DE102007027006A1 - Microbiological production of aldehydes, in particular of 3-hydroxypropionaldehyde - Google Patents

Abstract

The present invention relates to a cell which is capable of forming corrinoids and which has been genetically engineered with respect to its wild type in such a way that it contains more aldehydes of diols, triols or polyols, preferably more glycerol 3-hydroxypropionaldehyde, compared to its wild type. to be able to form. The invention also relates to a process for the production of a genetically modified cell, the genetically modified cell obtainable by these processes, processes for the preparation of aldehydes, preferably of 3-hydroxypropionaldehyde, and a process for the preparation of C <SUB> 3 </ SUB> - Compounds of 3-hydroxypropionaldehyde.

Description

The present invention relates to a genetically modified cell, a method for producing a genetically modified cell, the genetically modified cell obtainable by these methods, methods for producing aldehydes, in particular 3-hydroxypropionaldehyde, and a method for producing C ₃ compounds from 3-hydroxypropionaldehyde.

3-hydroxypropionaldehyde is a C ₃ compound which, by further oxidation, reduction or dehydration, be it biocatalytically or chemically-catalytically, is converted into the building blocks of 3-hydroxypropionic acid, 1,3-propanediol and acrolein, which are important for the chemical industry For example, acrolein is an important intermediate for the production of methionine (for use as a pharmaceutical amino acid or feed additive), acrylic acid (monomer of the superabsorbent polyacrylic acid) or methyl acrylates (monomers for high performance polymers).

It is already known from the prior art that 3-hydroxypropionaldehyde can be prepared efficiently by biotransformation from glycerol. The responsible enzyme is coenzyme B ₁₂ -dependent glycerol dehydratase, which was detected in a number of Gram-positive and Gram-negative bacteria. Among other things, 3-hydroxypropionaldehyde can be produced by biotransformation of glycerol with Lactobacillus reuteri cells (see for example Doleyres et al., Applied Microbiology and Biotechnology, Vol. 68 (4), 2005, pp. 467-474; Vollenweider and Lacroix in Applied Microbiology and Biotechnology, Vol. 64 (1), 2004, pages 16-27; Lüthi-Peng et al. In Applied Microbiology and Biotechnology, Vol. 60 (1-2), 2002, pages 73-80. )

The preparation of 3-hydroxypropionaldehyde with Lactobacillus reuteri has at least two serious disadvantages. Firstly, Lactobacillus reuteri is able to convert the formed 3-hydroxypropionaldehyde partially into 1,3-propanediol (with the aid of the enzyme 1,3-propanediol oxidoreductase or 1,3-propanediol dehydrogenase) ( See, for example, Vollenweider and Lacroix in Applied Microbiology and Biotechnology, Vol. 64 (1), 2004, pages 16-27) and 3-hydroxypropionic acid (using the enzyme 3-hydroxypropionaldehyde dehydrogenase) (see, for example, Chen et al. in Journal of Biomedical Materials Research, Vol. 61, 2002, pp. 360-369 ), thereby reducing the 3-hydroxypropionaldehyde yield. These by-products may further complicate the work-up of the 3-hydroxypropionaldehyde formed. On the other hand, Lactobacillus reuteri, like other lactobacilli, has a low biomass yield relative to the carbon source used. The cause is the inability of these cells to use oxygen as a terminal electron acceptor. Significant quantities of reduced fermentation products, such as lactic acid, are therefore formed. This formation of lactic acid in turn inhibits the growth of the cells and leads to a reduction of the carbon source-related biomass yield and to increased costs for the production of the biomass and thus of the biocatalyst.

In addition to Lactobacillus reuteri, other host cells, such as Klebsiella pneumoniae, Citrobacter freundii or Enterobacter agglomerans, for the fermentative production of 3-hydroxypropionaldehyde were used ( see, for example, Slininger et al. in Applied Environmental Microbiology, Vol. 46, 1983, pages 62-67, and for review Vollenweider and Lacroix in Applied Microbiology and Biotechnology, Vol. 64 (1), 2004, pages 16-27 ). The disadvantage of the use of such microorganisms for the production of 3-hydroxypropionaldehyde, however, is that due to the bactericidal effect of the microorganisms bactericidal activity of the resulting 3-hydroxypropionaldehyde semicarbazide must be added that by reaction with 3-hydroxypropionaldehyde to the corresponding semicarbazone 3-hydroxypropionaldehyde removed from the nutrient medium. In the absence of semicarbazide, only small amounts of 3-hydroxypropionaldehyde are formed. ( See, for example, Vollenweider and Lacroix in Applied Microbiology and Biotechnologist, Vol. 64 (1), 2004, pages 16-27 ). However, the addition of semicarbazide would be a significant cost factor in the case of large-scale application of the technology and would complicate the further work-up of the 3-hydroxypropionaldehyde.

Finally, recombinant cells capable of producing 3-hydroxypropionaldehyde have also been described (see for example EP-A-1 669 457 ). However, coenzyme B _{12 had to be} added here, that the enzyme glycerol dehydratase is needed as cofactor due to the radical reaction mechanism, but can not be formed by the cells used itself. Due to the high price of coenzyme B ₁₂ , this technology is unsuitable for large-scale application.

Of the Present invention was based on the object resulting from the Overcome prior art disadvantages.

In particular, the present invention has the object to provide cells which are even more efficient than the cells known from the prior art capable of producing aldehydes from suitable carbon compounds, but in particular 3-hydroxypropionaldehyde from glycerol. In this case, various C ₃ compounds, so-called by-products, should be produced from the glycerol to the smallest possible extent of 3-hydroxypropionaldehyde. The cells used for this purpose should be able to automatically produce the coenzyme B ₁₂ required by the enzyme glycerol dehydratase.

Farther the present invention was based on the object, a method for the preparation of aldehydes, in particular of 3-hydroxypropionaldehyde, to specify by means of which these aldehydes directly and in particular without prior reaction with reactive compounds, such as semicarbazide, can be produced.

a Contribute to the solution of the above mentioned tasks a cell capable of forming corrinoids and those opposite their wild type has been genetically engineered such that they compared to their wild type more aldehydes from diols, for example from 1,2-propanediol, triols or polyols, for example D-galactonate or D-mannonate, but preferably more 3-hydroxypropionaldehyde from glycerol, is able to form. By the ability of Cell to form coral itself, this is capable of Aldehydes, preferably 3-hydroxypropionaldehyde, also in a culture medium to form, which contains no Corrionide

Completely surprisingly, but no less advantageous, it has been found that genetically engineered cells capable of producing coenzyme B ₁₂ are suitable for efficiently producing, for example, 3-hydroxypropionaldehyde from glycerol. This was by no means foreseeable for a person skilled in the art since it was known from the prior art that 3-hydroxypropionaldehyde does not adapt to numerous microorganisms whose wild-type naturally does not form 3-hydroxypropionaldehyde and which, in contrast to Lactobacillus reuteri, does not adapt to the formation of this compound are bacteriostatic or bacteriocidal ( See, for example, Chen et al., Journal of Biomedical Materials Research, Vol. 61 (3), 2002, pp. 360-369 ). The use of preferably resting, genetically engineered cells capable of producing coenzyme B ₁₂ allows efficient fermentative production of 3-hydroxypropionaldehyde without the need to add reactive compounds such as semicarbazide to reduce its bacteriocidal or bacteriostatic effect.

A "corrionoid" is understood to mean a heterocyclic ring system which is related to the porphyrin ring in hemoglobin The ring system of the corrinoid consists of four reduced pyrrole subunits (tetrapyrrole), which on two opposite sides each have a -C (CH ₃ ) = Methine bridge, directly connected on one side via a -CH = methine bridge and on the last side Corrionoids which are preferred according to the invention are in particular vitamin B ₁₂ , coenzyme B ₁₂ and cobalamin.

Under "dormant Cells "within the meaning of the invention are understood to be cells which to be in a state in which the ability to divide cells due to a lack of metabolites required for division restricted, preferably not given at all is.

Under a "wild type" of a cell is preferably the one Cell whose genome is in a state like that naturally originated by evolution. Of the Term is used both for the entire cell and for single genes used. The term "wild-type" therefore falls in particular not such cells or genes whose gene sequences at least in part by humans using recombinant techniques have been changed.

As used herein, the term "3-hydroxypropionaldehyde" describes the corresponding compound in the form in which it is present in the nutrient medium after production by the cells The term "3-hydroxypropionaldehyde" therefore particularly includes the free monomer of FIG Hydroxypropionaldehydes (C ₃ O ₂ H ₆ ), the hydrate of 3-hydroxypropionaldehyde (C ₃ O ₃ H ₈ ), the cyclized dimer of 3-hydroxypropionaldehyde (C ₆ O ₄ H ₁₂ ), oligomers of 3-hydroxypropionaldehyde, as well as the "Reuterin" known mixture of the monomer, the hydrate of 3-hydroxypropionaldehyde and the cyclized dimer of 3-hydroxypropionaldehyde.

The formulation "that it is able to form more aldehydes from diols, triols or polyols, preferably more glycerol 3-hydroxypropionaldehyde compared to its wild type" also applies to the case that the wild-type of the genetically modified cell has no aldehydes or no 3-hydroxypropionaldehyde, but at least no detectable amounts of these compounds can form and detectable amounts of this compound can be formed only after the genetic modification.

Farther the phrase "more aldehydes from diols, triols or polyols, preferably more 3-hydroxypropionaldehyde from glycerol " clarify that the cells are the aldehydes or 3-hydroxypropionaldehyde directly from those offered via the nutrient medium Diols, triols or polyols, preferably from glycerol produce can, or that the cells have other carbon compounds, such as glucose, sucrose or other mono- and polysaccharides, first to diols, triols or polyols, preferably to Glycerol, and then these diols, triols or polyols, preferably the glycerol, then added the corresponding aldehydes, preferably to 3-hydroxypropionaldehyde, can implement.

In this connection, it is particularly preferred that the genetically engineered cell is genetically engineered such that it does so within a defined time interval, preferably within 2 hours, more preferably within 8 hours, and most preferably within 24 hours, at least 2 times , more preferably at least 10 times, more preferably at least 100 times, even more preferably at least 1,000 times, and most preferably at least 10,000 times more aldehydes, preferably more 3-hydroxypropionaldehyde than the wild type of the cell. The increase in product formation can be determined, for example, by culturing the cell according to the invention and the wild-type cell separately under the same conditions (same cell density, same nutrient medium, same culture conditions) for a specific time interval in a suitable nutrient medium and then the amount Target product (aldehyde, preferably monomer of 3-hydroxypropionaldehyde, hydrate of 3-hydroxypropionaldehyde, cyclized dimer of 3-hydroxypropionaldehyde, oligomer of 3-hydroxypropionaldehyde or reuterin) is determined in the nutrient medium. Methods for determining the amount of these compounds in a nutrient medium by GC-MS are, for example, by Chen et al. in the Journal of Biomedical Materials Research, Vol. 61 (3), 2002, pages 360-369 ,

The cells according to the invention can in principle be derived from all cells whose wild-type is already able to form corrinoids, preferably vitamin B ₁₂ , coenzyme B ₁₂ and cobalamin. However, it is also conceivable to use cells whose wild-type is not or is insufficiently capable of producing corrinoids, but which have been modified by recombinant techniques in such a way that they can form sufficient amounts of corrinoids.

According to a first particular embodiment of the cells according to the invention, the cells are recombinant cells of the genus Bacillus, in particular those of the species Bacillus megaterium. Bacillus megaterium is a Gram-positive, immobile, rod-shaped bacterium having a size of about 2 × 5 μm. The bacterium grows under aerobic conditions, is oxidase-negative and catalase-positive. Like all bacteria of the genus Bacillus, Bacillus megaterium is endospore-producing. For example, cells of the species Bacillus megaterium are available under the accession numbers DSM 32 ^T , DSM 90, DSM 319, DSM 321, DSM 322, DSM 333, DSM 337, DSM 339, DSM 344, DSM 509, DSM 510, DSM 786, DSM 1517, DSM 1668, DSM 1669, DSM 1670, DSM 1671, DSM 1804, DSM 2894, DSM 3228 and DSM 3641 deposited with the German Collection of Microorganisms and Cell Cultures GmbH.

According to one second particular embodiment of the invention Cells are the cells are recombinant cells of Genus Escherichia, in particular the species Escherichia coli or of the species Escherichia blattae. Escherichia blattae counts to the Enterobacteriaceae family and also grows under aerobic conditions. Cells of the species Escherichia blattae are included, for example, under the accession numbers DSM 4481 the German Collection of Microorganisms and Cell Cultures GmbH deposited.

It is inventively preferred that the cell according to the invention according to the first and also according to the second particular embodiment has a ge compared to their wild-type increased activity of an enzyme E ₁ , which catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde.

The following statements on increasing the enzyme activity in cells apply both to the increase in the activity of the enzyme E ₁ and to all the enzymes mentioned below, the activity of which may optionally be increased.

Basically, an increase in enzymatic activity can be achieved by increasing the copy number of the gene sequence (s) encoding the enzyme, using a strong promoter, or using a gene or allele encoding a corresponding enzyme with enhanced activity and, where appropriate, combining these measures. Genetically modified according to the invention The cells are generated, for example, by transformation, transduction, conjugation or a combination of these methods with a vector which contains the desired gene, an allele of this gene or parts thereof and a vector which enables the expression of the gene. In particular, heterologous expression is achieved by integration of the gene or alleles into the chromosome of the cell or an extrachromosomally replicating vector.

An overview of the possibilities for increasing the enzyme activity in cells using the example of pyruvate carboxylase gives DE-A-100 31 999 , which is hereby incorporated by reference, and the disclosure of which relates to the potential for increasing enzyme activity in cells forms part of the disclosure of the present invention.

The expression of the above and all of the enzymes or genes mentioned below can be detected in the gel with the aid of 1- and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration with appropriate evaluation software. If the increase in enzyme activity is based solely on an increase in the expression of the corresponding gene, the quantification of the increase in enzyme activity can be determined in a simple manner by comparing the 1- or 2-dimensional protein separations between wild type and genetically modified cell. A common method for preparing the protein gels in coryneform bacteria and for identifying the proteins is that described by Hermann et al. in Electrophoresis, Volume 22: 1712-23 (2001 ) procedure described. The protein concentration can also be determined by Western blot hybridization with an antibody specific for the protein to be detected ( Sambrook et al., Molecular Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989 ) and subsequent optical evaluation with appropriate software for concentration determination ( Lohaus and Meyer (1989) Biospektrum, 5: 32-39; Lottspeich (1999), Angewandte Chemie 111: 2630-2647 ) to be analyzed. The activity of DNA-binding proteins can be monitored by DNA band-shift assays ( also referred to as gel retardation) (Wilson et al., (2001), Journal of Bacteriology, 183: 2151-2155 ). The effect of DNA binding proteins on the expression of other genes can be demonstrated by various well-described methods of the reporter gene assay ( Sambrook et al., Molecular Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989 ). The intracellular enzymatic activities can be determined by various methods described ( Donahue et al. (2000) Journal of Bacteriology 182 (19): 5624-5627; Ray et al. (2000) Journal of Bacteriology 182 (8): 2277-2284; Freedberg et al. (1973) Journal of Bacteriology 115 (3): 816-823 ). Unless specific methods for the determination of the activity of a particular enzyme are specified in the following, the determination of the increase in the enzyme activity and also the determination of the reduction of an enzyme activity is preferably carried out by means of in Hermann et al., Electrophoresis, 22: 1712-23 (2001 ) Lohaus et al., Biospektrum 5 32-39 (1998), Lottspeich, Angewandte Chemie 111: 2630-2647 (1999) and Wilson et al., Journal of Bacteriology 183: 2151-2155 (2001 ) methods described.

Becomes the increase of enzyme activity by mutation of the endogenous gene, such Mutations generated either undirected by classical methods be such as by UV irradiation or by mutagenic Chemicals, or specifically by means of genetic engineering methods such as Deletion (s), insertion (s) and / or nucleotide exchange (s). By These mutations will be genetically engineered cells receive. Particularly preferred mutants of enzymes are in particular even those enzymes that are no longer or at least compared to Wild-type enzyme reduces feedback-inhibitable.

If the increase in the enzyme activity is accomplished by increasing the expression of an enzyme, for example, the copy number of the corresponding genes is increased or the promoter and regulation region or the ribosome binding site located upstream of the structural gene is mutated. In the same way, expression cassettes act, which are installed upstream of the structural gene. Inducible promoters also make it possible to increase expression at any time. Furthermore, the enzyme gene can also be assigned as regulatory sequences but also what are termed "enhancers" which likewise bring about increased gene expression via an improved interaction between RNA polymerase and DNA. Measures to extend the lifetime of mRNA also improve expression. Furthermore, by preventing degradation of the enzyme protein, enzyme activity is also enhanced. The genes or gene constructs are either present in plasmids with different copy numbers or are integrated and amplified in the chromosome. Alternatively, overexpression of the genes in question can be achieved by changing the composition of the medium and culture. For guidance, the skilled artisan will find, inter alia, Martin et al. (Bio / Technology 5, 137-146 (1987)), Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), Eikmanns et al. (Gene 102, 93-98 (1991)), in EP-A-0 472 869 , in the US 4,601,893 , Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991), Reinscheid et al .. (Applied and Environmental Microbiology 60, 126-132 (1994)), LaBarre et al .. (Journal of Bacteriology 175 , 1001-1007 (1993)), in WO-A-96/15246 in Malumbres et al. (Gene 134, 15-24 (1993), in JP-A-10-229891 , in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998) and in well-known textbooks of genetics and molecular biology.) The measures described above lead as well as the mutations to genetically modified cells.

To increase the expression of the respective genes, episomal plasmids are used, for example. Suitable plasmids are in particular those which are replicated in coryneform bacteria. Numerous known plasmid vectors, such as pZ1 (Menkel et al., Applied and Environmental Microbiology 64: 549-554 (1989)), pEKEx1 (Eikmanns et al., Gene 107: 69-74 (1991)) or pHS2- 1 (Sonnen et al., Gene 107: 69-74 (1991)) are based on the cryptic plasmids pHM1519, pBLI or pGAl. Other plasmid vectors, such as those based on pCG4 ( US 4,489,160 ) or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66: 119-124 (1990)), pAG1 ( US 5,158,891 ) or pWH1520 (Rygus and Rillen, Applied Microbiology and Biotechnology 35: 594-599 (1991)) can be used in the same way.

Also suitable are those plasmid vectors by means of which one can apply the method of gene amplification by integration into the chromosome, as it is, for example, of Reinscheid et al. (Applied and Environmental Microbiology 60: 126-132 (1994) ) has been described for duplication or amplification of the hom-thrB operon. In this method, the complete gene is cloned into a plasmid vector which is expressed in a host ( typically Escherichia coli), but can not be replicated in Corynebacterium glutamicum. Examples of vectors which are used are pSUP301 (Simon et al., Bio / Technology 1: 784-791 (1983). ), pK18mob or pK19mob ( Schäfer et al., Gene 145: 69-73 (1994) ), pGEM-T (Promega Corporation, Madison, Wisconius, USA), pCR2.1-TOPO (Shuman, Journal of Biological Chemistry 269: 32678-84 (1994) ), pCR® Blunt (Invitrogen, Groningen, Netherlands), pEMi ( Schrumpf et al., Journal of Bacteriology 173: 4510-4516) ) or pBGS8 (Spratt et al., Gene 41: 337-342 (1986) ) in question. The plasmid vector containing the gene to be amplified is then converted by conjugation or transformation into the desired strain of Corynebacterium glutamicum. The Method of conjugation is described, for example, in Schäfer et al., Applied and Environmental Microbiology 60: 756-759 (1994). Methods for transformation are described, for example, in Thierbach et al., Applied Microbiology and Biotechnology 29: 356-362 (1988), Dunican and Shivnan, Bio / Technology 7: 1067-1070 (1989), and Tauch et al., FEMS Microbiology Letters 123: 343-347 (1994) described. After homologous recombination by means of a cross-over event, the resulting strain contains at least two copies of the gene of interest.

The term "an activity of an enzyme Ex" which is increased in relation to its wild-type is preferably always one more than a factor of at least 2, more preferably at least 10, more preferably at least 100, and even more preferably of at least 1000 and most preferably of at least 10,000 increased activity of the respective enzyme E _x Furthermore, the cell according to the invention comprises "an increased activity of an enzyme E _x " compared to its wild type, in particular also a cell whose wild type is none or at least no detectable activity of this enzyme E _x and only after increasing the enzyme activity, for example by overexpression, a detectable activity of this enzyme Ex shows. In this context, the term "overexpression" or the expression "increase in expression" used in the following also encompasses the case that a starting cell, for example a wild-type cell, has no or at least no detectable expression and only by recombinant methods a detectable Expression of the gene coding for the enzyme ex is induced.

Under the term "reduced activity of an enzyme Ex "is accordingly preferably one by one Factor of at least 0.5, more preferably at least 0.1, moreover, preferably at least 0.01, above more preferably still at least 0.001 and most preferably understood of at least 0.0001 decreased activity. The reduction of the activity of a certain enzyme can, for example, by targeted mutation, by adding competitive or non-competitive inhibitors or by others skilled in the art Known measures to reduce the expression of a carried out for a particular enzyme encoding gene.

The enzyme E ₁ , which catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde, is preferably a glycerol dehydratase or a diol dehydratase (EC 4.2.1.30). The present invention therefore relates in particular to cells which are capable of forming corrinoids and in de the activity of glycerol dehydratase or diol dehydratase has been increased. This enzyme is preferably encoded by the genes selected from the group consisting of pduC, pduD, pduE, pddA, pddB, pddC, dhaB, dhaC, dhaE and the gldABC genes from Lactobacillus reuteri ATCC55730. Another advantage is the use of in WO-A-2004/056963 described glycerol dehydratase variant SHGDH22, which the in WO-A-2004/056963 disclosed SEQ ID NO. 322 has. The nucleotide sequence of these and other suitable genes for a glycerol dehydratase can be found for example in the "Kyoto Encyclopedia of Genes and Genomes" (KEGG database), the National Library of Biotechnology Information (NCBI) databases of the National Library of Medicine (Bethesda, MD. Furthermore, it may be advantageous according to the invention, in particular the glycerol dehydratase from the genera Klebsiella, Citrobacter, Clostridium, Lactobacillus, Enterobacter, Caloramator, Salmonella and Listeria, to be particularly advantageous in the cells preferred glycerol dehydratase from the species Klebsiella pneumoniae, Citrobacter pneumoniae, Clostridium pasteurianum, Lactobacillus leichmannii, Citrobacter intermedium, Lactobacillus reuteri, Lactobacillus buchneri, Lactobacillus brevis, Enterobacter agglomerans, Clostridium pasteurianum, Clostridium perfringens, Clostridium kluyveri, Caloramator vi terbensis, Lactobacillus collinoides, Lactobacillus hilgardii, Salmonella typhimurium, Listeria monocytogenes and Listeria innocua, wherein the expression of glycerol dehydratase from the cells of the species Lactobacillus reuteri in Bacillus megaterium, Escherichia coli or Escherichia blattae cells is most preferred , Particularly preferred is therefore a Bacillus megaterium, Escherichia coli or Escherichia blattae cell in which a glycerol dehydratase is expressed, which is encoded by the gene for the glycerol dehydratase from Lactobacillus reuteri. In connection with the diol dehydratase, it may be advantageous, in particular the diol dehydratase in the cells of the genera Klebsiella, Propionibacterium, Clostridium, Lactobacillus, Salmonella, Citrobacter, Flavobacterium, Acetobacterium, Brucella and Fusobacterium, particularly preferably from the species Klebsiella pneumoniae , Propionibacterium freudenreichii, Clostridium glycolicum, Lactobacillus brevis, Salmonella typhimurium, Citrobacter freundii, Lactobacillus buchneri, Brucella melitensis, Fusobacterium nucleatum, Klebsiella oxytoca, Salmonella typhimurium, Listeria monocytogenes and Listeria innocua.

Furthermore, it is preferred according to the invention that in addition to the activity of the enzyme E ₁ , the activity of an enzyme E ₂ which catalyzes the reactivation of inactivated enzyme E ₁ is increased in the cell, whereby this enzyme E _{2 is} preferably glycerol. Dehydratase reactivation factor or a diol dehydratase reactivation factor. The glycerol dehydratase reactivating factor or diol dehydratase reactivating factor is a factor capable of reactivating the inactivated center of the dehydratase after the reaction of glycerol to 3-hydroxypropionaldehyde is carried out. The active center of the glycerol dehydratase or the diol dehydratase comprises the coenzyme B ₁₂ , which is inactivated after the conversion of glycerol to 3-hydroxypropionaldehyde. The glycerol dehydratase reactivation factor or the diol dehydratase reactivation factor are able to replace this inactivated coenzyme B ₁₂ by an active coenzyme B ₁₂ . Preferably, the dehydratase reactivation factor comprises a large subunit of the glycerol dehydratase reactivation factor or the diol dehydratase reactivation factor and a small subunit of the glycerol dehydratase reactivation factor or the diol dehydratase reactivation factor, most preferably a large subunit of the glycerol dehydratase. Reactivation factor and a small subunit of the glycerol dehydratase reactivation factor or the diol dehydratase reactivation factor, most preferably a glycerol dehydratase reactivation factor and a small subunit of the glycerol dehydratase reactivation factor. Furthermore, for example, the large subunit of the diol dehydratase reactivation factor can be encoded by the ddrA gene and the large subunit of the glycerol dehydratase reactivation factor by the gdrA gene, while the small subunit of the dihydrogen dehydratase reactivation factor is encoded by the ddrB gene and the small subunit Subunit of the glycerol dehydratase reactivation factor from the gdrB gene can be encoded. Especially advantageous is the expression of the gldDE genes from Lactobacillus reuteri ATCC55730 in the cells according to the invention. For example, in the cells of the invention, the glycerol dehydratase reactivation factor can be expressed from cells of the genus Lactobacillus, Klebsiella, Citrobacter, Clostridium and Enterobacter, in particular from cells of the species Lactobacillus sp., Klebsiella pneumoniae, Lactobacillus leichmannii, Citrobacter freundii, Citrobacter intermedium, Lactobacillus reuteri, Lactobacillus buchneri, Lactobacillus brevis, Clostridium pasteurianum, Enterobacter agglomerans and Clostridium kluyveri and most preferably from cells of the species Lactobacillus reuteri. The diol dehydratase reactivation factor preferably originates from cells of the genus Klebsiella, Citrobacter, Propionibacterium, Lactobacillus, Flavobacterium and Acetobacterium, in particular from the species Klebsiella pneumoniae, Citrobacter freundii, Propionibacterium freudenreichii, Lactobacillus brevis, Lactobacillus buchneri, Flavobacterium sp. and Acetobacterium sp., wherein the diol dehydratase reactivation factor from the genus Lactobacillus, in particular from the species Lactobacillus brevis and Lactobacillus buchneri is most preferred.

Farther can in the case of expression of dehydratase and the Dehydratase reactivation factor in the inventive Cells these two components from the same species or else come from different species.

Furthermore, it is inventively preferred that in addition to the activity of the enzyme E ₁ and optionally also the activity of the enzyme E ₂ in the cell, the activity of an enzyme E _{3 is} increased, which facilitates the diffusion of glycerol into the cell. By doing so, the ability of the cell to take up glycerol provided by the nutrient medium and to convert it intracellularly to 3-hydroxypropionaldehyde can be improved. Preferably, the enzyme E ₃ is a glycerol channel or a glycerol facilitator (TC 1.A.8.1.1, TC 1.A.8.2.1 or TC 1.A.8.2.2), the preferably encoded by the glpF gene.

• – eines Enzyms E₄, welches die Umsetzung von Dihydroxyaceton-Phosphat zu Glycerin-3-Phosphat katalysiert;
• – eines Enzyms E₅, welches die Umsetzung von Glycerin-3-Phosphat zu Glycerin katalysiert;
• – eines Enzyms E₆, welches die Umsetzung von Dihydroxyaceton-Phosphat zu Dihydroxyaceton katalysiert;
• – eines Enzyms E₇, welches die Umsetzung von Dihydroxyaceton zu Glycerin katalysiert.

In the case of a cell according to the invention which can produce 3-hydroxypropionaldehyde from glycerol, it is basically also conceivable to modify the cell according to the invention by recombinant measures in such a way that it can not only take up glycerol provided by the nutrient medium and convert it intracellularly into 3-hydroxypropionaldehyde, but that they may also take on the nutrient medium provided glucose, sucrose or other mono- and polysaccharides, intracellularly convert to glycerol and then convert the glycerol intracellularly to 3-hydroxypropionaldehyde. Alternatively, the glycerol formed in the meantime may also accumulate extracellularly before being re-introduced into the cell and intracellularly converted to 3-hydroxypropionaldehyde. In this context, it may be particularly advantageous if in addition to the activity of the enzyme E ₁ and optionally of the enzymes E ₂ and E ₃ , the activity of at least one of the enzymes E ₄ , E ₅ , E ₆ or E _{7 is} increased in the cells:

• An enzyme E ₄ which catalyzes the conversion of dihydroxyacetone phosphate to glycerol 3-phosphate;
• - An enzyme E ₅ , which catalyzes the conversion of glycerol-3-phosphate to glycerol;
• An enzyme E ₆ which catalyzes the conversion of dihydroxyacetone phosphate to dihydroxyacetone;
• - An enzyme E ₇ , which catalyzes the conversion of dihydroxyacetone to glycerol.

Preferred cells are in particular those cells in which the activity of the following enzymes or combination of enzymes is increased: E ₁ E ₄ , E ₁ E ₅ , E ₁ E ₆ , E ₁ E ₇ , E ₁ E ₄ E ₅ , E ₁ E ₄ E ₆ , E ₁ E ₄ E ₇ , E ₁ E ₅ E ₆ , E ₁ E ₅ E ₇ , E ₁ E ₆ E ₇ , E ₁ E ₂ E ₄ , E ₁ E ₂ E ₅ , E ₁ E ₂ E ₆ , E ₁ E ₂ E ₇ and E ₁ E ₂ E ₄ E ₅ E ₆ E ₇ , the combination E ₁ E ₂ E ₄ E ₅ E ₆ E _{7 being} most preferred.

In this context, it is further preferred that the enzyme
E _{4 is} a glycerol-3-phosphate dehydrogenase (EC 1.1.1.8),
E _{5 is} a glycerol-3-phosphate phosphatase (EC 3.1.3.21),
E ₆ dihydroxyacetone phosphate phosphatase (EC 3.1.3.1), and
E ₇ a glycerol dehydrogenase (EC 1.1.1.6)
is.

The Glycerol-3-phosphate dehydrogenase is preferably selected from genes from the group comprising Gpo1, Gpd2, LOC607942, Gpdh and gpsA. The glycerol-3-phosphate phosphatase is preferably of the corresponding Gene of this enzyme from Escherichia coli encoded. The dihydroxyacetone phosphate phosphatase is preferably selected from genes selected from the group comprising phoA, ALPI, ALPL, ALPP, ALPPL2, Akp2, Akp3 and Akp5 encoded during the glycerol dehydrogenase is preferably encoded by the gldA gene.

The nucleotide sequences of other suitable genes of the enzymes E ₄ to E ₇ can be found in the KEGG database, the NCBI database or EMBL database.

• – eines Enzyms E₈, welches die Umsetzung von Glycerin zu Glycerol-3-Phosphat katalysiert;
• – eines Enzyms E₉, welches die Umsetzung von Glycerin zu Dihydroxyaceton katalysiert;
• – eines Enzyms E₁₀, welches die Umsetzung von 3-Hydroxypropionaldehyd zu 1,3-Propandiol katalysiert;
• – eines Enzyms E₁₁, welches die Umsetzung von 3-Hydroxypropionaldehyd zu 3-Hydroxypropionsäure katalysiert.

Furthermore, in connection with the cell according to the invention, it may also be advantageous if, in addition to increasing the enzyme E ₁ , increasing the enzyme E ₁ and E ₂ , increasing the enzymes E ₁ , E ₂ and one or more of the E ₃ until E _{7 has} a reduced activity of at least one of the enzymes E ₆ to E ₁₁ :

• - An enzyme E ₈ , which catalyzes the conversion of glycerol to glycerol-3-phosphate;
• An enzyme E ₉ which catalyzes the conversion of glycerol to dihydroxyacetone;
• An enzyme E ₁₀ which catalyzes the conversion of 3-hydroxypropionaldehyde to 1,3-propanediol;
• - An enzyme E ₁₁ , which catalyzes the conversion of 3-hydroxypropionaldehyde to 3-hydroxypropionic acid.

Particularly preferred cells are those cells in which the activity of the following enzymes or combination of enzymes is reduced: E ₈ , E ₉ , E ₁₀ , E ₁₁ , E ₈ E ₉ , E ₈ E ₁₀ , E ₈ E ₁₁ , E ₉ E ₁₀ , E ₉ E ₁₁ , E ₁₀ E ₁₁ and E ₈ E ₉ E ₁₀ E ₁₁ , the combination E ₈ E ₉ E ₁₀ E _{11 being} most preferred.

In this connection it is particularly preferred that the enzyme
E _{8 is} a glycerol kinase (EC 2.7.1.30),
E _{9 is} a glycerol dehydrogenase (EC 1.1.1.6 or EC 1.1.1.156 or EC 1.1.99.22) and
E ₁₀ a 1,3-propanediol dehydrogenase (EC 1.1.1.202)
E _{11 is} a 3-hydroxypropionaldehyde dehydrogenase (EC 1.2.1.3 or EC 1.2.1.5)
is.

A contribution to the solution of the above-mentioned objects is further provided by a method for producing a genetically modified cell which is capable of forming corrionoids, comprising the step of increasing the activity of an enzyme E ₁ which inhibits the intracellular conversion of diols, triols or polyols to aldehydes, preferably from glycerol to 3-hydroxypropionaldehyde. Preferred enzymes E ₁ are in particular those which have already been mentioned above in connection with the cells according to the invention.

Furthermore, according to a particular embodiment of the method according to the invention, it is preferred that the method additionally comprises the step of increasing the activity of an enzyme E ₂ , which catalyzes the reactivation of inactivated enzyme E ₁ , and optionally also increasing an enzyme E ₄ , which the Reaction of dihydroxyacetone phosphate catalyzed to glycerol-3-phosphate, and / or an enzyme E ₅ , which catalyzes the conversion of glycerol-3-phosphate to glycerol, and / or an enzyme E ₆ , which is the reaction of dihydroxyacetone phosphate Dihydroxyacetone catalyzes, and / or an enzyme E ₇ , which catalyzes the conversion of dihydroxyacetone to glycerol, wherein preferred enzymes E ₂ , E ₄ , E ₅ , E ₆ and E _{7 are} again those already mentioned in the beginning in connection with the Cell according to the invention have been mentioned.

Furthermore, the process according to the invention for the production of a genetically modified cell can also comprise the process step of reducing the activity of one or more of the enzymes E ₈ to E ₁₁ mentioned in connection with the cell according to the invention.

If the cells according to the invention can produce aldehydes from diols, for example propionaldehyde from 1,2-propanediol, it is preferred for the cell according to the invention to have an increased activity of an enzyme E ₁₂ which converts 1,2-diols into the corresponding ones Aldehydes catalyzes, wherein this enzyme E _{12 is} preferably a diol dehydratase. If the cells according to the invention can produce aldehydes from polyols, for example 2-dehydro-3-deoxy-D-galactonate from D-galactonate or 2-dehydro-3-deoxy-D-gluconate from D-mannonate, it is preferred that the inventive Cell has an increased activity of an enzyme E ₁₃ , which catalyzes the conversion of polyols to the corresponding aldehydes, wherein this enzyme E _{13 is} preferably a D-galactonate dehydratase or a D-mannonate dehydratase.

a Contribute to the solution of the above-mentioned tasks furthermore the one obtainable by the method described above Cell.

• i) in Kontakt bringen einer erfindungsgemäßen Zelle oder einer durch das erfindungsgemäße Verfahren erhältlichen Zelle mit einem Kulturmedium, welches die Bildung von Biomasse ermöglicht, wobei dieses in Kontakt bringen der Zelle mit dem Nährmedium vorzugsweise unter Bedingungen erfolgt, unter denen keine Aldehyde, vorzugswei se kein 3-Hydroxypropionaldehyd, in für die Zellen toxischer Konzentration gebildet werden;
• ii) gegebenenfalls Abtrennen der Zellen von dem Kulturmedium;
• iii) in Kontakt bringen der Zellen mit einem Kulturmedium, in welchem die pro Zeit- und Volumeneinheit gebildete Biomasse um mindestens 50 besonders bevorzugt um mindestens 90%, besonders bevorzugt um mindestens 99 gegenüber der pro Zeit- und Volumeneinheit im Verfahrensschritt i) gebildeten Biomasse reduziert ist und welches Diole, Triole oder Polyole, vorzugsweise Glycerin, beinhaltet, unter Bedingungen, unter denen die Zellen aus den Diolen, Triolen oder Polyolen Aldehyde, vorzugsweise aus dem Glycerin 3-Hydroxypropionaldehyd, bilden;
• iv) gegebenenfalls Aufreinigung der gebildeten Aldehyde, vorzugsweise des gebildeten 3-Hydroxypropionaldehyds, aus dem Kulturmedium.

A contribution to achieving the abovementioned objects is also made by a process for preparing aldehydes from diols, triols or polyols, preferably from 3-hydroxypropionaldehyde from glycerol, comprising the following process steps:

• i) contacting a cell according to the invention or a cell obtainable by the process according to the invention with a culture medium which allows the formation of biomass, this bringing into contact the cell with the nutrient medium preferably under conditions under which no aldehydes, vorzugswei no 3-hydroxypropionaldehyde, to be formed in cells of toxic concentration;
• ii) optionally separating the cells from the culture medium;
• iii) contacting the cells with a culture medium in which the biomass formed per unit time and volume unit is reduced by at least 50, more preferably by at least 90%, more preferably by at least 99, compared to the biomass formed per unit time and volume in method step i) and which contains diols, triols or polyols, preferably glycerol, under conditions in which the cells from the diols, triols or polyols form aldehydes, preferably from the glycerol 3-hydroxypropionaldehyde;
• iv) optionally purification of the aldehydes formed, preferably of the formed 3-hydroxypropionaldehyde, from the culture medium.

in the Process step i) of the process according to the invention is a cell of the invention or a through the method according to the invention available Cell with a culture medium, which is the formation of biomass allows the cells to be brought into contact. The phrase "which allows the formation of biomass from the cells " expressing that the cells in the process step i) culture medium are capable of sufficient to share.

The culture medium used in process step i) is preferably a nutrient medium containing as carbon source preferably carbohydrates, in particular glucose, sucrose or other mono- and polysaccharides, glycerol or a mixture of carbohydrates, in particular glucose, and glycerol. If the cells used in the process according to the invention for the preparation of aldehydes, preferably of 3-hydroxypropionaldehyde, are capable of producing aldehydes, in particular 3-hydroxypropionaldehyde, from carbohydrates such as glucose, sucrose or other mono- and polysaccharides, it is advantageous that the Activity of the enzyme E ₁ and optionally also the activity of the enzyme E ₂ during the implementation of the method step i) is not yet increased, to prevent already aldehydes, in particular 3-hydroxypropionaldehyde is formed in step i). This can be realized, for example, by the fact that the genes of the enzyme E ₁ and optionally of the enzyme E ₂ are in the cells under the control of suitable promoters and only in process step iii) are these promoters induced. If, for example, the lacZ promoter is used, the expression of the genes under the control of this promoter for the enzyme E ₁ and optionally also for the enzyme E _{2 could be} effected only in process step iii) by the addition of suitable inducers such as IPTG. It is also conceivable to use promoters which can be regulated by way of certain physical parameters, such as the temperature, in order to control the expression of the enzyme E ₁ and optionally also of the enzyme E ₂ .

The genetically modified cells according to the invention can be used continuously or discontinuously in the batch process (batch culturing) or in the fed-batch process (feed process) or repeated-fed-batch process (repetitive feed process) for the purpose of producing biomass in process step i) brought into contact with the nutrient medium and thus cultivated. Also conceivable is a semi-continuous process, as described in the GB-A-1009370 is described. A summary of known cultivation methods are in the textbook by Chmiel ("Bioprocess 1. Introduction to Bioprocess Engineering" (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas ("Bioreactors and Peripheral Facilities", Vieweg Verlag, Braunschweig / Wiesbaden, 1994).

The to be used culture medium in process step i) must in suitable To meet the requirements of the respective strains. Descriptions of culture media of various microorganisms are in the manual "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981).

When Carbon source can be carbohydrates such as glucose, Sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such. Soybean oil, Sunflower oil, peanut oil and coconut oil, fatty acids such as As palmitic acid, stearic acid and linoleic acid or glycerin are used. These substances can be sold individually or used as a mixture. Particularly preferred is the Use of carbohydrates, in particular of glucose, or of glycerol.

When Nitrogen source can be organic nitrogen-containing compounds such as peptone, yeast extract, meat extract, malt extract, corn steep liquor, Soybean meal and urea or inorganic compounds such as Ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate are used. The nitrogen sources can used singly or as a mixture.

When Phosphorus source can phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing ones Salts are used. The culture medium must continue to salts of Metals include such. As magnesium sulfate or iron sulfate, the necessary for growth. Finally, you can essential growth factors such as amino acids and vitamins in addition used for the above-mentioned substances. The culture medium In addition, suitable precursors may be added become. The stated feedstocks can be used to culture in Formed as a one-off approach or as appropriate be fed during cultivation.

to pH control of the culture are basic compounds such as sodium hydroxide, Potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid in suitable Way used. To control the foaming can Antifoams such. B. fatty acid polyglycol used become. To maintain the stability of plasmids can the medium suitable selective substances such as z. B. antibiotics are added. To aerobic conditions maintain oxygen or oxygen-containing gas mixtures, such as As air, registered in the culture. The temperature of the culture is usually 20 ° C to 80 ° C and preferably at 25 ° C to 40 ° C.

After this in process step i) a sufficient biomass of recombinant Bacillus megaterium, Escherichia coli or Escherichia blattae cells It can be produced, especially if the cells not immobilized on or in a substrate and in this Form in process step iii) for the preparation of aldehydes or 3-hydroxypropionaldehyde to be used, be advantageous the cells thus obtained in process step ii) of the used Separate culture medium. The separation of the cells takes place by means of the separation process known to those skilled in the art. It is conceivable in In this context, in particular a continuous separation the cells of the nutrient medium, for example by means of a suitable filter, for example by means of a filter with an exclusion variable in a range of 20 to 200 kDa. The use is also conceivable a centrifuge, a suitable sedimentation device or a combination of these devices, it being particularly preferred is, at least part of the microorganisms first separated by sedimentation and then the by The microorganisms partially freed fermentation broth an ultrafiltration or centrifugation device.

The In this way, separated cells become, if in several successive separation stages were obtained, optionally combined and can directly the process step iii) are supplied. However, it can be beneficial prove the cells before carrying out the process step iii) still to wash, this washing preferably already with that Culture medium, which is used in step iii) becomes. For washing the cells, for example, they can in a suitable volume of the method used in step iii) Culture medium resuspended and then through the separation processes described above, in particular by filtration, Sedimentation or centrifugation, or by a combination of these measures, to be freed from the culture medium. On This way, the cells can be separated from those components of the nutrient medium used in process step i), which in particular allow growth of the cells (nitrogen compounds, Phosphorus compounds, essential amino acids and the like) be freed.

in the Process step iii) of the process according to the invention the cells are then called "resting cells" with a culture medium, in which the related to a time and volume unit biomass formation at least 50%, more preferably at least 90%, especially preferably at least 99% higher than at a time and volume unit related biomass formation in the process step i) is reduced, and which diols, triols or polyols, preferably Glycerin, under conditions in which the cells from the diols, triols or polyols aldehydes, preferably from the glycerol 3-hydroxypropionaldehyde form, brought into contact. Most preferably, in step iii) the use a culture medium in which no detectable biomass formation more occurs, the cells hardly share much more. For example, a 50% reduction in biomass formation is achieved. if in the culture medium used in step i) below the cultivation conditions in process step i) 10 g of microorganisms per liter and per hour, while in the process step iii) by means of the culture medium used there under the same Cultivation conditions as in process step i) only 5 g of microorganisms be formed per liter and per hour.

In This culture medium are the invention Cells then able to be those offered over the culture medium Diols, triols or polyols, preferably the glycerol offered, in high yield to aldehydes, preferably to 3-hydroxypropionaldehyde, implement.

If, as described above, the activity of the enzyme E ₁ and optionally also of the enzyme E _{2 is increased,} for example by the use of suitable promoters, only in process step iii), the culture medium used in process step iii) contains, in addition to the glycerol, also suitable inducers of the promoter or , appropriate physical parameters, such as the temperature, in step iii) are adjusted accordingly to induce the expression of the enzymes.

The culture medium used in process step iii) can be any of the solvents or solutions known to the person skilled in the art, which are suitable for cultivating cells of the species Bacillus megaterium, Escherichia coli or Escherichia blattae, without these cells sharing significantly, however without appreciably affecting the vitality of the cells. Thus, the cells should, as far as possible again after a possibly in a further process step v) separation from the process step iii) culture medium used to be able to form again biomass in a resuspension in a nutrient medium enabling cell division.

Conceivable as a culture medium in step iii) is for example the Use of water, of buffered saline solutions such as PBS ("phosphate buffered saline") or other saline solutions, these media preferably being suitable pH buffer systems, such as include HEPES, MOPS or other buffer components.

It is preferred in accordance with the invention that the culture medium used in process step iii) contains no corrinoids, preferably no coenzyme B ₁₂ , no vitamin B ₁₂ and no cobalamin.

The aldehydes thus formed or the 3-hydroxypropionaldehyde thus formed can then be separated off in a further process step iv) from the culture medium used in process step iii) and optionally unreacted glycerol and thus purified, this purification by methods known in the art, such as for example, by crystallization, by lyophilization, by chromatographic methods or by a combination of these methods. Other possibilities for the purification of, for example, 3-hydroxypropionaldehyde and derivatives thereof include Vollenweider et al. in Journal of Agricultural and Food Chemistry, Vol. 51, 2003, pages 3.287-3.293 The disclosure of which is hereby incorporated by reference as a reference with regard to the possibilities for the purification of 3-hydroxypropionaldehyde from fermentation solutions and forms part of the disclosure of the present invention. After purification of the aldehydes can be obtained in the case of 3-hydroxypropionaldehyde a composition which, depending on the nature of the purification process, in addition to the monomer of 3-hydroxypropionaldehyde and the hydrate of 3-hydroxypropionaldehyde, the cyclized dimer of 3-hydroxypropionaldehyde and optionally other oligomers of 3-hydroxypropionaldehyde.

• I) in Kontakt bringen einer erfindungsgemäßen Zelle, in der neben der Aktivität des Enzyms E₁ und gegebenenfalls auch des Enzyms E₂ auch die Aktivität mindestens eines der, vorzugsweise aller Enzyme E₄ bis E₇ erhöht ist, mit einem Kulturmedium, welches eine Bildung von Biomasse aus den Zellen ermöglicht, wobei dieses in Kontakt bringen der Zelle mit dem Nährmedium vorzugsweise unter Bedingungen erfolgt, unter denen keine Aldehyde, vorzugsweise kein 3-Hydroxypropionaldehyd, in für die Zellen toxischer Konzentration gebildet werden;
• II) gegebenenfalls Abtrennen der Zellen von dem Kulturmedium;
• III) in Kontakt bringen der Zellen mit einem Kulturmedium, in welchem die pro Zeit- und Volumeneinheit gebildete Biomasse um mindestens 50%, besonders bevorzugt um mindestens 90 besonders bevorzugt um mindestens 99 gegenüber der pro Zeit- und Volumeneinheit im Verfahrensschritt I) gebildeten Biomasse reduziert ist und welches Glucose, Saccharose oder andere Mono- und Polysaccharide sowie gegebenenfalls auch Glycerin beinhaltet, unter Bedingungen, unter denen die Zellen aus der Glucose, Saccharose oder anderen Mono- und Polysacchariden über Glycerin als Zwischenprodukt und gegebenenfalls auch direkt aus Glycerin 3-Hydroxypropionaldehyd bilden;
• IV) gegebenenfalls Aufreinigung des gebildeten 3-Hydroxypropionaldehyds aus dem Kulturmedium.

A contribution to achieving the abovementioned objects is further provided by a process for the preparation of 3-hydroxypropionaldehyde from glucose, sucrose or other mono- and polysaccharides, comprising the following process steps:

• I) bring into contact a cell according to the invention, in addition to the activity of the enzyme E ₁ and optionally also of the enzyme E ₂ , the activity of at least one, preferably all enzymes E ₄ to E _{7 is} increased, with a culture medium, which is a formation of biomass from the cells, this contacting the cell with the nutrient medium preferably under conditions in which no aldehydes, preferably no 3-hydroxypropionaldehyde, are formed in cells of toxic concentration;
• II) optionally separating the cells from the culture medium;
• III) bringing the cells into contact with a culture medium in which the biomass formed per unit time and volume unit is reduced by at least 50%, particularly preferably by at least 90, more preferably by at least 99% compared to the biomass formed per unit time and volume in method step I) and which contains glucose, sucrose or other mono- and polysaccharides and optionally also glycerol, under conditions under which the cells from the glucose, sucrose or other mono- and polysaccharides via glycerol as intermediate and optionally also directly from glycerol form 3-hydroxypropionaldehyde ;
• IV) optionally purification of the 3-hydroxypropionaldehyde formed from the culture medium.

Regarding the details of this procedure will apply to the appropriate Embodiments in connection with the above Process for the preparation of 3-hydroxypropionaldehyde from glycerol, comprising the method steps i) to iv) referenced.

• A) Bereitstellung von 3-Hydroxypropionaldehyd mittels des vorstehend beschriebenen Verfahrens zur Herstellung von 3-Hydroxypropionaldehyd aus Glycerin oder des vorstehend beschriebenen Verfahrens zur Herstellung von 3- Hydroxypropionaldehyd aus Glucose, Saccharose oder anderen Mono- und Polysacchariden;
• B) chemische oder biokatalytische Umsetzung des 3-Hydroxypropionaldehys durch Reduktion, Oxidation oder Dehydratisierung unter Erhalt von C3-Verbindungen;
• C) gegebenenfalls die weitere chemische oder biokatalytische Umsetzung der im Verfahrensschritt II) erhaltenen C₃-Verbindung durch Reduktion, Oxidation oder Addition.

A further contribution to the solution of the abovementioned objects is also provided by a process for the preparation of C3 compounds from 3-hydroxypropionaldehyde, comprising the process steps:

• A) providing 3-hydroxypropionaldehyde by means of the above-described process for the preparation of 3-hydroxypropionaldehyde from glycerol or the above-described process for the preparation of 3-hydroxypropionaldehyde from glucose, sucrose or other mono- and polysaccharides;
• B) chemical or biocatalytic reaction of the 3-hydroxypropionaldehyde by reduction, oxidation or dehydration to give C3 compounds;
• C) optionally, the further chemical or biocatalytic reaction in step II) he retaining C ₃ compound by reduction, oxidation or addition.

First is in process step I) 3-hydroxypropionaldehyde by means of above-described process for the preparation of 3-hydroxypropionaldehyde from glycerol or the process for the preparation described above of 3-hydroxypropionaldehyde from glucose, sucrose or others Mono- and polysaccharides provided, this optionally, as described above in connection with these methods, may have been purified from the fermentation solution.

In process step II), 3-hydroxypropionaldehyde can then be chemically or biocatalytically obtained by reduction, oxidation or dehydration to give C ₃ compounds, in particular to give 1,3-propanediol (reduction), acrolein (dehydration) or 3-hydroxypropionic acid (oxidation) , be implemented. Here are details of the reaction of 3-hydroxypropionaldehyde to acrolein, inter alia Hall and Stern in Journal of the Chemical Society, 1950, pages 490-498 while details of the preparation of 1,3-propanediol from 3-hydroxypropionaldehyde include the US 5,334,778 can be removed. The preparation of 3-hydroxypropionic acid from 3-hydroxypropionaldehyde in turn is inter alia in US 6,852,517 described.

Optionally, the C ₃ compounds obtained in process step C) can be further reacted chemically or microbiologically by reduction, oxidation or addition in a further process step D). Particularly suitable here is the reaction of acrolein obtained in process step C) by oxidation to acrylic acid, which can then optionally be reacted in a still further process step D) radically to form acrylic acid-based polymers. In particular, the free-radical polymerization of optionally partially neutralized acrylic acid in the presence of suitable crosslinkers to form water-absorbing polyacrylates, which are also referred to as "superabsorbers." The chemical oxidation of acrolein to acrylic acid and the subsequent free radical polymerization of the resulting acrylic acid is, inter alia, in of the WO 2006/136336 described.

The The present invention will now be described by way of non-limiting examples explained in more detail:

example 1

(Comparative analysis of the resistance of resting bacterial cells towards 3-hydroxypropionaldehyde)

• - To analyze the resistance of resting bacterial cells to 3-hydroxypropionaldehyde (3-hydroxypropionaldehyde), a 3-hydroxypropionaldehyde aqueous solution was prepared in a two-step process. 10 ml of MRS medium (Difco, Heidelberg), which additionally contained 20 mM glycerol, were inoculated with 1% (v / v) of a L. reuteri freezing culture, incubated for 18 h without shaking at 37 ° C., and this preculture was completely added 40 ml fresh MRS medium and incubated for a further 6 h at 37 ° C without shaking. The second preculture was also completely transferred to 1 L fresh MRS medium and cultured for 15 h at 37 ° C without shaking in a 1000 ml Schott flask. Subsequently, the cells were harvested (10 min, 4000 g, RT), washed with 0.1 M potassium phosphate buffer and used directly for the production of 3-hydroxypropionaldehyde. The cell pellet was resuspended in 100 ml of 250 mM glycerol to start the biotransformation of glycerol to 3-hydroxypropionaldehyde. After 60 min, the reaction was again centrifuged (10 min, 12000 g, 4 ° C, the resulting supernatant contained 160 mM 3-hydroxypropionaldehyde.) Quantification of 3-hydroxypropionaldehyde was determined by a colorimetric assay according to Doleyres et al. "Production of 3-hydroxypropionaldehyde using a two-step process with Lactobacillus reuteri, Applied Microbiology and Biotechnology, Vol. 68, 2005, pp. 467-474 ) carried out.
• For the analysis of host resistance, 5 ml of LB medium (according to Miller, Merck KGaA, Darmstadt) were inoculated with B. megaterium DSM319 or E. blattae DSM4481 cells from a fresh LB plate (Miller, Merck KGaA, Darmstadt) and incubated for approx. 8 h at 37 ° C. and at 180 rpm. Subsequently, 100 ml of LB medium were inoculated with 1.5% (v / v) of the preculture and the cells were cultured until the stationary phase (approximately 16 h) at 37 ° C., 180 rpm. L. reuteri cells were cultured in MRS medium (without glycerol) as described above. Subsequently, the cells were harvested by centrifugation (10 min, 5292 g, 4 ° C), washed once with 0.1 M potassium phosphate buffer (pH 7) and resuspended in 5 ml of the 160 mM 3-hydroxypropionaldehyde solution described above. The biomass concentration was ~ 1 × 10 ¹⁰ cells per ml.
• The mixtures were incubated for 1 h at room temperature and after 10 min each 100 ul samples taken to determine the viable cell count. For this purpose, suitable dilutions of the cell suspension in dilution solution (0.85% (w / v) NaCl, 0.1% (w / v) peptone from casein) were plated on MRS agar plates and incubated anaerobically for 48 h at 37 ° C. Subsequently, the cell colonies were counted and the viable cell count determined. It has been observed that as with Lactobacillus reuteri ATCC55730 incubation ru The presence of B. megaterium DSM319 or E. blattae DSM4481 cells with 3-hydroxypropionaldehyde does not result in a significant reduction in the number of live cells within the analyzed period. The 3-hydroxypropionaldehyde concentration did not decrease significantly over the analyzed period.

Example 2

(Preparation of a recombinant B. megaterium cell, in which the activity of glycerol dehydratase has been increased is)

• - For overexpression of Lactobacillus reuteri ATCC55730 genes gldABC (Enzyme E ₁ ) and gldDE (Enzyme E ₂ , see also GenBank entries DQ233724-DQ233720) (SEQ.ID.NO.01) in Escherichia blattae DSM4481 and Bacillus megaterium DSM319 was the Plasmid pWH1520-gldABCDE constructed (SEQ ID NO: 02). Chromosomal DNA from Lactobacillus reuteri ATCC55730 served as a template for a PCR with the Expand ^™ High Fidelity PCR Kit from Roche Diagnostics (Mannheim), according to the manufacturer's instructions The coding range of the glycerol dehydratase (gldABC) and the associated Reactivation factor (gldDE) coding genes, including 34 bp before the gldA start codon, were isolated from chromosomal DNA using the oligonucleotides Bm GDfw XmaI (SEQ ID NO: 03) and Bm GDRFrv SphI (SEQ ID NO: 04) of Lactobacillus reuteri ATCC55730 amplified in a PCR ( "PCR protocols: A guide to methods and applications", 1990, Academic Press ) and provided in this way at the 5 'end or 3' end with an XmaI or a SphI interface. The following oligonucleotides were used for this:
• Bm_GDfw_XmaI: 5'-TCC C CC CGG G CA TTA AGT AGA TCT GGA AGG AGG A-3 '(SEQ ID NO: 02, containing an XmaI recognition sequence at the 5' end)
• Bm_GDRFrv_SphI: 5'-ACA T GC ATG CAA ATC ACC TGT TTA CCA TTT CCT T-3 '(SEQ ID NO: 03, containing a SphI recognition sequence at the 5' end)
• The PCR product (5,212 base pairs) was purified using the QIAquick PCR purification kit (Qiagen, Hilden) according to the instructions of the manufacturer and then by means of the restriction endonucleases XmaI and SphI (according to the manufacturer's instructions, New England Biolabs, Schwalbach) cut. The now 5.202 bp fragment was inserted into the XmaI / SphI-cut expression vector pWH1520 ( 7,896 base pairs; by Rygus and Rillen in Applied Microbiology and Biotechnology, Vol. 35 (5), 1991, pp. 594-599 ) ligated. The resulting plasmid pWH1520-gldABCDE (SEQ ID NO: 02) is 13,098 base pairs in size. The ligation and the transformation of chemically competent E. coli cells (New England Biolabs, Schwalbach) were carried out in a manner known to the person skilled in the art. The authenticity of the insert was checked by DNA sequence analysis.
• The plasmid pWH1520-gldABCDE as well as the empty vector pWH1520 were analyzed by protoplast transformation according to Biedendieck ( Biedendieck, "Versatile Tools for Production, Secretion and Purification of Recombinant Proteins", 2006, Dissertation, pp. 45-47 ) in B. megaterium DSM319. The resulting strain was named B. megaterium / pWH1520-gldABCDE.

Example 3

(Preparation of a recombinant E. blattae cell, in which increases the activity of glycerol dehydratase has been)

• To test the suitability of E. blattae for the production of 3-hydroxypropionaldehyde, the B. megaterium xylA promoter in plasmid pWH1520-g1dABCDE was first replaced by the lacZ promoter from E. coli. For this, the lacZ promoter was amplified by PCR according to Innis et al. using pUC19 DNA (GenBank entry L09137) as template and the following oligonucleotides: PlacZ-fw: 5'-TAT ATA GCT AGC CTC ATT AGG CAC CCC AGG C-3 '(SEQ ID NO: 05, including a NheI recognition sequence at the 5'-end) PlacZ-rv: 5'-TAT ATA CCC GGG CAA TTC CAC ACA ACA TAC GAG CC-3 '(SEQ ID NO: 06, containing an XmaI recognition sequence at the 5' end). End) The PCR product (84 base pairs) was purified using the QIA-quick PCR purification kit from Qiagen, Hilden, according to the manufacturer's instructions. The purified PCR product was digested with NheI and XmaI (also as described by the manufacturer of restriction endonucleases, New England Biolabs, Schwalbach) and into the NheI / XmaI-cut vector pWH1520-gldABCDE (Example 2) to obtain the vector pWH1520-gldABCDE lacZ (10,896 base pairs, SEQ ID NO: 07). The AU The thenticity of the insert was determined by means of DNA sequencing. Ligation of the expression vector and the production and transformation of chemically competent E. coli cells were carried out in a manner known to the person skilled in the art. In order to test the suitability of glycerol dehydratases for the production of 3-hydroxypropionaldehyde improved via directed evolution, the in patent application WO-A-2004/056963 under SEQ ID NO. 322 listed Klebsiella pneumoniae glycerol dehydratase variant SHGDH22 used. This variant is distinguished from the wild-type biocatalyst by almost six times increased conversion of glycerol to 3-hydroxypropionaldehyde in the presence of 600 mM 1,3-propanediol. The corresponding coding sequence (SEQ ID NO: 08) was synthesized by GeneArt AG (Regensburg). To construct a synthetic operon from the glycerol dehydratase variant SHGDH22 coding sequence and the coding sequences of the associated reactivation factor (large and small subunit), first the glycerol dehydratase variant SHGDH22 coding sequence by PCR (according to Innis et al. ) with the gene provided by GeneArt, which contains as an insert the glycerol dehydratase variant SHGDH22 coding sequence amplified as template and the following oligonucleotides: GD-fw: 5'-TAT ATA CCC GGG AAG GAG GAC TAC TTT ATT ATG AAA AGA TCA AAA CGA TTT GCA G-3 '(SEQ ID NO: 09, containing an XmaI recognition sequence at the 5' end) GD rv: 5'-GAC ATG GTA TAT CTC CTT AGC TTC CTT TAC GTA GCT TAT GCC- 3 '(SEQ ID NO: 10) The PCR product (2,738 base pairs) was purified using the QIAquick PCR purification kit from Qiagen, Hilden, according to the manufacturer's instructions. At the same time, this was in patent application EP-A-1 669 457 described synthetic operon from the sequences encoding the major and minor subunit of the associated reactivation factor from Klebsiella pneumoniae ATCC25955 using vector 2 (SEQ.ID.NO. 02 in EP-A-1 669 457 ) were amplified with the following oligonucleotides: GDRF-fw: 5'-GGA AGC TAA GGA GAT ATA CCA TGT CGC TTT CAC C-3 '(SEQ ID NO: 11) GDRF-rv: 5'-TAT ATA GCA TGC TTA ATT CGC CTG ACC GGC CAG-3 '(SEQ ID NO: 12, containing a SphI recognition sequence at the 5' end) The PCR product (2,234 base pairs) was amplified using the QIAquick PCR purification kit from Qiagen, Hilden, according to In the next step, the glycerol dehydratase variant SHGDH22 was used as a crossover PCR (according to Innis et al.) Using the two primary PCR products, the second and the third, respectively, of the corresponding reactivation factor For this purpose, 1 ng each of the two primary PCR products were combined and used together as a template for a PCR with the oligonucleotides GD-fw (SEQ ID NO: 09) and GDRF-rv (SEQ. ID No. 12) The PCR product (4,938 base pairs) was amplified using the QIAquick PCR Prep cleaning kits from Qiagen, Hilden, according to the manufacturer's instructions. The purified PCR product was digested by XmaI and SphI (also as described by the manufacturer of restriction endonucleases, New England Biolabs, Schwalbach) and into the XmaI / SphI-cut vector pWH1520-gldABCDE (Example 2) to obtain the vector pWH1520-SHGDH22 (10,118 base pairs, SEQ ID NO: 13). The authenticity of the insert was determined by DNA sequencing. Ligation of the expression vector and the production and transformation of chemically competent E. coli cells were carried out in a manner known to the person skilled in the art.
• The plasmids pWH1520, pWH1520-gldABCDE-lacZ and pWH1520-SHGDH22 were introduced by electroporation into E. blattae DSM4481 and the resulting strains E. blattae / pWH1520, E. blattae / pWH1520-gldABCDE and E. blattae / pWH1520-SHGDH22 respectively , For the preparation of electrocompetent E. blattae cells, 1 l of LB medium (Miller, Merck KGaA, Darmstadt) was inoculated with 10 ml of an overnight culture, cultured to reach an OD ₆₀₀ of 0.7 at 37 ° C., and then the cells 30 min incubated on ice. All further steps were done on ice. After centrifugation (15 minutes, 5292 g, 4 ° C), the cells were washed with 1 liter of ice-cold water and again with 500 ml of ice-cold water. This was followed by another washing step with 10 ml glycerol solution (10%, w / v, 15 min, 3300 g, 4 ° C). The cell pellet was resuspended in 2 ml of 10% glycerol solution and 40 μl aliquots stored at -80 ° C.
• - For electroporation, the to be transformed DNA (1-2 μg) into an aliquot frozen, competent E. blattae cells and thawed on ice (maximum 10 min). Subsequently, the approach was in a pre-cooled Transferred electroporation cuvette. The Cells were exposed to a current pulse, with the following parameters were adjusted: voltage: 1.8 kV; Resistance: 200 Ω; Capacity: 25 μF. Immediately after the electroporation 1 ml of LB medium was added and the mixture was incubated for 1 h at 37 ° C and incubated at 700 rpm. Thereafter, the cells were grown on selective LB plates (100 μg / ml ampicillin) plated out.

Example 4

(HPLC-based quantification of glycerol, 1,3-propanediol and 3-hydroxypropionaldehyde)

• - For the quantification of glycerol, 1,3-propanediol and 3-hydroxypropionaldehyde were each in 1 ml of cell suspension the biomass by centrifugation (10 min, 16,100 g, 4 ° C) separated and 40 ul of the culture supernatant under Using an Agilent Technologies 1200 LC system (High Performance Autosampler, G136B; Thermostat for autosampler, G1330B; Micro-Vacuum Degasser, G1379B; Binary pump, G1312A; Column thermostat, G1316A; Refractive index detector, G1362A, Agilent Technologies, Waldbronn) with an Aminex HPX-87H 300 × 7.8 mm separation column (Bio-Rad Laboratories GmbH, Munich) with 9 microns Particle size analyzed. The to be analyzed Substances were isocratically with a mobile phase of 10 mM Sulfuric acid at a flow rate of 0.6 ml / min for 20 min eluted at 40 ° C. Glycerine, 1,3-propanediol and 3-hydroxypropionaldehyde were compared by comparison with the residence time of reference substances (Glycerol and 1,3-Propanediol: Sigma-Aldrich Chemie GmbH, Steinheim; 3-hydroxypropionaldehyde: own preparation). The Concentration of the three compounds in the culture supernatants was using a comparison of peak areas with calculated using a calibration standard previously created with external standards.

Example 5

(Production of 3-hydroxypropionaldehyde with recombinant E. blattae cells that have the activity the glycerol dehydratase has been increased)

• The E. blattae strains E. blattae / pWH1520, E. blattae / pWH1520-gldABCDE or E. blattae / pWH1520-SHGDH22 constructed in Example 3 were initially added overnight in 5 ml LB medium containing 100 μg / ml ampicillin Cultured at 37 ° C and 180 rpm. The next morning, 2 ml of these precultures were used to inoculate 200 ml LB each with 2% (w / v) glucose, 20 mM glycerol and 100 μg / ml ampicillin in 1 L baffled flasks. Both flasks were incubated at 37 ° C and 180 rpm. Growth of these cultures was monitored by apparent optical density (OD ₆₀₀ ). At ~ ₆₀₀ OD600, the cells were harvested, washed twice with 200 ml saline (0.9% (w / v) NaCl) and resuspended in 20 ml 250 mM glycerol. Samples were taken every 10 min for a period of 1 h and the concentration of glycerol, 1,3-propanediol and 3-hydroxypropionaldehyde was analyzed by HPLC (Example 4). While the control strain E. blattae / pWH1520 did not produce any 3-hydroxypropionaldehyde, in the case E. blattae / pWH1520-gldABCDE and E. blattae / pWH1520-SHGDH22 the formation of 3-hydroxypropionaldehyde could be detected.

Example 6

(Production of 3-hydroxypropionaldehyde with recombinant B. megaterium cells in which the activity of the Glycerol dehydratase has been increased)

• The B. megaterium strain B. megaterium / pWH1520-gldABCDE constructed in Example 2 and the corresponding control strain B. megaterium / pWH1520 were initially mixed in 5 ml of A5 medium (Malten, M., Hollmann, R., Deckwer, WD, Jahn, D. Production and secretion of recombinant Leuconostoc mesenteroides dextransucrase DsrS in Bacillus megaterium Biotechnol. Bioeng., 2005. 89 (2): 206-18.) With 4 g / l yeast extract and 10 μg / ml tetracycline overnight at 37 ° C and cultured 180 rpm. The next morning, 2 ml of these precultures were used to inoculate 200 ml each of A5 medium containing 20 mM glycerol and 10 μg / ml tetracycline in 1 L baffled flasks. Both flasks were incubated at 37 ° C and 180 rpm. Growth of these cultures was monitored by apparent optical density (OD ₆₀₀ ). At an OD ₆₀₀ of ~ 0.4, 0.5% (w / v) D-xylose was added to the cultures to induce biocatalyst formation. At an OD ₆₀₀ of ~ 4, the cells were harvested, washed twice with 200 ml of saline (0.9% (w / v) NaCl) and resuspended in 250 mM glycerol 20 ml. Samples were taken every 10 min for a period of 1 h and the concentration of glycerol, 1,3-propanediol and 3-hydroxypropionaldehyde was analyzed by HPLC (Example 4). While the control strain B. megaterium / pWH1520 pWH1520 did not produce any 3-hydroxypropionaldehyde, the formation of 3-hydroxypropionaldehyde was detected in the case of B. megaterium / pWH1520-gldABCDE.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• - EP 1669457 A [0006, 0074, 0074]
• - DE 10031999 A [0025]
• EP 0472869 A [0028]
• US 4601893 [0028]
• WO 96/15246 A [0028]
• JP 10-229891 A [0028]
• US 4489160 [0029]
• US 5158891 [0029]
• WO 2004/056963 A [0033, 0033, 0074]
• - GB 1009370 A [0053]
• US 5334778 [0072]
• - US 6852517 [0072]
• WO 2006/136336 A [0073]

Cited non-patent literature

• Doleyres et al., Applied Microbiology and Biotechnology, Vol. 68 (4), 2005, pp. 467-474; Vollenweider and Lacroix in Applied Microbiology and Biotechnology, Vol. 64 (1), 2004, pages 16-27; Lüthi-Peng et al. In Applied Microbiology and Biotechnology, Vol. 60 (1-2), 2002, pages 73-80. [0003]
• See, for example, Vollenweider and Lacroix in Applied Microbiology and Biotechnology, Vol. 64 (1), 2004, pages 16-27) and 3-hydroxypropionic acid (using the enzyme 3-hydroxypropionaldehyde dehydrogenase) (see, for example, Chen et al in Journal of Biomedical Materials Research, Vol. 61, 2002, pp. 360-369 [0004]
• see, for example, Slininger et al. in Applied Environmental Microbiology, Vol. 46, 1983, pp. 62-67, and for review Vollenweider and Lacroix in Applied Microbiology and Biotechnology, Vol. 64 (1), 2004, pp. 16-27 [0005]
• See, for example, Vollenweider and Lacroix in Applied Microbiology and Biotechnologist, Vol. 64 (1), 2004, pages 16-27 [0005]
• see, for example, Chen et al. in Journal of Biomedical Materials Research, Vol. 61 (3), 2002, pp. 360-369 [0011]
• - Chen et al. in the Journal of Biomedical Materials Research, Vol. 61 (3), 2002, pages 360-369. [0018]
• A common method for the preparation of the protein gels in coryneform bacteria and for the identification of the proteins is that described by Hermann et al. in Electrophoresis, Volume 22: 1712-23 (2001 )
• Sambrook et al., Molecular Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989. [0026]
• Lohaus and Meyer (1989) Biospektrum, 5: 32-39; Lottspeich (1999), Angewandte Chemie 111: 2630-2647 [0026]
• also referred to as gel retardation) (Wilson et al., (2001) Journal of Bacteriology, 183: 2151-2155 [0026]
• Sambrook et al., Molecular Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989. [0026]
• Donahue et al. (2000) Journal of Bacteriology 182 (19): 5624-5627; Ray et al. (2000) Journal of Bacteriology 182 (8): 2277-2284; Freedberg et al. (1973) Journal of Bacteriology 115 (3): 816-823 [0026]
• Hermann et al., Electrophoresis, 22: 1712-23 (2001) [0026]
• Lohaus et al., Biospektrum 5 32-39 (1998), Lottspeich, Angewandte Chemie 111: 2630-2647 (1999) and Wilson et al., Journal of Bacteriology 183: 2151-2155 (2001 [0026]
• Reinscheid et al. (Applied and Environmental Microbiology 60: 126-132 (1994) [0030]
• - typically Escherichia coli), but can not be replicated in Corynebacterium glutamicum. Examples of vectors which are used are pSUP301 (Simon et al., Bio / Technology 1: 784-791 (1983) [0030]
• Schäfer et al., Gene 145: 69-73 (1994) [0030]
• - (Shuman, Journal of Biological Chemistry 269: 32678-84 (1994) [0030]
• Schrumpf et al., Journal of Bacteriology 173: 4510-4516). [0030]
• - (Spratt et al., Gene 41: 337-342 (1986) [0030]
• Method of conjugation is described, for example, in Schäfer et al., Applied and Environmental Microbiology 60: 756-759 (1994). Methods for transformation are described, for example, in Thierbach et al., Applied Microbiology and Biotechnology 29: 356-362 (1988), Dunican and Shivnan, Bio / Technology 7: 1067-1070 (1989), and Tauch et al., FEMS Microbiology Letters 123: 343-347 (1994) [0030]
• - et al. in Journal of Agricultural and Food Chemistry, Vol. 51, 2003, pp. 3.287-3.293 [0067]
• Hall and star described in Journal of the Chemical Society, 1950, pages 490-498. [0072]
• "Production of 3-hydroxypropionaldehydes using a two-step process with Lactobacillus reuteri, Applied Microbiology and Biotechnology, Vol. 68, 2005, pages 467-474 [0074]
• - "PCR protocols: A guide to methods and applications", 1990, Academic Press [0074]
• - 7,896 base pairs; described by Rygus and Rillen in Applied Microbiology and Biotechnology, Vol. 35 (5), 1991, pp. 594-599 [0074]
• - Biedendieck, "Versatile Tools for Production, Secretion and Purification of Recombinant Proteins", 2006, Dissertation, p. 45-47 [0074]

Claims (19)

A cell that is capable of corrinoids too form and genetically modified compared to their wild type became more aldehydes compared to their wild-type Diols, triols or polyols, preferably more 3-hydroxypropionaldehyde from glycerol, is able to form. The cell of claim 1, wherein the corrinoids are selected from the group consisting of coenzyme B ₁₂ , vitamin B ₁₂ and cobalamin. The cell of claim 1 or 2, wherein the cell selected from a cell is made up of the group from Escherichia coli, Escherichia blattae and Bacillus megaterium. The cell according to any one of the preceding claims, wherein the cell has an increased activity of an enzyme E ₁ , which compared to its wild-type, which catalyzes the conversion of diols, triols or polyols to aldehydes, preferably from glycerol to 3-hydroxypropionaldehyde. The cell according to claim 4, wherein the enzyme E _{1 is} a glycerol dehydratase or a diol dehydratase (EC 4.2.1.30). The cell according to one of the preceding claims, wherein in addition to the activity of the enzyme E ₁ , the activity of an enzyme E ₂ which catalyzes the reactivation of inactivated enzyme E ₁ is also increased in the cell. The cell of claim 5, wherein the enzyme E _{2 is} a glycerol dehydratase reactivation factor or a diol dehydratase reactivating factor. The cell according to any one of the preceding claims, wherein in addition to the enzyme E ₁ and optionally the enzyme E ₂ , the activity of an enzyme E ₃ which facilitates the diffusion of glycerol into the cell is also increased in the cell. The cell according to claim 8, wherein the enzyme E _{3 is} a glycerol channel or a glycerol facilitator (TC 1.A.8.1.1, TC 1.A.8.2.1 or TC 1.A.8.2.2) , The cell according to any one of claims 1 to 9, wherein in addition to the activity of the enzyme E ₁ and optionally of the enzyme E ₂ , the activity of at least one of the enzymes E ₄ , E ₅ , E ₆ or E _{7 is} increased in the cell: - one Enzyme E ₄ , which catalyzes the conversion of dihydroxyacetone phosphate to glycerol 3-phosphate; - An enzyme E ₅ , which catalyzes the conversion of glycerol-3-phosphate to glycerol; An enzyme E ₆ which catalyzes the conversion of dihydroxyacetone phosphate to dihydroxyacetone; - An enzyme E ₇ , which catalyzes the conversion of dihydroxyacetone to glycerol. The cell according to claim 10, wherein the enzyme E _{4 is} a glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), E _{5 is} a glycerol-3-phosphate phosphatase (EC 3.1.3.21), E _{6 is} a dihydroxyacetone-phosphate Phosphatase (EC 3.1.3.1), and E _{7 is} a glycerol dehydrogenase (EC 1.1.1.6). The cell of any one of the preceding claims, wherein the cell has a reduced activity of at least one of the enzymes E ₈ to E ₁₁ - an enzyme E ₈ which catalyzes the conversion of glycerol to glycerol 3-phosphate; An enzyme E ₉ which catalyzes the conversion of glycerol to dihydroxyacetone; An enzyme E ₁₀ which catalyzes the conversion of 3-hydroxypropionaldehyde to 1,3-propanediol; - An enzyme E ₁₁ , which catalyzes the conversion of 3-hydroxypropionaldehyde to 3-hydroxypropionic acid. The cell according to claim 12, wherein the enzyme E _{8 is} a glycerol kinase (EC 2.7.1.30), E _{9 is} a glycerol dehydrogenase (EC 1.1.1.6 or EC 1.1.1.156 or EC 1.1.99.22), E _{10 is} a 1, 3-propanediol dehydrogenase (EC 1.1.1.202) and E _{11 is} a 3-hydroxypropionaldehyde dehydrogenase (EC 1.2.1.3 or EC 1.2.1.5). A method of producing a genetically engineered cell capable of producing corrinoids comprising the step of increasing the activity of an enzyme E ₁ which inhibits intracellular conversion of diols, triols or polyols to aldehydes, preferably glycerol. Hydroxypropionaldehyde, catalyzed. The method of claim 14, wherein the method additionally comprises the step of increasing the activity of an enzyme E ₂ which catalyzes the reactivation of inactivated enzyme E ₁ . A cell, obtainable by a process according to claim 14 or 15. A process for the preparation of aldehydes Diols, triols or polyols, preferably 3-hydroxypropionaldehyde of glycerin, comprising the following process steps: i) contacting a cell according to any one of the claims 1 to 9 or 12 and 13 with a culture medium, which is a formation of biomass from the cells allows; ii) if appropriate Separating the cells from the culture medium; iii) bring in contact the cells with a culture medium in which the per-time and Volume unit formed biomass by at least 50 compared the per time and volume unit formed in step i) Biomass is reduced, and which diols, triols or polyols, preferably glycerol, under conditions under which the cells from the diols, triols or polyols aldehydes, preferably from glycerol 3-hydroxypropionaldehyde; iv) if appropriate Purification of the formed aldehydes, preferably of the formed 3-hydroxypropionaldehyde, from the culture medium. A process for the preparation of 3-hydroxypropionaldehyde from glucose, sucrose or other mono- and polysaccharides, comprising the following method steps: I) a cell according to any one of claims 10 to 13 with a Culture medium which allows biomass to be generated from the cells; II) optionally separating the cells from the culture medium; III) contacting the cells with a culture medium in which the biomass formed per unit time and volume by at least 50 versus the per unit time and volume in the process step I) biomass is reduced and which glucose, sucrose or other mono- and polysaccharides, under conditions under which the cells from the glucose, sucrose or other Mono- and polysaccharides via glycerol as an intermediate Form 3-hydroxypropionaldehyde; IV) optionally purification of the formed 3-hydroxypropionic aldehyde from the culture medium. A process for the preparation of C ₃ compounds from 3-hydroxypropionaldehyde comprising the steps of: A) providing 3-hydroxypropionaldehyde by a process according to claim 17 or 18; B) chemical or biocatalytic reaction of the 3-hydroxypropionaldehyde by reduction, oxidation or dehydration to give C ₃ compounds; C) optionally further chemical or biocatalytic reaction of the obtained in step B) C ₃ compound by reduction, oxidation or addition.