 1, that the actual start locations of several of the genes could differ from the start locations shown in the table, because of the presence in frame codons for methionine or valine in close proximity to the codon indicated as the start codon. The actual start codon can be confirmed by amino acid sequencing of the proteins expressed from the genes.
- 6260 10493 epoB gene encodes module 1 , the NRPS module
- ORF 8 (putative oxidoreductase membrane subunit)
- the epothilone PKS is multiprotein complex composed of the gene products of the epoA, epoB, epoC, epoD, epoE, and epoF genes.
- epoA, epoB, epoC, epoD, epoE, and epoF genes are provided by the present invention.
- epothilone and other PKS enzymes may be referred to as a single entity herein, these enzymes are typically multisubunit proteins.
- PKS a PKS that differs from a naturally occurring PKS by deletion or mutation
- hybrid PKS a PKS that is composed of portions of two different PKS enzymes
- the post-PKS modification or tailoring of epothilone includes multiple steps mediated by multiple enzymes. These enzymes are referred to herein as tailoring or modification enzymes.
- Epothilones G and H lack the C-12-C-13 ⁇ -bond of epothilones C and D and the C-12-C-13 epoxide of epothilones A and B, having instead a hydrogen and hydroxyl group at C- 13, a single bond between C-12 and C-13, and a hydrogen and H or methyl group at C-12.
- the dehydration reaction that mediates the formation of this double bond may be due to the action of an as yet unrecognized domain of the epothilone PKS (for example, dehydration could occur in the next module, which possesses an active DH domain and could generate a conjugated diene precursor prior to its dehydrogenation by an ER domain) or an endogenous enzyme in the heterologous host cells (Streptomyces coelicolor) in which it was observed.
- epothilones G and H may be produced in Sorangium cellulosum or other host cells and, to be converted to epothilones C and D, by the action of a dehydratase, which may be encoded by the epoL gene.
- epothilones C and D are converted to epothilones A and B by an epoxidase encoded by the epoK gene.
- Epothilones A and B are converted to epothilones E and F by a hydroxylase gene, which may be encoded by one of the ORFs identified above or by another gene endogenous to Sorangium cellulosum.
- a hydroxylase gene which may be encoded by one of the ORFs identified above or by another gene endogenous to Sorangium cellulosum.
- the present invention provides a wide variety of recombinant DNA compounds and host cells for expressing the naturally occurring epothilones A, B, C, and D and derivatives thereof.
- the invention also provides recombinant host cells, particularly Sorangium cellulosum host cells that produce epothilone derivatives modified in a manner similar to epothilones E and F.
- the invention provides host cells that can produce the heretofore unknown epothilones G and H, either by expression of the epothilone PKS genes in host cells that do not express the dehydratase that converts epothilones G and H to C and D or by mutating or altering the PKS to abolish the dehydratase function, if it is present in the epothilone PKS.
- the macrolide compounds that are products of the PKS cluster can thus be modified in various ways.
- the PKS products can be glycosylated, hydroxylated, dehydroxylated, oxidized, methylated and demethylated using appropriate enzymes.
- additional compounds within the scope of the invention can be produced by additional enzyme-catalyzed activity either provided by a host cell in which the polyketide synthases are produced or by modifying these cells to contain additional enzymes or by additional in vitro modification using purified enzymes or crude extracts or, indeed, by chemical modification.
- the present invention also provides a wide variety of recombinant DNA compounds and host cells that make epothilone derivatives. As used herein, the phrase
- epothilone derivative refers to a compound that is produced by a recombinant epothilone PKS in which at least one domain has been either rendered inactive, mutated to alter its catalytic function, or replaced by a domain with a different function or in which a domain has been inserted.
- the "epothilone derivative PKS” functions to produce a compound that differs in structure from a naturally occurring epothilone but retains its ring backbone structure and so is called an “epothilone derivative.”
- an “epothilone derivative” a compound that differs in structure from a naturally occurring epothilone but retains its ring backbone structure and so is called an “epothilone derivative.”
- the loading domain of the epothilone PKS includes an inactive KS domain, KS Y , an AT domain specific for malonyl CoA (which is believed to be decarboxylated by the KS Y domain to yield an acetyl group), and an ACP domain.
- the present invention provides recombinant DNA compounds that encode the epothilone loading domain.
- the loading domain coding sequence is contained within an -8.3 kb EcoRI restriction fragment of cosmid pKOS35-70.8 A3.
- the KS domain is referred to as inactive, because the active site region "T AYS SSL" of the KS domain of the loading domain has a Y residue in place of the cysteine required for ketosynthase activity; this domain does have decarboxylase activity. See Witkowski et al, 1 Sep. 1999, Biochem. 38(36): 11643- 11650, inco ⁇ orated herein by reference.
- the presence of the Y residue in place of a Q residue may make the KS domain less efficient at decarboxylation.
- the present invention provides a recombinant epothilone PKS loading domain and corresponding DNA sequences that encode an epothilone PKS loading domain in which the Y residue has been changed to a Q residue by changing the codon therefor in the coding sequence of the loading domain.
- the present invention also provides recombinant PKS enzymes comprising such loading domains and host cells for producing such enzymes and the polyketides produced thereby.
- recombinant loading domains include those in which just the Y residue has been changed, those in which amino acids surrounding and including the Y domain have been changed, and those in which the complete KS Y domain has been replaced by a complete KS Q domain.
- the latter embodiment includes but is not limited to a recombinant epothilone loading domain in which the KS Y domain has been replaced by the KS Q domain of the oleandolide PKS or the narbonolide PKS (see the references cited below in connection with the oleandomycin, narbomycin, and picromycin PKS and modification enzymes).
- the epothilone loading domain also contains an AT domain believed to bind malonyl CoA.
- the sequence "QTAFTQPALFTFEYALAALW. . . GHSIG" in the AT domain is consistent with malonyl CoA specificity.
- the malonyl CoA is believed to be decarboxylated by the KS Y domain to yield acetyl CoA.
- the present invention provides recombinant epothilone derivative loading domains or their encoding DNA sequences in which the malonyl specific AT domain or its encoding sequence has been changed to another specificity, such as methylmalonyl CoA, ethylmalonyl CoA, and 2-hydroxymalonyl CoA.
- the present invention provides recombinant PKS enzymes comprising such loading domains and host cells for producing such enzymes and the polyketides produced thereby.
- an AT domain that is specific for 2- hydroxymalonyl CoA will result in a polyketide with a hydroxyl group at the corresponding location in the polyketide produced, and that the hydroxyl group can be methylated to yield a methoxy group by polyketide modification enzymes. See, e.g., the patent applications cited in connection with the FK-520 PKS in the table below. Consequently, reference to a PKS that has a 2-hydroxymalonyl specific AT domain herein similarly refers to polyketides produced by that PKS that have either a hydroxyl or methoxyl group at the corresponding location in the polyketide.
- the loading domain of the epothilone PKS also comprises an ER domain. While, this ER domain may be involved in forming one of the double bonds in the thiazole moiety in epothilone (in the reverse of its normal reaction), or it may be non-functional. In either event, the invention provides recombinant DNA compounds that encode the epothilone PKS loading domain with and without the ER region, as well as hybrid loading domains that contain an ER domain from another PKS (either active or inactive, with or without accompanying KR and DH domains) in place of the ER domain of the epothilone loading domain. The present invention also provides recombinant PKS enzymes comprising such loading domains and host cells for producing such enzymes and the polyketides produced thereby.
- a DNA compound comprising a sequence that encodes the epothilone loading domain is coexpressed with the proteins of a heterologous PKS.
- a heterologous modular PKS or to the coding sequence therefor refers to all or part of a PKS, including each of the multiple proteins constituting the PKS, that synthesizes a polyketide other than an epothilone or epothilone derivative (or to the coding sequences therefor). This coexpression can be in one of two forms.
- the epothilone loading domain can be coexpressed as a discrete protein with the other proteins of the heterologous PKS or as a fusion protein in which the loading domain is fused to one or more modules of the heterologous PKS. In either event, the hybrid PKS formed, in which the loading domain of the heterologous PKS is replaced by the epothilone loading domain, provides a novel PKS.
- heterologous PKS examples include but are not limited to DEBS and the picromycin (narbonolide), oleandolide, rapamycin, FK-506, FK- 520, rifamycin, and avermectin PKS enzymes and their corresponding coding sequences.
- a nucleic acid compound comprising a sequence that encodes the epothilone loading domain is coexpressed with the proteins that constitute the remainder of the epothilone PKS (i.e., the epoB, epoC, epoD, epoE, and epoF gene products) or a recombinant epothilone PKS that produces an epothilone derivative due to an alteration or mutation in one or more of the epoB, epoC, epoD, epoE, and epoF genes.
- the proteins that constitute the remainder of the epothilone PKS i.e., the epoB, epoC, epoD, epoE, and epoF gene products
- a recombinant epothilone PKS that produces an epothilone derivative due to an alteration or mutation in one or more of the
- reference to an epothilone or a PKS that produces an epothilone derivative refers to all or any one of the proteins that comprise the PKS (or to the coding sequences therefor).
- the invention provides recombinant nucleic acid compounds that encode a loading domain composed of part of the epothilone loading domain and part of a heterologous PKS.
- the invention provides, for example, either replacing the malonyl CoA specific AT with a methylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT.
- This replacement like the others described herein, is typically mediated by replacing the coding sequences therefor to provide a recombinant DNA compound of the invention; the recombinant DNA is used to prepare the corresponding protein.
- Such changes may be referred to herein either at the DNA or protein level.
- the compounds of the invention also include those in which both the KS Y and AT domains of the epothilone loading domain have been replaced but the ACP and/or linker regions of the epothilone loading domain are left intact.
- Linker regions are those segments of amino acids between domains in the loading domain and modules of a PKS that help form the tertiary structure of the protein and are involved in correct alignment and positioning of the domains of a PKS.
- These compounds include, for example, a recombinant loading domain coding sequence in which the KS Y and AT domain coding sequences of the epothilone PKS have been replaced by the coding sequences for the KS Q and AT domains of, for example, the oleandolide PKS or the narbonolide PKS.
- PKS enzymes that do not employ a KS Q domain but instead merely utilize an AT domain that binds acetyl CoA, propionyl CoA, or butyryl CoA (the DEBS loading domain) or isobutyryl CoA (the avermectin loading domain).
- the compounds of the invention also include, for example, a recombinant loading domain coding sequence in which the KS Y and AT domain coding sequences of the epothilone PKS have been replaced by an AT domain of the DEBS or avermectin PKS.
- the present invention also provides recombinant DNA compounds encoding loading domains in which the ACP domain or any of the linker regions of the epothilone loading domain has been replaced by another ACP or linker region. Any of the above loading domain coding sequences is coexpressed with the other proteins that constitute a PKS that synthesizes epothilone, an epothilone derivative, or another polyketide to provide a PKS of the invention.
- the loading domain coding sequence is typically expressed as a discrete protein, as is the loading domain in the naturally occurring epothilone PKS. If the product desired is produced by the loading domain of the invention and proteins from one or more non-epothilone PKS enzymes, then the loading domain is expressed either as a discrete protein or as a fusion protein with one or more modules of the heterologous PKS.
- the present invention also provides hybrid PKS enzymes in which the epothilone loading domain has been replaced in its entirety by a loading domain from a heterologous PKS with the remainder of the PKS proteins provided by modified or unmodified epothilone PKS proteins.
- the present invention also provides recombinant expression vectors and host cells for producing such enzymes and the polyketides produced thereby.
- the heterologous loading domain is expressed as a discrete protein in a host cell that expresses the epoB, epoC, epoD, epoE, and epoF gene products.
- the heterologous loading domain is expressed as a fusion protein with the epoB gene product in a host cell that expresses the epoC, epoD, epoE, and epoF gene products.
- the present invention provides recombinant epothilone PKS enzymes in which the loading domain has been deleted and replaced by an NRPS module and corresponding recombinant DNA compounds and expression vectors.
- the recombinant PKS enzymes thus produce an epothilone derivative that comprises a dipeptide moiety, as in the compound leinamycin.
- the invention provides such enzymes in which the remainder of the epothilone PKS is identical in function to the native epothilone PKS as well as those in which the remainder is a recombinant PKS that produces an epothilone derivative of the invention.
- the present invention also provides reagents and methods useful in deleting the loading domain coding sequence or any portion thereof from the chromosome of a host cell, such as Sorangium cellulosum, or replacing those sequences or any portion thereof with sequences encoding a recombinant loading domain.
- a recombinant vector that comprises DNA complementary to the DNA including and/or flanking the loading domain coding sequence in the Sorangium chromosome, one can employ the vector and WO 00/31247 _ 2i _ PCT/XJS99/27438
- pu ⁇ oses One pu ⁇ ose is to construct a host cell that does not make a naturally occurring epothilone or epothilone derivative.
- a host cell that has been modified to not produce a naturally occurring epothilone may be particularly preferred for making epothilone derivatives or other polyketides free of any naturally occurring epothilone.
- Another purpose is to replace the deleted gene with a gene that has been altered so as to provide a different product or to produce more of one product than another.
- the resulting host cell will produce a non-functional epothilone PKS.
- This PKS could still bind and process extender units, but the thiazole moiety of epothilone would not form, leading to the production of a novel epothilone derivative. Because this derivative would predictably contain a free amino group, it would be produced at most in low quantities.
- provision of a heterologous or other recombinant loading domain to the host cell would result in the production of an epothilone derivative with a structure determined by the loading domain provided.
- the loading domain of the epothilone PKS is followed by the first module of the PKS, which is an NRPS module specific for cysteine.
- This NRPS module is naturally expressed as a discrete protein, the product of the epoB gene.
- the present invention provides the epoB gene in recombinant form.
- the recombinant nucleic acid compounds of the invention that encode the NRPS module of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications.
- a nucleic acid compound comprising a sequence that encodes the epothilone NRPS module is coexpressed with genes encoding one or more proteins of a heterologous PKS.
- the NRPS module can be expressed as a discrete protein or as a fusion protein with one of the proteins of the heterologous PKS.
- the resulting PKS in which at least a module of the heterologous PKS is replaced by the epothilone NRPS module or the NRPS module is in effect added as a module to the heterologous PKS, provides a novel PKS.
- a DNA compound comprising a sequence that encodes the epothilone NRPS module is coexpressed with the other epothilone PKS proteins or modified versions thereof to provide a recombinant epothilone PKS that produces an epothilone or an epothilone derivative.
- hybrid PKS enzymes provided by the invention illustrate this aspect. Both hybrid PKS enzymes are hybrids of DEBS and the epothilone NRPS module.
- the first hybrid PKS is composed of four proteins: (i) DEBSl; (ii) a fusion protein composed of the KS domain of module 3 of DEBS and all but the KS domain of the loading domain of the epothilone PKS; (iii) the epothilone NRPS module; and (iv) a fusion protein composed of the KS domain of module 2 of the epothilone PKS fused to the AT domain of module 5 of DEBS and the rest of DEBS3.
- This hybrid PKS produces a novel polyketide with a thiazole moiety inco ⁇ orated into the macrolactone ring and a molecular weight of 413.53 when expressed in Streptomyces coelicolor. Glycosylated, hydroxylated, and methylated derivatives can be produced by expression of the hybrid PKS in Saccharopolyspora erythraea.
- the structure of the product is:
- the second hybrid PKS illustrating this aspect of the invention is composed of five proteins: (i) DEBSl; (ii) a fusion protein composed of the KS domain of module 3 of DEBS and all but the KS domain of the loading domain of the epothilone PKS; (iii) the epothilone NRPS module; and (iv) a fusion protein composed of the KS domain of module 2 of the epothilone PKS fused to the AT domain of module 4 of DEBS and the rest of DEBS2; and (v) DEBS3.
- This hybrid PKS produces a novel polyketide with a thiazole moiety inco ⁇ orated into the macrolactone ring and a molecular weight of 455.61 when expressed in Streptomyces coelicolor. Glycosylated, hydroxylated, and methylated derivatives can be produced by expression of the hybrid PKS in Saccharopolyspora erythraea.
- the structure of the product is:
- a portion of the NRPS module coding sequence is utilized in conjunction with a heterologous coding sequence.
- the invention provides, for example, changing the specificity of the NRPS module of the epothilone PKS from a cysteine to another amino acid. This change is accomplished by constructing a coding sequence in which all or a portion of the epothilone PKS NRPS module coding sequences have been replaced by those coding for an NRPS module of a different specificity.
- the specificity of the epothilone NRPS module is changed from cysteine to serine or threonine.
- the recombinant PKS produces an epothilone derivative in which the thiazole moiety of epothilone (or an epothilone derivative) is changed to an oxazole or 5-methyloxazole moiety, respectively.
- the present invention provides recombinant PKS enzymes composed of the products of the epo A, epoC, epoD, epoE, and epoF genes (or modified versions thereof) without an NRPS module or with an NRPS module from a heterologous PKS.
- the heterologous NRPS module can be expressed as a discrete protein or as a fusion protein with either the epoA or epoC genes.
- the invention also provides methods and reagents useful in changing the specificity of a heterologous NRPS module from another amino acid to cysteine. This change is accomplished by constructing a coding sequence in which the sequences that determine the specificity of the heterologous NRPS module have been replaced by those that specify cysteine from the epothilone NRPS module coding sequence.
- the resulting heterologous NRPS module is typically coexpressed in conjunction with the proteins constituting a heterologous PKS that synthesizes a polyketide other than epothilone or an epothilone derivative, although the heterologous NRPS module can also be used to produce epothilone or an epothilone derivative.
- the invention provides recombinant epothilone PKS enzymes and corresponding recombinant nucleic acid compounds and vectors in which the NRPS module has been inactivated or deleted. Such enzymes, compounds, and vectors are constructed generally in accordance with the teaching for deleting or inactivating the epothilone PKS or modification enzyme genes above.
- Inactive NRPS module proteins and the coding sequences therefore provided by the invention include those in which the peptidyl carrier protein (PCP) domain has been wholly or partially deleted or otherwise rendered inactive by changing the active site serine (the site for phosphopantetheinylation) to another amino acid, such as alanine, or the adenylation domains have been deleted or otherwise rendered inactive. In one embodiment, both the loading domain and the NRPS have been deleted or rendered inactive. In any event, the resulting epothilone PKS can then function only if provided a substrate that binds to the KS domain of module 2 (or a subsequent module) of the epothilone PKS or a PKS for an epothilone derivative. In a method provided by the invention, the thus modified cells are then fed activated acylthioesters that are bound by preferably the second, but potentially any subsequent, module and processed into novel epothilone derivatives.
- PCP peptidyl carrier protein
- the invention provides Sorangium and non-Sorangium host cells that express an epothilone PKS (or a PKS that produces an epothilone derivative) with an inactive NRPS.
- the host cell is fed activated acylthioesters to produce novel epothilone derivatives of the invention.
- the host cells expressing, or cell free extracts containing, the PKS can be fed or supplied with N-acylcysteamine thioesters (NACS) of novel precursor molecules to prepare epothilone derivatives.
- NACS N-acylcysteamine thioesters
- the second (first non-NRPS) module of the epothilone PKS includes a KS, an AT specific for methylmalonyl CoA, a DH, a KR, and an ACP. This module is encoded by a sequence within an -13.1 kb EcoRI-Nsil restriction fragment of cosmid pKOS35-70.8A3.
- the recombinant nucleic acid compounds of the invention that encode the second module of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications.
- the second module of the epothilone PKS is produced as a discrete protein by the epoC gene.
- the present invention provides the epoC gene in recombinant form.
- a DNA compound comprising a sequence that encodes the epothilone second module is coexpressed with the proteins constituting a heterologous PKS either as a discrete protein or as a fusion protein with one or more modules of the heterologous PKS.
- the resulting PKS in which a module of the heterologous PKS is either replaced by the second module of the epothilone PKS or the latter is merely added to the modules of the heterologous PKS, provides a novel PKS.
- a DNA compound comprising a sequence that encodes the second module of the epothilone PKS is coexpressed with the other proteins constituting the epothilone PKS or a recombinant epothilone PKS that produces an epothilone derivative.
- the invention provides, for example, either replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2- hydroxymalonyl CoA specific AT; deleting either the DH or KR or both; replacing the DH or KR or both with a DH or KR or both that specify a different stereochemistry; and/or inserting an ER.
- any reference herein to inserting or replacing a PKS KR, DH, and/or ER domain includes the replacement of the associated KR, DH, or ER domains in that module, typically with corresponding domains from the module from which the inserted or replacing domain is obtained.
- the KS and/or ACP can be replaced with another KS and/or ACP.
- the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a gene for a PKS that produces a polyketide other than epothilone, or from chemical synthesis.
- the resulting heterologous second module coding sequence can be coexpressed with the other proteins that constitute a PKS that synthesizes epothilone, an epothilone derivative, or another polyketide.
- Illustrative recombinant PKS genes of the invention include those in which the AT domain encoding sequences for the second module of the epothilone PKS have been altered or replaced to change the AT domain encoded thereby from a methylmalonyl specific AT to a malonyl specific AT.
- Such malonyl specific AT domain encoding nucleic acids can be isolated, for example and without limitation, from the PKS genes encoding the narbonolide PKS, the rapamycin PKS (i.e., modules 2 and 12), and the FK-520 PKS (i.e., modules 3, 7, and 8).
- the resulting epothilone derivative produced is a 16-desmethyl epothilone derivative.
- the invention provides DNA compounds and vectors encoding recombinant epothilone PKS enzymes and the corresponding recombinant proteins in which the KS domain of the second (or subsequent) module has been inactivated or deleted. In a preferred embodiment, this inactivation is accomplished by changing the codon for the active site cysteine to an alanine codon.
- the resulting recombinant epothilone PKS enzymes are unable to produce an epothilone or epothilone derivative unless supplied a precursor that can be bound and extended by the remaining domains and modules of the recombinant PKS enzyme.
- Illustrative diketides are described in Example 6, below.
- the third module of the epothilone PKS includes a KS, an AT specific for malonyl CoA, a KR, and an ACP. This module is encoded by a sequence within an -8 kb Bglll- Nsil restriction fragment of cosmid pKOS35-70.8A3.
- the recombinant DNA compounds of the invention that encode the third module of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications.
- the third module of the epothilone PKS is expressed in a protein, the product of the epoD gene, which also contains modules 4, 5, and 6.
- the present invention provides the epoD gene in recombinant form.
- the present invention also provides recombinant DNA compounds that encode each of the epothilone PKS modules 3, 4, 5, and 6, as discrete coding sequences without coding sequences for the other epothilone modules.
- a DNA compound comprising a sequence that encodes the epothilone third module is coexpressed with proteins constituting a heterologous PKS.
- the third module of the epothilone PKS can be expressed either as a discrete protein or as a fusion protein fused to one or more modules of the heterologous PKS.
- the resulting PKS in which a module of the heterologous PKS is either replaced by that for the third module of the epothilone PKS or the latter is merely added to the modules of the heterologous PKS, provides a novel PKS.
- a DNA compound comprising a sequence that encodes the third module of the epothilone PKS is coexpressed with proteins comprising the remainder of the epothilone PKS or a recombinant epothilone PKS that produces an epothilone derivative, typically as a protein comprising not only the third but also the fourth, fifth, and sixth modules.
- all or a portion of the third module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
- the invention provides, for example, either replacing the malonyl CoA specific AT with a methylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting the KR; replacing the KR with a KR that specifies a different stereochemistry; and/or inserting a DH or a DH and an ER.
- the reference to inserting a DH or a DH and an ER includes the replacement of the KR with a DH and KR or an ER, DH, and KR.
- the KS and/or ACP can be replaced with another KS and/or ACP.
- the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a coding sequence for a PKS that produces a polyketide other than epothilone, or from chemical synthesis.
- the resulting heterologous third module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes epothilone, an epothilone derivative, or another polyketide.
- Illustrative recombinant PKS genes of the invention include those in which the AT domain encoding sequences for the third module of the epothilone PKS have been altered or replaced to change the AT domain encoded thereby from a malonyl specific AT to a methylmalonyl specific AT.
- Such methylmalonyl specific AT domain encoding nucleic acids can be isolated, for example and without limitation, from the PKS genes encoding DEBS, the narbonolide PKS, the rapamycin PKS, and the FK-520 PKS.
- the recombinant PKS produces the 14-methyl epothilone derivatives of the invention.
- the KR domain of the third module of the PKS is responsible for forming the hydroxyl group involved in cyclization of epothilone. Consequently, abolishing the KR domain of the third module or adding a DH or DH and ER domains will interfere with the cyclization, leading either to a linear molecule or to a molecule cyclized at a different location than is epothilone.
- the fourth module of the epothilone PKS includes a KS, an AT that can bind either malonyl CoA or methylmalonyl CoA, a KR, and an ACP.
- This module is encoded by a sequence within an -10 kb Nsil-Hindlll restriction fragment of cosmid pKOS35-70.1A2.
- the recombinant DNA compounds of the invention that encode the fourth module of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications.
- a DNA compound comprising a sequence that encodes the epothilone fourth module is inserted into a DNA compound that comprises the coding sequence for one or more modules of a heterologous PKS.
- the resulting construct encodes a protein in which a module of the heterologous PKS is either replaced by that for the fourth module of the epothilone PKS or the latter is merely added to the modules of the heterologous PKS. Together with other proteins that constitute the heterologous PKS, this protein provides a novel PKS.
- a DNA compound comprising a sequence that encodes the fourth module of the epothilone PKS is expressed in a host cell that also expresses the remaining modules and proteins of the epothilone PKS or a recombinant epothilone PKS that produces an epothilone derivative.
- the recombinant fourth module is usually expressed in a protein that also contains the epothilone third, fifth, and sixth modules or modified versions thereof.
- all or a portion of the fourth module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
- the invention provides, for example, either replacing the malonyl CoA and methylmalonyl specific AT with a malonyl CoA, methylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting the KR; and/or replacing the KR, including, optionally, to specify a different stereochemistry; and/or inserting a DH or a DH and ER.
- the KS and/or ACP can be replaced with another KS and/or ACP.
- the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a gene for a PKS that produces a polyketide other than epothilone, or from chemical synthesis.
- the resulting heterologous fourth module coding sequence is inco ⁇ orated into a protein subunit of a recombinant PKS that synthesizes epothilone, an epothilone derivative, or another polyketide.
- the recombinant fourth module is typically expressed as a protein that also contains the third, fifth, and sixth modules of the epothilone PKS or modified versions thereof.
- the invention provides recombinant PKS enzymes for epothilones and epothilone derivatives in which the entire fourth module has been deleted or replaced by a module from a heterologous PKS.
- the invention provides recombinant DNA compounds comprising the coding sequence for the fourth module of the epothilone PKS modified to encode an AT that binds methylmalonyl CoA and not malonyl CoA.
- These recombinant molecules are used to express a protein that is a recombinant derivative of the epoD protein that comprises the modified fourth module as well as modules 3, 5, and 6, any one or more of which can optionally be in derivative form, of the epothilone PKS.
- the invention provides recombinant DNA compounds comprising the coding sequence for the fourth module of the epothilone PKS modified to encode an AT that binds malonyl CoA and not methylmalonyl CoA.
- These recombinant molecules are used to express a protein that is a recombinant derivative of the epoD protein that comprises the modified fourth module as well as modules 3, 5, and 6, any one or more of which can optionally be in derivative form, of the epothilone PKS.
- Sorangium cellulosum produced epothilones A, B, C, D, E, and F and that epothilones A, C, and E had a hydrogen at C-12, while epothilones B, D, and F had a methyl group at this position.
- Unappreciated prior to the present invention was the order in which these compounds were synthesized in S. cellulosum, and the mechanism by which some of the compounds had a hydrogen at C- 12 where others had a methyl group at this position.
- epothilones A and B are derived from epothilones C and D by action of the epoK gene product and that the presence of a hydrogen or methyl moiety at C-12 is due to the AT domain of module 4 of the epothilone PKS.
- This domain can bind either malonyl or methylmalonyl CoA and, consistent with its having greater similarity to malonyl specific AT domains than to methylmalonyl specific AT domains, binds malonyl CoA more often than methylmalonyl CoA.
- the invention provides recombinant DNA compounds and expression vectors and the corresponding recombinant PKS in which the hybrid fourth module with a methylmalonyl specific AT has been inco ⁇ orated.
- the methylmalonyl specific AT coding sequence can originate, for example and without limitation, from coding sequences for the oleandolide PKS, DEBS, the narbonolide PKS, the rapamycin PKS, or any other PKS that comprises a methylmalonyl specific AT domain.
- the hybrid fourth module expressed from this coding sequence is inco ⁇ orated into the epothilone PKS (or the PKS for an epothilone derivative), typically as a derivative epoD gene product.
- epothilone H (or an epothilone H derivative) if there is no dehydratase activity to form the C-12-C-13 alkene
- epothilone D or an epothilone D derivative
- epothilone B or an epothilone B derivative
- epothilone F or an epothilone F derivative
- the cell will produce the corresponding epothilone derivative if there have been other changes to the epothilone PKS.
- the recombinant PKS comprising the hybrid methylmalonyl specific fourth module is expressed in, for example, Sorangium cellulosum
- the appropriate modifying enzymes are present (unless they have been rendered inactive in accordance with the methods herein), and epothilones D, B, and/or F are produced.
- Such production is typically carried out in a recombinant S. cellulosum provided by the present invention in which the native epothilone PKS is unable to function at all or unable to function except in conjunction with the recombinant fourth module provided.
- the recombinant vector can exist as an extrachromosomal element or as a segment of DNA integrated into the host cell chromosome.
- the invention provides that one can simply integrate the recombinant methylmalonyl specific module 4 coding sequence into wild- type S. cellulosum by homologous recombination with the native epoD gene to ensure that only the desired epothilone is produced.
- the invention provides that the S.
- cellulosum host can either express or not express (by mutation or homologous recombination of the native genes therefor) the dehydratase, epoxidase, and/or oxidase gene products and thus form or not form the corresponding epothilone D, B, and F compounds, as the practitioner elects.
- Sorangium cellulosum modified as described above is only one of the recombinant host cells provided by the invention.
- the recombinant methylmalonyl specific epothilone fourth module coding sequences are used in accordance with the methods of invention to produce epothilone D, B, and F (or their corresponding derivatives) in heterologous host cells.
- the invention provides reagents and methods for introducing the epothilone or epothilone derivative PKS and epothilone dehydratase, epoxidase, and hydroxylase genes and combinations thereof into heterologous host cells.
- the invention provides a hybrid fourth module coding sequence in which, in addition to the replacement of the endogenous AT coding sequence with a coding sequence for an AT specific for methylmalonyl Co A, coding sequences for a DH and KR for, for example and without limitation, module 10 of the rapamycin PKS or modules 1 or 5 of the FK-520 PKS have replaced the endogenous KR coding sequences.
- the gene product comprising the hybrid fourth module and epothilone PKS modules 3, 5, and 6 (or derivatives thereof) encoded by this coding sequence is inco ⁇ orated into a PKS comprising the other epothilone PKS proteins (or derivatives thereof) produced in a host cell
- the cell makes either epothilone D or its trans stereoisomer (or derivatives thereof), depending on the stereochemical specificity of the inserted DH and KR domains.
- the invention provides recombinant DNA compounds comprising the coding sequence for the fourth module of the epothilone PKS modified to encode an AT that binds malonyl CoA and not methylmalonyl CoA.
- the invention provides recombinant DNA compounds and vectors and the corresponding recombinant PKS in which this hybrid fourth module has been inco ⁇ orated into a derivative epoD gene product.
- the resulting recombinant epothilone PKS produces epothilones C, A, and E, depending, again, on whether epothilone modification enzymes are present.
- the practitioner of the invention can produce one or more of the epothilone G, C, A, and E compounds and derivatives thereof using the compounds, host cells, and methods of the invention.
- the fifth module of the epothilone PKS includes a KS, an AT that binds malonyl CoA, a DH, an ER, a KR, and an ACP.
- This module is encoded by a sequence within an -12.4 kb Nsil-Notl restriction fragment of cosmid pKOS35-70.1A2.
- a DNA compound comprising a sequence that encodes the epothilone fifth module is inserted into a DNA compound that comprises the coding sequence for one or more modules of a heterologous PKS.
- the resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the fifth module of the epothilone PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS can be inco ⁇ orated into an expression vector and used to produce the recombinant protein encoded thereby.
- a novel PKS is produced.
- a DNA compound comprising a sequence that encodes the fifth module of the epothilone PKS is inserted into a DNA compound that comprises coding sequences for the epothilone PKS or a recombinant epothilone PKS that produces an epothilone derivative.
- the epothilone fifth module is typically expressed as a protein comprising the third, fourth, and sixth modules of the epothilone PKS or derivatives thereof.
- a portion of the fifth module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module coding sequence and the hybrid module encoded thereby.
- the invention provides, for example, either replacing the malonyl CoA specific AT with a methylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting any one, two, or all three of the ER, DH, and KR; and/or replacing any one, two, or all three of the ER, DH, and KR with either a KR, a DH and KR, or a KR, DH, and ER, including, optionally, to specify a different stereochemistry.
- the KS and/or ACP can be replaced with another KS and/or ACP.
- the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a coding sequence for a PKS that produces a polyketide other than epothilone, or from chemical synthesis.
- the resulting hybrid fifth module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes epothilone, an epothilone derivative, or another polyketide.
- the fifth module of the epothilone PKS can be deleted or replaced in its entirety by a module of a heterologous PKS to produce a protein that in combination with the other proteins of the epothilone PKS or derivatives thereof constitutes a PKS that produces an epothilone derivative.
- Illustrative recombinant PKS genes of the invention include recombinant epoD gene derivatives in which the AT domain encoding sequences for the fifth module of the epothilone PKS have been altered or replaced to change the AT domain encoded thereby from a malonyl specific AT to a methylmalonyl specific AT.
- Such methylmalonyl specific AT domain encoding nucleic acids can be isolated, for example and without limitation, from the PKS genes encoding DEBS, the narbonolide PKS, the rapamycin PKS, and the FK-520 PKS.
- Another recombinant epoD gene derivative provided by the invention includes not only this altered module 5 coding sequence but also module 4 coding sequences that encode an AT domain that binds only methylmalonyl CoA.
- the recombinant epoD gene derivative product leads to the production of 10-methyl epothilone B and/or D derivatives.
- illustrative recombinant epoD gene derivatives of the invention include those in which the ER, DH, and KR domain encoding sequences for the fifth module of the epothilone PKS have been replaced with those encoding (i) a KR and DH domain; (ii) a KR domain; and (iii) an inactive KR domain.
- recombinant epoD gene derivatives of the invention are coexpressed with the epo A, epoB, epoC, epoE, and epoF genes to produce a recombinant PKS that makes the corresponding (i) C-l 1 alkene, (ii) C-l 1 hydroxy, and (iii) C-l 1 keto epothilone derivatives.
- These recombinant epoD gene derivatives can also be coexpressed with recombinant epo genes containing other alterations or can themselves be further altered to produce a PKS that makes the corresponding C-l 1 epothilone derivatives.
- one recombinant epoD gene derivative provided by the invention also includes module 4 coding sequences that encode an AT domain that binds only methylmalonyl CoA.
- the recombinant epoD gene derivative product leads to the production of the corresponding C-l 1 epothilone B and/or D derivatives.
- Functionally similar epoD genes for producing the epothilone C-l 1 derivatives can also be made by inactivation of one, two, or all three of the ER, DH, and KR domains of the epothilone fifth module.
- the preferred mode for altering such domains in any module is by replacement with the complete set of desired domains taken from another module of the same or a heterologous PKS coding sequence. In this manner, the natural architecture of the PKS is conserved.
- KR and DH or KR, DH, and ER domains that function together in a native PKS are preferably used in the recombinant PKS.
- Illustrative replacement domains for the substitutions described above include, for example and without limitation, the inactive KR domain from the rapamycin PKS module 3 to form the ketone, the KR domain from the rapamycin PKS module 5 to form the alcohol, and the KR and DH domains from the rapamycin PKS module 4 to form the alkene.
- Other such inactive KR, active KR, and active KR and DH domain encoding nucleic acids can be isolated from, for example and without limitation, the PKS genes encoding DEBS, the narbonolide PKS, and the FK-520 PKS.
- the sixth module of the epothilone PKS includes a KS, an AT that binds methylmalonyl CoA, a DH, an ER, a KR, and an ACP. This module is encoded by a sequence within an -14.5 kb Hindlll-Nsil restriction fragment of cosmid pKOS35- 70.1A2.
- a DNA compound comprising a sequence that encodes the epothilone sixth module is inserted into a DNA compound that comprises the coding sequence for one or more modules of a heterologous PKS.
- the resulting protein encoded by the construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the sixth module of the epothilone PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS provides a novel PKS when coexpressed with the other proteins comprising the PKS.
- a DNA compound comprising a sequence that encodes the sixth module of the epothilone PKS is inserted into a DNA compound that comprises the coding sequence for modules 3, 4, and 5 of the epothilone PKS or a recombinant epothilone PKS that produces an epothilone derivative and coexpressed with the other proteins of the epothilone or epothilone derivative PKS to produce a PKS that makes epothilone or an epothilone derivative in a host cell.
- a portion of the sixth module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
- the invention provides, for example, either replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting any one, two, or all three of the ER, DH, and KR; and/or replacing any one, two, or all three of the ER, DH, and KR with either a KR, a DH and KR, or a KR, DH, and ER, including, optionally, to specify a different stereochemistry.
- the KS and/or ACP can be replaced with another KS and/or ACP.
- the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a coding sequence for a PKS that produces a polyketide other than epothilone, or from chemical synthesis.
- the resulting heterologous sixth module coding sequence can be utilized in conjunction with a coding sequence for a protein subunit of a PKS that makes epothilone, an epothilone derivative, or another polyketide.
- the hybrid sixth module is typically expressed as a protein comprising modules 3, 4, and 5 of the epothilone PKS or derivatives thereof.
- the sixth module of the epothilone PKS can be deleted or replaced in its entirety by a module from a heterologous PKS to produce a PKS for an epothilone derivative.
- Illustrative recombinant PKS genes of the invention include those in which the AT domain encoding sequences for the sixth module of the epothilone PKS have been altered or replaced to change the AT domain encoded thereby from a methylmalonyl specific AT to a malonyl specific AT.
- Such malonyl specific AT domain encoding nucleic acids can be isolated from, for example and without limitation, the PKS genes encoding the narbonolide PKS, the rapamycin PKS, and the FK-520 PKS.
- the recombinant epoD gene of the invention encoding such a hybrid module 6 is coexpressed with the other epothilone PKS genes, the recombinant PKS makes the 8-desmethyl epothilone derivatives.
- This recombinant epoD gene derivative can also be coexpressed with recombinant epo gene derivatives containing other alterations or can itself be further altered to produce a PKS that makes the corresponding 8-desmethyl epothilone derivatives.
- one recombinant epoD gene provided by the invention also includes module 4 coding sequences that encode an AT domain that binds only methylmalonyl CoA.
- the recombinant epoD gene product leads to the production of the 8- desmethyl derivatives of epothilones B and D.
- illustrative recombinant epoD gene derivatives of the invention include those in which the ER, DH, and KR domain encoding sequences for the sixth module of the epothilone PKS have been replaced with those that encode (i) a KR and DH domain; (ii) a KR domain; and (iii) an inactive KR domain.
- These recombinant epoD gene derivatives of the invention when coexpressed with the other epothilone PKS genes make the corresponding (i) C-9 alkene, (ii) C-9 hydroxy, and (iii) C-9 keto epothilone derivatives.
- one recombinant epoD gene derivative provided by the invention also includes module 4 coding sequences that encode an AT domain that binds only methylmalonyl CoA.
- the recombinant epoD gene product leads to the production of the C-9 derivatives of epothilones B and D.
- Functionally equivalent sixth modules can also be made by inactivation of one, two, or all three of the ER, DH, and KR domains of the epothilone sixth module.
- the preferred mode for altering such domains in any module is by replacement with the complete set of desired domains taken from another module of the same or a heterologous PKS coding sequence.
- Illustrative replacement domains for the substitutions described above include but are not limited to the inactive KR domain from the rapamycin PKS module 3 to form the ketone, the KR domain from the rapamycin PKS module 5 to form the alcohol, and the KR and DH domains from the rapamycin PKS module 4 to form the alkene.
- inactive KR, active KR, and active KR and DH domain encoding nucleic acids can be isolated from for example and without limitation the PKS genes encoding DEBS, the narbonolide PKS, and the FK-520 PKS.
- PKS genes encoding DEBS, the narbonolide PKS, and the FK-520 PKS.
- Each of the resulting PKSs produces a polyketide compound that comprises a functional group at the C-9 position that can be further derivatized in vitro by standard chemical methodology to yield semi- synthetic epothilone derivatives of the invention.
- the seventh module of the epothilone PKS includes a KS, an AT specific for methylmalonyl CoA, a KR, and an ACP. This module is encoded by a sequence within an -8.7 kb Bglll restriction fragment from cosmid pKOS35-70.4.
- the recombinant DNA compounds of the invention that encode the seventh module of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications.
- the seventh module of the epothilone PKS is contained in the gene product of the epoE gene, which also contains the eighth module.
- the present invention provides the epoE gene in recombinant form, but also provides
- DNA compounds that encode the seventh module without coding sequences for the eighth module as well as DNA compounds that encode the eighth module without coding sequences for the seventh module.
- a DNA compound comprising a sequence that encodes the epothilone seventh module is inserted into a DNA compound that comprises the coding sequence for one or more modules of a heterologous PKS.
- the resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the seventh module of the epothilone PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS provides a novel PKS coding sequence that can be expressed in a host cell.
- the epothilone seventh module can be expressed as a discrete protein.
- a DNA compound comprising a sequence that encodes the seventh module of the epothilone PKS is expressed to form a protein that, together with other proteins, constitutes the epothilone PKS or a PKS that produces an epothilone derivative.
- the seventh module is typically expressed as a protein comprising the eighth module of the epothilone PKS or a derivative thereof and coexpressed with the epo A, epoB, epoC, epoD, and epoF genes or derivatives thereof to constitute the PKS.
- a portion or all of the seventh module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
- the invention provides, for example, either replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting the KR; replacing the KR with a KR that specifies a different stereochemistry; and/or inserting a DH or a DH and an ER.
- the KS and/or ACP can be replaced with another KS and/or ACP.
- the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a coding sequence for a PKS that produces a polyketide other than epothilone, or from chemical synthesis.
- the resulting heterologous seventh module coding sequence is utilized, optionally in conjunction with other coding sequences, to express a protein that together with other proteins constitutes a PKS that synthesizes epothilone, an epothilone derivative, or another polyketide.
- the seventh module is typically expressed as a protein comprising the eighth module or derivative thereof and coexpressed with the epoA, epoB, epoC, epoD, and epoF genes or derivatives thereof to constitute the PKS.
- the coding sequences for the seventh module in the epoE gene can be deleted or replaced by those for a heterologous module to prepare a recombinant epoE gene derivative that, together with the epo A, epoB, epoC, epoD, and epoF genes, can be expressed to make a PKS for an epothilone derivative.
- Illustrative recombinant epoE gene derivatives of the invention include those in which the AT domain encoding sequences for the seventh module of the epothilone PKS have been altered or replaced to change the AT domain encoded thereby from a methylmalonyl specific AT to a malonyl specific AT.
- Such malonyl specific AT domain encoding nucleic acids can be isolated from for example and without limitation the PKS genes encoding the narbonolide PKS, the rapamycin PKS, and the FK-520 PKS.
- the eighth module of the epothilone PKS includes a KS, an AT specific for methylmalonyl CoA, inactive KR and DH domains, a methyltransferase (MT) domain, and an ACP. This module is encoded by a sequence within an -10 kb Notl restriction fragment of cosmid pKOS35-79.85.
- a DNA compound comprising a sequence that encodes the epothilone eighth module is inserted into a DNA compound that comprises the coding sequence for one or more modules of a heterologous PKS.
- the resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the eighth module of the epothilone PKS or the latter is merely added to coding sequences for modules of the heterologous PKS provides a novel PKS coding sequence that is expressed with the other proteins constituting the PKS to provide a novel PKS.
- the eighth module can be expressed as a discrete protein that can associate with other PKS proteins to constitute a novel PKS.
- a DNA compound comprising a sequence that encodes the eighth module of the epothilone PKS is coexpressed with the other proteins constituting the epothilone PKS or a PKS that produces an epothilone derivative.
- the eighth module is typically expressed as a protein that also comprises the seventh module or a derivative thereof.
- a portion or all of the eighth module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
- the invention provides, for example, either replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting the inactive KR and/or the inactive DH; replacing the inactive KR and/or DH with an active KR and/or DH; and/or inserting an ER.
- the KS and/or ACP can be replaced with another KS and/or ACP.
- the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a coding sequence for a PKS that produces a polyketide other than epothilone, or from chemical synthesis.
- the resulting heterologous eighth module coding sequence is expressed as a protein that is utilized in conjunction with the other proteins that constitute a PKS that synthesizes epothilone, an epothilone derivative, or another polyketide.
- the heterologous or hybrid eighth module is typically expressed as a recombinant epoE gene product that also contains the seventh module.
- the coding sequences for the eighth module in the epoE gene can be deleted or replaced by those for a heterologous module to prepare a recombinant epoE gene that, together with the epo A, epoB, epoC, epoD, and epoF genes, can be expressed to make a PKS for an epothilone derivative.
- the eighth module of the epothilone PKS also comprises a methylation or methyltransferase (MT) domain with an activity that methylates the epothilone precursor.
- This function can be deleted to produce a recombinant epoD gene derivative of the invention, which can be expressed with the other epothilone PKS genes or derivatives thereof that makes an epothilone derivative that lacks one or both methyl groups, depending on whether the AT domain of the eighth module has been changed to a malonyl specific AT domain, at the corresponding C-4 position of the epothilone molecule.
- MT methylation or methyltransferase
- the present invention provides recombinant DNA compounds that encode a polypeptide with this methylation domain and activity and a variety of recombinant PKS coding sequences that encode recombinant PKS enzymes that inco ⁇ orate this polypeptide.
- the availability of this MT domain and the coding sequences therefor provides a significant number of new polyketides that differ from known polyketides by the presence of at least an additional methyl group.
- the MT domain of the invention can in effect be added to any PKS module to direct the methylation at the corresponding location in the polyketide produced by the PKS.
- the present invention provides the recombinant nucleic acid compounds resulting from inserting the coding sequence for this MT activity into a coding sequence for any one or more of the six modules of the DEBS enzyme to produce a recombinant DEBS that synthesizes a 6-deoxyerythronolide B derivative that comprises one or more additional methyl groups at the C-2, C-4, C-6, C-8, C-10, and/or C-12 positions.
- the MT domain can be inserted adjacent to the AT or the ACP.
- the ninth module of the epothilone PKS includes a KS, an AT specific for malonyl CoA, a KR, an inactive DH, and an ACP. This module is encoded by a sequence within an -14.7 Hindlll-Bglll kb restriction fragment of cosmid pKOS35-79.85.
- the recombinant DNA compounds of the invention that encode the ninth module of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications.
- the ninth module of the epothilone PKS is expressed as a protein, the product of the epoF gene, that also contains the TE domain of the epothilone PKS.
- the present invention provides the epoF gene in recombinant form, as well as DNA compounds that encode the ninth module without the coding sequences for the TE domain and DNA compounds that encode the TE domain without the coding sequences for the ninth module.
- a DNA compound comprising a sequence that encodes the epothilone ninth module is inserted into a DNA compound that comprises the coding sequence for one or more modules of a heterologous PKS.
- the resulting construct in which the coding sequence for a module of the heterologous PKS is either replaced by that for the ninth module of the epothilone PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS protein coding sequence that when coexpressed with the other proteins constituting a PKS provides a novel PKS.
- the ninth module coding sequence can also be expressed as a discrete protein with or without an attached TE domain.
- a DNA compound comprising a sequence that encodes the ninth module of the epothilone PKS is expressed as a protein together with other proteins to constitute an epothilone PKS or a PKS that produces an epothilone derivative.
- the ninth module is typically expressed as a protein that also contains the TE domain of either the epothilone PKS or a heterologous PKS.
- a portion or all of the ninth module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
- the invention provides, for example, either replacing the malonyl CoA specific AT with a methylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxy malonyl CoA specific AT; deleting the KR; replacing the KR with a KR that specifies a different stereochemistry; and/or inserting a DH or a DH and an ER.
- the KS and/or ACP can be replaced with another KS and/or ACP.
- the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a coding sequence for a PKS that produces a polyketide other than epothilone, or from chemical synthesis.
- the resulting heterologous ninth module coding sequence is coexpressed with the other proteins constituting a PKS that synthesizes epothilone, an epothilone derivative, or another polyketide.
- the present invention provides a PKS for an epothilone or epothilone derivative in which the ninth module has been replaced by a module from a heterologous PKS or has been deleted in its entirety.
- the TE domain is expressed as a discrete protein or fused to the eighth module.
- the ninth module of the epothilone PKS is followed by a thioesterase domain.
- This domain is encoded in the -14.7 kb Hindlll-Bglll restriction comprising the ninth module coding sequence.
- the present invention provides recombinant DNA compounds that encode hybrid PKS enzymes in which the ninth module of the epothilone PKS is fused to a heterologous thioesterase or one or more modules of a heterologous PKS are fused to the epothilone PKS thioesterase.
- a thioesterase domain coding sequence from another PKS can be inserted at the end of the ninth module ACP coding sequence in recombinant DNA compounds of the invention.
- Recombinant DNA compounds encoding this thioesterase domain are therefore useful in constructing DNA compounds that encode a protein of the epothilone PKS, a PKS that produces an epothilone derivative, and a PKS that produces a polyketide other than epothilone or an epothilone derivative.
- the present invention thus provides a hybrid PKS and the corresponding recombinant DNA compounds that encode the proteins constituting those hybrid PKS enzymes.
- a hybrid PKS is a recombinant PKS that comprises all or part of one or more modules, loading domain, and thioesterase/cyclase domain of a first PKS and all or part of one or more modules, loading domain, and thioesterase/cyclase domain of a second PKS.
- the first PKS is most but not all of the epothilone PKS
- the second PKS is only a portion or all of a non-epothilone PKS.
- hybrid PKS includes an epothilone PKS in which the natural loading domain has been replaced with a loading domain of another PKS.
- Another example of such a hybrid PKS is an epothilone PKS in which the AT domain of module four is replaced with an AT domain from a heterologous PKS that binds only methylmalonyl CoA.
- the first PKS is most but not all of a non-epothilone PKS, and the second PKS is only a portion or all of the epothilone PKS.
- An illustrative example of such a hybrid PKS includes an erythromycin PKS in which an AT specific for methylmalonyl CoA is replaced with an AT from the epothilone PKS specific for malonyl CoA.
- Another example is an erythromycin PKS that includes the MT domain of the epothilone PKS.
- DNA synthesis allows the artisan to construct de novo DNA compounds of size sufficient to construct a useful portion of a PKS module or domain. For pu ⁇ oses of the present invention, such synthetic DNA compounds are deemed to be a portion of a PKS.
- Streptomyces avermitilis genes encoding the avermectin polyketide synthase.
- Preferred PKS enzymes and coding sequences for the proteins which constitute them for pu ⁇ oses of isolating heterologous PKS domain coding sequences for constructing hybrid PKS enzymes of the invention are the soraphen PKS and the PKS described as a Sorangium PKS in the above table.
- Example 1 To summarize the functions of the genes cloned and sequenced in Example 1:
- the hybrid PKS-encoding DNA compounds of the invention can be and often are hybrids of more than two PKS genes. Even where only two genes are used, there are often two or more modules in the hybrid gene in which all or part of the module is derived from a second (or third) PKS gene.
- Illustrative examples of recombinant epothilone derivative PKS genes of the invention which are identified by listing the specificities of the hybrid modules (the other modules having the same specificity as the epothilone PKS), include:
- module 4 with methylmalonyl specific AT (mm AT) and a KR and module 2 with a malonyl specific AT (m AT) and a KR;
- module 4 with mM AT and a KR and module 3 with mM AT and a KR;
- module 4 with mM AT and a KR and module 5 with mM AT and a ER, DH, and KR;
- module 4 with mM AT and a KR and module 5 with mM AT and a DH and
- module 4 with mM AT and a KR and module 5 with mM AT and a KR;
- module 4 with mM AT and a KR and module 5 with mM AT and an inactive KR;
- module 4 with mM AT and a KR and module 6 with m AT and a DH and KR;
- module 4 with mM AT and a KR and module 6 with m AT and a KR (i) module 4 with mM AT and a KR and module 6 with m AT and a KR; (j) module 4 with mM AT and a KR and module 6 with m AT and an inactive KR;
- a hybrid PKS of the invention includes but is not limited to a PKS of any of the following types: (i) an epothilone or epothilone derivative PKS that contains a module in which at least one of the domains is from a heterologous module; (ii) an epothilone or epothilone derivative PKS that contains a module from a heterologous PKS; (iii) an epothilone or epothilone derivative PKS that contains a protein from a heterologous PKS; and (iv) combinations of the foregoing.
- the present invention also provides recombinant epothilone PKS genes in which there is no second PKS gene sequence present but which differ from the epothilone PKS gene by one or more deletions.
- the deletions can encompass one or more modules and/or can be limited to a partial deletion within one or more modules.
- a deletion encompasses an entire module other than the NRPS module
- the resulting epothilone derivative is at least two carbons shorter than the compound produced from the PKS from which the deleted version was derived.
- the deletion can also encompass the NRPS module and/or the loading domain, as noted above.
- the deletion When a deletion is within a module, the deletion typically encompasses a KR, DH, or ER domain, or both DH and ER domains, or both KR and DH domains, or all three KR, DH, and ER domains.
- the catalytic properties of the domains and modules of the epothilone PKS and of epothilone modification enzymes can also be altered by random or site specific mutagenesis of the corresponding genes.
- a wide variety of mutagenizing agents and methods are known in the art and are suitable for this pu ⁇ ose.
- the technique known as DNA shuffling can also be employed. See, e.g., U.S. Patent Nos. 5,830,721; 5,811,238; and 5,605,793; and references cited therein, each of which is inco ⁇ orated herein by reference.
- the full complement of genes can be assembled and manipulated more readily for heterologous expression, and each of the segments of the gene can be altered, and various altered segments can be combined in a single host cell to provide a recombinant PKS of the invention.
- This technique makes more efficient the construction of large libraries of recombinant PKS genes, vectors for expressing those genes, and host cells comprising those vectors.
- the genes encoding the desired PKS are not only present on two or more vectors, but also can be ordered or arranged differently than in the native producer organism from which the genes were derived.
- this technique as applied to the epothilone PKS are described in the Examples below.
- the epo A, epoB, epoC, and epoD genes are present on a first plasmid, and the epoE and epoF and optionally either the epoK or the epoK and epoL genes are present on a second (or third) plasmid.
- the recombinant nucleic acid compounds of the invention are expression vectors.
- expression vector refers to any nucleic acid that can be introduced into a host cell or cell-free transcription and translation medium.
- An expression vector can be maintained stably or transiently in a cell, whether as part of the chromosomal or other DNA in the cell or in any cellular compartment, such as a replicating vector in the cytoplasm.
- An expression vector also comprises a gene that serves to produce RNA that is translated into a polypeptide in the cell or cell extract.
- the vector typically includes a promoter to enhance gene expression but alternatively may serve to inco ⁇ orate the relevant coding sequence under the control of an endogenous promoter.
- expression vectors may typically contain additional functional elements, such as resistance-conferring genes to act as selectable markers and regulatory genes to enhance promoter activity.
- an expression vector can vary widely, depending on the intended use of the vector. In particular, the components depend on the host cell(s) in which the vector will be used or is intended to function.
- Vector components for expression and maintenance of vectors in E. coli are widely known and commercially available, as are vector components for other commonly used organisms, such as yeast cells and Streptomyces cells.
- the vectors of the invention are used to transform Sorangium host cells to provide the recombinant Sorangium host cells of the invention.
- U.S. Pat. No. 5,686,295, inco ⁇ orated herein by reference describes a method for transforming Sorangium host cells, although other methods may also be employed.
- Sorangium is a convenient host for expressing epothilone derivatives of the invention in which the recombinant PKS that produces such derivatives is expressed from a recombinant vector in which the epothilone PKS gene promoter is positioned to drive expression of the recombinant coding sequence.
- the epothilone PKS gene promoter is provided in recombinant form by the present invention and is an important embodiment thereof.
- the promoter is contained within an -500 nucleotide sequence between the end of the transposon sequences and the start site of the open reading frame of the epoA gene.
- the recombinant vector need drive expression of only a portion of the PKS containing the altered sequences.
- a vector may comprise only a single altered epothilone PKS gene, with the remainder of the epothilone PKS polypeptides provided by the genes in the host cell chromosomal DNA. If the host cell naturally produces an epothilone, the epothilone derivative will thus be produced in a mixture containing the naturally occurring epothilone(s).
- Sorangium host cells in which one or more genes involved in epothilone biosynthesis have been rendered inactive.
- the invention provides such Sorangium host cells, which may be preferred host cells for expressing epothilone derivatives of the invention so that complex mixtures of epothilones are avoided.
- Particularly preferred host cells of this type include those in which one or more of any of the epothilone PKS gene ORFs has been disrupted, and/or those in which any or more of the epothilone modification enzyme genes have been disrupted.
- Such host cells are typically constructed by a process involving homologous recombination using a vector that contains DNA homologous to the regions flanking the gene segment to be altered and positioned so that the desired homologous double crossover recombination event desired will occur.
- Homologous recombination can thus be used to delete, disrupt, or alter a gene.
- the present invention provides a recombinant epothilone producing Sorangium cellulosum host cell in which the epoK gene has been deleted or disrupted by homologous recombination using a recombinant DNA vector of the invention.
- This host cell unable to make the epoK epoxidase gene product is unable to make epothilones A and B and so is a preferred source of epothilones C and D.
- Homologous recombination can also be used to alter the specificity of a PKS module by replacing coding sequences for the module or domain of a module to be altered with those specifying a module or domain of the desired specificity.
- the present invention provides a recombinant epothilone producing Sorangium cellulosum host cell in which the coding sequence for the AT domain of module 4 encoded by the epoD gene has been altered by homologous recombination using a recombinant DNA vector of the invention to encode an AT domain that binds only methylmalonyl CoA.
- This host cell unable to make epothilones A, C, and E is a preferred source of epothilones B, D, and F.
- the invention also provides recombinant Sorangium host cells in which both alterations and deletions of epothilone biosynthetic genes have been made.
- the invention provides recombinant Sorangium cellulosum host cells in which both of the foregoing alteration and deletion have been made, producing a host cell that makes only epothilone D.
- the present invention provides a wide variety of recombinant Sorangium cellulosum host cells that make less complex mixtures of the epothilones than do the wild type producing cells as well as those that make one or more epothilone derivatives.
- host cells include those that make only epothilones A, C, and E; those that make only epothilones B, D, and F, those that make only epothilone D; and those that make only epothilone C.
- the present invention provides expression vectors and recombinant Myxococcus, preferably M.
- xanthus host cells containing those expression vectors that express a recombinant epothilone PKS or a PKS for an epothilone derivative.
- vectors that replicate extrachromosomally in M. xanthus are not known.
- Mx8, Mx9, Mx81, and Mx82 a number of phage known to integrate into M. xanthus chromosomal DNA, including Mx8, Mx9, Mx81, and Mx82.
- the integration and attachment function of these phages can be placed on plasmids to create phage-based expression vectors that integrate into the M. xanthus chromosomal DNA.
- phage Mx9 and Mx8 are preferred for pu ⁇ oses of the present invention.
- Plasmid pPLH343 described in Salmi et al, Feb. 1998, Genetic determinants of immunity and integration of temperate Myxococcus xanthus phage Mx8, J. Bact. 180(3): 614-621, is a plasmid that replicates in E. coli and comprises the phage Mx8 genes that encode the attachment and integration functions.
- the promoter of the epothilone PKS gene functions in Myxococcus xanthus host cells.
- the present invention provides a recombinant promoter for use in recombinant host cells derived from the promoter of the Sorangium cellulosum epothilone PKS gene.
- the promoter can be used to drive expression of one or more epothilone PKS genes or another useful gene product in recombinant host cells.
- the invention also provides an epothilone PKS expression vector in which one or more of the epothilone PKS or epothilone modification enzyme genes are under the control of their own promoter.
- Another preferred promoter for use in Myxococcus xanthus host cells for pu ⁇ oses of expressing a recombinant PKS of the invention is the promoter of the pilA gene of M. xanthus. This promoter, as well as two M.
- xanthus strains that express high levels of gene products from genes controlled by the pilA promoter, a pilA deletion strain and a pilS deletion strain, are described in Wu and Kaiser, Dec. 1997, Regulation of expression of the pilA gene in Myxococcus xanthus, J. Bact. 179(24):7748-7758, inco ⁇ orated herein by reference.
- the invention provides recombinant Myxococcus host cells comprising both the pilA and pilS deletions.
- Another preferred promoter is the starvation dependent promoter of the sdcK gene.
- Selectable markers for use in Myxococcus xanthus include kanamycin, tetracycline, chloramphenicol, zeocin, spectinomycin, and streptomycin resistance conferring genes.
- the recombinant DNA expression vectors of the invention for use in Myxococcus typically include such a selectable marker and may further comprise the promoter derived from an epothilone PKS or epothilone modification enzyme gene.
- the present invention provides preferred expression vectors for use in preparing the recombinant Myxococcus xanthus expression vectors and host cells of the invention. These vectors, designated plasmids pKOS35-82.1 and pKOS35-82.2 ( Figure 3), are able to replicate in E. coli host cells as well as integrate into the chromosomal DNA of
- the vectors comprise the Mx8 attachment and integration genes as well as the pilA promoter with restriction enzyme recognition sites placed conveniently downstream.
- the two vectors differ from one another merely in the orientation of the pilA promoter on the vector and can be readily modified to include the epothilone PKS and modification enzyme genes of the invention.
- the construction of the vectors is described in Example 2.
- Especially preferred Myxococcus host cells of the invention are those that produce an epothilone or epothilone derivative or mixtures of epothilones or epothilone derivatives at equal to or greater than 20 mg/L, more preferably at equal to or greater than 200 mg/L, and most preferably at equal to or greater than 1 g/L.
- M. xanthus host cells that produce at these levels.
- M. xanthus host cells that can be employed for pu ⁇ oses of the invention include the DZ1 (Campos et al, 1978, J. Mol. Biol. 119: 167- 178, inco ⁇ orated herein by reference), the TA-producing cell line ATCC 31046, DK1219 (Hodgkin and Kaiser, 1979, Mol. Gen. Genet. 171 : 177-191, inco ⁇ orated herein by reference), and the DK1622 cell lines (Kaiser, 1979, Proc. Natl. Acad. Sci. USA 76: 5952- 5956, inco ⁇ orated herein by reference).
- the present invention provides expression vectors and recombinant Pseudomonas fluorescens host cells that contain those expression vectors and express a recombinant PKS of the invention.
- a plasmid for use in constructing the P. fluorescens expression vectors and host cells of the invention is plasmid pRSFlOlO, which replicates in E. coli and P. fluorescens host cells (see Scholz et al, 1989, Gene 75:271-8, inco ⁇ orated herein by reference). Low copy number replicons and vectors can also be used.
- the invention also provides the promoter of the Sorangium cellulosum epothilone PKS and epothilone modification enzyme genes in recombinant form.
- the promoter can be used to drive expression of an epothilone PKS gene or other gene in P. fluorescens host cells.
- the promoter of the soraphen PKS genes can be used in any host cell in which a Sorangium promoter functions.
- the present invention provides an epothilone PKS expression vector for use in P. fluorescens host cells.
- the expression vectors of the invention are used to construct recombinant Streptomyces host cells that express a recombinant PKS of the invention.
- Streptomyces host cells useful in accordance with the invention include S. coelicolor, S. lividans, S. venezuelae, S. ambofaciens, S. fradiae, and the like.
- Preferred Streptomyces host cell/vector combinations of the invention include S. coelicolor CH999 and S. lividans K4-114 and K4-155 host cells, which do not produce actinorhodin, and expression vectors derived from the pRMl and pRM5 vectors, as described in U.S. Patent No.
- Streptomyces host cells of the invention are those that produce an epothilone or epothilone derivative or mixtures of epothilones or epothilone derivatives at equal to or greater than 20 mg/L, more preferably at equal to or greater than 200 mg/L, and most preferably at equal to or greater than 1 g/L.
- S. coelicolor and S. lividans host cells that produce at these levels.
- species of the closely related genus Saccharopolyspora can be used to produce epothilones, including but not limited to S. erythraea.
- the present invention provides a wide variety of expression vectors for use in Streptomyces.
- the origin of replication can be, for example and without limitation, a low copy number replicon and vectors comprising the same, such as SCP2* (see Hopwood et al, Genetic Manipulation of Streptomyces: A Laboratory manual (The John Innes Foundation, Norwich, U.K., 1985); Lydiate et al, 1985, Gene 35: 223- 235; and Kieser and Melton, 1988, Gene 65: 83-91, each of which is inco ⁇ orated herein by reference), SLP1.2 (Thompson et al, 1982, Gene 20: 51-62, inco ⁇ orated herein by reference), and pSG5(ts) (Muth et al, 1989, Mol.
- SCP2* see Hopwood et al, Genetic Manipulation of Streptomyces: A Laboratory manual (The John Innes Foundation, Norwich, U.K., 1985); Lydiate e
- E coli origin of replication such as from pUC, plP, pll, and pBR.
- E coli origin of replication such as from pUC, plP, pll, and pBR.
- the phage phiC31 and its derivative KC515 can be employed (see Hopwood et al, supra).
- plasmid pS ⁇ T152, plasmid pSAM, plasmids pSElOl and pSE211 all of which integrate site-specifically in the chromosomal DNA of S. lividans, can be employed.
- the expression vector will comprise one or more marker genes by which host cells containing the vector can be identified and/or selected.
- Useful antibiotic resistance conferring genes for use in Streptomyces host cells include the ermE (confers resistance to erythromycin and lincomycin), tsr (confers resistance to thiostrepton), aadA (confers resistance to spectinomycin and streptomycin), aacC4 (confers resistance to apramycin, kanamycin, gentamicin, geneticin (G418), and neomycin), hyg (confers resistance to hygromycin), and vph (confers resistance to viomycin) resistance conferring genes.
- the recombinant PKS gene on the vector will be under the control of a promoter, typically with an attendant ribosome binding site sequence.
- a preferred promoter is the actl promoter and its attendant activator gene actII-ORF4, which is provided in the pRMl and pRM5 expression vectors, supra. This promoter is activated in the stationary phase of growth when secondary metabolites are normally synthesized.
- Other useful Streptomyces promoters include without limitation those from the ermE gene and the melCl gene, which act constitutively, and the tipA gene and the merA gene, which can be induced at any growth stage.
- the T7 RNA polymerase system has been transferred to Streptomyces and can be employed in the vectors and host cells of the invention.
- the coding sequence for the T7 RNA polymerase is inserted into a neutral site of the chromosome or in a vector under the control of the inducible merA promoter, and the gene of interest is placed under the control of the T7 promoter.
- one or more activator genes can also be employed to enhance the activity of a promoter.
- Activator genes in addition to the actII-ORF4 gene discussed above include dnrl, redD, and ptpA genes (see U.S. patent application Serial No. 09/181,833, supra), which can be employed with their cognate promoters to drive expression of a recombinant gene of the invention.
- the present invention also provides recombinant expression vectors that drive expression of the epothilone PKS and PKS enzymes that produce epothilone or epothilone derivatives in plant cells.
- Such vectors are constructed in accordance with the teachings in U.S. patent application Serial No. 09/114,083, filed 10 July 1998, and PCT patent publication No. 99/02669, each of which is inco ⁇ orated herein by reference. Plants and plant cells expressing epothilone are disease resistant and able to resist fungal infection.
- an epothilone or epothilone derivative in any heterologous host cells including plant, Myxococcus, Pseudomonas, and Streptomyces host cells
- any heterologous host cells including plant, Myxococcus, Pseudomonas, and Streptomyces host cells
- U.S. patent application Serial No. 08/728,742 filed 11 Oct. 1996
- PCT patent publication No. 97/13845 both of which are inco ⁇ orated herein by reference.
- the present invention also provides, as discussed above, DNA compounds that encode epothilone modification enzyme genes.
- the present invention provides recombinant expression vectors and host cells transformed with those vectors that express any one or more of those genes and so produce the corresponding epothilone or epothilone derivative.
- the present invention provides the epoK gene in recombinant form and host cells that express the gene product thereof, which converts epothilones C and D to epothilones A and B, respectively.
- the present invention provides vectors for disrupting the function of any one or more of the epoL, epoK, and any of the ORFs associated with the epothilone PKS gene cluster in Sorangium cells.
- the invention also provides recombinant Sorangium host cells lacking (or containing inactivated forms of) any one or more of these genes. These cells can be used to produce the corresponding epothilones and epothilone derivatives that result from the absence of any one or more of these genes.
- the invention also provides non-Sorangium host cells that contain a recombinant epothilone PKS or a PKS for an epothilone derivative but do not contain (or contain nonfunctional forms of) any epothilone modification enzyme genes.
- These host cells of the invention are expected produce epothilones G and H in the absence of a dehydratase activity capable of forming the C-12-C-13 alkene of epothilones C and D. This dehydration reaction is believed to take place in the absence of the epoL gene product in Streptomyces host cells.
- the host cells produce epothilones C and D (or the corresponding epothilone C and D derivative) when the dehydratase activity is present and the P450 epoxidase and hydroxylase (that converts epothilones A and B to epothilones E and F, respectively) genes are absent.
- the host cells also produce epothilones A and B (or the corresponding epothilone A and B derivatives) when the hydroxylase gene only is absent.
- Preferred for expression in these host cells is the recombinant epothilone PKS enzymes of the invention that contain the hybrid module 4 with an AT specific for methylmalonlyl CoA only, optionally in combination with one or more additional hybrid modules.
- Also preferred for expression in these host cells is the recombinant epothilone PKS enzymes of the invention that contain the hybrid module 4 with an AT specific for malonyl CoA only, optionally in combination with one or more additional hybrid modules.
- the recombinant host cells of the invention can also include other genes and corresponding gene products that enhance production of a desired epothilone or epothilone derivative.
- the epothilone PKS proteins require phosphopantetheinylation of the ACP domains of the loading domain and modules 2 through 9 as well as of the PCP domain of the NRPS. Phosphopantethein-ylation is mediated by enzymes that are called phosphopantetheinyl transferases (PPTases).
- the host cells of the invention can be grown and fermented under conditions known in the art for other pu ⁇ oses to produce the compounds of the invention.
- the compounds of the invention can be isolated from the fermentation broths of these cultured cells and purified by standard procedures. Fermentation conditions for producing the compounds of the invention from Sorangium host cells can be based on the protocols described in PCT patent publication Nos. 93/10121, 97/19086, 98/22461, and 99/42602, each of which is inco ⁇ orated herein by reference.
- the novel epothilone analogs of the present invention, as well as the epothilones produced by the host cells of the invention can be derivatized and formulated as described in PCT patent publication Nos. 93/10121, 97/19086, 98/08849, 98/22461, 98/25929, 99/01124, 99/02514, 99/07692, 99/27890,
- Preferred compounds of the invention include the 14-methyl epothilone derivatives
- preferred epothilone derivative compounds of the invention are those that can be produced by altering the epothilone PKS genes as described herein and optionally by action of epothilone modification enzymes and/or by chemically modifying the resulting epothilones produced when those genes are expressed.
- the present invention provides compounds of the formula:
- A is a substituted or unsubstituted straight, branched chain or cyclic alkyl, alkenyl or alkynyl residue optionally containing 1-3 heteroatoms selected from O, S and N; or wherein A comprises a substituted or unsubstituted aromatic residue;
- R represents H,H, or H,lower alkyl, or lower alkyl,lower alkyl;
- X 5 O or a derivative thereof, or H,OH or H,NR 2 wherein R is H, or alkyl, or acyl or H,OCOR or H,OCONR 2 wherein R is H or alkyl, or is H,H;
- R 6 represents H or lower alkyl, and the remaining substituent on the corresponding carbon is H;
- X 7 represents OR, NR 2 , wherein R is H, or alkyl or acyl or is OCOR, or OCONR 2
- R is H or alkyl or X taken together with X forms a carbonate or carbamate cycle, and wherein the remaining substituent on the corresponding carbon is H;
- R 8 represents H or lower alkyl and the remaining substituent on the carbon is H;
- R 10 is H,H or H,lower alkyl, or lower alkyl,lower alkyl;
- X 1 ' O or a derivative thereof, or is H,OR, or H,NR wherein R is H, or alkyl or acyl or is H,OCOR or H,OCONR 2 wherein R is H or alkyl, or is H,H or wherein X 1 ' in combination with X 9 may form a cyclic carbonate or carbamate;
- R is H,H, or H,lower alkyl, or lower alkyl,lower alkyl
- X O or a derivative thereof, or H,OR or H,NR 2 wherein R is H, alkyl or acyl or is H,OCOR or H,OCONR 2 wherein R is H or alkyl;
- R 14 is H,H, or H,lower alkyl, or lower alkyl,lower alkyl
- R 16 is H or lower alkyl; and wherein optionally H or another substituent may be removed from positions 12 and 13 and/or 8 and 9 to form a double bond, wherein said double bond may optionally be converted to an epoxide.
- a substituent which "comprises an aromatic moiety" contains* least one aromatic ring, such as phenyl, pyridyl, pyrimidyl, thiophenyl, or thiazolyl.
- B. substituent may also include fused aromatic residues such as naphthyl, indolyl, benzothiazolyl, and the like.
- the aromatic moiety may also be fused to a nonaromati ⁇ ing and/or may be coupled to the remainder of the compound in which it is a substituent through a nonaromatic, for example, alkylene residue.
- the aromatic moiety may be substituted or unsubstituted as may the remainder of the substituent.
- alkyl refers to a C]-C 8 saturated, straight or branched chain hydrocarbon radical derived from a hydrocarbon moiety by removal of a single hydrogen atom.
- alkenyl and alkynyl refer to the corresponding unsaturated forms. Examples of alkyl include but are not limited to methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, i-hexyl, n-heptyl, n-octyl.
- Lower alkyl (or alkenyl or alkynyl) refers to a 1-4C radical.
- Methyl is preferred.
- Acyl refers to alkylCO, alkenylCO or alkynylCO.
- halo and halogen as used herein refer to an atom selected from fluorine, chlorine, bromine, and iodine.
- haloalkyl as used herein denotes an alkyl group to which one, two, or three halogen atoms are attached to any one carbon and includes without limitation chloromethyl, bromoethyl, trifluoromethyl, and the like.
- heteroaryl refers to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.
- heterocyle includes but is not limited to pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, mo ⁇ holinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.
- substituted refers to a group substituted by independent replacement of any of the hydrogen atoms thereon with, for example, Cl, Br, F, I, OH, CN, alkyl, alkoxy, alkoxy substituted with aryl, haloalkyl, alkylthio, amino, alkylamino, dialkylamino, mercapto, nitro, carboxaldehyde, carboxy, alkoxycarbonyl, or carboxamide. Any one substituent may be an aryl, heteroaryl, or heterocycloalkyl group.
- substituents at positions 2, 4, 6, 8, 10, 12, 14 and 16 in formula (1) is determined at least initially by the specificity of the AT catalytic domain of modules 9, 8, 7, 6, 5, 4, 3 and 2, respectively. Because AT domains that accept malonyl CoA, methylmalonyl CoA, ethylmalonyl CoA (and in general, lower alkyl malonyl CoA), as well as hydroxymalonyl CoA, are available, one of the substituents at these positions may be H, and the other may be H, lower alkyl, especially methyl and ethyl, or OH.
- ⁇ -carbonyl modifications can readily be controlled by modifying the epothilone PKS gene cluster to include the appropriate sequences in the corresponding positions of the epothilone gene cluster which will or will not contain active KR, DH and/or ER domains.
- X 5 , X 7 , X 9 , X 1 ' and X synthetically available are numerous, including the formation of ⁇ -bonds with the adjacent ring positions.
- Positions occupied by OH are readily converted to ethers or esters by means well known in the art; protection of OH at positions not to be derivatized may be required. Further, a hydroxyl may be converted to a leaving group, such as a tosylate, and replaced by an amino or halo substituent. A wide variety of "hydroxyl derivatives" such as those discussed above is known in the art.
- ring positions which contain oxo groups may be converted to "carbonyl derivatives" such as oximes, ketals, and the like. Initial reaction products with the oxo moieties may be further reacted to obtain more complex derivatives. As described in
- Example 8 such derivatives may ultimately result in a cyclic substituent linking two ring positions.
- the enzymes useful in modification of the polyketide initially synthesized can be supplied endogenously by a host cell when the polyketide is synthesized intracellularly, by modifying a host to contain the recombinant materials for the production of these modifying enzymes, or can be supplied in a cell-free system, either in purified forms or as relatively crude extracts.
- the epoxidation of the ⁇ -bond at position 12- 13 may be effected using the protein product of the epoK gene directly in vitro.
- A is most conveniently controlled by employing an epothilone PKS which comprises an inactivated module 1 NRPS (using a module 2 substrate) or a KS2 knockout (using a module 3 substrate) as described in Example 6, hereinbelow.
- Limited variation can be obtained by altering the AT catalytic specificity of the loading module; further variation is accomplished by replacing the NRPS of module 1 with an NRPS of different specificity or with a conventional PKS module.
- variants are more readily prepared by feeding the synthetic module 2 substrate precursors and module 3 substrate precursors to the appropriately altered epothilone PKS as described in Example 6.
- the compounds can be readily formulated to provide the pharmaceutical compositions of the invention.
- the pharmaceutical compositions of the invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid, or liquid form.
- This preparation will contain one or more of the compounds of the invention as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application.
- the active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, pessaries, solutions, emulsions, suspensions, and any other form suitable for use.
- the carriers which can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semi-solid, or liquified form.
- auxiliary stabilizing, thickening, and coloring agents and perfumes may be used.
- the compounds of the invention may be utilized with hydroxypropyl methylcellulose essentially as described in U.S. Patent No. 4,916,138, inco ⁇ orated herein by reference, or with a surfactant essentially as described in EPO patent publication No. 428,169, inco ⁇ orated herein by reference.
- Oral dosage forms may be prepared essentially as described by Hondo et al, 1987, Transplantation Proceedings XIX, Supp. 6: 17-22, inco ⁇ orated herein by reference.
- Dosage forms for external application may be prepared essentially as described in EPO patent publication No. 423,714, inco ⁇ orated herein by reference.
- the active compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the disease process or condition.
- a compound of the invention may be administered orally, topically, parenterally, by inhalation spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvant, and vehicles.
- parenteral includes subcutaneous injections, and intravenous, intrathecal, intramuscular, and intrasternal injection or infusion techniques.
- Dosage levels of the compounds of the present invention are of the order from about 0.01 mg to about 100 mg per kilogram of body weight per day, preferably from about 0.1 mg to about 50 mg per kilogram of body weight per day.
- the dosage levels are useful in the treatment of the above-indicated conditions (from about 0.7 mg to about 3.5 mg per patient per day, assuming a 70 kg patient).
- the compounds of the present invention may be administered on an intermittent basis, i.e., at semi-weekly, weekly, semi-monthly, or monthly intervals.
- the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
- a formulation intended for oral administration to humans may contain from 0.5 mg to 5 gm of active agent compounded with an appropriate and convenient amount of carrier material, which may vary from about 5 percent to about 95 percent of the total composition.
- Dosage unit forms will generally contain from about 0.5 mg to about 500 mg of active ingredient.
- the compounds of the invention may be formulated within the range of, for example, 0.00001% to 60% by weight, preferably from 0.001% to 10% by weight, and most preferably from about 0.005% to 0.8% by weight.
- the specific dose level for any particular patient will depend on a variety of factors. These factors include the activity of the specific compound employed; the age, body weight, general health, sex, and diet of the subject; the time and route of administration and the rate of excretion of the drug; whether a drug combination is employed in the treatment; and the severity of the particular disease or condition for which therapy is sought.
- factors include the activity of the specific compound employed; the age, body weight, general health, sex, and diet of the subject; the time and route of administration and the rate of excretion of the drug; whether a drug combination is employed in the treatment; and the severity of the particular disease or condition for which therapy is sought.
- the DNA was packaged in lambda phage as recommended by the manufacturer and the mixture then used to infect E. coli XL 1 -Blue MR cells. This procedure yielded approximately 3,000 isolated colonies on LB-ampicillin plates. Because the size of the S. cellulosum genome is estimated to be circa 10 7 nucleotides, the DNA inserts present among 3000 colonies would correspond to circa 10 S. cellulosum genomes.
- KS domains were used to design oligonucleotide primers for a PCR with Sorangium cellulosum genomic DNA as template. The fragment generated was then used as a probe to screen the library. This approach was chosen, because it was found, from the examination of over a dozen PKS genes, that KS domains are the most highly conserved (at the amino acid level) of all the PKS domains examined. Therefore, it was expected that the probes produced would detect not only the epothilone PKS genes but also other PKS gene clusters represented in the library.
- the fragment was removed from the gel and cloned in the Hindi site of pUCl 18 (which is a derivative of pUC18 with an insert sequence for making single stranded DNA).
- plasmid DNA from ten independent clones was isolated and sequenced. The analysis revealed nine unique sequences that each corresponded to a common segment of KS domains in PKS genes. Of the nine, three were identical to a polyketide synthase gene cluster previously isolated from this organism and determined not to belong to the epothilone gene cluster from the analysis of the modules. The remaining six KS fragments were excised from the vector, pooled, end-labeled with P and used as probe in hybridizations with the colonies containing the cosmid library under high stringency conditions.
- the screen identified 15 cosmids that hybridized to the pooled KS probes.
- DNA was prepared from each cosmid, digested with Notl, separated on an agarose gel, and transferred to a nitrocellulose membrane for Southern hybridization using the pooled KS fragments as probe.
- the blot was stripped of the label and re-probed, under less stringent conditions, with labeled DNA containing the sequence corresponding to the enoylreductase domain from module four of the DEBS gene cluster.
- Sequence from one of the cosmids that hybridized strongly to the ER probe showed homology to NRPSs and, in particular, to the adenylation domain of an NRPS. Because it was anticipated that the thiazole moiety of epothilone might be derived from the formation of an amide bond between an acetate and cysteine molecule (with a subsequent cyclization step), the presence of an NRPS domain in a cosmid that also contained ER domain(s) supported the prediction that this cosmid might contain all or part of the epothilone PKS gene cluster.
- each of the Notl restriction fragments from the four cosmids was cloned into the Notl site of the commercially available pBluescript plasmid.
- Initial sequencing was performed on the ends of each of the clones. Analysis of the sequences allowed the prediction, before having the complete sequence, that there would be 10 modules in this PKS gene cluster, a loading domain plus 9 modules.
- the nucleotide sequence was determined for a linear segment corresponding to -72 kb. Analysis revealed a PKS gene cluster with a loading domain and nine modules. Downstream of the PKS sequence is an ORF, designated epoK, that shows strong homology to cytochrome P450 oxidase genes and encodes the epothilone epoxidase.
- the nucleotide sequence of 15 kb downstream of epo K has also been determined: a number of additional ORFs have been identified but an ORF that shows homology to any known dehydratase has not been identified.
- the epoL gene may encode a dehydratase activity, but this activity may instead be resident within the epothilone PKS or encoded by another gene.
- the PKS genes are organized in 6 open reading frames. At the polypeptide level, the loading domain and modules 1 , 2, and 9 appear on individual polypeptides; their corresponding genes are designated epoA, epoB, epoC and epoF respectively. Modules 3, 4, 5, and 6 are contained on a single polypeptide whose gene is designated epoD, and modules 7 and 8 are on another polypeptide whose gene is designated epoE . It is clear from the spacing between ORFs that epoC, epoD, epoE and epoF constitute an operon.
- the epo A, epoB, and epoK gene may be also part of the large operon, but there are spaces of approximately 100 bp between epoB and epoC and 115 bp between epoF and epoK which could contain a promoter.
- the present invention provides the intergenic sequences in recombinant form. At least one, but potentially more than one, promoter is used to express all of the epothilone genes.
- the epothilone PKS gene cluster is shown schematically below.
- the sequence of the AT domain in the loading module and in modules 3, 4, 5, and 9 shows similarity to the consensus sequence for malonyl loading domains, consistent with the presence of an H side chain at C-l 4, C-12 (epothilones A and C), C-10, and C-2, respectively, as well as the loading region.
- the AT domains in modules 2, 6, 7, and 8 resemble the consensus sequence for methylmalonyl specifying AT domains, again consistent with the presence of methyl side chains at C-l 6, C-8, C-6, and C-4 respectively.
- the loading module contains a KS domain in which the cysteine residue usually present at the active site is instead a tyrosine.
- This domain is designated as KS y and serves as a decarboxylase, which is part of its normal function, but cannot function as a condensing enzyme.
- the loading domain is expected to load malonyl CoA, move it to the ACP, and decarboxylate it to yield the acetyl residue required for condensation with cysteine.
- Module 1 is the non-ribosomal peptide synthetase that activates cysteine and catalyzes the condensation with acetate on the loading module.
- the sequence contains segments highly similar to ATP-binding and ATPase domains, required for activation of amino acids, a phosphopantotheinylation site, and an elongation domain.
- module 1 shows very high similarity to a number of previously identified peptide synthetases.
- Module 2 determines the structure of epothilone at C-l 5 - C-l 7.
- the presence of the DH domain in module 2 yields the C-l 6- 17 dehydro moiety in the molecule.
- the domains in module 3 are consistent with the structure of epothilone at C-l 4 and C-l 5; the OH that comes from the action of the KR is employed in the lactonization of the molecule.
- Module 4 controls the structure at C-12 and C-13 where a double bond is found in epothilones C and D, consistent with the presence of a DH domain.
- the sequence of the AT domain appears to resemble those that specify malonate loading, it can also load methylmalonate, thereby accounting in part for the mixture of epothilones found in the fermentation broths of the naturally producing organisms.
- module 4 A significant departure from the expected array of functions was found in module 4. This module was expected to contain a DH domain, thereby directing the synthesis of epothilones C and D as the products of the PKS. Rigorous analysis revealed that the space between the AT and KR domains of module 4 was not large enough to accommodate a functional DH domain. Thus, the extent of reduction at module 4 does not proceed beyond the ketoreduction of the beta-keto formed after the condensation directed by module 4. Because the C-l 2, 13 unsaturation has been demonstrated (epothilones C and D), there must be an additional dehydratase function that introduces the double bond, and this function is believed to be in the PKS itself or resident in an ORF in the epothilone biosynthetic gene cluster.
- the action of the dehydratase could occur either during the synthesis of the polyketide or after cyclization has taken place.
- the compounds produced at the end of acyl chain growth would be epothilones C and D. If the C-l 2, 13 dehydration were a post-polyketide event, the completed acyl chain would have a hydroxyl group at C-13, as shown below.
- the names epothilones G and H have been assigned to the 13-hydroxy compounds produced in the absence of or prior to the action of the dehydratase.
- Modules 5 and 6 each have the full set of reduction domains (KR, DH and ER) to yield the methylene functions at C-l 1 and C-9.
- Modules 7 and 9 have KR domains to yield the hydroxyls at C-7 and C-3, and module 8 does not have a functional KR domain, consistent with the presence of the keto group at C-5.
- Module 8 also contains a methyltransferase (MT) domain that results in the presence of the geminal dimethyl function at C-4.
- Module 9 has a thioesterase domain that terminates polyketide synthesis and catalyzes ring closure.
- the genes, proteins, modules, and domains of the epothilone PKS are summarized in the Table hereinabove.
- Plasmid pKOS35-77 serves as a convenient plasmid for expressing recombinant PKS genes of the invention under the control of the epothilone PKS gene promoter.
- the entire epothilone PKS gene with its homologous promoter is inserted in one or more fragments into the plasmid to yield an expression vector of the invention.
- the present invention also provides expression vectors in which the recombinant PKS genes of the invention are under the control of a Myxococcus xanthus promoter.
- the promoter of the pilA gene of M. xanthus was isolated as a PCR amplification product. Plasmid pSWU357, which comprises the pilA gene promoter and is described in Wu and Kaiser, Dec. 1997, J. Bact. 179(24):7748-7758, was mixed with PCR primers Seql and Mxpill primers: Seql : 5'-AGCGGATAACAATTTCACACAGGAAACAGC-3'; and
- Mxpill 5'-TTAATTAAGAGAAGGTTGCAACGGGGGGC-3', and amplified using standard PCR conditions to yield an -800 bp fragment.
- This fragment was cleaved with restriction enzyme Kpnl and ligated to the large Kpnl-EcoRV restriction fragment of commercially available plasmid pLitmus 28 (New England Biolabs).
- the resulting circular DNA was designated plasmid pKOS35-71B.
- the promoter of the pilA gene from plasmid pKOS35-71B was isolated as an -800 bp EcoRV-SnaBI restriction fragment and ligated with the large Mscl restriction fragment of plasmid pKOS35-77 to yield a circular DNA -6.8 kb in size. Because the -800 bp fragment could be inserted in either one of two orientations, the ligation produced two plasmids of the same size, which were designated as plasmids pKOS35-82.1 and pKOS35- 82.2. Restriction site and function maps of these plasmids are presented in Figure 3.
- Plasmids pKOS35-82.1 and pKOS35-82.2 serve as convenient starting materials for the vectors of the invention in which a recombinant PKS gene is placed under the control of the Myxococcus xanthus pilA gene promoter.
- These plasmids comprise a single Pad restriction enzyme recognition sequence placed immediately downstream of the transcription start site of the promoter.
- the entire epothilone PKS gene without its homologous promoter is inserted in one or more fragments into the plasmids at the Pad site to yield expression vectors of the invention.
- the sequence of the pilA promoter in these plasmids is shown below.
- M. xanthus cells are grown in CYE media (Campos and Zusman, 1975, Regulation of development in Myxococcus xanthus: effect of 3': 5'-cyclic AMP, ADP, and nutrition, Proc. Natl. Acad. Sci. USA 72: 518-522) to a Klett of 100 at 30°C at 300 rpm. The remainder of the protocol is conducted at 25°C unless otherwise indicated.
- the cells are then pelleted by centrifugation (8000 ⁇ m for 10 min. in an SS34 or SA600 rotor) and resuspended in deionized water. The cells are again pelleted and resuspended in 1/100th of the original volume.
- DNA (one to two ⁇ L) is electroporated into the cells in a 0.1 cm cuvette at room temperature at 400 ohm, 25 ⁇ FD, 0.65 V with a time constant in the range of 8.8 - 9.4.
- the DNA should be free of salts and so should be resuspended in distilled and deionized water or dialyzed on a 0.025 ⁇ m Type VS membrane (Millipore). For low efficiency electroporations, spot dialyze the DNA, and allow outgrowth in CYE.
- the recombinant DNA expression vectors of the invention are electroporated into Myxococcus host cells that express recombinant PKSs of the invention and produce the epothilone, epothilone derivatives, and other novel polyketides encoded thereby.
- BAC Bacterial Artificial Chromosome
- Myxococcus xanthus To express the epothilone PKS and modification enzyme genes in a heterologous host to produce epothilones by fermentation, Myxococcus xanthus, which is closely related to Sorangium cellulosum and for which a number of cloning vectors are available, can also be employed in accordance with the methods of the invention. Because both M. xanthus and S. cellulosum are myxobacteria, it is expected that they share common elements of gene expression, translational control, and post translational modification (if any), thereby enhancing the likelihood that the epo genes from S. cellulosum can be expressed to produce epothilone in M. xanthus. Secondly, M.
- xanthus has been developed for gene cloning and expression.
- DNA can be introduced by electroporation, and a number of vectors and genetic markers are available for the introduction of foreign DNA, including those that permit its stable insertion into the chromosome.
- M. xanthus can be grown with relative ease in complex media in fermentors and can be subjected to manipulations to increase gene expression, if required.
- the epothilone gene cluster can be built into the chromosome by using cosmids of the invention and homologous recombination to assemble the complete gene cluster.
- the complete epothilone gene cluster can be cloned on a bacterial artificial chromosome (BAC) and then moved into M. xanthus for integration into the chromosome.
- BAC bacterial artificial chromosome
- the first plasmid, pKOS35-154 is linearized with Dral and electroporated into M. xanthus.
- This plasmid contains two regions of the dev locus flanking two fragments of the epothilone gene cluster. Inserted in between the epo gene regions are the kanamycin resistance marker and the galK gene. Kanamycin resistance arises in colonies if the DNA recombines into the dev region by a double recombination using the dev sequence as regions of homology.
- This strain, K35-159 contains small regions of the epothilone gene cluster that will allow for recombination of pKOS35-79.85. Because the resistance markers on pKOS35-79.85 are the same as that for K35-159, a tetracycline transposon was transposed into the cosmid, and cosmids that contain the transposon inserted into the kanamycin marker were selected. This cosmid, pKOS90-23, was electroporated into K35-159, and oxytetracycline resistant colonies were selected to create strain K35-174.
- the plasmid pKOS90-22 is linearized with Dral and electroporated into K35-175 to create K35-180.
- This strain is electroporated with the tetracycline resistant version of pKOS35-70.1A2, pKOS90-38, and colonies resistant to oxytetracycline are selected.
- Recombinants that now have the whole epothilone gene cluster are selected by resistance to 1% galactose. This results in strain K35-188.
- This strain contains all the epothilone genes as well as all potential promoters. This strain is fermented and tested for the production of epothilones A and B.
- BAC bacterial artificial chromosome
- SMP44 cells are embedded in agarose and lysed according to the BIO-RAD genomic DNA plug kit.
- DNA plugs are partially digested with restriction enzyme, such as Sau3AI or Hindlll, and electrophoresed on a FIGE or CHEF gel.
- DNA fragments are isolated by electroeluting the DNA from the agarose or using gelase to degrade the agarose.
- the method of choice to isolate the fragments is electroelution, as described in Strong et al, 1997, Nucleic Acids Res. 19: 3959-3961, inco ⁇ orated herein by reference.
- the DNA is ligated into the BAC (pBeloBACII) cleaved with the appropriate enzyme.
- pBeloBACII A map of pBeloBACII is shown below.
- the DNA is electroporated into DH10B cells by the method of Sheng et al, 1995, Nucleic Acids Res. 23: 1990-1996, inco ⁇ orated herein by reference, to create an S. cellulosum genomic library. Colonies are screened using a probe from the NRPS region of the epothilone cluster. Positive clones are picked and DNA is isolated for restriction analysis to confirm the presence of the complete gene cluster. This positive clone is designated pKOS35-178.
- a plasmid that contains regions of homology that are upstream and downstream of the epothilone gene cluster flanked by the dev locus and containing the kanamycin resistance galK cassette, analogous to plasmids pKOS90-22 and pKOS35-154.
- This plasmid is linearized with Dral and electroporated into M. xanthus, in accordance with the method of Kafeshi et al, 1995, Mol. Microbiol. 15: 483-494, to create K35-183.
- the plasmid pKOS35-178 can be introduced into K35-183 by electroporation or by transduction with bacteriophage PI and chloramphenicol resistant colonies are selected.
- a version of pKOS35-178 that contains the origin of conjugative transfer from pRP4 can be constructed for transfer of DNA from E. coli to K35-183.
- This plasmid is made by first constructing a transposon containing the oriT region from RP4 and the tetracycline resistance maker from pACYC184 and then transposing the transposon in vitro or in vivo onto pKOS35-178. This plasmid is transformed into S17-1 and conjugated into M. xanthus.
- This strain K35-190, is grown in the presence of 1% galactose to select for the second recombination event.
- This strain contains all the epothilone genes as well as all potential promoters. This strain will be fermented and tested for the production of epothilones A and B.
- a transposon is constructed that contains the integration genes and att site from either Mx8 or Mx9 along with the tetracycline gene from pACYC184. Alternative versions of this transposon may have only the attachment site.
- the integration genes are then supplied in trans by coelectroporation of a plasmid containing the integrase gene or having the integrase protein expressed in the electroporated strain from any constitutive promoter, such as the mgl promoter (see Magrini et al, Jul. 1999, J. Bact.
- transposon is constructed, it is transposed onto pKOS35-178 to create pKOS35-191.
- This plasmid is introduced into Myxococcus xanthus as described above.
- This strain contains all the epothilone genes as well as all potential promoters. This strain is fermented and tested for the production of epothilones A and B.
- the present invention provides recombinant expression vectors for the heterologous expression of modular polyketide synthase genes in Streptomyces hosts.
- These vectors include expression vectors that employ the actl promoter that is regulated by the gene actll ORF4 to allow regulated expression at high levels when growing cells enter stationary phase.
- the vectors available are plasmids pRMl and pRM5, and derivatives thereof such as pCK7, which are stable, low copy plasmids that carry the marker for thiostrepton resistance in actinomycetes.
- Such plasmids can accommodate large inserts of cloned DNA and have been used for the expression of the DEBS PKS in S. coelicolor and S.
- S. lividans does not make the tRNA that recognizes the TTA codon for leucine until late-stage growth and that if production of a protein is desired earlier, then appropriate codon modifications can be made.
- Plasmid pCK7 Another vector is a derivative of plasmid pSET152 and comprises the actll ORF4- Pactl expression system but carries the selectable marker for apramycin resistance. These vectors contain the attP site and integrase gene of the actinophage phiC31 and do not replicate autonomously in Streptomyces hosts but integrate by site specific recombination into the chromosome at the attachment site for phiC31 after introduction into the cell. Derivatives of pCK7 and pSET152 have been used together for the heterologous production of a polyketide, with different PKS genes expressed from each plasmid. See U.S. patent application Serial No. 60/129,731, filed 16 Apr. 1999, inco ⁇ orated herein by reference.
- Plasmid pKOS010-153 a pSET152 Derivative
- the need to develop expression vectors for the epothilone PKS that function in Streptomyces is significant.
- the epothilone compounds are currently produced in the slow growing, genetically intractable host Sorangium cellulosum or are made synthetically.
- the streptomycetes, bacteria that produce more than 70% of all known antibiotics and important complex polyketides, are excellent hosts for production of epothilones and epothilone derivatives.
- S. lividans and S. coelicolor have been developed for the expression of heterologous PKS systems.
- These organisms can stably maintain cloned heterologous PKS genes, express them at high levels under controlled conditions, and modify the corresponding PKS proteins (e.g. phosphopantetheinylation) so that they are capable of production of the polyketide they encode.
- these hosts contain the necessary pathways to produce the substrates required for polyketide synthesis, e.g. malonyl CoA and methylmalonyl CoA.
- a wide variety of cloning and expression vectors are available for these hosts, as are methods for the introduction and stable maintenance of large segments of foreign DNA. Relative to the slow growing Sorangium host, S. lividans and S.
- the epothilone PKS including the NRPS module
- the epothilone PKS including the NRPS module
- the two plasmids are introduced into S. lividans employing selection for thiostrepton and apramycin resistance.
- the pCK7 derivative replicates autonomously whereas the pKOS010-153 derivative is integrated in the chromosome.
- expression of the epothilone genes is from the actl promoter resident within the plasmid.
- the two epothilone PKS encoding segments were cloned as translational fusions with the N-terminal segment of the KS domain of module 5 of the ery PKS.
- High level expression has been demonstrated from this promoter employing KS5 as the first translated sequence, see Jacobsen et al, 1998, Biochemistry 37: 4928-4934, inco ⁇ orated herein by reference.
- a convenient BsaBI site is contained within the DNA segment encoding the amino acid sequence EPIAV that is highly conserved in many KS domains including the KS-encoding regions of epoA and of module 7 in epoE.
- the expression vector for the loading domain and modules one through six of the epothilone PKS was designated pKOS039-124, and the expression vector for modules seven through nine was designated pKOS039-126.
- pKOS039-124 The expression vector for modules one through six of the epothilone PKS was designated pKOS039-124
- pKOS039-126 The expression vector for modules seven through nine was designated pKOS039-126.
- preferred expression vectors of the invention described below and derived from pKOS039-124 and pKOS039-126, have been deposited under the terms of the Budapest Treaty, only a summary of the construction of plasmids pKOS039-124 and pKOS039-126 is provided below.
- the eryKS5 linker coding sequences were cloned as an -0.4 kb Pacl-Bglll restriction fragment from plasmid pKOS10-153 into pKOS039-98 to construct plasmid pKOS039-l 17.
- the coding sequences for the eryKS5 linker were linked to those for the epothilone loading domain by inserting the -8.7 kb EcoRI-Xbal restriction fragment from cosmid pKOS35-70.1A2 into EcoRI-Xbal digested plasmid pLItmus28.
- the final pKOS039- 124 expression vector was constructed by ligating the -34 kb Xbal-Avrll restriction fragment of cosmid pKOS35-70.1A2 with the -21.1 kb Avrll-Xbal restriction fragment of pKOS039-123.
- the plasmid pKOS039-126 expression vector was constructed as follows. First the coding sequences for module 7 were linked from cosmids pKOS35-70.4 and pKOS35- 79.85 by cloning the -6.9 kb Bglll-Notl restriction fragment of pKOS35-70.4 and the -5.9 kb Notl-Hindlll restriction fragment of pKOS35-79.85 into Bglll-Hindlll digested plasmid pLitmus28 to construct plasmid pKOS039-l 19.
- the -12 kb Ndel-Nhel restriction fragment of cosmid pKOS35-79.85 was cloned into Ndel-Xbal digested plasmid pKOS039-l 19 to construct plasmid pKOS039-122.
- the -1 kb BsaBI-Bglll restriction fragment derived from cosmid pKOS35-70.4 was cloned into BsaBI-BclI digested plasmid ⁇ KOS039-l 17 to construct plasmid pKOS039- 121.
- the -21.5 kb Avrll restriction fragment from plasmid pKOS039-122 was cloned into Avrll-Xbal digested plasmid pKOS039-121 to construct plasmid pKOS039-125.
- the -21.8 kb PacI-EcoRI restriction fragment of plasmid pKOS039-125 was ligated with the -9 kb PacI-EcoRI restriction fragment of plasmid pKOS039-44 to construct pKOS039- 126.
- Plasmids pKOS039-124 and pKOS126 were introduced into S. lividans K4-114 sequentially employing selection for the corresponding drug resistance marker. Because plasmid pKOS039-126 does not replicate autonomously in streptomycetes, the selection is for cells in which the plasmid has integrated in the chromosome by site-specific recombination at the attB site of phiC31. Because the plasmid stably integrates, continued selection for apramycin resistance is not required. Selection can be maintained if desired. The presence of thiostrepton in the medium is maintained to ensure continued selection for plasmid pKOS039-124.
- Plasmids pKOS039-124 and pKOS039-126 were transformed into Streptomyces lividans K4-114, and transformants containing the plasmids were cultured and tested for production of epothilones. Initial tests did not indicate the presence of an epothilone. To improve production of epothilones from these vectors, the eryKS5 linker sequences were replaced by epothilone PKS gene coding sequences, and the vectors were introduced into Streptomyces coelicolor CH999.
- oligonucleotides primers were used: N39-73, 5'-GCTTAATTAAGGAGGACACATATGCCCGTCGTGGCGGATCGTCC-3' ; and N39-74, 5'-GCGGATCCTCGAATCACCGCCAATATC-3' .
- the template DNA was derived from cosmid pKOS35-70.8A3.
- the -0.8 kb PCR product was digested with restriction enzymes Pad and BamHI and then ligated with the -2.4 kb BamHI-Notl and the -6.4 kb Pad-Notl restriction fragments of plasmid pKOS039-120 to construct plasmid pKOS039-136.
- the -5 kb Pacl-Avrll restriction fragment of plasmid pKOS039- 136 was ligated with the -50 kb Pacl-Avrll restriction fragment of plasmid pKOS039-124 to construct the expression plasmid pKOS039-124R.
- Plasmid pKOS039-124R has been deposited with the ATCC under the terms of the Budapest Treaty and is available under accession number .
- N39-67A 5 ' -GCTTAATTAAGGAGGACACATATGACCGACCGAGAAGGCCAGCTC-CTGGA-3 '
- N39-68 5'-GGACCTAGGCGGGATGCCGGCGTCT-3' .
- the template DNA was derived from cosmid pKOS35-70.1A2.
- the -0.4 kb amplification product was digested with restriction enzymes Pad and Avrll and ligated with either the -29.5 kb Pacl-Avrll restriction fragment of plasmid pKOS039-126 or the -23.8 kb Pacl-Avrll restriction fragment of plasmid pKOS039-125 to construct plasmid pKOS039-126R or plasmid pKOS039-125R, respectively.
- Plasmid pKOS039-126R was deposited with the ATCC under the terms of the Budapest Treaty and is available under accession number .
- the plasmid pair pKOS039-124R and pKOS039-126R (as well as the plasmid pair pKOS039-124 and pKOS039-126) contain the full complement of epoA, epoB, epoC, epoD, epoE, epoF, epoK, and epoL genes.
- Plasmid pKOS039-126R (as well as plasmid pKOS039-126); however, to ensure that these genes were expressed at high levels, another expression vector of the invention, plasmid pKOS039-141 ( Figure 8), was constructed in which the epoK and epoL genes were placed under the control of the ermE* promoter.
- the epoK gene sequences were amplified by PCR using the oligonucleotide primers:
- Plasmid pXH106 (described in J.
- the -2.8 kb Ncol-Avrll restriction fragment of the resulting plasmid was ligated to the -18 kb PacI-BspHI restriction fragment of plasmid pKOS039-125R and the -9 kb Spel-Pad restriction fragment of plasmid pKOS039-42 to construct plasmid pKOS045-12.
- Plasmid pKOS039-141 was partially digested with restriction enzyme Ndel, the -9 kb Ndel restriction fragment was isolated, and the fragment then circularized by ligation to yield plasmid pKOS039-l 50.
- the various expression vectors described above were then transformed into Streptomyces coelicolor CH999 and S. lividans K4-114 in a variety of combinations, the transformed host cells fermented on plates and in liquid culture (R5 medium, which is identical to R2YE medium without agar). Typical fermentation conditions follow.
- a seed culture of about 5 mL containing 50 ⁇ g/L thiostrepton was inoculated and grown at 30°C for two days.
- about 1 to 2 mL of the seed culture was used to inoculate a production culture of about 50 mL containing 50 ⁇ g/L thiostrepton and 1 mM cysteine, and the production culture was grown at 30°C for 5 days.
- the seed culture was used to prepare plates of cells (the plates contained the same media as the production culture with 10 mM propionate), which were grown at 30°C for nine days.
- Streptomyces coelicolor cultures and culture broths were analyzed for production of epothilones.
- the liquid cultures were extracted with three times with equal volumes of ethyl acetate, the organic extracts combined and evaporated, and the residue dissolved in acetonitrile for LC/MS analysis.
- the agar plate media was chopped and extracted twice with equal volumes of acetone, and the acetone extracts were combined and evaporated to an aqueous slurry, which was extracted three times with equal volumes of ethyl acetate.
- the organic extracts were combined and evaporated, and the residue dissolved in acetonitrile for LC/MS analysis.
- epothilone A shows [M+H] at 494.4 amu, with observed fragments at 476.4, 318.3, and 306.4 amu.
- Epothilone B shows [M+H] at 508.4 amu, with observed fragments at 490.4, 320.3, and 302.4 amu.
- Transformants containing the vector pairs pKOS039-124R and pKOS039-126R or pKOS039-124 and pKOS039-126R produced detectable amounts of epothilones A and B.
- Transformants containing these plasmid pairs and the additional plasmid pKOS039-141 produced similar amounts of epothilones A and B, indicating that the additional copies of the epoK and epoL genes were not required for production under the test conditions employed.
- Transformants containing the plasmid pair pKOS039-124R and pKOS045-12 produced epothilones C and D, as did transformants containing this plasmid pair and the additional plasmid pKOS039-150. These results showed that the epoL gene was not required under the test conditions employed to form the C-12-C-13 double bond. These results indicate that either the epothilone PKS gene alone is able to form the double bond or that Streptomyces coelicolor expresses a gene product able to convert epothilones G and H to epothilones C and D. Thus, these transformants produced epothilones C and D when recombinant epoA, epoB, epoC, epoD, epoE, and epoF genes were present.
- the heterologous expression of the epothilone PKS described herein is believed to represent the recombinant expression of the largest proteins and active enzyme complex that have ever been expressed in a recombinant host cell.
- the epothilone producing Streptomyces coelicolor transformants exhibited growth characteristics indicating that either the epothilone PKS genes, or their products, or the epothilones inhibited cell growth or were somewhat toxic to the cells. Any such inhibition or toxicity could be due to accumulation of the epothilones in the cell, and it is believed that the native Sorangium producer cells may contain transporter proteins that in effect pump epothilones out of the cell.
- Such transporter genes are believed to be included among the ORFs located downstream of the epoK gene and described above.
- the present invention provides Streptomyces and other host cells that include recombinant genes that encode the products of one or more, including all, of the ORFs in this region.
- each ORF can be cloned behind the ermE* promoter, see Stassi et al, 1998, Appl. Microbiol. Biotechnol. 49: 725-731, inco ⁇ orated herein by reference, in a pSAM2 -based plasmid that can integrate into the chromosome of Streptomyces coelicolor and S. lividans at a site distinct from attB of phage phiC31, see Smokvina et al, 1990, Gene 94: 53-59, inco ⁇ orated herein by reference.
- a pSAM2 -based vector carrying the gene for hygromycin resistance is modified to carry the ermE* promoter along with additional cloning sites.
- Each ORF downstream is PCR cloned into the vector which is then introduced into the host cell (also containing pKOS039-124R and pKOS039-126R or other expression vectors of the invention) employing hygromycin selection.
- Clones carrying each individual gene downstream from epo K are analyzed for increased production of epothilones. Additional fermentation and strain improvement efforts can be conducted as illustrated by the following.
- the levels of expression of the PKS genes in the various constructs can be measured by assaying the levels of the corresponding mRNAs (by quantitative RT PCR) relative to the levels of another heterologous PKS mRNA (e.g. picromycin) produced from genes cloned in similar expression vectors in the same host. If one of the epothilone transcripts is unde ⁇ roduced, experiments to enhance its production by cloning the corresponding DNA segment in a different expression vector are conducted, for example, multiple copies of any one or more of the epothilone PKS genes can be introduced into a cell if one or more gene products are rate limiting for biosynthesis.
- Another method to increase the yield of epothilones produced is to change the KS Y domain of the loading domain of the epothilone PKS to a KS Q domain.
- Such altered loading domains can be constructed in any of a variety of ways, but one illustrative method follows. Plasmid pKOS39-124R of the invention can be conveniently used as a starting material.
- N39-83 5 ' -CCGGTATCCACCGCGACACACGGC-3 '
- N39-84 5 ' -GCCAGTCGTCCTCGCTCGTGGCCGTTC-3 '
- N39-73 and N39-74 which have been described above.
- the PCR fragment generated with N37-73 and N39-83 and the PCR fragment generated with N39-74 and N39-84 are treated with restriction enzymes Pad and BamHI, respectively, and ligated with the -3.1 kb Pad-BamHI fragment of plasmid pKOS39-120 to construct plasmid pKOS039-148.
- the -0.8 kb Pad-BamHI restriction fragment of plasmid pKOS039-148 (comprising the two PCR amplification products) is ligated with the -2.4 kb BamHI-Notl restriction fragment and the -6.4 kb Pad-Notl restriction fragment of plasmid pKOS39-120 to construct pKOS39-136Q.
- the -5 kb Pacl-Avrll restriction fragment of plasmid pKOS039- 136Q is ligated to the -50 kb Pacl-Avrll restriction fragment of plasmid pKOS039-124 to construct plasmid pKOS39-124Q. Plasmids pKOS039-124Q and pKOS039-126R are then transformed into Streptomyces coelicolor CH999 for epothilone production.
- epoA through epoF optionally with epoK or with epoK plus epoL, genes cloned and expressed are sufficient for the synthesis of epothilone compounds, and the distribution of the C-12 H to C-12 methyl congeners appears to be similar to that seen in the natural host (A:B::2:1).
- This ratio reflects that the AT domain of module 4 more closely resembles that of the malonyl rather than methylmalonyl specifying AT consensus domains.
- epothilones D and B are produced at lower quantities than their C-12 unmethylated counte ⁇ arts C and A.
- the invention provides PKS genes that produce epothilone D and/or B exclusively.
- methylmalonyl CoA specifying AT domains from a number of sources can be used to replace the naturally occurring at domain in module 4.
- the exchange is performed by direct cloning of the incoming DNA into the appropriate site in the epothilone PKS encoding DNA segment or by gene replacement through homologous recombination.
- the donor sequence to be exchanged is placed in a delivery vector between segments of at least 1 kb in length that flank the AT domain of epo module 4 encoding DNA. Crossovers in the homologous regions result in the exchange of the epo AT4 domain with that on the delivery vector. Because pKOS039-124 and pKOS039-124R contain AT4 coding sequences, they can be used as the host DNA for replacement.
- the adjacent DNA segments are cloned in one of a number of E. coli plasmids that are temperature sensitive for replication.
- the heterologous AT domains can be cloned in these plasmids in the correct orientation between the homologous regions as cassettes enabling the ability to perform several AT exchanges simultaneously.
- the reconstructed plasmid (pKOS039-124* or pKOS039-124R*) is tested for ability to direct the synthesis of epothilone B and/or by introducing it along with pKOS039-126 or pKOS039-126R in Streptomyces coelicolor and/or S. lividans.
- the invention provides a number of heterologous methylmalonyl CoA specifying AT domains to ensure that production of epothilone D at titers equivalent to that of the C and D mixture produced in the Streptomyces coelicolor host described above.
- larger segments of the donor genes can be used for the replacements, including, in addition to the AT domain, adjacent upstream and downstream sequences that correspond to an entire module.
- the KS, methylmalonyl AT, DH, KR, ACP - encoding DNA segment can be obtained from for example and without limitation the DNA encoding the tenth module of the rapamycin PKS, or the first or fifth modules of the FK-520 PKS.
- Example 5 Heterologous Expression of EpoK and Conversion of Epothilone D to Epothilone B This Example describes the construction of E. coli expression vectors for epoK.
- the epoK gene product was expressed in E. coli as a fusion protein with a polyhistidine tag (his tag).
- the fusion protein was purified and used to convert epothilone D to epothilone B.
- Plasmids were constructed to encode fusion proteins composed of six histidine residues fused to either the amino or carboxy terminus of EpoK. The following oligos were used to construct the plasmids:
- Plasmid pKOS35-83.5 contains the -5 kb Notl fragment comprising the epoK gene ligated into pBluescriptSKII+ (Stratagene).
- PCR products were cleaved with restriction enzymes BamHI and Ndel and ligated into the BamHI and Ndel sites of pET22b (Invitrogen). Both plasmids were sequenced to verify that no mutations were introduced during the PCR amplification. Protein gels were run as known in the art.
- EpoK Purification of EpoK was performed as follows. Plasmids pKOS55-121 and pKOS55-129 were transformed into BL21(DE3) containing the groELS expressing plasmid pREP4-groELS (Caspers et al, 1994, Cellular and Molecular Biology 40(5): 635-644). The strains were inoculated into 250 mL of M9 medium supplemented with 2 mM MgSO4, 1% glucose, 20 mg thiamin, 5 mg FeCl , 4 mg CaCl 2 and 50 mg levulinic acid. The cultures were grown to an OD 600 between 0.4 and 0.6, at which point IPTG was added to 1 mM, and the cultures were allowed to grow for an additional two hours.
- the cells were harvested and frozen at -80°C.
- the frozen cells were resuspended in 10 ml of buffer 1 (5 mM imidazole, 500 mM NaCl, and 45 mM Tris pH 7.6) and were lysed by sonicating three times for 15 seconds each on setting 8.
- the cellular debris was pelleted by spinning in an SS-34 rotor at 16,000 ⁇ m for 30 minutes.
- the supernatant was removed and spun again at 16,000 ⁇ m for 30 minutes.
- the supernatant was loaded onto a 5 mL nickel column (Novagen), after which the column was washed with 50 mL of buffer 1 (Novagen).
- EpoK was eluted with a gradient from 5 mM to 1M imidazole. Fractions containing EpoK were pooled and dialyzed twice against 1 L of dialysis buffer ( 45 mM Tris pH7.6, 0.2 mM DTT, 0.1 mM EDTA, and 20% glycerol). Aliquots were frozen in liquid nitrogen and stored at -80°C. The protein preparations were greater than 90% pure. The EpoK assay was performed as follows (See Betlach et al, Biochem (1998) 37:14937, inco ⁇ orated herein by reference).
- reactions consisted of 50 mM Tris (pH7.5), 21 ⁇ M spinach ferredoxin, 0.132 units of spinach ferredoxin: NADP + oxidoreductase, 0.8 units of glucose-6-phosphate dehydrogenase, 1.4 mM NADP, and 7.1 mM glucose-6-phosphate, 100 ⁇ M or 200 ⁇ M epothilone D (a generous gift of S.
- epothilone D eluted at 11.6 min and epothilone B at 9.3 min.
- the LC/MS spectra were obtained using an atmosphere pressure chemical ionization source with orifice and ring voltages set at 20 V and 250 V, respectively, at a mass resolution of 1 amu.
- epothilone E shows an [M+H] at m/z 493, with observed fragments at 405 and 304.
- Epothilone B shows an [M+H] at m/z 509, with observed fragments at 491 and 320.
- the reactions containing EpoK and epothilone D contained a compound absent in the control that displayed the same retention time, molecular weight, and mass fragmentation pattern as pure epothilone B.
- an epothilone D concentration of 100 ⁇ M the amino and the carboxy terminal his tagged EpoK was able to convert 82% and 58% to epothilone B, respectively.
- conversion was 44% and 21 %, respectively.
- This Example describes a series of thioesters provided by the invention for production of epothilone derivatives via chemobiosynthesis.
- the DNA sequence of the biosynthetic gene cluster for epothilone from Sorangium cellulosum indicates that priming of the PKS involves a mixture of polyketide and amino acid components. Priming involves loading of the PKS-like portion of the loading domain with malonyl CoA followed by decarboxylation and loading of the module one NRPS with cysteine, then condensation to form enzyme-bound N-acetylcysteine. Cyclization to form a thiazoline is followed by oxidation to form enzyme bound 2-methylthiazole-4-carboxylate, the product of the loading domain and NRPS. Subsequent condensation with methylmalonyl CoA by the ketosynthase of module 2 provides the substrate for module, as shown in the following diagram.
- the present invention provides methods and reagents for chemobiosynthesis to produce epothilone derivatives in a manner similar to that described to make 6-dEB and erythromycin analogs in PCT Pat. Pub. Nos. 99/03986 and 97/02358.
- Two types of feeding substrates are provided: analogs of the NRPS product, and analogs of the module 3 substrate.
- the module 2 substrates are used with PKS enzymes with a mutated NRPS- like domain, and the module 3 substrates are used with PKS enzymes with a mutated KS domain in module 2.
- module 2 substrates (as N-acetyl cysteamine thioesters) for use as substrates for epothilone PKS with modified inactivated NRPS:
- the module 2 substrates are prepared by activation of the corresponding carboxylic acid and treatment with N-acetylcysteamine. Activation methods include formation of the acid chloride, formation of a mixed anhydride, or reaction with a condensing reagent such as a carbodiimide.
- Exemplary module 3 substrates also as NAc thioesters for use as substrates for epothilone PKS with KS2 knockout are:
- Ethyl 2-methyl-3-(3-thienyl)acrylate A mixture of thiophene-3- carboxaldehyde (1.12 g) and (carbethoxyethylidene)triphenylphosphorane (4.3 g) in dry tetrahydrofuran (20 mL) was heated at reflux for 16 hours. The mixture was cooled to ambient temperature and concentrated to dryness under vacuum. The solid residue was suspended in 1 : 1 ether/hexane and filtered to remove triphenylphosphine oxide. The filtrate was filtered through a pad of SiO using 1 : 1 ether/hexane to provide the product (1.78 g, 91%) as a pale yellow oil.
- the above compounds are supplied to cultures of host cells containing a recombinant epothilone PKS of the invention in which either the NRPS or the KS domain of module 2 as appropriate has been inactivated by mutation to prepare the corresponding epothilone derivative of the invention.
- the present invention provides a variety of recombinant Sorangium cellulosum host cells that produce less complex mixtures of epothilones than the naturally occurring epothilone producers as well as host cells that produce epothilone derivatives.
- This Example illustrates the construction of such strains by describing how to make a strain that produce only epothilones C and D without epothilones A and B. To construct this strain, an inactivating mutation is made in epoK.
- plasmid pKOS35-83.5 which contains a Notl fragment harboring the epoK gene
- the kanamycin and bleomycin resistance markers from Tn5 are ligated into the Seal site of the epoK gene to construct pKOS90-55.
- the orientation of the resistance markers is such that transcription initiated at the kanamycin promoter drives expression of genes immediately downstream of epoK. In other words, the mutation should be nonpolar.
- oriT origin of conjugative transfer
- This plasmid can be introduced into SI 7-1 and conjugated into SMP44.
- the transconjugants are selected on phleomycin plates as previously described.
- electroporation of the plasmid can be achieved using conditions described above for Myxococcus xanthus.
- the epo K mutation is constructed in M. xanthus by linearizing plasmid pKOS90-55 and electroporating into M. xanthus. Kanamycin resistant colonies are selected and have a gene replacement of epoK. This strain is infected with Mx9, Mx8, Mx4 tsl8 hft hrm phages to make phage lysates. These lysates are then individually infected into SMP44 and phleomycin resistant colonies are selected. Once the strain is constructed, standard fermentation procedures, as described below, are employed to produce epothilones C and D.
- S42 medium contains tryptone, 0.5 g/L; MgSO 4; 1.5 g/L; HEPES, 12 g/L; agar, 12 g/L, with deionized water.
- the pH of S42 medium is set to 7.4 with KOH.
- baffle flask instead of a baffle flask, use a 250 ml coiled spring flask with a foil cover. Include 2-3 drops of antifoam B, and incubate in a coffin shaker for 7 days at 37°C and 250 RPM. Subculture the entire 50 mL into 500 mL of fresh medium in a baffled narrow necked Fernbach flask with a 38 mm silicone foam closure. Include 0.5 ml of antifoam to the culture. Incubate under the same conditions for 2-3 days. Use at least a 10% inoculum for a bioreactor fermentation. To culture on solid media, the following protocol is used.
- the fermentation can be conducted in a B. Braun Biostat MD-1 5L bioreactor.
- Prepare 4 L of production medium (same as the soymeal medium for the seed culture without HEPES buffer).
- For the sample port be sure that the tubing that will come into contact with the culture broth has a small opening to allow the XAD to pass through into the vial for collecting daily samples. Stir the mixture completely before autoclaving to evenly distribute the components.
- Sorangium cellulosum cells are rod shaped like a pill, with 2 large distinct circular vacuoles at opposite ends of the cell. Length is approximately 5 times that of the width of the cell.
- Samples (50 ⁇ L) are analyzed by HPLC/MS on a system comprised of a Beckman System Gold HPLC and PE Sciex API100LC single quadrapole MS-based detector equipped with an atmospheric pressure chemical ionization source. Ring and orifice voltages are set to 75V and 300V, respectively, and a dual range mass scan from m/z 290-330 and 450-550 is used.
- HPLC conditions Metachem 5 ⁇ ODS-3 Inertsil (4.6 X 150 mm); 100%) H 2 O for 1 min, then to 100% MeCN over 10 min a 1 mL/min. Epothilone A elutes at 0.2 min under these conditions and gives characteristic ions at m/z 494 (M+H), 476 (M+H-H 2 O), 318, and 306.
- Example 8 Epothilone Derivatives as Anti-Cancer Agents
- the novel epothilone derivatives shown below by Formula (1) set forth above are potent anti-cancer agents and can be used for the treatment of patients with various forms of cancer, including but not limited to breast, ovarian, and lung cancers.
- Epothilone derivative compounds A-H differ from epothilone by modifications in the less sensitive portion of the molecule and so possess good biological activity and offer better pharmacokinetic characteristics, having improved lipophilic and steric profiles.
- novel derivatives can be prepared by altering the genes involved in the biosynthesis of epothilone optionally followed by chemical modification.
- the 9-hydroxy- epothilone derivatives prepared by genetic engineering can be used to generate the carbonate derivatives (compound D) by treatment with triphosgene or 1,1 ' carbonyldiimidazole in the presence of a base.
- the 9, 11 -dihydroxy- epothilone derivative upon proper protection of the C-7 hydroxyl group if it is present, yields the carbonate derivatives (compound F).
- a solution of 254 mg epothilone D in 5 mL of methylene chloride is added. It is cooled by an ice bath, and 0.3 mL of triethyl amine is then added. To this solution, 104 mg of triphosgene is added. The ice bath is removed, and the mixture is stirred under nitrogen for 5 hours. The solution is diluted with 20 mL of methylene chloride and washed with dilute sodium bicarbonate solution. The organic solution is dried over magnesium sulfate and filtered. Upon evaporation to dryness, the epothilone D-7, 9 - cyclic carbonate is isolated.
Abstract
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JP2000584057A JP4662635B2 (en) | 1998-11-20 | 1999-11-19 | Recombinant methods and materials for producing epothilone and epothilone derivatives |
CA002350189A CA2350189A1 (en) | 1998-11-20 | 1999-11-19 | Recombinant methods and materials for producing epothilone and epothilone derivatives |
CNB998156787A CN100381566C (en) | 1998-11-20 | 1999-11-19 | Recombinant methods and materials for producing epothilone and eopthilone derivatives |
IL14306999A IL143069A0 (en) | 1998-11-20 | 1999-11-19 | Recombinant methods and materials for producing epothilone and epothilone derivatives |
AU17377/00A AU768220B2 (en) | 1998-11-20 | 1999-11-19 | Recombinant methods and materials for producing epothilone and epothilone derivatives |
NZ511722A NZ511722A (en) | 1998-11-20 | 1999-11-19 | Recombinant methods and materials for producing epothilone and epothilone derivatives |
EP99960500A EP1135470A2 (en) | 1998-11-20 | 1999-11-19 | Recombinant methods and materials for producing epothilone and epothilone derivatives |
US09/560,367 US6410301B1 (en) | 1998-11-20 | 2000-04-28 | Myxococcus host cells for the production of epothilones |
IL143069A IL143069A (en) | 1998-11-20 | 2001-05-10 | Recombinant methods and materials for producing epothilone and epothilone derivatives |
US10/191,694 US7067286B2 (en) | 1998-11-20 | 2002-07-08 | Cystobacterineae host cells containing heterologous PKS genes for the synthesis of polykedtides |
US11/231,735 US20060223151A1 (en) | 1998-11-20 | 2005-09-20 | Production of polyketides |
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WO2020089310A1 (en) | 2018-10-31 | 2020-05-07 | Bio Even | Flavin adenine dinucleotide (fad) for use in the prevention and/or treatment of cancer |
Also Published As
Publication number | Publication date |
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KR20010093103A (en) | 2001-10-27 |
JP4662635B2 (en) | 2011-03-30 |
AU768220B2 (en) | 2003-12-04 |
IL143069A0 (en) | 2002-04-21 |
US6583290B1 (en) | 2003-06-24 |
US6858411B1 (en) | 2005-02-22 |
CN1333820A (en) | 2002-01-30 |
KR100851418B1 (en) | 2008-08-08 |
CA2350189A1 (en) | 2000-06-02 |
EP1135470A2 (en) | 2001-09-26 |
US7129071B1 (en) | 2006-10-31 |
US7402421B2 (en) | 2008-07-22 |
KR20070087132A (en) | 2007-08-27 |
KR100716272B1 (en) | 2007-05-09 |
AU1737700A (en) | 2000-06-13 |
US6921650B1 (en) | 2005-07-26 |
CN100381566C (en) | 2008-04-16 |
IL143069A (en) | 2008-06-05 |
US7732186B2 (en) | 2010-06-08 |
US6303342B1 (en) | 2001-10-16 |
KR20060124789A (en) | 2006-12-05 |
US20090170172A1 (en) | 2009-07-02 |
NZ511722A (en) | 2004-05-28 |
WO2000031247A3 (en) | 2000-12-07 |
JP2007097595A (en) | 2007-04-19 |
US20040253697A1 (en) | 2004-12-16 |
JP2002530107A (en) | 2002-09-17 |
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