WO2003093488A1 - Method and micro-organisms for the microbial production of pyruvate from carbohydrates and alcohols - Google Patents

Abstract

The invention relates to a method and micro-organisms for the microbial production of pyruvate from carbohydrates and alcohols. Known chemical methods for producing pyruvate are technically complex and cost-intensive. Known biological methods and micro-organisms have a drawback in that the substrates are not 100 % converted into pyruvate thereby resulting in a deterioration of the product yield. In addition, a complex control of the necessary vitamin concentration is required for the microbial pyruvate production from glucose. The inventive method and micro-organisms enable the microbial production of pyruvate with a nearly 100 % conversion of the substrate. An increased pyruvate production could be achieved by using micro-organisms that have been genetically modified, i.e. deletion, insertion or low expression of the ldhA gene sequence, which codes for the lactate dehydrogenase.

Description

 Description

Processes and microorganisms for the microbial production of pyruvate from carbohydrates and alcohols

The invention relates to a method and microorganisms for the microbial production of pyruvate from carbohydrates and alcohols.

A classic method of producing pyruvate (the salt of pyruvic acid) is the pyrolysis (ie the burning) of grape acid. Heavy metals and temperatures of around 200 ° C are required (Römp Chemie Lexikon, 1995). The following microbial processes for pyruvate synthesis from lactate are also known: Production of pyruvate from lactate with permeabilized cells of the methylotrophic yeasts Hansenula polymorpha or Pichia pastoris, which express the gene for the (S) -hydroxy acid oxidase from spinach (DiCosi o, R.; Eisenberg, A.; Seip, JE; Gavagan JE; Payne, M. S.; Anton DL (1997): Pyruvic acid production using methylotrophic yeast transformants as catalyst. J. Mol. Cat. B: Enzymatic Volume 2, 223-232; U.S. Patent 5,538,875). The H ₂ 0 ₂ formed in the lactate oxidation is converted to H ₂ 0 and 0 ₂ by endogenous catalase. With this process, a 1 M solution of sodium or ammonium lactate could be converted to pyruvate to over 98%. In addition, the possibility of cell-free oxidation of lactate to pyruvate with immobilized (S) -hydroxy acid oxidase and catalase is also known (Burdick, BA; Schaeffer, JR (1987): Co-immobilized coupled enzyme systems on nylon mesh capable of gluco- never and pyruvic aeid production. Biotechnol. Lett. 9: 253-258). Another method is the conversion of lactate to pyruvate with resting, anaerobically grown cells from Proteus mirabilis or Proteus vulgaris (Simon, H.; Schinschel, C. (1993): Preparation of pyruvate from (R) -lactate with Proteus species. J. Biotechnol. 31: 191-203). With this system, a 0.65 M lactate solution could be converted to pyruvate to 94% in 1 h.

As an alternative to lactate, glucose is used as a substrate for pyruvate production by means of full-line biotransformation by yeasts or bacteria, pyruvate being formed primarily by the reactions of glycolysis. For example, Torulopsis glabrata strains with multiple vitamin auxotrophy have been described which produced 1.17 mol pyruvate / mol glucose (Yonehara, T .; Miyata, R. (1994): Fermentative production of pyruvate from glucose by Torulopsis glabrata. J. Ferm. Bioeng. 78: 155-159). The maximum pyruvate concentration achieved with these strains in fed-batch fermentations was 67.8 g / L (0.77 M). A comparable method for pyruvate production from glucose was also used for lipoic acid auxotrophic strains of Enterobacter aerogenes (Yokota A. (1989): Pyruvic aeid production by lipoie aeid auxotrophs of Enterobacter aerogenes. Agric. Biol. Chem. 53: 705- 711) and Escherichia coli (Yokota A.; Shimizu, H .; Terasawa Y .; Takaoka, N.; Tomita. F. (1994b): Pyruvic aeid production by a lipoie aeid auxotroph of Escherichia coli W1485. Appl. Microbiol. Biotechnol. 41: 638-643). When grown on glucose under lipoic acid deficiency conditions, the Enterobacter aerogenes strains maximally produced 0.8 mol pyruvate per mol glucose, the Escherichia coli strains up to 1.2 mol pyruvate / mol glucose (Yokata A. Shimizu, H .; Terasawa Y.; Takaoka, N.; Tomita. F. (1994a): Pyruvic aeid production by an Fl-ATPase- defective mutant of Escherichia coli W1485lip2. Biosci. Biotechnol. Biochem. 58: 2164-2167; Yokota, A.; Henmi, M.; Takaoka, N.; Hayashi, C .; Takezawa Y. ; Fukumori, Y .; Tomita, F. (1997): Enhancement of glucose metabolism in pyruvic aeid-hyperproducing Escherichia coli mutant defective in Fl-ATPase activity. J. Ferm. Bioeng. 83: 132-138). The production of pyruvate by pyrolysis of grape acid has the disadvantage that 1. a high energy consumption (boiling point of grape acid: approx. 200 ° C.) arises for the process and 2. heavy metals are required and 3. grape acid is only available to a limited extent. The disadvantage of the microbial production of pyruvate from lactate is that the substrate must first be produced, e.g. B. with the help of lactic acid bacteria from glucose. The disadvantage of the processes for microbial pyruvate formation from glucose is that the yields achieved are significantly below the maximum theoretically achievable yield. In addition, complex optimization of the vitamin concentrations must be carried out (Li, Y .; Chen, J.; Lun, S.-Y .; Rui, X.-S. (2001) Efficient pyruvate production by a multi-vitamin auxotroph of Torulopsis glabrata: key role and optimization of vitamin levels. Appl. Microbiol. Biotechnol. 55: 680-685). The inventors have already developed a process for the fermentative production of pyruvate using an Escherichia coli strain YYC202 in DE 101 29 711.4 which, under suitable test conditions, has an almost completely constant conversion of glucose into pyruvate allowed (≥1.9 mole pyruvate / mole glucose).

This method is based on an Escherichia coli strain YYC202, which has a deletion of the genes aceEF for the proteins Ei and E _{2 of} the pyruvate dehydrogenase, and mutations in the genes poxB for the pyruvate oxidase, pfB for the pyruvate formate lyase and pps for the PEP synthetase. Pyruvate dehydrogenase is responsible for the oxidation of pyruvate to acetyl-CoA under aerobic conditions. The pyruvate formate lyase catalyzes the conversion of pyruvate to acetyl-CoA and formate under anaerobic conditions and is completely inactivated even at low oxygen concentrations. Pyruvate oxidase catalyzes the oxidation of pyruvate to acetate and transfers the electrons to ubiquinone. The enzyme is increasingly formed in the stationary phase and its synthesis is dependent on a sigma factor S (RpoS). PEP synthetase catalyzes the formation of phosphoenolpyruvate, AMP and Pi from pyruvate and ATP. The strain YYC202 is in aerobic growth in glucose minimal medium in acetate auxotroph (Ace ^" ). This Ace ^" phenotype is based on the fact that the lack of pyruvate dehydrogenase, in particular, means that acetyl groups can no longer be provided for biosynthesis. E. coli mutants lacking only pyruvate dehydrogenase show a similar phenotype, but can form microcolonies after 6-8 days of incubation on glucose minimal medium plates. Pyruvate oxidase is responsible for the low acetate production that enables the formation of these microcolonies. If the poxB gene is additionally inactivated in a pyruvate dehydrogenase mutant no more micro-colonies formed. The strain E. coli YYC202 likewise did not form any microcolonies on glucose minimal medium plates without acetate after 6-8 days.

With aerobic growth in minimal medium with glucose and acetate, E. coli YYC202 converts a large part of the glucose to pyruvate (up to 1.7 mol pyruvate / mol glucose) and excretes it into the medium. Under non-growing conditions, E. coli YYC202 could even produce ≥1.9 mole pyruvate / mole glucose. Both under growing and non-growing conditions, the NADH formed in glycolysis is reoxidized to NAD ⁺ by the respiratory chain with oxygen as the terminal electron acceptor. Under anaerobic, fermenting conditions, however, a conversion of glucose to pyruvate is not possible due to the lack of possibility for reoxidation of NADH.

By transforming E. coli YYC202 with plasmid pGS87 (obtained from JR Guest, University of Sheffield, Great Britain), which carries the genes aceEF and lpd (coding for the third subunit of pyruvate dehydrogenase) from E. coli the acetate auxotrophy is abolished and the strain no longer produces pyruvate. This confirms that the deletion of the genes aceE and aceF is responsible for pyruvate formation by E. coli YYC202.

All previously described methods for microbial pyruvate formation from glucose with the exception of DE 101 297 11.4 are based on vitamin auxotrophs, in particular thiamine auxotrophic strains, e.g. B. from Toru lopsis glabrata or from E. coli. The activity of the pyruvate-converting enzymes, e.g. B. the pyruvate decarboxylase or pyruvate dehydrogenase, controlled by the availability of the vitamins. In contrast, the strain E. coli YYC202 according to DE 101 297 11.4 has no vitamin auxotrophies and only requires acetate as a supplement for growth in minimal glucose medium.

From US 5,869,301 it is known that the E. coli mutant AFP-111 with a defect in the genes which code for the pyruvate formate lyase and the lactate dehydrogenase, and a further, unknown mutation, to an increased extent under anaerobic conditions Malate, fumarate and succinate forms, but not pyruvate. It is generally indicated in US Pat. No. 5,869,301 that measures which lead to the deactivation of enzyme systems for pyruvate degradation theoretically also cause an accumulation of the pyruvate. However, no indication of which enzymes need to be specifically switched off in order to specifically increase pyruvate production is disclosed. In particular, however, it is not taken into account that continuous pyruvate formation from glucose is not possible under anaerobic, fermenting conditions, since the reoxidation of the NADH formed in the glycolysis only with the conversion of pyruvate to other, reduced compounds, such as, for. B. lactate is possible.

As described in DE 101 297 11.4, by switching off pyruvate-degrading enzymes, an almost complete conversion of glucose into pyruvate was achieved. den (> 1.9 moles pyruvate / mole glucose). When aerobic cultivation of E. coli YYC202 in the presence of high glucose concentrations, lactate was formed in addition to pyruvate. This reduces the yield of pyruvate and also makes it more difficult to purify it.

It is therefore an object of the invention to provide a method which no longer has the disadvantages mentioned above.

Starting from the preamble of claim 1, the object is achieved according to the invention with the features specified in the characterizing part of claim 1. The object is further achieved, starting from the preamble of claim 12, with the features specified in the characterizing part of claim 12. Furthermore, starting from the preamble of claim 13, the object is achieved with the features specified in the characterizing part of claim 13.

With the method and the microorganisms according to the invention, it is now possible to produce an increased concentration of pyruvate compared to conventional methods. The inventors surprisingly found that when using microorganisms in which the pyruvate dehydrogenase, the phosphoenolpyruvate synthetase and the pyruvate oxidase are inactivated or have a reduced activity, the additional deactivation of the enzyme activity of the NAD ⁺ -dependent D- Lactate dehydrogenase leads to a further increase in pyruvate production. This D-lactate dehydrogenase is also called below Designation LDH summarized. The term “switching off” used in the following includes the complete inhibition of the synthesis of LDH or the inactivation of the gene coding for D-lactate dehydrogenase or the reduction or switching off of the intracellular activity of the enzymes mentioned.

The gene sequence coding for the LDH is already known from the literature (Bunch, PK, Mat-Jan, F., Lee, N. and Clark, DP (1997) The IdhÄ gene encoding the fermentative lactate dehydrogenase of Escherichia coli. Microbiology 143: 187-195.). This gene is summarized below under the name "IdiiÄ gene sequence". With the aid of directed recombinant DNA techniques, nucleotide sequences coding for an LDH can be removed or their expression reduced. With the aid of these methods, for example, the 1 dhA gene sequence can be deleted in the chromosome. Suitable methods for this are known to the person skilled in the art, for example, from Link AJ, Philips D., Church GM (1997) Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame charaterization. J. Bacteriol. 179: 6228-6237, or also from Datsenko KA, Wanner WL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad USA 97: 6640-6645, also only parts of the 1 dhA gene sequence can be deleted or mutated fragments of the 2 dhA gene sequence can be exchanged desire or a reduction in LDH activity. By transduction with the phage Pl, the intact 2di-Ä gene of E. coli YYC202 could be replaced by a defective 2di-ü gene from the E. coli strain NZN117 (obtained from DP Clark, Dep. of Microbiology, Southern Illinois University, Carbondale, USA). The corresponding protocols are well established for E. coli (Silhavy, J., Berman, M. and Enquist, L. (1984) Experiments with gene fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.). By inactivating the 2dήΛ gene, a complete loss of LDH activity is achieved.

An example of such a mutant is the Escherichia coli strain YYC2022di-A, deposited on March 12, 2002 under the name DSM 14863, which carries an insertion in the 2 dhA gene sequence.

Another possibility to weaken or switch off the activity of LDH are mutagenesis procedures. These include undirected processes that use chemical reagents such as. B. use N-methyl-N-nitro-N-nitrosoguanidine or UV radiation for mutagenesis. Methods for mutation triggering and mutant search are generally known and can be found, inter alia, in Miller (A Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria (Cold Spring Harbor Laboratory Press, 1992)) or in the manual "Manual of Methods for General Bacteriology "of the American Society for Bacteriology (Washington DC, USA, 1981).

In addition, the expression of the 2d A gene sequence can also be reduced. In this way, the promoter and regulatory region located upstream of the structural gene can be mutated. Work in the same way Expression regulation cassettes which are installed upstream of the structural gene. With adjustable promoters, it is also possible to reduce expression in the course of fermentative pyruvate production. In addition, regulation of translation is also possible, for example, by reducing the stability of the m-RNA. Furthermore, genes can be used which code for the corresponding enzyme with low activity. Alternatively, a reduced expression of the 2dhA gene sequence can also be achieved by changing the media composition and culture management.

The LDH catalyzes the reduction of pyruvate to lactate with NADH as coenzyme and is activated by pyruvate allosterically (Tarmy, EM and Kaplan, NO (1968) Kinetics of Escherichia coli B D-lactate dehydrogenase and evidence for pyruvate-controlled change in confor - Mation. J ^" . Biol. Chem. 243: 2587-2596). The apparent iζ _n value for pyruvate is, depending on the pH value, between 4.4 M (pH 6.7) and 7.2 mM (pH 7.5) (Tarmy, EM and Kaplan, NO (1968) Kinetics of Escherichia coli B D-lactate dehydrogenase and evidence for pyruvate-controlled change in conformation. \ Biol. Chem. 243: 2587-2596.). These concentrations are found in E. coli wild-type strains The expression of the 2di ^* ιÄ gene is induced under anaerobic conditions by a low pH, under aerobic conditions there is a pH-independent expression (Jiang, GR, Nikolova, S. and Clark DP (2001 ) Regulation of the I hA gene, encoding the fermentative lactate dehydrogenase of Escherichia CO2i. Microbiology 147: 2437-2446.). Pyruvate stimulates the expression of the 2diιÄ gene 2 to 4 times (Jiang et al. 2001). The significant formation of lactate when cultivating E. coli YYC202 with high glucose concentrations can be explained as follows: The blocking of pyruvate degradation leads to an accumulation of pyruvate, which on the one hand causes an increased expression of the 2diιÄ gene and on the other hand stimulates the activity of the enzyme. Significant lactate formation is observed in shake flask batches from a glucose concentration of 50 mM, while in fermentations it is already observed from glucose concentrations of approximately 5 mM. To prevent lactate formation, a derivative of the strain Escherichia coli YYC202 was constructed in the present invention, for example, in which the 2diιÄ gene was inactivated by inserting a kanamycin resistance gene into the open reading frame of the 2di-A gene sequence.

Escherichia coli YYC202 is described in the literature with regard to its genetic properties

(Chang, YY.; Cronan, JE (1982): Mapping nonselectable genes of Escherichia coli by using transposon TnlO: location of a gene affecting pyruvate oxidase. J. Bacteriol. 151: 1279-1289; Chang, YY .; Cronan , JE

(1983): Genetic and biochemical analyzes of Escherichia coli strains having a mutation in the structural gene

(poxB) for pyruvate oxidase. J. Bacteriol. 169 (5): 756-762; Georgiou, D. Ch.; Fang, H _.; Gennis, RB

(1987): Identification of the cydC locus required for expression of the functional form of the cytochrome d terminal oxidase complex in Escherichia coli. J. Bacte- riol. 169: 2107-2112). Bacteria and yeasts, in particular gram-negative bacteria, preferably from the Enterobacteriaceae family, such as, for. B. Escherichia coli. The strain Escherichia coli YYC2022d A, deposited on March 12, 2002 with the German Collection of Microorganisms and Cell Cultures (DSMZ) under DSM number 14863, has proven to be particularly suitable and was modified as described above.

The process according to the invention prevented the formation of lactate as the end product of the metabolism and significantly improved the conversion of high glucose concentrations into pyruvate by switching off the enzymes pyruvate dehydrogenase, pyruvate oxidase and phosphoenolpyruvate synthetase and additionally switching off the LDH. The microorganisms that are used in the method according to the invention can, for example, pyruvate from carbohydrates such as. As glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose, or from alcohols such as. B. produce glycerin. These substances can be used individually or as a mixture.

Continuous or discontinuous batch processes (batch cultivation) or fed-batch (feed process) or repeated fed-batch processes (repetitive feed process) for the purpose of pyruvate production can be used as cultivation methods. The culture medium to be used must be suitable meet the requirements of the microorganisms. The fermentations are carried out aerobically.

Advantageous further developments are specified in the subclaims.

The figures show an overview of the pyruvate pathway and experimental results of the method according to the invention.

It shows :

Fig. 1: Scheme of the conversion of glucose in pyruvate in E. coli YYC202ldhA. After glucose has been taken up by the phosphoenolpyruvate-dependent phosphotransferase system (PTS), glucose-6-phosphate is converted to pyruvate in the reactions of glycolysis. Due to the genetic defects in the genes aceEF, PflB, poxB, and IdhA, pyruvate cannot be converted further and is excreted into the medium.

Fig. 2: Southern blot analysis (Southern, EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J ^" . Mo. Biol. 98: 503-517.) By E. coli YYC202 ldhA.

Lanes 1, 5 and 6 each contain 75 ng digoxigenin-labeled DNA standard. Lanes 2 and 7 contain chroraosomal DNA from E. coli YYC202, lanes 3 and 8 chromosomal DNA from E. coli YYC202 IdhA and lanes 4 and 9 chromosomal DNA from E. coli NZN117. 15 μg of chromosomal DNA, which had been digested with BcoRI and Hinllll, were separated per lane. The blot lanes 1-5 were used to hybridize with a digoxigenin-labeled 2153 bp DNA fragment to the E. coli ldhA gene, the blot with lanes 6-10 to a digoxigenin-labeled 668 bp fragment with the camamycin Resistance gene from E. coli NZN117. The DNA fragments were separated on a 1% agarose gel and, after denaturation, blotted onto a nylon membrane. Labeling of the probes with digoxigenin, hybridization, washing and detection of the probes with CDP-Star were carried out using the DIG chemlink labeling and detection kit (Röche Diagnostics) in accordance with the manufacturer's instructions. As expected, an 11 kb BcoRI-ffindlll fragment was detected with the 2 hA probe in E. coli YYC202, whereas fragments of 6.2 kb and 6.8 kb were detected in E. coli YYC202 IdhA and E. coli NZN117. As expected, no signal was detected in the E. coli YYC202 with the Jan probe, but fragments of 6.2 kb and 6.8 kb in turn were detected in YYC202 IdhA and NZN117.

3: Conversion of glucose (■) to pyruvate (A) and possibly lactate (•) by cell suspensions of E. coli YYC202 (A) and E. coli YYC202 ldhA (B).

The abscissa X indicates the time in hours and the ordinate Y the concentration of glucose, pyruvate or lactate in mM.

The cells were precultivated in M9 minimal medium with glucose and acetate, then washed with 0.9% NaCl solution and then to an OD ₆₀ o of 7.4 (YYC202) or 3.1 (YYC202 IdhA) in 500 mM MOPS buffer pH 7.0 resuspended with 100 mM or 88 mM glucose. The cell suspensions were incubated in shake flasks at 37 ° C and 140 rpm. The glucose concentration was in a coupled enzymatic assay with hexokinase and glucose-6-phosphate dehydrogenase determined (Bergmeyer, HU (1984) Methods of Enzymatic Analysis, 3 edition Vol VI ^rd:.. Metabolites. 1: Carbohydrates, pages 163-172, Verlag Chemie, Weinheim. ). Pyruvate and lactate were determined by HPLC analysis with an Aminex HPX-87H column (Bio-Rad) and UV detection at 215 nm.

Fig. 4: Fed-batch fermentation of E. coli YYC202 and E. coli YYC202 IdhA.

The abscissa X shows the fermentation time in hours and the ordinate Y in A, the optical density OD ₆ oo or B in the concentration of pyruvate and lactate in mM. The strains were cultivated in a 7.5 1 laboratory fermenter under aerobic conditions (p02> 40% saturation) at 37 ° C and constant pH 7 (pH regulation) in a synthetic medium. The glucose concentration was kept constant at about 5 mM using an “on-line general analyzer”. The acetate was added in excess, the CO ₂ concentration in the exhaust gas being used for regulation. The growth of E coli YYC202 (•) and E. coli YYC202 IdhA (■). In B the pyruvate formation is represented by E. coli YYC202 (•) and E. coli YYC202 2dhÄ (■) and the lactate formation by E. coli YYC202 (O) and E. coli YYC202 IdhA (□) are shown.

The invention will be described below by way of example.

By using microorganisms whose activity has reduced or completely inactivated the LDH, could implement a high z. B. from DE 101 29 711.4 known methods, increased pyruvate production can be achieved. With resting cells, the yield of pyruvate could be increased by 20% when 100 mM glucose was converted. In the case of fermentations using the fed-batch process, the pyruvate concentration reached after 37 h could be increased by 40%. Both yeasts and bacteria can be used as microorganisms. Microorganisms from the Enterobacteriaceae family, particularly from the Escherichia genus, have proven to be particularly suitable. The organism Escherichia coli YYC202 has proven to be very suitable. This bacterial strain has some genetic changes compared to the wild type strain (WT). So z. B. from DE 101 29 711.4 it is known that the following gene sequences have been changed: aceEF - coding for the subunits El and E2 of pyruvate dehydrogenase, poxB - coding for the pyruvate oxidase; pps - coding for phosphoenol pyruvate synthetase; pfB - coding for the pyruvate formate lyase. Surprisingly, the additional switching off of the LDH in the form of z. B. an insertion of a resistance gene in the open reading frame of the 2diιA gene sequence, as described for Escherichia coli YYC202ldhA, a further significant increase in pyruvate production. Switching off the gene sequences coding for the pyruvate dehydrogenase, the pyruvate oxidase, the phosphoenolpyruvate synthetase and the LDH is considered to be particularly advantageous for improved pyruvate production.

With resting cells, the substrate could be converted into pyruvate, which was considerably higher than that Yields achieved with previously known processes. Due to the genotype, the organisms are unable to convert pyruvate to acetyl-CoA, acetate or phosphoenol pyruvate. This acetate auxotrophy allows growth and product formation to be regulated by dosing the essential co-substrate acetate. It could be shown in DE 101 29 711.4 that the acetate consumption rate is identical to the C0 ₂ formation rate. This correlation can be used to determine and regulate the acetate consumption rate based on the online measurable C0 ₂ concentration in the exhaust gas. In the presence of acetate and other nutrients such as. B. Nitrogen in the fermentation medium can take place due to metabolism cell division / growth. However, since part of the carbon source is used for the biosynthesis of new cell material during growth, a maximum pyruvate yield with growing cells is not to be expected. This is possible with cells which, due to the media conditions, do not carry out biosynthesis for the purpose of forming new cell material, ie “resting” cells. These cells are obtained by culturing in a medium in which a high cell concentration can be formed. The cells then become Harvested, for example, by centrifugation and placed in a medium which contains no acetate and no other nutrients required for growth, such as nitrogen, under which conditions an even higher pyruva yield can be achieved , in particular hexoses, and alcohols In order to ensure the economic viability of the process, preference is given to substrates which are obtained as waste materials or are directly available, ie do not have to be pretreated or produced in a special way for further processing for the method according to the invention.

The process according to the invention can furthermore be used, for example, to produce labeled pyruvate in high yields, for example by B. ¹³ C-labeled carbon sources can be used. The economy of this process is significantly improved by the high product yields. The method according to the invention can additionally have a positive effect on the production of further metabolic products derived from pyruvate. So z. B. by introducing a gene for L-alanine dehydrogenase and in the presence of high ammonium concentrations under anaerobic conditions, a homoalanine fermentation can be established, in which 1 mole of glucose and 2 moles of ammonium is converted to 2 moles of L-alanine.

The present invention is explained in more detail below on the basis of exemplary embodiments.

1. Construction of a derivative of E. coli YYC202 with a defective IdhA gene

To inactivate the 2d Ä gene, the open reading frame was interrupted by a kanamycin resistance gene. The corresponding mutation was determined by Pl transduction (Silhavy, J., Berman, M. and Enquist, L. (1984) Experiments with gene fusions. Co2d Spring Harbor Laboratory, Cold Spring Harbor, New York.) From the donor strain E. coli NZN117 (Bunch, PK, Mat-Jan, F., Lee, N. and Clark, DP (1997) The IdhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli. Microbiology 143: 187-195.) Transferred to E. coli YYC202. The successful transduction was confirmed by Southern blot analysis (FIG. 2). The lactate dehydrogenase activity (LDH activity) in the parent strain YYC202 and in the derivative YYC202 IdhA was determined to be 0.4% (w / v) glucose after cultivation in LB medium for 6 hours. For this purpose, cell-free extracts were prepared, the membrane fraction was removed by ultracentrifugation and the soluble fraction was used in an enzyme test in which the pyruvate-dependent oxidation of NADH to NAD ^{+ was} measured spectrophotometrically on the basis of the decrease in absorbance at 340 nm (Bunch, PK, Mat-Jan, F., Lee, N. and Clark, DP (1997) The IdhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli. Microbiology 143: 187-195). E. coli YYC202 had a high lactate dehydrogenase activity of 3.1 μmol min ^"1 mg protein ^{" 1} , while E. coli YYC202ldhA showed an activity of <0.01 μmol min ^"1 mg protein ^{" 1} .

2. Glucose conversion by resting cells from E. coli YYC202 and E. coli YYC2021dhA

The strains E. coli YYC202 and E. coli YYC202ldhA were precultivated in minimal medium with glucose and acetate. The cells were then washed, resuspended in a buffer (500 mM MOPS, pH 7.0) with approximately 100 mM glucose, and incubated in shaking flasks at 37 ° C. and 140 rpm. As shown in Fig. 3A, the glucose in the YYC202 strain was initially converted to pyruvate and lactate at approximately the same rate. After this When the glucose was consumed, the lactate formed was slowly reoxidized to pyruvate. In contrast, in the YYC202 IdhA strain, the glucose was converted to pyruvate at a constant rate, but no lactate was formed (FIG. 3B).

3. Growth and product formation in fed-batch cultivation of Ξ. coli YYC202 and E. coli YYC202 IdhA in minimal glucose medium

For fed-batch fermentation, the strains were cultivated in a 7.5 1 laboratory fermenter under aerobic conditions (p02> 40% saturation) at 37 ° C and pH 7 (controlled) in a synthetic medium (1.5 g NaH ₂ P0 ₄ per liter x H ₂ 0; 3.25 g KH ₂ P0 ₄ ; 2.5 g K ₂ HP0 ₄ ; 0.2 g NH ₄ C1; 2 g (NH ₄ ) S0 ₄ ; 0.5 g MgS0 ₄ ; 10 mg CaCl ₂ x 2H ₂ 0; 0.5 mg ZnS0 ₄ x 7H ₂ 0 l ^"1 ; 0.25 mg CuCl ₂ x 2H ₂ 0; 2.5 mg MnS0 ₄ x H ₂ 0; 1.75 mg CoCl ₂ x 6H ₂ 0; 1.25 mg H ₃ B0 ₃ ; 2.5 mg AlCl ₃ x 6H ₂ 0; 0.5 mg Na ₂ Mo0 x 2H ₂ 0; 10 mg FeS0 ₄ ). The glucose concentration was kept constant at about 5 mM using an "On-line General Analyzer". The acetate was added in excess, the C0 ₂ concentration in the exhaust gas being used for regulation. It had previously been shown that the acetate consumption rate is equal to the CO ₂ formation rate (German patent application 101 29 711.4). In the experiments shown in FIGS. 4A and 4B, both strains showed approximately the same growth behavior, but differed significantly in terms of pyruvate and lactate formation. The starting strain YYC202 had formed 487 mM pyruvate and 341 mM lactate after 37 h, while the strain YYC2022d Ä after 37 h 673 M pyruvate, but no lactate had formed.

4. Control experiment with E. coli NZN117

In order to rule out that the 2 iιA mutation in the E. coli YYC202-ldhA strain is responsible for pyruvate formation, the E. coli NZN117 donor strain (Bunch, PK, Mat-Jan, F., Lee, N. and Clark , DP (1997) The 2dhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli. Microbiology 143: 187-195.) In minimal medium with 10 mM glucose and 2 mM acetate at 37 ° C and 140 rpm. At no point during growth and in the stationary phase could pyruvate be detected in the culture supernatant. This shows that a 2diiA mutation does not lead to the excretion of pyruvate under the conditions mentioned. In E. coli YYC202ldhA, the deletion of the aceEF genes for two subunits of pyruvate dehydrogenase and the inactivation of the poxB gene for pyruvate oxidase are responsible.

Claims

Patent claim 1. Process for the microbial production of pyruvate from carbohydrates and alcohols using microorganisms in which the pyruvate dehydrogenase, the phosphoenolpyruvate synthetase and the pyruvate oxidase are inactivated and / or have a reduced activity, characterized in that the D- Lactate dehydrogenase is turned off and these microorganisms are used. 2. The method according to claim 1, characterized in that the expression of the IdiiA gene sequence is reduced and / or the IdhA gene sequence is changed such that no expression takes place or the IdhA gene sequence is deleted. 3. The method according to any one of claims 1 to 2, characterized in that a resistance gene is incorporated into the open reading frame of the 2d A gene sequence. 4. The method according to any one of claims 1 to 3, characterized in that Microorganisms from the group of Enterobacteriaceae are used. 5. The method according to any one of claims 1 to 4, characterized in that Microorganisms from the genus Escherichia are used. 6. The method according to any one of claims 1 to 5, characterized in that Escherichia coli YYC2022d-aA, deposited under the name DSM 14863, is used. 7. The method according to any one of claims 1 to 6, characterized in that carbohydrates or alcohols are used as the substrate in the fermentation. 8. The method according to any one of claims 1 to 7, characterized in that Acetate is used in fermentation with growing cells. 9. The method according to any one of claims 1 to 8, characterized in that no acetate is used in fermentations with resting cells. 10. The method according to any one of claims 1 to 9, characterized in that a C0 ₂ measurement analysis is used in the fermentations. 11. The method according to any one of claims 1 to 10, characterized in that an aerobic fermentation is carried out. 12. A process for the production of metabolic products derived from pyruvate, characterized in that a process according to claims 1 to 11 is carried out and an anaerobic fermentation is carried out. 13. Microorganism for the microbial production of pyruvate from carbohydrates and alcohols in which the pyruvate dehydrogenase, the phosphoenolpyruvate synthetase and the pyruvate oxidase are inactivated and / or have a reduced activity, characterized in that it has an deactivated D-lactate dehydrogenase having . 14. Microorganism according to claim 13, characterized in that the expression of the ldhA gene sequence is reduced and / or the ldhA gene sequence is changed such that no expression takes place or the ldhA gene sequence is deleted. 15. Microorganism according to one of claims 13 to 14, characterized in that it has an insertion in the open reading frame of the IdhA gene sequence. 16. Microorganism according to one of claims 13 to 15, characterized in that it comprises the insertion of a resistance gene in the open reading frame of the 2dA gene sequence. 17. Microorganism according to one of claims 13 to 16, characterized in that it comes from the family Enterobacteriacea. 18. Microorganism according to one of claims 13 to 17, characterized in that it comes from the genus Escherichia. 19. Microorganism according to one of claims 13 to 18, deposited with the DSMZ under the DSM no. 14,863th