RU2466186C2 - BACTERIUM Escherichia coli - SUCCINIC ACID PRODUCER AND METHOD FOR PREPARING SUCCINIC ACID - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to microbiological industry and concerns the bacterium Escherichia coli that is a succinic acid producer, and a method for preparing succinic acid with the use of such bacteria. The described bacteria is modified by changing a nucleotide sequence of a promoter and a ribosome-binding site controlling expression of operon aceEF-lpdA genes in a bacterial chromosome so that expression of the aceE, aceF and lpdA genes in said bacteria is amplified. A method for preparing succinic acid consists in culture of such bacterium in a nutrient medium and recovery of succinic acid from a culture fluid.

EFFECT: invention provides producing a higher amount of succinic acid by microbiological procedure.

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Description

Technical field

The present invention relates to the microbiological industry, in particular, to a method for producing succinic acid using bacteria of the genus Escherichia, modified in such a way that the expression of aceEF-lpdA operon genes encoding the proteins of the component of the NAD ⁺ -reducing pyruvate dehydrogenase enzyme complex is enhanced in the bacterium.

Description of the Related Art

Succinic acid, traditionally obtained by petrochemical synthesis, is one of the compounds whose production from renewable raw materials is most in demand due to rising oil prices and limited reserves. Succinic acid is one of the most important building blocks for the production of a wide range of high value-added chemicals that are also currently synthesized from petroleum products. The expected market for succinic acid is more than 300,000 tons / year.

The most effective microbiological process for the production of succinic acid and its salts is anaerobic fermentation of sugars, in particular glucose, with the formation of the target substance in the reduction part of the tricarboxylic acid cycle. In this process, the maximum possible conversion rate of the substrate into the product based on carbon is 2 mol / mol. This is due to the fixation of CO ₂ at the stage of formation from glycolytic intermediates of oxaloacetate, the precursor in the anaerobic pathway of glucose fermentation to succinic acid. However, the feasibility of this process is hindered by its redox imbalance. The conversion of each of the two oxaloacetate molecules obtained from glycolytic glucose utilization intermediates to succinic acid requires 2 reduced equivalents (NADH), while only half of the required equivalents are formed during glycolysis (1 NADH per oxaloacetate molecule).

This leads to the fact that the best indicators of succinic acid production by specially obtained recombinant strains are achieved in biosynthesis processes, which are a combination of two biochemical pathways - the reducing part of the tricarboxylic acid cycle and glyoxylate shunt. The ratio of the contributions of each of the pathways to the biosynthesis of succinic acid determines the final yield of the target substance.

The glyoxylate shunt, consuming less NADH for each synthesized succinic acid molecule, acts in this case as a donor of "residual" glycolytic NADH for highly productive formation of the target substance in the reduction part of the tricarboxylic acid cycle. However, since 2 Acetyl-CoA molecules formed from a glycolytic precursor with CO ₂ loss are involved in the glyoxylate shunt, along with oxaloacetate, the contribution of this process to the total biosynthesis of succinic acid leads to a decrease in the total yield of the product.

The balance of carbon fluxes towards the biosynthesis of succinic acid along the indicated paths is determined by both the metabolic status of the cell and the effect of directed modifications introduced into the biochemical apparatus of the target microorganism.

The rational design of recombinant microbial producers of succinic acid suggests the need for inactivation of competitive, in relation to the target, biochemical pathways of utilization of key metabolic precursors and reduced equivalents of NADH. These competitive paths are:

A) the conversion, under the action of lactate dehydrogenase, of pyruvic acid, which is the precursor of Acetyl-CoA biosynthesis and a potential precursor in oxaloacetate biosynthesis, to lactic acid with the consumption of the reduced equivalent of NADH;

B) the conversion, under the action of pyruvate oxidase, pyruvic acid into acetic acid;

B) the conversion, under the action of aldehyde / alcohol dehydrogenase, Acetyl-CoA, a substrate of glyoxylate shunt, into ethanol with a flow rate of two restored equivalents of NADH;

D) the conversion, under the action of phosphotransacetylase and acetate kinase, Acetyl-CoA into acetic acid.

It should be noted that inactivation of the corresponding pathways not only “saves” metabolic precursors and reduced equivalents for the target pathways of succinic acid biosynthesis, but also excludes the possibility of accumulation in the medium of by-products (acetic and lactic acids, as well as ethanol), which are, along with succinic acid , the main products of microbial mixed acid fermentation. As a result, succinic acid becomes the main product of the fermentation process, which facilitates its further isolation and purification.

The study of the metabolism of Escherichia coli cells and the availability of powerful genetic engineering tools led to the fact that the best microbial producers of succinic acid to date are obtained on the basis of this particular microorganism.

Changes in the functioning of the metabolic site of phosphoenolpyruvate-oxaloacetate-pyruvate-Acetyl-CoA, a key branching point that determines the distribution of carbon fluxes between the reducing part of the tricarboxylic acid cycle and the glyoxylate shunt in E. coli cells, can be made in various ways.

In this regard, it should be noted that in E. coli, the conversion of pyruvic acid into Acetyl-CoA, under anaerobic conditions, is not accompanied by the formation of NADH under pyruvate format lyase, under aerobic conditions, under the action of pyruvate dehydrogenase, an additional NADH molecule is formed on each formed Acetyl-CoA . However, gene expression of pyruvate dehydrogenase complex under anaerobic conditions was repressed in this microorganism (see, for example, Soini J. et al., Norvaline is accumulated after a down-shift of oxygen in Escherichia coli W3110. Microb Cell Fact., 2008, 7: 30, doi: 10.1186 / 1475-2859-7-30).

One of the standard approaches to modifying the functioning of the metabolic site of phosphoenolpyruvate-oxaloacetate-pyruvate-Acetyl-CoA is to inactivate pyruvate formatliase. In this case, the main anaerobic pathway for the formation of Acetyl-CoA is blocked, the residual biosynthesis of which occurs due to the basal activity of the pyruvate dehydrogenase complex. As a result of this, conditions are created for the potentially dominant participation in the succinic acid biosynthesis of the reduction part of the tricarboxylic acid cycle due to an artificial decrease in the activity of the glyoxylate shunt. However, the oxidation / reduction balance of the cell does not undergo significant changes capable of providing the reduction part of the tricarboxylic acid cycle with the reduced equivalents of NADH that are insufficient for effective succinic acid biosynthesis. In addition, this approach has several disadvantages. Inactivation of the main anaerobic pathway of Acetyl-CoA biosynthesis in recombinant producers in most cases leads to the impossibility of their growth under anaerobic conditions and necessitates the use of a two-stage aerobic / anaerobic fermentation process. Also, the inability to efficiently convert pyruvic acid to Acetyl-CoA in a number of similar recombinant producers leads to undesirable accumulation of this substance in the culture medium.

This approach has been used in the construction of recombinant succinic acid producers such as E. coli KJ060 (K. Jantama et al., Combining metabolic engineering and metabolic evolution to develop nonrecombinant strains of Escherichia coli C that produce succinate and malate. Biotechnol Bioeng., 2008, 99 (5), 1140-1153), E. coli AFP184 (Andersson C. et al., Effect of different carbon sources on the production of succinic acid using metabolically engineered Escherichia coli. Biotechnol Prog. 2007, 23 (2), 381- 388), E. coli NZN111 (Stols L, Donnelly MI., Production of succinic acid through overexpression of NAD (+) - dependent malic enzyme in an Escherichia coli mutant. Appi Environ Microbiol. 1997, 63 (7), 2695-2701 ) and others.

An alternative approach to regulating the distribution of carbon fluxes between the two pathways of succinic acid biosynthesis as a result of targeted interference with the functioning of the metabolic unit of phosphoenolpyruvate-oxaloacetate-pyruvate-acetyl-CoA is to provide the target recombinant with increased activity of pyruvate carboxylase in the presence of intact pyruvate format lyase. Both enzymes use pyruvic acid as a substrate, however, the carboxylating effect of the former leads to the formation of oxaloacetate, while the action of the latter leads to the formation of Acetyl-CoA, resulting in decarboxylation. Thus, due to the action of pyruvate carboxylase competitive in relation to pyruvate formatliase, an increase in the concentration of oxaloacetate with respect to Acetyl-CoA is created. This, in turn, contributes to the possibility of a preferred biosynthesis of succinic acid in the reducing branch of the tricarboxylic acid cycle.

However, even in this case, the oxidation / reduction balance of the cell does not undergo significant changes, providing the generation of additional NADH equivalents for the succinate-forming reduction part of the tricarboxylic acid cycle.

Using this approach, for example, a recombinant succinic acid producer E. coli SBS550MG / pyc (Sanchez AM et al., Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity was obtained. Metab Eng. 2005 , 7 (3), 229-239.). When constructing E. coli strain AFP111 / pyc (Vemuri GN et al., Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli. Appi Environ Microbiol. 2002, 68 (4), 1715-1727) Both of the above approaches were applied.

In the framework of these approaches, directed modifications of the metabolism were not used that could provide anaerobic generation of reduced NADH equivalents additional to glycolytic. However, such generation can be achieved at the stage of conversion of pyruvic acid into Acetyl-CoA if it is carried out under the influence of enzymes of the NAD ⁺ -reducing pyruvate dehydrogenase complex, whose activity is practically not manifested under anaerobic conditions due to reduced expression of the corresponding genes. The redistribution of carbon fluxes between the two pathways of succinic acid biosynthesis will be carried out under the influence of the effect of precision metabolic regulation with negative feedback, since the activity of the pyruvate dehydrogenase complex is NADH-repressible (Schmincke-Ott, E. and Bisswanger H. Dihydrolipoamide dehydrogenase component of the pyruvate dehydrogenase complex from Escherichia coli K12. Comparative characterization of the free and the complex-bound component. Eur. J. Biochem. 1981, 114, 413-420; Shen L. C. and Atkinson DE Regulation of pyruvate dehydrogenase from Escherichia coli. Interactions of adenylate energy charge and other regulatory parameters. J. Biol. Chem. 1970, 245, 5974-597 8). The generation of NADH, as a result of the action of the pyruvate dehydrogenase complex, will contribute to the possibility of effective biosynthesis of succinic acid precisely in the reducing part of the tricarboxylic acid cycle, while reducing, due to NADH-inhibited synthesis of Acetyl-CoA, the contribution of the glyoxylate shunt. Exhaustion of additionally generated NADH equivalents in the reduction part of the tricarboxylic acid cycle will again lead to the temporary activation of the pyruvate dehydrogenase complex, the formation of equivalents, and a relative increase in the contribution to the biosynthesis of succinic acid of the glyoxylate shunt. Thus, the process of redistributing carbon fluxes between the pathways of succinic acid biosynthesis in this case becomes cyclical, dynamic, and auto-regulated. As a result, the combination of the effect of metabolic regulation and adaptive capabilities of the biochemical apparatus of the cell can help achieve a favorable balance of the two pathways of succinic acid biosynthesis and ensure its high efficiency.

Enzymes of the NAD ⁺ reducing pyruvate dehydrogenase complex are encoded by the aceEF-lpdA operon genes. Thus, the activity of the NAD ⁺ reducing pyruvate dehydrogenase complex can be increased by enhancing the expression of the aceEF-lpdA operon genes.

However, there are currently no reports describing the use of amplification of gene expression of the aceEF-lpdA operon to produce succinic acid.

Description of the invention

Objectives of the present invention include increasing the productivity of succinic acid producing strains and providing a method for producing succinic acid using these strains.

The above objectives were achieved by discovering that enhancing the expression of the aceEF-lpdA operon genes can lead to an increase in succinic acid production.

The object of the present invention is a bacterium of the genus Escherichia, with the ability to increased production of succinic acid.

Also an object of the present invention is a bacterium of the genus Escherichia, a succinic acid producer, wherein said bacterium is modified in such a way that the expression of the aceEF-lpdA operon genes in said bacterium is enhanced.

Also an object of the present invention is the bacterium described above, in which said expression is enhanced by modifying the sequence controlling the expression of the aceEF-lpdA operon genes.

Also an object of the present invention is the bacterium described above, wherein said bacterium belongs to the species Escherichia coli.

Also an object of the present invention is a bacterium as described above, wherein said bacterium is further modified so that it acquires pyruvate carboxylase activity.

Another object of the present invention is the bacterium described above, wherein the activity of pyruvate carboxylase is ensured by the presence in the bacterium of a DNA molecule containing a gene encoding pyruvate carboxylase.

Another object of the present invention is a method for producing succinic acid, comprising the steps of culturing the bacteria described above in a nutrient medium; and isolating the produced and accumulated succinic acid from the culture fluid.

Another object of the present invention is the above-described method in which the process of cultivating the bacterium includes an aerobic stage of biomass accumulation and an anaerobic stage of production of succinic acid.

In more detail, the present invention is described below.

BEST MODE FOR CARRYING OUT THE INVENTION

1. Bacteria

A bacterium is a bacterium producer of succinic acid of the genus Escherichia, modified in such a way that the expression of the aceEF-lpdA operon genes in the specified bacterium is enhanced.

The term "succinic acid producing bacterium" may mean a bacterium capable of producing and secreting succinic acid into a culture medium when the bacterium is grown in said culture medium.

The ability to produce succinic acid can be given to bacteria as a result of selection, mutagenesis, or genetic engineering manipulations.

The term "succinic acid producing bacterium" can also mean a bacterium that is capable of producing succinic acid and causes the accumulation of succinic acid in a fermentation medium in quantities greater than the natural or parent E. coli strain, such as E. coli K strain -12, and may mean that the microorganism is capable of accumulating succinic acid in the medium in an amount of not less than 0.4 g / l.

The term "bacterium belonging to the genus Escherichia" may mean that the bacterium belongs to the genus Escherichia in accordance with the classification known to the person skilled in the field of microbiology. As an example of a microorganism belonging to the genus Escherichia, the bacterium Escherichia coli (E. coli) may be mentioned.

The term "enhancing gene expression" may mean that gene expression is higher than in an unmodified strain, for example, in a wild-type strain. Examples of such modifications may include increasing the number of copies of an expressed gene in a cell, enhancing gene expression, etc. The number of copies of the expressed gene is determined, for example, by restriction of the chromosomal DNA followed by Southern blotting using a probe constructed based on the gene sequence, in situ fluorescence hybridization (FISH), and the like. The level of gene expression can be determined by various known methods, including Northern blotting, quantitative RT-PCR, etc. In addition, strains that can be used as a control include, for example, Escherichia coli K-12.

The genes aceE, aceF, and lpdA (synonyms: b0114, ECC0113; b0115, ECC0114; b0116, ECC0115) encode the ACEE, AceF, and LpdA proteins, the components of the enzymatic complex of the NAD ⁺ -reducing pyruvate dehydrogenase. The aceEF-lpdA genes of the operon (nucleotides homologous to nucleotides 123017 to 129336; in sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990) are located between the pdhR and uACH genes on the E. coli K-12 chromosome. The nucleotide sequences of the aceEF-lpdA gene of the operon and the amino acid sequences of the AcEE, AceF and LpdA proteins encoded by the ACEE, aceF and lpdA genes are presented in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, respectively.

Since representatives of various strains of the genus Escherichia may have some variations in the nucleotide sequences, the aceEF-lpdA genes of the operon, aceE, aceF and lpdA, are not limited to the genes whose sequences are given in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 but may also include genes homologous to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3. Therefore, the variants of the proteins encoded by the aceEF-lpdA gene of the operon — acEE, aceF and lpdA — can have a homology of at least 80%, or at least 90%, or at least 95% with respect to the complete amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, provided that they have the activity of AceE, AceF, and LpdA proteins. The term "protein variant" can mean a protein with changes in the sequence, whether deletions, insertions, additions or substitutions of amino acids. The number of changes in a protein variant depends on the position or type of amino acid residue in the tertiary structure of the protein. It can be from 1 to 30, or from 1 to 15 in another example, or from 1 to 5 in another example, in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6. These variations in variants may occur in areas not critical to protein function. These changes are possible because some amino acids have a high homology to each other, therefore, such changes do not affect the tertiary structure.

Homology between two amino acid sequences can be determined using known methods, for example, the BLAST 2.0 computer program, which counts three parameters: count, identity, and similarity.

Substitution, deletion, insertion or addition of one or more amino acid residues is a conservative mutation (s) if activity persists. A typical example of a conservative mutation is a conservative substitution. Examples of conservative substitutions include replacing Ala with Ser or Thr, replacing Arg with Gln, His or Lys, replacing Asn with Glu, Gln, Lys, His or Asp, replacing Asp with Asn, Glu or Gln, replacing Cys with Ser or Ala, replacing Gln with Asn, Glu, Lys, His, Asp or Arg, replacing Glu with Asn, Gln, Lys or Asp, replacing Gly with Pro, replacing His with Asn, Lys, Gln, Arg or Tur, replacing Il with Leu, Met, Val or Phe, replace Leu with Ile, Met, Val or Phe, replace Lys with Asn, Glu, Gln, His or Arg, replace Met with Ile, Leu, Val or Phe, replace Phe with Trp, Tyr, Met, Ile or Leu, replacing Ser with Thr or Ala, replacing Thr with Ser or Ala, replacing Trp with Phe or Tur, replacing Tur with His, Phe or Trp, and replacing Val with Met, Ile, or Leu.

In this regard, the aceEF-lpdA genes of the operon - acEE, aceF and lpdA can be variants that hybridize under stringent conditions with the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or c probe, which can be synthesized based on the specified nucleotide sequence, provided that they encode the functional proteins AceE, AceF and LpdA. "Stringent conditions" include those conditions under which specific hybrids, for example, hybrids with homology of at least 60%, 70%, 80%, 90% or 95% in different examples, are formed, and non-specific hybrids, for example, hybrids with less homology than the above are not formed. A practical example of harsh conditions is a single or multiple washing, or two or three times in another example, at a salt concentration of 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, at 60 ° C. The duration of washing depends on the type of membrane used for blotting and, as a rule, is as recommended by the manufacturer. For example, the recommended washing time for Hybond ™ N ⁺ nylon membrane (Amersham) under severe conditions is 15 minutes. Washing can be done two or three times. The length of the probe can be selected depending on the hybridization conditions, in this particular case, it can be about 100-1000 bp.

Methods that can be used to enhance gene expression include increasing the number of copies of the gene. The introduction of a gene into a vector capable of functioning in bacteria of the genus Escherichia may increase the number of copies of the gene. Low copy vectors may be used. Examples of low copy vectors include, but are not limited to, pSC101, pMW118, pMW119, and the like. The term "low copy vector" is used for vectors, the number of copies of which in a cell reaches five. Enhanced gene expression can also be achieved by introducing multiple copies of the gene into the bacterial chromosome, for example, by homologous recombination, Mu integration, etc. For example, one act of Mu integration allows up to 3 copies of a gene to be introduced into the bacterial chromosome.

The number of copies of a gene can also be increased by introducing multiple copies of the gene into the chromosomal DNA of the bacterium. To introduce multiple copies of a gene into a bacterial chromosome, homologous recombination can be performed using the target sequences present on the chromosome in multiple copies. Multiple copy sequences in chromosomal DNA include, but are not limited to, repetitive DNA or inverted repeats at the ends of transposed elements. It is also possible to incorporate the gene into the transposon and provide for its transfer to introduce multiple copies of the gene into the chromosomal DNA.

Enhanced gene expression can also be achieved by substituting a DNA fragment under the control of a strong promoter. Examples of strong promoters are the lac promoter, trp promoter, trc promoter, P _R or P _L phage λ promoters. Using a strong promoter can be combined with an increase in gene copies.

On the other hand, the action of the promoter can be enhanced, for example, by introducing a mutation into the promoter, leading to an increase in the level of transcription of the gene localized behind the promoter. In addition, it is known that the replacement of several nucleotides in the region between the ribosome binding site (RBS) and the start codon, especially in the sequence immediately before the start codon, significantly affects the translational ability of mRNA. For example, a 20-fold variation in expression level was found depending on the nature of the three nucleotides preceding the start codon (Gold et al., Annu. Rev. Microbiol., 35, 365-403, 1981; Hui et al., EMBO J, 3 623-629, 1984).

In addition, it is possible to introduce a nucleotide substitution in the region of the promoter of the gene on the bacterial chromosome, resulting in an increase in the function of the promoter. Changing the expression control sequence can be accomplished, for example, in the same way as replacing a gene using a temperature-sensitive plasmid, as disclosed in WO 00/18935 and JP 1-215280 A.

The bacterium according to the present invention is also the bacterium described above, further modified so that it acquires pyruvate carboxylase activity. Pyruvate carboxylase activity can be achieved, in particular, by the presence in the bacterium of a DNA molecule containing a gene encoding pyruvate carboxylase.

Pyruvate carboxylase is an enzyme capable of catalyzing the carboxylation reaction of pyruvic acid to form oxaloacetic acid: ATP + pyruvate + HCO ₃ ^- = ADP + phosphate + oxaloacetate. The indicated activity is classified as E.C. 6.4.1.1. The presence of pyruvate carboxylase activity can be determined, for example, using the method described by Peters-Wendisch PO et al. (Pyruvate carboxylase from Corynebacterium glutamicum: characterization, expression and inactivation of the rus gene. Microbiology, 1998; 144; 915-927). An example of an enzyme having pyruvate carboxylase activity is RusA protein from Bacillus subtilis (sequence with accession number NP_389369.1 in the GenBank database, gi: 16078550) encoded by the RusA gene (nucleotides 1554185 to 1557631 in sequence with accession number NC_000964.3 в GenBank Database, gi: 255767013). The nucleotide sequence of the RusA gene and the amino acid sequence of the RusA protein encoded by it are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

The term "protein variant" is also applicable to pyruvate carboxylase. The term "protein variant" is interpreted in the same way as described above.

In this regard, the RusA gene can also exist in the form of variants that hybridize under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 7, or with a probe that can be synthesized based on the specified nucleotide sequence, provided that they encode functional protein RusA. The term "stringent conditions" is interpreted in the same way as described above.

A DNA molecule containing the gene encoding pyruvate carboxylase can be introduced into the bacterium as part of a plasmid vector or integrated into the bacterial chromosome by homologous recombination or Mu integration. The rusA gene can be obtained by PCR using the body chromosome naturally containing this gene and primers synthesized in accordance with the target nucleotide sequence as a matrix.

Methods for preparing plasmid DNA, DNA restriction and ligation, transformation, selection of nucleotides as a primer, etc. may be conventional methods known to a person skilled in the art. These methods are described, for example, in Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).

Succinic acid producing bacterium

As a bacterium according to the present invention, modified so that the expression of the aceEF-lpdA operon genes in said bacterium is enhanced, a bacterium capable of producing succinic acid can be used.

The bacterium of the present invention can be obtained by enhancing the expression of the aceEF-lpdA operon genes in bacteria already capable of producing succinic acid. On the other hand, the bacterium according to the present invention can be obtained by imparting a bacterium in which the expression of the aceEF-lpdA operon genes is already enhanced, the ability to produce succinic acid.

Similarly, as a bacterium according to the present invention, modified in such a way that the expression of the aceEF-lpdA operon genes in said bacterium is enhanced and wherein said bacterium is further modified so that it acquires pyruvate carboxylase activity, a bacterium capable of producing succinic acid can be used.

The bacterium of the present invention can be obtained by enhancing the expression of the aceEF-lpdA operon genes in a bacterium already possessing the ability to produce succinic acid and having pyruvate carboxylase activity. On the other hand, the bacterium according to the present invention can be obtained by providing pyruvate carboxylase activity in a bacterium already capable of producing succinic acid and modified so that the expression of the aceEF-lpdA operon genes in it is enhanced.

The ability to produce succinic acid can be given to bacteria as a result of selection, mutagenesis, or genetic engineering manipulations, as well as a combination of these approaches. The results of these manipulations can be, but not be limited to, inactivation of genes encoding the components of the phosphoenolpyruvate-dependent phosphotransferase sugar transport system - ptsG, ptsH, pstI, crr; inactivation of the pta and ackA genes encoding phosphotransacetylase and acetate kinase, which catalyze the conversion of Acetyl-CoA to acetic acid; inactivation of the rohB gene encoding pyruvate oxidase, which catalyzes the reaction of the conversion of pyruvic acid to acetic acid; inactivation of the ldhA gene encoding lactate dehydrogenase, catalyzing the conversion of pyruvic acid to lactic acid; inactivation of the adhE gene encoding a CoA-dependent aldehyde / alcohol dehydrogenase catalyzing the conversion of Acetyl-CoA to ethanol; inactivation of the pflB gene encoding pyruvate format lyase catalyzing the conversion of pyruvic acid to Acetyl-CoA and formic acid; inactivation of iclR and fadR genes encoding protein repressors of gene expression encoding glyoxylate shunt enzymes; increasing the expression level of the ppc gene encoding phosphoenolpyruvate carboxylase catalyzing the conversion of phosphoenolpyruvate to oxaloacetate; increasing the expression level of the pckA gene encoding phosphoenolpyruvate carboxykinase catalyzing the reversible interconversion reaction of phosphoenolpyruvate and oxaloacetate in any combination.

In this invention, E. coli strain SGM0.1, constructed as a result of genetic engineering manipulations, was used as a model producer of succinic acid, so that pta, ackA, poxB, and ldhA genes encoding proteins catalyzing the formation reactions were inactivated acetic and lactic acids.

The method of obtaining succinic acid

The method according to the present invention includes the production of succinic acid by growing the bacteria according to the present invention in a nutrient medium providing production of succinic acid, the accumulation of succinic acid in the culture fluid and the separation of succinic acid or its salt from the culture fluid.

According to the present invention, the cultivation, isolation and purification of succinic acid or its salt from a culture or similar liquid can be carried out in a manner similar to traditional fermentation methods in which succinic acid is produced using microorganisms. The nutrient medium used for growing can be either synthetic or natural, provided that the medium contains sources of carbon, nitrogen, mineral additives and, if necessary, the appropriate amount of nutrient additives necessary for the growth of microorganisms. Carbon sources include various carbohydrates, such as glucose and sucrose, as well as various organic polyols, such as glycerin. Various inorganic ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds, such as amines, natural nitrogen sources, such as peptone, cereal or bean hydrolysates, and fermentolizate of microorganisms can be used as a nitrogen source. As mineral additives, potassium phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium chloride and the like can be used. If necessary, additional nutrients can be added to the culture medium. The nutrient medium may also contain additional sources of CO ₂ and the HCO ₃ ^- ion introduced into the medium due to gas exchange or dissolution of the corresponding salts.

Cultivation and incubation are carried out at a temperature in the range from 30 ° C to 37 ° C, preferably in the range from 35 ° C to 37 ° C. The pH of the medium is maintained in the range from 5 to 9, preferably from 6.5 to 7.2. The pH of the medium can be adjusted by ammonia, calcium carbonate, various acids, bases and buffer solutions. Usually, growing for 1 to 5 days leads to the accumulation of succinic acid in the culture fluid.

The process of culturing a microorganism in the method according to the present invention may include aerobic and anaerobic stages.

After growth, solid residues, such as cells, can be removed from the culture fluid by centrifugation or filtration through a membrane, and then succinic acid or its salt can be isolated and purified by ion exchange chromatography, reverse phase chromatography, concentration and / or crystallization.

Examples

The present invention will be described in more detail below with reference to the following non-limiting Examples.

Example 1. Construction of bacterial strains and plasmids

1. Construction of strain SGM0.1

The strain SGM0.1 was obtained on the basis of E. coli strain MG1655 as a result of inactivation of the ackA-pta, poxB and ldhA genes by recombination engineering based on a method using λRed-dependent homologous recombination. The principle of the method is described in detail, for example, in (Sawitzke J. et al., Recombineering: In Vivo Genetic Engineering in E. coli, S. enterica, and Beyond. Methods in Enzymology. 2007, 421, 171-199), and examples thereof practical application, for example, in (RU 2330883).

2. Construction of strain SGM0.1 P _L -aceEF-lpdA

1. Obtaining strain E. coli MG1655 attR-cat-attL-P _L -aceEF-lpdA

To enhance the expression of the aceEF-lpdA operon genes, the P _L promoter linked to the Shine-Dalgarno sequence (SD sequence) of the aceF gene, which is more efficient than the ACEE gene SD sequence, was integrated upstream of the coding region of the aceE gene located first in the corresponding operon, on the chromosome of E. coli strain MG1655 using a technique developed by Datsenko, KA and Wanner, BL (Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640-6645), known as "Red-dependent integration" . Also, an artificial DNA fragment integrated into the chromosome contained a Cm ^R marker encoded by the cat gene.

The construction of the aforementioned artificial DNA fragment integrated into the corresponding section of the bacterial chromosome was carried out in several stages. At the first stage, PCR was used to obtain a DNA fragment containing at the beginning the BglII recognition site, the P _L promoter, the aceF gene SD sequence, and finally 36 nucleotides complementary to the 5'-end of the coding region of the acee gene. The fragment was obtained in two stages. At the first stage, using the lambda phage genomic DNA as a template (sequence with accession number NC_001416.1 in the GenBank database, gi: 9626243), a DNA fragment was obtained containing at the beginning the BglII recognition site, the P _L promoter, and the SD gene sequence aceF. PCR was performed using primers P1 (SEQ ID NO: 9) and P2 (SEQ ID NO: 10). Primer P1 contains a BglII recognition site and a region homologous to the 5'-end of the P _L promoter. Primer P2 contains the aceF SD sequence and region complementary to the 3'-end of the P _L promoter. The following temperature profile for PCR was used: denaturation at 95 ° C for 5 min; subsequent 25 cycles: 30 sec at 95 ° C, 30 sec at 51 ° C, 60 sec at 72 ° C; and final polymerization: 7 min at 72 ° C. Used Pfu DNA polymerase, the appropriate buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania).

The resulting PCR product, purified on an agarose gel and isolated according to the manufacturer's recommendations using the Qiagen QIAquick Gel Extraction Kit, was used as a template in the next round of PCR. Primers P1 (SEQ ID NO: 9) and P3 (SEQ ID NO: 11) were used. Primer P3 contains a region complementary to the 3'-end of the P _L promoter, the aceF SD sequence and 36 nucleotides from the reading frame of the aceE gene. The following temperature profile for PCR was used: denaturation at 95 ° C for 5 min; subsequent 25 cycles: 30 sec at 95 ° C, 30 sec at 53 ° C, 60 sec at 72 ° C; and final polymerization: 7 min at 72 ° C. Used Pfu DNA polymerase, the appropriate buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania).

In parallel, the second stage of constructing the DNA fragment was carried out. The DNA fragment containing the Cm ^R marker encoded by the cat gene was obtained by PCR using primers P4 (SEQ ID NO: 12) and P5 (SEQ ID NO: 13) and plasmid pMW118-attL-Cm-attR (Katashkina J. I. et al., 2005. Directional change in the level of gene expression in the bacterial chromosome, Mol. Biol., 39 (5), 823-831) as a matrix. Primer P4 contains 36 nucleotides homologous to the DNA region immediately preceding the coding region of the ACEE gene and a DNA region of 28 nucleotides complementary to the DNA region located at the 3 ′ end of the attR region. Primer P5 contains a BglII recognition site and a 28-nucleotide-sized DNA region homologous to the DNA region located at the 5'-end of the attL region. The following temperature profile for PCR was used: denaturation at 95 ° C for 5 min; subsequent 25 cycles: 30 sec at 95 ° C, 30 sec at 52 ° C, 1 min at 72 ° C; and final polymerization: 7 min at 72 ° C. Used Taq DNA polymerase, the appropriate buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania).

The resulting DNA fragments were purified on an agarose gel and isolated, according to the manufacturer's recommendations, using a Qiagen QIAquick Gel Extraction Kit, followed by ethanol precipitation.

The two DNA fragments obtained were treated with BglII restriction endonuclease followed by ligation using T4 DNA ligase (Maniatis T., Fritsch EF, Sambrook, J .: Molecular Cloning: A Laboratory Manual. 2 ^nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989).

The ligation product was amplified by PCR using primers P3 (SEQ ID NO: 11) and P4 (SEQ ID NO: 12). The following temperature profile for PCR was used: denaturation at 98 ° C for 1 min; the next 30 cycles: 15 seconds at 98 ° C, 15 seconds at 55 ° C, 1 min at 72 ° C; and final polymerization: 7 min at 72 ° C. Used Phusion DNA polymerase and the appropriate buffer manufactured by Finnzymes; deoxynucleoside triphosphates manufactured by Fermentas (Lithuania). The resulting 1970 bp PCR product, purified on an agarose gel and isolated according to the manufacturer's recommendations using the Qiagen QIAquick Gel Extraction Kit, was used for electroporation into E. coli strain MG1655 containing plasmid pKD46 with a heat-sensitive replicon. Plasmid pKD46 (Datsenko, KA and Wanner, BL, Proc. Natl. Acad. Sci. USA, 2000, 97 (12): 6640-6645) contains a 2154 bp DNA fragment of phage λ. (positions 31088 through 33241 of the nucleotide sequence with accession number J02459 in the GenBank database), and also contains the genes of the λ Red-homologous recombination system (γ, β, exo genes) under the control of the _araB -induced P _araB promoter. Plasmid pKD46 is required for integration of the PCR product into the chromosome of strain MG1655.

Electrocompetent cells of E. coli strain MG1655 containing plasmid pKD46 were prepared as follows: overnight culture of E. coli strain MG1655 containing plasmid pKD46 was grown at 30 ° C in LB medium supplemented with ampicillin (100 mg / L), diluted 100 times by adding 10 ml of SOB medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) containing ampicillin and L-arabinose (10 mM), The resulting culture was grown with stirring at 30 ° C until the OD ₆₀₀ ≈0.8, after which the cells were made electrocompetent by concentrating 100-fold and three-fold laundering edyanoy deionized H ₂ O. Electroporation was performed using 70 l of cells and ≈300 ng of PCR product. After electroporation, the cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37 ° C for 2.5 hours, after which they were plated on L plates. agar containing 30 μg / ml chloramphenicol and grown at 37 ° C to select Cm ^R recombinants. Then, to remove the plasmid pKD46, the clones were seeded to separate colonies on agarized LB at 37 ° C, and Cm ^R Ap ^S variants were selected.

The fact that the proposed and experimentally obtained chromosome structure corresponded to the selected Cm ^R Ap ^S colonies was confirmed by PCR analysis using locus-specific primers P6 (SEQ ID NO: 14) and P7 (SEQ ID NO: 15). The following temperature profile was used for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 52 ° C, 1 min at 72 ° C; final step: 7 min at 72 ° C. The length of the PCR product obtained by the reaction using the parental strain MG1655 as a matrix of cells is 201 bp. The length of the PCR product obtained by the reaction using a mutant strain as a matrix of cells is 2099 bp

On the chromosomes of the obtained clones, the correspondence between the planned and experimentally obtained nucleotide sequence of the new regulatory element introduced before the coding region of the ACEE gene was confirmed by sequencing. Thus, strain MG1655 attR-cat-attL-P _L -aceEF-lpdA was obtained.

2. Obtaining a strain of E. coli SGM0.1 P _L -aceEF-lpdA

DNA fragment from the chromosome of the previously described MG1655 strain attR-cat-attL-P _L -aceEF-lpdA containing the aceEF-lpdA region of the operon using P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press , Plainview, NY) was transferred to E. coli strain SGM0.1 to obtain strain SGM0.1 attR-cat-attL-P _L -aceEF-lpdA.

Next, the Cm resistance gene (cat gene) was eliminated from the chromosome of strain SGM0.1 attR-cat-attL-P _L -aceEF-lpdA using the int-xis system. To this end, the strain SGM0.1 attR-cat-attL-P _L -aceEF-lpdA was transformed with the plasmid pMWts-Int / Xis (WO 2007 013638; RU 2005 123423). The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The plates were incubated overnight at 30 ° C. The cat gene was removed from transformed clones by growing individual colonies at 37 ° C (at this temperature, the CIts repressor is inactivated to some extent, while the int / xis genes are activated), followed by selection of Cm ^S Ap ^R variants. Removal of the cat gene from the chromosome of the strains was confirmed by PCR using locus-specific primers P6 (SEQ ID NO: 14) and P7 (SEQ ID NO: 15). The PCR conditions were the same as described above. The length of the PCR product obtained in the case of cells with the deleted cat gene was 502 bp.

The strain was isolated from the pMWts-Int / Xis helper plasmid containing the heat-sensitive replicon from Cm ^S Ap ^R variants by re-sieving to individual colonies at 37 ° C and selection of Cm ^S Ap ^S clones.

On the chromosomes of the obtained clones, the correspondence between the planned and experimentally obtained nucleotide sequence of the new regulatory element introduced before the coding region of the ACEE gene was confirmed by sequencing. Thus, the strain SGM0.1 λattB-P _L -aceEF-lpdA was obtained, in short - SGM0.1 P _L -aceEF-lpdA.

3. Obtaining plasmids pPYC

1. Obtaining strain E. coli MG1655 Δppc

Plasmid pPYC contained the RusA gene from Bacillus subtilis encoding pyruvate carboxylase. To select plasmid variants containing the rusA gene encoding functionally active pyruvate carboxylase, the E. coli strain MG1655 Δ ppc was previously obtained in which the ppc gene encoding phosphoenolpyruvate carboxylase was inactivated. PPC mutants of E. coli are known to be incapable of synthesizing oxaloacetate and are auxotrophs in the intermediates of the tricarboxylic acid cycle; as a result, they are not able to grow on media containing sugar or glycerol as carbon sources [Sauer U. and Eikmanns B. 2005 The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiol. Rew., 29, 765-794]. Thus, the presence in the E. coli strain MG1655 Δppc of the pPYC plasmid containing the RusA gene encoding the functionally active pyruvate carboxylase catalyzing the reaction of formation of oxaloacetate from pyruvic acid should restore the growth ability of the strain on a minimal medium containing carbon sources, for example, glucose.

The ppc gene deletion was performed using a technique developed by Datsenko, K.A. and Wanner, BL (Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640-6645), known as "Red-Dependent Integration." A DNA fragment containing the Cm ^R marker encoded by the cat gene was obtained by PCR using primers P8 (SEQ ID NO: 16) and P9 (SEQ ID NO: 17) and plasmid pMW118-attL-Cm-attR as a template. Primer P8 contains a 36 bp DNA region homologous to the DNA region located at the 5 ′ end of the ppc gene and a 28 bp DNA region complementary to the DNA region located at the 3 ′ end of the attR region. Primer P9 contains a 36 bp DNA region complementary to the DNA region located at the 3 ′ end of the ppc gene, and a 28 bp DNA region homologous to the DNA region located at the 5 ′ end of the attL region. The following temperature profile for PCR was used: denaturation at 95 ° C for 3 min; the first two cycles: 1 min at 95 ° C, 30 sec at 55 ° C, 40 sec at 72 ° C; subsequent 25 cycles: 30 sec at 95 ° C, 30 sec at 54 ° C, 40 sec at 72 ° C; and final polymerization: 5 min at 72 ° C. The obtained 1700 bp PCR product, purified on an agarose gel and isolated, according to the manufacturer's recommendations, using the Qiagen QIAquick Gel Extraction Kit, was used for electroporation into E. coli strain MG1655 containing plasmid pKD46 with a thermosensitive replicon. Plasmid pKD46 is required for integration of the PCR product into the chromosome of strain MG1655.

Electrocompetent cells of E. coli strain MG1655 containing plasmid pKD46 were obtained as described above. Electroporation was performed using 70 μl of cells and ≈300 ng of PCR product. After electroporation, the cells were incubated in 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37 ° C for 2.5 hours, after which they were plated on L plates. agar containing 30 μg / ml chloramphenicol and grown at 37 ° C to select Cm ^R recombinants. Then, to remove plasmid pKD46, the clones were seeded to separate colonies on agarized LB at 37 ° C, and Cm ^R Ap ^S variants were selected.

The fact that the putative and predicted chromosome structures corresponded to the selected Cm ^R Ap ^S colonies was confirmed by PCR analysis using locus-specific primers P10 (SEQ ID NO: 18) and PI 1 (SEQ ID NO: 19). The following temperature profile was used for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 53 ° C, 1 min at 72 ° C; final step: 7 min at 72 ° C. The length of the PCR product obtained by the reaction using the parental strain MG1655 as a matrix of cells is 3003 bp The length of the PCR product obtained by the reaction using a mutant strain as a matrix of cells is 2050 bp. The mutant strain was named MG1655 ppc :: attR-cat-att.

Next, the Cm resistance gene (cat gene) was eliminated from the chromosome of strain MG1655 ppc :: attR-cat-att using the int-xis system. To this end, strain MG1655 ppc :: attR-cat-att was transformed with the plasmid pMWts-Int / Xis (WO 2007 013638; RU 2005 123423). The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The plates were incubated overnight at 30 ° C. The cat gene was removed from transformed clones by growing individual colonies at 37 ° C (at this temperature, the CIts repressor is inactivated to some extent, while the int / xis genes are activated), followed by selection of Cm ^S Ap ^R variants. Removal of the cat gene from the chromosome of the strains was confirmed by PCR using locus-specific primers P10 (SEQ ID NO: 18) and P11 (SEQ ID NO: 19). The PCR conditions were the same as described above. The length of the PCR product obtained in the case of cells with the deleted cat gene was 453 bp.

The strain was isolated from the pMWts-Int / Xis helper plasmid containing the heat-sensitive replicon from Cm ^S Ap ^R variants by re-sieving to individual colonies at 37 ° C and selection of Cm ^S Ap ^S clones.

Thus, the MG1655 ppc :: λattB strain was obtained, abbreviated MG1655 Δppc.

2. Construction of the plasmid pPYC *

Plasmid pMW119 (sequence with accession number AB005476.2 in the GenBank database, gi: 67968376) was used as the base vector for creating pPYC.

The DNA fragment containing the RusA gene with its own regulatory region was obtained by PCR using the B. subtilis 168 strain and primers P12 (SEQ ID NO: 20) and P13 (SEQ ID NO: 21) as the chromosome matrix. Primer P12 contains an XbaI recognition site and a nucleotide sequence homologous to the region lying above the 5'-end of the coding sequence of the B.subtilis RusA gene. Primer P13 contains a KpnI recognition site and a nucleotide sequence homologous to the region below the 3'-end of the coding sequence of the B. subtilis RusA gene. The following temperature profile for PCR was used: denaturation at 98 ° C for 1 min; the next 30 cycles: 15 sec at 98 ° C, 15 sec at 53 ° C, 3 min at 72 ° C; and final polymerization: 7 min at 72 ° C. Used Phusion DNA polymerase and the appropriate buffer manufactured by Finnzymes; deoxynucleoside triphosphates manufactured by Fermentas (Lithuania).

The resulting 4438 bp PCR product, purified on an agarose gel and isolated, according to the manufacturer's recommendations, using the Qiagen QIAquick Gel Extraction Kit, was subsequently subsequently treated with XbaI and KpnI restriction endonucleases. In parallel with the restriction endonucleases XbaI and KpnI, the plasmid pMW119 was processed. The obtained linear DNA fragments containing the rusA gene and the larger XbaI-KpnI fragment of plasmid pMW119, purified on an agarose gel and isolated, according to the manufacturer's recommendations, using Qiagen QIAquick Gel Extraction Kit, were ligated using T4 DNA ligase (Maniatis T. , Fritsch EF, Sambrook, J .: Molecular Cloning: A Laboratory Manual. 2 ^nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989). The E. coli strain MG1655 was transformed with the resulting ligase mixture, followed by selection of Ap ^R transformants on LB agar medium containing 100 mg / L ampicillin.

The plasmid isolated by standard methods from the corresponding transformants was named pPYC *.

3. Selection of plasmid pPYC containing the gene RusA encoding functionally active pyruvate carboxylase

Cells of strain MG1655 Δpc were transformed with the preparation of plasmid pPYC *. Transformants were selected on agarized M9 minimal medium (Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989) in the presence of 0.2% glucose as the sole carbon source. Individual transformant clones capable of growth on this medium contained a plasmid containing the RusA gene encoding the functionally active pyruvate carboxylase. The plasmid isolated by standard methods from the corresponding transformants was named pPYC.

4. Obtaining strains of SGM0.1 [pPYC] and SGM0.1 P _L -aceEF-lpdA [pPYC]

The strains SGM0.1 [pPYC] and SGM0.1 P _L -aceEF-lpdA [rRUS] were obtained by transforming the strains SGM0.1 and SGM0.1 P _L -aceEF-lpdA with the plasmid pPYC according to the standard method. Similarly, the transformation strains SGM0.1 [pMW119] and SGM0.1 P _L -aceEF-lpdA [pMW119] were obtained by transformation of the strains SGM0.1 and SGM0.1 P _L -aceEF-lpdA with plasmid pMW119 by the standard method.

Example 2. Production of succinic acid engineered strains

Cells of strains SGM0.1, SGM0.1 P _L -aceEF-lpdA, SGM0.1 [pMW119], SGM0.1 R _L -aceEF-lpdA [pMW119], SGM0.1 [pPYC] and SGM0.1 P _L -aceEF -lpdA [pPYC] was grown overnight in M9 medium containing 2 g / l glucose at 37 ° C. 1 ml of the resulting overnight cultures was diluted 50 times by adding 49 ml of M9 medium containing 10 g / l glucose. The resulting cultures were grown in 750 ml flasks with vented plugs at 37 ° C on a rotary shaker at 250 rpm for 6 hours. The resulting cell suspensions were centrifuged for 15 minutes at 4000 rpm at 4 ° C. Precipitation was resuspended in 15 ml of M9 medium containing 20 g / l glucose. Subsequently, the resulting cell cultures were incubated for 24 hours in 15 ml tubes, closed by unventilated caps, at 37 ° C on a rotary shaker at 250 rpm. For strains SGM0.1 [pMW119], SGM0.1 P _L -aceEF-lpdA [pMW119], SGM0.1 [pPYC] and SGM0.1 P _L -aceEF-lpdA [pPYC], the media used contained an additional 100 mg / l ampicillin. The concentration of succinic acid and residual glucose in culture fluids freed from the cell mass by centrifugation was determined by high performance liquid chromatography using the Waters HPLC system (Waters, Milford, MA, USA). To measure succinic acid concentrations, a ReproSil-Pur C18-AQ reverse phase column (4 × 250 mm, 5 μm, Dr. Maisch, Ammerbuch, Germany) was used. Detection was carried out at a wavelength of 210 nm. An aqueous solution of phosphoric acid (100 mM) with acetonitrile and methanol (each in a volume concentration of 0.5%) with a flow rate of 1 ml / min was used as the mobile phase. To measure glucose concentration, the Waters HPLC system was equipped with a Waters 2414 refractory detector and a Spherisorb-NH2 column (4.6 × 250 mm, 5 μm, Waters, USA). A mixture of acetonitrile / ethyl acetate / water (volume ratio 76/4/20) with a flow rate of 1 ml / min was used as the mobile phase. Substances were identified by comparing their retention times with the retention times of their respective standards.

The results of the corresponding in vitro fermentations are presented in Table 1. The succinic acid production indices of the strains SGM0.1 [pMW119] and SGM0.1 P _L -aceEF-lpdA [pMW119] did not differ from those for strains SGM0.1 and SGM0.1 P _L -aceEF -lpdA, respectively.

The ability of the strains SGM0.1, SGM0.1 P _L -aceEF-lpdA, SGM0.1 [pMW119], SGM0.1 P _L -aceEF-lpdA [pMW119], SGM0.1 [pPYC] and SGM0.1 P was similarly evaluated. _L -aceEF-lpdA [pPYC] produce succinic acid in the presence of an additional source of HCO ₃ ^- ion in the medium. Fermentation was carried out as described above, except that the medium used in the final stage additionally contained 1.5 g / l NaHCO ₃ . The concentration of succinic acid and residual glucose in culture fluids freed from the cell mass by centrifugation was determined as described previously.

The results of the corresponding in vitro fermentations are presented in Table 2. The succinic acid production indices of the strains SGM0.1 [pMW119] and SGM0.1 P _L -aceEF-lpdA [pMW119] did not differ from those for strains SGM0.1 and SGM0.1 P _L -aceEF -lpdA, respectively.

As follows from Table 1 and Table 2, the strain SGM0.1 P _L -aceEF-lpdA with enhanced gene expression of the aceEF-lpdA operon in both cases accumulated a greater amount of succinic acid and with a higher molar yield than the parent strain SGM0.1. It also follows from Table 1 and Table 2 that the SGM0.1 P _L -aceEF-lpdA [pPYC] strain with enhanced gene expression of the aceEF-lpdA operon and additionally having pyruvate carboxylase activity in both cases accumulated a greater amount of succinic acid and with a large molar yield, than the control strains SGM0.1 P _L -aceEF-lpdA and SGM0.1 [pPYC].

Table 1 Strain Glucose consumed The amount of succinic acid Succinic acid molar yield g / l mm g / l mm % SGM0.1 4.50 ± 0.01 24.98 ± 0.01 0.46 ± 0.01 3.85 ± 0.01 15.4 ± 0.1 SGM0.1 P _L -aceEF-lpdA 4.60 ± 0.01 25.53 ± 0.01 0.52 ± 0.01 4.42 ± 0.01 17.3 ± 0.1 SGM0.1 [pPYC] 4.30 ± 0.01 23.87 ± 0.01 0.50 ± 0.01 4.19 ± 0.01 17.6 ± 0.1 SGM0.1 P _L -aceEF-lpdA [pPYC] 4.30 ± 0.01 23.87 ± 0.01 1.44 ± 0.01 12.22 ± 0.01 51.2 ± 0.1

table 2 Strain Glucose consumed The amount of succinic acid Succinic acid molar yield g / l mm g / l mm % SGM0.1 5.53 ± 0.01 30.69 ± 0.01 0.97 ± 0.01 8.17 ± 0.01 26.6 ± 0.1 SGM0.1 P _L -aceEF-lpdA 5.52 ± 0.01 30.64 ± 0.01 1.28 ± 0.01 10.84 ± 0.01 35.4 ± 0.1 SGM0.1 [pPYC] 5.52 ± 0.01 30.64 ± 0.01 1.13 ± 0.01 9.53 ± 0.01 31.1 ± 0.1 SGM0.1 P _L -aceEF-lpdA [pPYC] 6.04 ± 0.01 33.53 ± 0.01 2.27 ± 0.01 19.23 ± 0.01 57.3 ± 0.1

Claims (4)

1. The bacterium Escherichia coli is a producer of succinic acid, modified by changing the nucleotide sequence of the promoter and the ribosome binding site that controls the expression of the aceEF-lpdA operon genes in the bacterial chromosome, so that the expression of the aceE, aceF and lpdA genes in the specified bacterium is enhanced. 2. The bacterium according to claim 1, characterized in that the expression is enhanced by replacing the nucleotide sequence of the natural promoter controlling the expression of the aceEF-lpdA operon genes in the chromosome of the bacterium with a stronger promoter. 3. The bacterium according to claim 1, characterized in that said bacterium is additionally modified in such a way that it acquires pyruvate carboxylase activity by introducing into the bacterium a DNA molecule containing a gene encoding an enzyme having an activity classified as K. F. 6.4.1.1. 4. A method for producing succinic acid by culturing the bacterium according to any one of claims 1 to 3 in a nutrient medium and isolating succinic acid from the culture fluid.

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