RU2573936C1 - Strain of bacteria escherichia coli - producer of fumaric acid and method of obtaining fumaric acid using this strain - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: group of inventions relates to the strain of bacteria Escherichia coli FUM1.0 which is a producer of fumaric acid and to a method of obtaining fumaric acid using this strain. The strain is obtained by the inactivation of the genes ackA, pta, poxB, ldhA, adhE, pflB, frdA, frdB, sdhA, sdhB, aceA, aceB, and ptsG and amplification of the expression of the genes galP and glk in the strain Escherichia coli K-12 MG1655. The method of obtaining fumaric acid provides the cultivation of the named strain in suitable conditions with the subsequent release of fumaric acid from cultural liquid. The factor of conversion of glucose in fumaric acid at the cultivation of the offered strain amounts 1.31 mol/mol that is 65.5% of the theoretically possible maximum.

EFFECT: group of inventions provides the high yield of fumaric acid.

3 cl, 1 tbl, 2 ex

Description

The present invention relates to biotechnology, in particular, to a strain of bacteria Escherichia coli - a producer of fumaric acid, capable of efficiently producing fumaric acid from glucose, as well as to a method for producing fumaric acid using this strain, with an improved rate of substrate conversion to target product.

Fumaric acid, unsaturated four-carbon dicarboxylic acid, widely used in various sectors of the chemical, food and pharmaceutical industries.

Currently, the global market for fumaric acid is estimated at 90,000 tons / year. Fumaric acid is used as an intermediate in the production of paper (~ 31500 t / year), polyester resins (~ 13500 t / year), alkyd plastics (~ 5500 t / year) and plasticizers (~ 4500 t / year), as well as in cosmetic (~ 15,000 t / year) and food industry (~ 20,000 t / year) (Roa Engel, S.A. et al., 2008, Fumaric acid production by fermentation. Appl. Microbiol. Biotechnol. 78, 379-389). The non-toxic nature of fumaric acid allows its use in the production of beverages and pastries, as well as wheat and rye bread, fruit juices, jelly desserts (Yang, ST et al., 2011, Fumaric acid. In: Moo-Young, Bulter Michael, editors. Comprehensive biotechnology , 2nd Edition, 3, 163-77). Fumaric acid is added to cattle diets, resulting in a significant reduction of animal methane emissions of more than 70% (Mcginn, SM et al., 2004, Methane emissions from beef cattle: Effects of monensin, sunflower oil, enzymes, yeast and fumaric acid. J. Anim. Sci. 82, 3346-3356). In medicine, fumaric acid esters such as monoethyl fumarate, monomethyl fumarate and diethyl and dimethyl fumarates are used in the treatment of psoriasis (Moharregh-Khiabani, D. et al., 2009, Fumaric acid and its esters: an emerging treatment for multiple sclerosis. Curr. Neuropharmacol. 7, 60-64), significantly improving the quality of life of patients.

Fumaric acid is also considered as a promising “building block” for producing a wide range of high value-added chemicals synthesized from petroleum products. The most significant of these is 1,4-butanediol (BDO). BDO is used in such industries as: electronics (as a solvent and solution for removing photoresist, degreasing and cleaning); pharmaceuticals (as a solvent and extraction agent); fine organic synthesis (as a precursor in the synthesis of tetrahydrofuran (THF) and γ-butyrolactone (GBL), polybutylene terephthalate rubbers for the automotive industry, elastane, etc.) (Paster, M. et al., 2003, Industrial bioproducts: today and tomorrow. Energetics Inc., Columbia, MD, USA).

Currently, fumaric acid is produced by petrochemical synthesis during cis-trans isomerization of maleic acid, which, in turn; obtained from maleic anhydride. Industrial synthesis of maleic anhydride is carried out during the oxidation of benzene and a number of other aromatic compounds. However, due to the increased price of benzene, n-butane has been used as a feedstock in many enterprises (Lorences, MJ et al., 2003, Butane oxidation to maleic anhydride: kinetic modeling and byproducts. Ind. Eng. Chem. Res 42, 6730-6742). The reaction proceeds in the gas phase on bismuth catalysts with the removal of water vapor during partial condensation. The absorption of maleic anhydride from the gas phase is carried out in pairs of organic solvents, followed by fractional distillation of the resulting mixture to isolate the target product and return the solvent to the sorption column. Maleic acid obtained by hydrolysis of maleic anhydride undergoes cis-trans isomerization to fumaric acid using inorganic acids, peroxides, or thiourea as catalysts (Lohbeck, K. et al., 1990, Maleic and fumaric Acids. Ullmann's Encyclopedia of Industrial Chemistry, vol. A16. VCH, Weinheim, Germany, 53-62). Isolation and purification of the product is usually carried out by crystallization from the aqueous phase.

The isomerization of maleic acid to fumaric acid using chemical catalysts is limited by the equilibrium constant of the reaction. In addition, the corresponding chemical transformations occur at elevated temperatures, which cause the formation of by-products of thermal decomposition of maleic and fumaric acids, and thereby reduce the final yield of the target product below the equilibrium value (US 5783428). These shortcomings of the chemical process for producing fumaric acid have led to interest in alternative methods for the preparation of this substance.

Fumaric acid is an intermediate in the central metabolism of many organisms. However, the spectrum of organisms with the currently established natural ability to noticeable production of fumaric acid is very narrow. Microorganisms with a relatively pronounced ability to form detectable amounts of fumaric acid in cultivation media include individual representatives of the genera Rhizopus, Mucor, Cunninghamella and Circinella. Moreover, such representatives of the genus Rhizopus as R. nigricans, R. arrhizus, R. oryzae and R. formosa possess the best indicators of the conversion of the substrate to fumaric acid. (Roa Engel, C. A. et al., 2008. Fumaric acid production by fermentation. Appl. Microbiol. Biotechnol. 78, 379-389) Despite the fact that the ability of Rhizopus strains to produce fumaric acid was discovered at the beginning of 20 up to the present century, the physiological characteristics of this microorganism and the complexity of modifying its metabolism have not allowed to develop an economically viable process of microbiological synthesis of fumaric acid using Rhizopus cells (Straathof AJJ, and van Gulik WM, 2012, Production of Fumaric Acid by Fermentation. In X. Wang et al. (Eds.), Reprogramming Microbial Metabolic Pathways, 11, 225-240).

Taking into account that in the price structure of finished products of microbiological synthesis, the cost of raw materials is about 40%, it is obvious that the cost of the substrate and the rate of conversion of the substrate to the final product have a significant impact on the cost of the target substance. Due to the relative cheapness and significant production volumes, glucose is the preferred substrate for the microbiological production of fumaric acid (Straathof AJJ, and van Gulik WM, 2012, Production of Fumaric Acid by Fermentation. In X. Wang et al. (Eds.), Reprogramming Microbial Metabolic Pathways, 11, 225-240).

The optional anaerobic bacterium Escherichia coli is not capable of forming appreciable amounts of tricarboxylic acid cycle intermediates, in particular fumaric acid, during glucose utilization. Nevertheless, the study of metabolism and the development of accessible genetic engineering tools allowed the creation of highly efficient recombinant producers of succinic and malic acids based on E. coli cells, which, like fumaric acid, are not natural waste products of the bacterium under consideration (Cao, Y. et al ., 2011, Metabolically engineered Escherichia coli for biotechnological production of four-carbon 1,4-dicarboxylic acids. J Ind Microbiol Biotechnol. 38 (6), 649-656; Lin, H. et al., 2005, Genetic Reconstruction of the Aerobic Central Metabolism in Escherichia coli for the Absolute Aerobic Production of Succinate. Biotechnol Bioeng. 89, 148-156; Zhang, X. et al., 2011, L-malate production by metabolically engineered Escherichia coli. Appl Environ Microbiol. 77 (2), 427-434; Thakker, C. et al., 2012, Succinate production in Escherichia coli. Biotechnol J. 7 (2), 213-224).

Like most microorganisms, cells Ε. coli form fumaric acid in a limited set of biochemical processes. Among them are the oxidative cycle of tricarboxylic acids, reactions involving the glyoxylate shunt, and one of the branches of mixed acid fermentation, called the reducing branch of the tricarboxylic acid cycle. The main metabolic precursors in these biochemical processes are pyruvic and oxaloacetic acids, as well as phosphoenolpyruvate and acetyl-coenzyme A (acetyl-CoA). In the utilization of glucose, these substances are derivatives that are formed in reactions involving terminal glycolysis products, or are direct products of this process.

The physiological conditions for the functioning of the corresponding paths are different. The oxidative cycle of tricarboxylic acids is active under aeration conditions, the reducing branch of the cycle requires anaerobiosis conditions, while the glyoxylate shunt is able to function regardless of aeration conditions.

In the conversion of glucose to fumaric acid involving the oxidative part of the tricarboxylic acid cycle, one fumaric acid molecule is formed from one glucose molecule and, thus, the conversion rate of glucose to fumaric acid is 1/1 or 1.0 mol / mol.

When glucose is converted to fumaric acid with the formation of the target substance in a process involving the glyoxylate shunt, the conversion rate of the substrate into the target product is also 1/1 or 1.0 mol / mol.

The combination of these two pathways for the formation of fumaric acid from glucose is characterized by the formation of two fumaric acid molecules from two glucose molecules and causes a conversion factor of 2/2 or 1 mol / mol.

In contrast to the processes of biosynthesis of fumaric acid from glucose proceeding under aerobic conditions, the anaerobic formation of fumaric acid with the participation of the reducing branch of the tricarboxylic acid cycle requires maintaining the balance of the NAD + and NADH equivalents reduced and oxidized during the corresponding reactions. The processes of aerobic biosynthesis of fumaric acid from glucose are based on oxidation reactions and, as a result, are accompanied by the concomitant formation of a significant amount of reduced equivalents. In the presence of oxygen, excess reduced equivalents are reoxidized in the respiratory electron transport chain. Under conditions of anaerobiosis, the electron transport chain is inactive and the reoxidation of the reduced equivalents formed during glucose utilization should be associated with fermentation reactions. The formation of fumaric acid in the reducing branch of the tricarboxylic acid cycle, which is part of the processes of mixed acid fermentation, or anaerobic respiration, is associated with the consumption of reduced equivalents formed during glycolysis.

Achieving the intracellular redox balance during anaerobic biosynthesis of fumaric acid from glucose with the involvement of acetyl CoA in the formation of the target substance, i.e., involving the reactions of the oxidative part of the tricarboxylic acid cycle and glyoxylate shunt, is impossible.

The direct biosynthesis of fumaric acid from glucose in the reducing part of tricarboxylic acids is NAD + / NADH balanced and is able to provide a conversion ratio of 2 mol / mol. This is due, on the one hand, to the fixation of CCB at the stage of formation of oxaloacetate from the glycolytic intermediates, the precursor in the anaerobic pathway of glucose fermentation to fumaric acid, and, on the other hand, because the conversion of each of the two oxaloacetate molecules obtained from glycolytic glucose utilization intermediates , fumaric acid requires one reduced equivalent (NADH), during glycolysis, a strictly corresponding number of reduced equivalents is formed (2 NADH per glucose molecule, t. E. Strictly 1 NADH per molecule of oxaloacetate).

It should be noted that the increasing emission of greenhouse gases and, in particular, СО ₂ , is one of the main problems of modern technological civilization. The possibility of fixing CO ₂ in the processes of microbiological synthesis of substances with high added value is regarded as a major advantage. The location of fumaric acid production at the existing facilities for the production of alcoholic fermentation products (ethanol), accompanied by the emission of CO ₂ , will exclude the cost of transporting this component from the structure of the product price.

Thus, anaerobic fermentation of glucose, with the formation of the target substance in the reducing part of the tricarboxylic acid cycle, seems to be the most effective microbiological process for the preparation of fumaric acid or its salts.

Approaches to the construction of recombinant strains producing beneficial metabolites include several fundamental principles. These principles are: activation of key pathways of biosynthesis of the target substance; activation of biochemical pathways of biosynthesis of precursor metabolites; inactivation of competitive utilization pathways of precursors in secondary biochemical pathways; and inactivation of product degradation pathways. In the case of the creation of anaerobic producers of fermentation products, these principles are supplemented by the need to inactivate the pathways of inappropriate reoxidation of the reduced equivalents and maintain the intracellular redox balance.

However, since the biochemical pathways of the formation of fumaric acid in E. coli cells differ in physiological functioning conditions, modifications of the bacterial metabolism aimed at achieving effective fumaric acid formation in specific biochemical processes are different.

Song et al. (Song, CW et al., 2013, Metabolic engineering of Escherichia coli for the production of fumaric acid. Biotechnol. Bioeng. 110, 2025-2034) described the construction of recombinant strain Ε. coli for aerobic production of fumaric acid from glucose. The authors initially attempted to obtain a strain for the anaerobic production of fumaric acid through deletions of the a dhE genes encoding alcohol / aldehyde dehydrogenase, ldhA encoding lactate dehydrogenase, and frdABCD encoding the components of the fumarate reductase enzymatic complex. It was assumed that as a result of these modifications in the strain, the inappropriate use of metabolites of the precursors, acetyl-CoA and pyruvic acid, in ways that are side by side with the biosynthesis of fumaric acid, will be prevented, and the possibility of anaerobic conversion of fumaric acid to succinic acid will be eliminated. However, the corresponding strain CWF0 (W3110 Δ a dhE ΔldhA ΔfrdABCD) did not produce fumaric acid and was not able to grow under conditions of anaerobiosis. As a result of the subsequent multi-stage construction, the strain CWF811 (W3110 ΔiclR ΔfumC ΔfumA ΔfumB Δ a rcA ΔptsG Δ a spA Δl c I) / pTac15kppc, with inactivated genes encoding the repressor protein of the glyoxylate transcriptor repressor gene, was obtained the main glucose permease is phosphoenolpyruvate-dependent sugar transport system, ammonium aspartate lyase, lactose operon gene repressor protein, and having increased expression of phosphoenolpyruvate carboxylase. Strain CWF811 is capable of aerobic production of fumaric acid from glucose with a conversion coefficient of 0.65 mol / mol, i.e. 65% of the theoretically possible value for the corresponding process occurring under aeration conditions. A further attempt to optimize the production of the target substance was made by overexpression of the galP gene encoding a low affinity H + -symactor galactose, also capable of glucose transport. However, the conversion rate of the substrate into the target product, demonstrated by the corresponding strain CWF812 (W3110 ΔiclR ΔfumC ΔfumA ΔfumB Δ a rcA ΔptsG Δ a spA ΔlacI PgalP :: Ptrc) / pTac15kppc, not only did not increase, but also found a slight decrease to 0.6 m2 / mol. Acetic acid was the main by-product of glucose utilization by both strains, which is apparently due to the presence of intact poxB and pt a - a ckA genes encoding pyruvate oxidase, phosphotransacetylase, and acetate kinase and responsible for the formation of acetic acid from pyruvic acid and acetyl-CoA , respectively.

CN 102618570 describes the phased construction of an E. coli strain for the production of fumaric acid under anaerobic conditions. The final strain of HH5 (ΔfumA, ΔfumB, ΔfumC, Δ a rcA, ΔfrdB, ΔpflB, ΔldhA) was obtained by directed deletion of the following genes:

- fumA, fumB, fumC, encoding proteins of the fumarase isoenzymes,

- a rcA encoding the transcriptional regulator of respiratory metabolism genes,

- frdB encoding one of the catalytic components of reductase fumarate,

- pflB encoding pyruvate format lyase and

- ldhA encoding lactate dehydrogenase.

Due to the division of the fumA, fumB, fumC genes in the HH5 strain, the possibility of interconversion of malic and fumaric acids in both the aerobic and anaerobic branches of the tricarboxylic acid cycle is excluded. . Inactivation of the fumarate of the reductase enzymatic complex, due to the deletion of the frdB gene, excluded one of the possibilities of converting fumaric acid into succinic. The deletion of the pflB gene prevented the anaerobic conversion of pyruvic acid into acetyl-CoA and, thus, was supposed to reduce the efficiency of by-products of the strain of acetic acid and ethanol. Inactivation of the IdhA gene, which encodes lactate dehydrogenase, which catalyzes the formation of lactic acid from pyruvic acid with the oxidation of an equimolar amount of NADH, contributed both to the conservation of the reduced equivalents necessary for fumaric acid biosynthesis and also prevented the inappropriate use of one of the precursor metabolites. The molar conversion rate of glucose to fumaric acid, demonstrated by the HH5 strain, was 0.7, three times lower than the theoretically possible maximum.

CN 101240259 also describes the construction of an E. coli strain for anaerobic production of fumaric acid. The E. coli strain NZN111 (W1485 Δpfl :: Cam ΔldhA :: K a n) with inactivated ldhA and pflB genes, not able to ferment glucose anaerobically, was used as a base strain for producing a fumaric acid producer. After the fumarate of the reductase enzyme complex was inactivated and the sfcA gene encoding the malate enzyme was introduced on the plasmid, the resulting strain JM125 (W1485 Δpfl :: Cam ΔldhA :: K a n Δfrd) / pTrc99a-sfcA restored the ability of anaerobic acid to ferment glucose to ferment glucose . Nevertheless, the molar conversion rate of glucose to fumaric acid, demonstrated by strain JM125, was 0.72, with a maximum conversion coefficient of 2.0.

From the foregoing, it follows that modifications leading to the production of strains of E. coli, producers of fumaric acid, may vary in their set. A rational choice of target genes, based only on analysis of available information on the functioning of target biochemical pathways, cannot reliably determine success in improving fumaric acid production by engineered strains. Moreover, it is not obvious that the engineered strains retain the ability to anaerobically utilize glucose when they combine genetic modifications that affect the global coordination of carbon metabolism and the redox balance of the cell under conditions of anaerobiosis.

E. coli strains known from sources of information, anaerobic producers of fumaric acid, synthesize the target substance from glucose with a yield not exceeding the lower threshold value characteristic of the anaerobic process, i.e. less than 1 mol / mol.

Objectives of the present invention include:

- design of E. coli strain capable of producing fumaric acid with increased efficiency;

- development of a method for producing fumaric acid using this strain.

Tasks solved by

- constructing a strain of Escherichia coli FUM1.0, a producer of fumaric acid having inactivated genes a ckA, pt a , poxB, ldhA, a dhE, pflB, frdA, frdB, sdhA, sdhB, a ceA, and ceB and ptsG encoding acetate , phosphotransacetylase, pyruvate oxidase, lactate dehydrogenase, alcohol / aldehyde dehydrogenase, pyruvate format lyase, catalytic subunits of the fumarate reductase enzyme complex, catalytic subunits of succinate dehydrogenase enzyme complex, isolitase lyase, isocitrate lyase, expression of the galP and glk genes encoding the H + -symporter of galactose and glucokinase;

- development of a method for producing fumaric acid by culturing the inventive strain in suitable conditions, followed by isolation of fumaric acid from the culture fluid.

The object of the present invention is a bacterial strain of the species Escherichia coli, with the ability to efficiently produce fumaric acid.

Also an object of the present invention is a strain of the bacteria Escherichia coli with inactivated genes as SKA, pt a, rohV, ldhA, a dhE, pflB, frdA, frdB, sdhA, sdhB, a ceA, and sowing and ptsG, and enhanced expression of genes g a lP and glk is a producer of fumaric acid.

Another object of the present invention is a bacterial strain Escherichia coli, in which the expression of g a lP and glk genes is enhanced by replacing the nucleotide sequences of natural promoters controlling the expression of g a lP and glk genes on the chromosome with stronger promoters.

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

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

The strain according to the present invention is a strain of bacteria of the species Escherichia coli, constructed as a result of targeted genetic engineering manipulations.

The term "strain of bacteria Escherichia coli with inactivated genes of a ckA, pt a, rohV, ldhA, a dhE, pflB, frdA, frdB, sdhA, sdhB, and CEA and CEB and ptsG» means that the bacterium has been modified so that as a result of modification, such a bacterium contains a reduced amount of AckA, Pta, PoxB, LdhA, AdhE, PflB, FrdA, FrdB, SdhA, SdhB, AceA, AceB, and PtsG proteins compared to an unmodified bacterium, or the specified bacterium is not capable of synthesizing AckA proteins, Pta, PoxB, LdhA, AdhE, PflB, FrdA, FrdB, SdhA, SdhB, AceA, AcB and PtsG.

The term “inactivation of the a skA, pt a , pohB, ldhA, a dhE, pflB, frdA, frdB, sdhA, sdhB, a ceA, and ceB and ptsG genes” means that the natural expression of modified DNA regions is impossible due to deletions of these genes or parts thereof, or modifications of regions adjacent to genes that include sequences that control the expression of appropriate genes, such as promoters, enhancers, attenuators, etc.

The presence or absence of a ckA, pt a , pohB, ldhA, a dhE, pflB, frdA, frdB, sdhA, sdhB, and ceA, and ceB and ptsG genes on the chromosome of the bacterium can be determined by known methods, including PCR, Southern blotting, and etc. In addition, gene expression levels can be estimated by determining the amount of RNA transcribed from the gene using various known methods, including Northern blotting, quantitative RT-PCR, and the like. The amounts or molecular weights of the proteins encoded by the genes can be determined by known methods, including SDS-ΠΑΑΓ-electrophoresis, followed by PCR immunoblotting (Western blotting), etc.

The ASKA gene (synonyms: ESK2290, b2296) encodes an AckA protein (synonym: B2296), which exhibits acetate kinase activity, classified as K.F. 2.7.2.1. The a skA gene (nucleotides from 2,411,492 to 2,412,694 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the open reading frame of yfbV and the pta gene on the chromosome of E. coli K-12 strain. The nucleotide sequence of the gene a ckA and amino acid sequence AckA protein encoded by the genome of a ckA, are listed in the List of sequences numbered 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2), respectively. The pt a gene (synonyms: ESK2291, b2297) encodes a Pta protein (synonym: B2297), which exhibits phosphotransacetylase activity, classified as K.F. 2.3.1.8. The pt a gene (nucleotides 2,412,769 through 2,414,913 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the a ckA gene and the open reading frame yfcC on the chromosome of E. coli K-12 strain. The nucleotide sequence of the pt a gene and the amino acid sequence of the Pt a protein encoded by the pt a gene are shown in the Sequence Listing Numbers 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4), respectively. The RohB gene (synonyms: ESK0862, b0871) encodes the PohB protein (synonym: B0871), which exhibits pyruvate oxidase activity, classified as K.F. 1.2.5.1. The RohB gene (nucleotides complementary to nucleotides 908.554 through 910.272 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the ltaE gene and the hcr gene on the chromosome of E. coli K-12 strain. The nucleotide sequence of the RohB gene and the amino acid sequence of the PohB protein encoded by the RohB gene are shown in the Sequence Listing Numbers 5 (SEQ ID NO: 5) and 6 (SEQ ID NO: 6), respectively. The ldhA gene (synonyms: ECK1377, b1380) encodes an LdhA protein (synonym: B1380), exhibiting lactate dehydrogenase activity, classified as K.F. 1.1.1.28. The ldhA gene (nucleotides complementary to nucleotides 1,439,878 to 1,440,867 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the hslJ gene and the open ydbH reading frame on the chromosome of E. coli K-12 strain. The nucleotide sequence of the ldhA gene and the amino acid sequence of the ldhA protein encoded by the ldhA gene are shown in the Sequence Listing Numbers 7 (SEQ ID NO: 7) and 8 (SEQ ID NO: 8), respectively. The a dhE gene (synonyms: ESK1235, b1241) encodes an AdhE protein (synonym: B1241), which exhibits alcohol dehydrogenase activity and dehydrogenase aldehyde, classified as K.F. 1.1.1.1. and K.F. 1.2.1.3. The a dhE gene (nucleotides complementary to nucleotides 1,294,669 to 1,297,344 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the open reading frame ychG and the open reading frame ychE on the chromosome of E. coli K-12 strain. The nucleotide sequence of the a dhE gene and the amino acid sequence of the AdhE protein encoded by the a dhE gene are shown in the Sequence Listing Nos. 9 (SEQ ID NO: 9) and 10 (SEQ ID NO: 10), respectively. The pflB gene (synonyms: ESK0894, b0903) encodes a PflB protein (synonym: B0903), exhibiting pyruvate format lyase activity, classified as K.F. 2.3.1.54. The pflB gene (nucleotides complementary to nucleotides 950,495 to 952,777 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the pflA gene and the focA gene on the chromosome of E. coli K-12 strain. The nucleotide sequence of the pflB gene and the amino acid sequence of the PflB protein encoded by the pfl gene are shown in the sequence Listing numbers 11 (SEQ ID NO: 11) and 12 (SEQ ID NO: 12), respectively. The frdA and frdB genes (synonyms: ESK4150, b4154; ESK4149, b4153) encode the FrdA and FrdB proteins - subunits of the fumarate of the reductase enzymatic complex. The frdA and frdB genes (nucleotides complementary to nucleotides 4,378,533 to 4,380,341, and nucleotides complementary to nucleotides 4,377,806 to 4,378,540, respectively, in the sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990) are located between genes chromosome E. coli K-12. The nucleotide sequences of the frdA and frdB genes and the amino acid sequences of the FrdA and FrdB proteins encoded by the frdA and frdB genes are presented in SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 14, SEQ ID NO: 16, respectively. The sdhA and sdhB genes (synonyms: ECK0712, b0723; ECK0713, b0724) encode the SdhA and SdhB proteins, the catalytic subunits of the succinate dehydrogenase enzyme complex. The sdhA and sdhB genes (nucleotides 755,130 to 756,896, and nucleotides 756,912 to 757,628, respectively, in sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990) are located between the sdhD and sucA genes on the E. coli K chromosome -12. The nucleotide sequences of the sdhA and sdhB genes and the amino acid sequences of the SdhA and SdhB proteins encoded by the sdhA and sdhB genes are presented in SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 18, SEQ ID NO: 20, respectively. The aceA and aceB genes (synonyms: ESK4007, b4015; ESK4006, b4014) encode the AcAA and AceB proteins, which exhibit, respectively, the activity of lyase isocytrate and malate synthase, classified as K.F. 4.1.3.1. and K.F. 2.3.3.9. The ACEA and ACE genes (nucleotides from 4,215,132 to 4,216,436, and nucleotides from 4,213,501 to 4,215,102, respectively, in sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990) are located between the metA and a ceK genes on the chromosome of strain E. coli K-12. The nucleotide sequences of the a CeA and a CeB genes and the amino acid sequences of the AceA and AceB proteins encoded by the a CeA and a CeB genes are presented in SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO: 22, SEQ ID NO: 24, respectively . The ptsG gene (synonyms: ECK1087, b1101) encodes a PtsG protein (synonym: B1101), which exhibits glucose permease activity of phosphoenolpyruvate dependent sugar transport system. The ptsG gene (nucleotides 1,157,092 to 1,158,525 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the open reading frame ycfH and the fhuE gene on the chromosome of E. coli K-12 strain. The nucleotide sequence of the ptsG gene and the amino acid sequence of the PtsG protein encoded by the ptsG gene are shown in the Sequence Listing Nos. 25 (SEQ ID NO: 25) and 26 (SEQ ID NO: 26), respectively.

Since representatives of various strains of the Escherichia coli species may have some variations in the nucleotide sequences, the concept of an inactivated gene is not limited to genes whose sequences are listed in the Sequence Listing Numbers 1 (SEQ ID No: 1), 3 (SEQ ID No: 3), 5 ( SEQ ID No: 5), 7 (SEQ ID No: 7), 9 (SEQ ID No: 9), 11 (SEQ ID No: 11), 13 (SEQ ID NO: 13), 15 (SEQ ID No: 15 ), 17 (SEQ ID No: 17), 19 (SEQ ID No: 19), 21 (SEQ ID NO: 21), 23 (SEQ ID No: 23), 25 (SEQ ID NO: 25), but can also include genes homologous to SEQ ID No: 1, SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID No: 9, SEQ ID No: 11, SEQ ID NO: 13, SEQ ID No: : 15, SEQ ID No: 17, SEQ ID No: 19, SEQ ID NO: 21, SEQ ID No: 23, SEQ ID NO: 25 encoding variants of the proteins AckA, Pta, PoxB, LdhA, AdhE, PflB, FrdA, FrdB, SdhA, SdhB, AcA, AcB and PtsG. The term "protein variant" in the sense in which it is used in the present invention, means a protein having changes in the amino acid sequence, namely the division, insertion, addition or replacement of amino acids. The number of changes in the protein variant depends on the position of the amino acid residue in the three-dimensional structure or the type of amino acid residue. The number of changes can be from 1 to 30, preferably from 1 to 15, and most preferably from 1 to 5 changes in the sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 26. These changes in protein variants are conservative mutations in which protein function is maintained. In other words, these changes can occur in areas of the protein that are uncritical of its three-dimensional structure. This is possible due to the fact that some amino acids have a high homology to each other, and therefore the tertiary structure is not violated during such substitutions. A conservative mutation is a mutation in which mutual substitutions occur among Phe, Trp, Tyr, if the substitution site is an aromatic amino acid; among Leu, Ile, Val, if the substitution site is a hydrophobic amino acid; between Gln, Asn, if the substitution site is a positively charged amino acid; among Lys, Arg, His, if the substitution site is a basic amino acid; between Asp, Glu, if the substitution site is an acidic amino acid, and between Ser, Thr, if it is an amino acid with a hydroxyl group. Conservative substitutions are typical conservative mutations. Examples of conservative substitutions include replacing Ala with Ser or Thr, replacing Arg with Gin, His or Lys, replacing Asn with Glu, Gin, Lys, His or Asp, replacing Asp with Asn, Glu or Gin, replacing Cys with Ser or Ala, replacing Gin with Asn, Glu, Lys, His, Asp or Arg, replacing Glu with Asn, Gin, Lys or Asp, replacing Gly with Pro, replacing His with Asn, Lys, Gin, Arg or Tur, replacing Ile with Leu, Met, Val or Phe, replacing Leu with Ile, Met, Val or Phe, replacing Lys with Asn, Glu, Gin, His or Arg, replacing Met with Ile, Leu, Val or Phe, replacing Phe with Trp, Tur, Met, He or Leu, replacing Ser with Thr or Ala, replacing Thr with Ser or Ala, replacing Trp with Phe or Tyr, replacing Tyr with His, Phe or Trp, and replacing Val with Met, Ile or Leu. The substitutions, deletions, insertions, additions, permutations, etc. described above. one or more amino acid residues include natural mutations (mutant or variant) depending on species differences or individual differences in microorganisms containing the genes a ckA, pt a , poxB, ldhA, a dhE, pflB, frdA, frdB, sdhA, sdhB, a ceA , and seB and ptsG. Such genes can be obtained by modifying the nucleotide sequence shown in SEQ ID No: 1, SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID No: 9, SEQ ID No: 11, SEQ ID NO : 13, SEQ ID No: 15, SEQ ID No: 17, SEQ ID No: 19, SEQ ID NO: 21, SEQ ID No: 23 and SEQ ID NO: 25 using, for example, site-specific mutagenesis, thus that the I site-specific amino acid residue in the corresponding encoded protein includes substitutions, deletions, insertions or additions.

Consequently, variants of the proteins encoded by the a skA, pt a , pohB, ldhA, a dhE, pflB, frdA, frdB, sdhA, sdhB, a ceA, and ceB and ptsG genes can have a homology of at least 80%, preferably at least 90 %, and most preferably at least 95%, with respect to the complete amino acid sequence shown in the sequence Listing under the numbers 2 (SEQ ID NO: 2), 4 (SEQ ID NO: 4), 6 (SEQ ID NO: 6), 8 (SEQ ID NO: 8), 10 (SEQ ID NO: 10), 12 (SEQ ID NO: 12), 14 (SEQ ID NO: 14), 16 (SEQ ID NO: 16), 18 (SEQ ID NO : 18), 20 (SEQ ID NO: 20), 22 (SEQ ID NO: 22), 24 (SEQ ID NO: 24) and 26 (SEQ ID NO: 26), respectively.

In this regard, the genes a scA, pt a , poxB, ldhA, a dhE, pflB, frdA, frdB, sdhA, sdhB, a ceA, and ceB and ptsG can be variants that hybridize under stringent conditions with the nucleotide sequences given in The sequence listing under numbers 1 (SEQ ID No: 1), 3 (SEQ ID No: 3), 5 (SEQ ID No: 5), 7 (SEQ ID No: 7), 9 (SEQ ID No: 9), 11 (SEQ ID No: 11), 13 (SEQ ID NO: 13), 15 (SEQ ID No: 15), 17 (SEQ ID No: 17), 19 (SEQ ID No: 19), 21 (SEQ ID NO: 21), 23 (SEQ ID No: 23), 25 (SEQ ID NO: 25), or with a probe that can be synthesized based on the indicated nucleotide sequence. "Stringent conditions" include those conditions under which specific hybrids, such as hybrids with homology! not less than 60%, preferably not less than 70%, more preferably not less than 80%, even more preferably not less than 90%, and most preferably not less than 95%, nonspecific hybrids are formed, for example, hybrids with less homology than indicated above , - are not formed. A practical example of harsh conditions is a single wash, preferably two or three times, 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, as recommended by the manufacturer. For example, the recommended washing time for Hybond ™ N + nylon membrane (Amersham) under stringent conditions is 15 minutes. Two to three times washing is preferred. The length of the probe can be selected depending on hybridization conditions, usually from 100 n.p. up to 1000 n.p.

Homology between amino acid sequences can be determined using known methods, for example, the BLAST 2.0 computer program.

Gene expression of a skA, pt a , poxB, ldhA, a dhE, pflB, frdA, frdB, sdhA, sdhB, a ceA, a ceB and ptsG genes can be weakened by introducing a mutation into the corresponding gene on the chromosome, in which the intracellular activity of the protein encoded genome is reduced or absent compared to an unmodified strain. Such a gene mutation may be the insertion of an antibiotic resistance gene, or a deletion of a gene or part thereof (Qiu, Ζ. And Goodman, MF, J., 1997, Biol. Chem., 272, 8611-8617; Kwon, DH et al., 2000, J. Antimicrob. Chemother., 46, 793-796). The expression of a ckA, pt a , pohB, ldhA, a dhE, pflB, frdA, frdB, sdhA, sdhB, a ceA, and ceB and ptsG genes can also be attenuated by modification of regulatory sequences, such as a promoter or Shine-Dalgarno (SD ) (PCT application WO 95/34672; Carrier, T.A. and Keasling, JD, 1999, Biotechnol Prog 15, 58-64).

The following methods can be used to introduce mutations by gene recombination. A mutant gene is constructed, and the bacterium for its modification is transformed with a DNA fragment containing the mutant gene. Then the native gene on the chromosome is replaced by homologous recombination with the mutant gene, and the resulting strain is selected. Such gene substitution using homologous recombination can be carried out using a linear DNA method known as "Red-dependent integration" or "Red-system integration" (Datsenko, K.Α., Wanner, BL, 2000, Proc. Natl. Acad. Sci. USA, 97, 12, 6640-6645) or a method using a plasmid whose replication is temperature sensitive (US Pat. No. 6,303,383 or Japanese Patent Application JP 05-007491 A). Further, the introduction of a site-specific mutation by replacing a gene using the aforementioned homologous recombination can also be carried out using a plasmid with reduced replication ability in the host cell.

Gene expression can also be attenuated by inserting a transposon or IS factor into the coding region of the gene (US Pat. No. 5,175,107), or by conventional methods such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine).

Gene inactivation can also be carried out by traditional methods, such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine), site-specific mutagenesis, gene destruction using homologous recombination, and / or mutagenesis deletion insert (Yu, D. et al., 2000, Proc. Natl. Acad. Sci. USA, 97:12: 5978-83 and Datsenko, KA and Wanner, BL, 2000, Proc. Natl. Acad. Sci. USA, 97:12: 6640-45) also called "Red-Dependent Integration".

The above description regarding protein variants, gene inactivation, and other methods may be applicable to other proteins, genes, and bacterial constructs given below.

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 on the basis of a gene sequence, in situ fluorescence hybridization (FISH), etc. The level of gene expression can be determined by various known methods, including Northern blotting, quantitative RT-PCR, and the like. In addition, strains that can be used as controls include, for example, Escherichia coli K-12.

The glk gene (synonyms: ESK2384, b2388) encodes a Glk protein (synonym: B2388), exhibiting glucokinase activity, classified as K.F. 2.7.1.2. The glk gene (nucleotides complementary to nucleotides from 2,506,483 to 2,507,448 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the fryB gene and the open yfeO reading frame on the chromosome of E. coli K-12 strain. The nucleotide sequence of the glk gene and the amino acid sequence of the Glk protein encoded by the glk gene are shown in the Sequence Listing Nos. 27 (SEQ ID NO: 27) and 28 (SEQ ID NO: 28), respectively. The galP gene (synonyms: ESK2938, L2943) encodes a GalP protein (synonym: B2943), which exhibits the activity of the H + -salactor of galactose. The galP gene (nucleotides 3,086,306 to 3,087,700 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the metK gene and the open yggl reading frame on the chromosome of E. coli K-12 strain. The nucleotide sequence of the galP gene and the amino acid sequence of the GalP protein encoded by the galP gene are shown in the Sequence Listing Nos. 29 (SEQ ID NO: 29) and 30 (SEQ ID NO: 30), respectively.

The term "protein variant" is also applicable to Glk and GalP proteins encoding glucokinase and the H + -symptor of galactose. The term "protein variant" is interpreted in the same way as described above.

In this regard, the genes glk and galP can also exist as variants that hybridize under stringent conditions with the nucleotide sequences presented in SEQ ID NO: 27, SEQ ID NO: 29, or with probes that can be synthesized based on these nucleotide sequences, provided that they encode the functional proteins Glk and GalP. The term "stringent conditions" is interpreted in the same way as described above.

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 Escherichia coli species can 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 sequences present on the chromosome in multiple copies as target sequences. 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 ensure 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, tac 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 the expression level was found depending on the nature of the three nucleotides preceding the start codon (Gold et al., 1981, Annu. Rev. Microbiol., 35, 365-403; Hui et al., 1984, EMBO J ., 3, 623-629).

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.

Methods for preparing plasmid DNAs, DNA restriction and ligation, transformation, selection of nucleotides as primers, etc. may be the usual 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).

A method of obtaining fumaric acid in General.

The inventive method for the production of fumaric acid is carried out by culturing the inventive strain of bacteria Escherichia coli in a nutrient medium that provides production, the accumulation of fumaric acid in the culture fluid and the isolation of fumaric acid or its salt from the culture fluid.

According to the claimed method, the cultivation of a strain, the isolation and purification of fumaric acid or its salt from a culture or similar liquid is carried out in a manner similar to traditional fermentation methods in which fumaric acid is produced using microorganisms. As a nutrient medium, both synthetic and natural are used, provided that the medium contains sources of carbon, nitrogen, mineral additives and, if necessary, an 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. As a nitrogen source, 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, fermentolizate of microorganisms, are used. As mineral additives, potassium phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium chloride and the like are used. If necessary, additional sources of CO ₂ and / or HCO ₃ ^- and CO ₃ ^2- ions are added to the nutrient medium, introduced into the medium due to gas exchange or dissolution of the corresponding salts.

Cultivation and incubation is 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 is regulated by ammonia, calcium carbonate, various acids, bases and buffer solutions. Cultivation for 1 to 5 days leads to the accumulation of fumaric acid in the culture fluid.

The inventive method includes aerobic growth and anaerobic productive stages.

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

Example 1. Construction of strains of bacteria Escherichia coli.

The following strains were designed and used -

MSG0.1 (E. coli MG1655 Δ a ckA, Δpt a , ΔroxB, ΔldhA, Δ a dhE, ΔptsG);

MSG0.2 (Ε. Coli MG1655 Δ a ckA, Δpt a , ΔroxB, ΔldhA, Δ a dhE, ΔptsG, P _tac- galP);

MSG1.0 (Ε. Coli MG1655 Δ a ckA, Δpt a , ΔroxB, ΔldhA, Δ a dhE, ΔptsG, P _tac- galP, P _L -glk);

FUM1.0 (E. coli MG1655 Δ a ckA , Δpt a, ΔpoxB, ΔldhA, Δ a dhE, ΔpflB, ΔfrdA, ΔfrdB, ΔsdhA, ΔsdhB, Δ a ceA, Δ a ceB, ΔptsG, P tac -galP, P L -glk).

The wild-type strain Escherichia coli K-12 MG1655 (VKPM B-6195) was used as the base for the construction of all the strains used.

1.1. Construction of the strain MSG0.1.

The strain MSG0.1 (E. coli MG1655 Δ a ckA, Δpt a , ΔroxB, ΔldhA, ΔadhE, ΔptsG) was obtained on the basis of strain E. coli MG1655 as a result of inactivation of the genes a ckA-pt a , roxB, ldhA, a dhE and ptsG using 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., 2007, Recombineering: In Vivo Genetic Engineering in col. Coli, S. enterica, and Beyond. Methods in Enzymology., 421, 171-199), and examples ero practical application, for example, in (RU 2330883).

Initially, all chromosomal modifications were obtained individually using E. coli strain MG1655 as a recipient. Based on selected individual colonies of strains carrying marked target chromosome modifications, preparations of the corresponding Ρ1-transducing phages were obtained. Next, a series of sequential transductions of the resulting modifications are combined on the chromosome of the target strain. After each round of transductions, the antibiotic resistance marker flanked by the lambda phage sites was removed from the chromosome of the obtained strains using the plasmid pMWts-Int / Xis (WO 2007 013638; RU 2005123423), including the Int / Xis genes of the dependent site-specific recombination system phage lambda.

DNA fragments were originally obtained for the inactivation of the a ckA-pt a , poxB, ldhA, a dhE, and ptsG genes.

Linear DNA fragments for deletions of the target genes were obtained by PCR using primer pairs P1 (SEQ ID NO: 31) and P2 (SEQ ID NO: 32), P5 (SEQ ID NO: 33) and P6 (SEQ ID NO: 34) , P9 (SEQ ID NO: 35) and PI0 (SEQ ID NO: 36), P13 (SEQ ID NO: 37) and P14 (SEQ ID NO: 38), P17 (SEQ ID NO: 39) and P18 (SEQ ID NO: 40) and plasmids pMW118-attL-Cm-attR (Katashkina Zh.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 .

Primers P1, P5, P9, P13, and P17 contain 36 nucleotide DNA regions homologous to the DNA regions located at the 5′-ends of the a ckA, pohB, ldhA, a dhE and ptsG genes, and 28 nucleotide regions of DNA complementary to the region DNA located at the 3′-end of the a ttR region. Primers P2, P6, PIO, PI4, and PI8 contain 36 nucleotide DNA regions complementary to the DNA regions located at the 3 ′ ends of the pt a , poxB, ldhA, a dhE, and ptsG genes, as well as homologous 28 nucleotide regions a DNA site located at the 5′-end of a ttL region. Temperature profile for PCR: 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 deoxyribonucleoside triphosphates manufactured by Fermentas (Lithuania).

Received PCR products with a length of 1700 bp To inactivate the genes a ckA-pt a , poxB, ldhA, a dhE, and ptsG, were individually purified by agarose gel electrophoresis and isolated, according to the manufacturer's recommendations, using the Qiagen QIAquick Gel Extraction Kit (Qiagen, USA). The resulting fragments contain the chloramphenicol resistance gene (c a t gene encoding chloramphenicol acetyl transferase) flanked by the a ttL and a ttR sites of the lambda phage, as well as 36 linking regions of flank homology with coding regions of the target genes.

Integration of the obtained linear DNA fragments into the chromosome of E. coli strain MG1655 was carried out using the system of Red-dependent homologous recombination of phage λ.

For this, recombinant DNAs were introduced by electrotransformation into individual clones of E. coli strain MG1655 containing the plasmid pKD46 with a heat-sensitive replicon. Plasmid pKD46 (Datsenko, K.A. and Wanner, BL, 2000, Proc. Natl. Acad. Sci. USA, 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 the integration of linear DNA into the chromosome of the strain.

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) and 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 OD ₆₀₀ ≈0.8 was reached, after which the cells were given electrocompetence properties by 100-fold concentration and three times washing with ice-cold deionized H ₂ O. Electroporation was performed using 70 μl of cells and ~ 300 ng individual PCR product. After electroporation, 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 plates with L-agar containing 30 μg / ml chloramphenicol and grown at 37 ° C to select Cm ^R recombinants. The pKD46 helper plasmid was removed by sieving the clones to individual colonies on LB agar medium with selection of chloramphenicol-resistant and ampicillin-sensitive Cm ^R Ap ^S variants.

Correspondence of the putative and experimentally obtained chromosome structures of the selected Cm ^R Ap ^S colonies was confirmed by PCR analysis using pairs of locus-specific primers P3 (SEQ ID NO: 41) and P4 (SEQ ID NO: 42), P7 (SEQ ID NO: 43) and P8 (SEQ ID NO: 44), P11 (SEQ ID NO: 45) and P12 (SEQ ID NO: 46), P15 (SEQ ID NO: 47) and P16 (SEQ ID NO: 48), P19 (SEQ ID NO: 49) and P20 (SEQ ID NO: 50) for the operon a ckA-pt a rohV and gene, ldhA, a dhE and ptsG, respectively. Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C.

The length of PCR products resulting from reactions using the parental strain MG1655 as a matrix of cells was 3351 bp for the intact operon a ckA-pt a , 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact a dhE gene and 1494 bp for the intact ptsG gene. The length of PCR products resulting from the reaction using mutant strains as a matrix of cells was 1920 bp. for inactivated operon a ckA-pta, 1852 bp for inactivated RohB gene, 2008 bp for the inactivated ldhA gene, 1926 bp for the inactivated a dhE gene and 1759 bp for the inactivated ptsG gene.

On the basis of the selected individual Cm ^R Ap ^S colonies of strains replaced gene c a t operon ackA-pt a and genes rohV, ldhA, a dhE and ptsG by standard methods (Sambrook et al, "Molecular Cloning :. A Laboratory Manual, Second Edition ", Cold Spring Harbor Laboratory Press, 1989) formulated the corresponding P1 transducing phages.

Strain Ε. coli genes deleted from a ckA-pt a received transfer into the chromosome of E. coli MG1655 chromosome fragment operon with a ckA-pt a, gene replaced c a t, flanked attechment sites of lambda phage, using the preparation transducing phage carrying the labeled target genetic modification. For this, Strain E. coli MG1655 cells were grown overnight at 37 ° C in LB medium (Sambrook et al., "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989). Cells from 500 μl of the overnight culture of the recipient strain were collected by centrifugation, resuspended in 100 μl of buffer containing 0.1 M MgSO ₄ and 5 mM CaCl ₂ , a phage transducing preparation was added at a dilution of 10 ⁹ phage particles / ml, and incubated for 20 min at 37 ° C. Cells were pelleted by centrifugation, washed with 1 ml of sterile water, plated on a L-agar plate containing 30 μg / ml chloramphenicol, and grown at 37 ° C to select Cm ^R transductants. Purification of the resulting transductants from residual phage particles was carried out by sieving the clones to individual colonies on an agarized LB medium, followed by selection of variants resistant to chloramphenicol Cm ^R. In the chromosomes of the clones forming the fastest growing colonies of the correct form, the replacement of the operon a ckA-pt a genes with the c a t gene, flanked by attachment sites of the lambda phage, by PCR analysis using locus-specific primers P3 (SEQ ID NO: 41) and P4 ( SEQ ID NO: 42). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. Used Taq DNA polymerase, the appropriate buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania). The length of PCR products resulting from reactions using the parental strain MG1655 as a matrix of cells was 3351 bp for the intact operon a ckA-pt a . The length of the PCR products obtained as a result of the reaction using the obtained transductants as a matrix of cells was 1920 bp. for the operon a ckA-pt a replaced by the c a t gene flanked by attachment sites of the lambda phage. In order to remove the antibiotic resistance marker from the chromosomes of transductants, the selected clones were transformed with the plasmid pMWts-Int / Xis (WO 2007 013638; RU 2005123423). The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The c a t gene was removed from transformed clones by growing separate strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Deleting gene c a t transductants of chromosomes and the deletion of these genes in the operon a ckA-pt a confirmed by PCR using appropriate locus-specific primers P3 and P4. Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. The length of PCR products resulting from reactions using the parental strain MG1655 as a matrix of cells was 3351 bp for the intact operon a ckA-pt a . The length of the PCR products obtained by the reaction using the ckA-pt a strain of the deleted cell operon a as a matrix of cells was 323 bp As a result of subsequent treatment of the clones from the plasmid helper pMWts-Int / Xis, carried out by re-sieving to individual colonies on plates with LB medium and selection of Cm ^S Ap ^S variants, E. coli strain MG1655 (Δ a ckA-pt a ) with the deleted genes a ckA-pt a .

The E. coli strain with deleted a ckA-pt a and poxB genes was obtained by transferring the chromosome fragment to the chromosome of E. coli strain MG1655 (Δ a ckA-pt a ), with the poxB gene replaced by the c a t gene flanked by attachment sites of the lambda phage, using a transducing phage preparation that carries the targeted labeled genetic modification as described above. Purification of the resulting transductants from residual phage particles was carried out by sieving the clones to individual colonies on an agarized LB medium, followed by selection of variants resistant to chloramphenicol Cm ^R. In the chromosomes of the clones that form the fastest growing colonies of the correct shape, the replacement of the poxB gene with the c a t gene, flanked by attachment sites of the lambda phage, by PCR analysis using locus-specific primers P7 (SEQ ID NO: 43) and P8 (SEQ ID NO: 44) was confirmed ) Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. Used Taq DNA polymerase, the appropriate buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania). The length of PCR products resulting from reactions using the parental strain MG1655 (Δ a ckA-pt a ) as a matrix of cells was 1872 bp for the intact rohB gene. The length of the PCR products obtained by the reaction using the obtained transductants as a matrix of cells was 1852 bp. for the roxB gene replaced by the c a t gene flanked by attachment sites of the lambda phage. In order to remove the antibiotic resistance marker from the chromosomes of transductants, the selected clones were transformed with the plasmid pMWts-Int / Xis (WO 2007013638; RU 2005123423). The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The c a t gene was removed from transformed clones by growing separate strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removal of the c a t gene from the transductant chromosomes and deletion of the ckA-pt a operon a and roxB gene in them was confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 41), P4 (SEQ ID NO: 42), and P7 (SEQ ID NO: 43), P8 (SEQ ID NO: 44). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. The length of the PCR products obtained as a result of reactions using the original strain MG1655 as a matrix of cells was 3351 bp for the intact operon a ckA-pt a and 1872 bp for the intact rohB gene. The length of the corresponding PCR products obtained as a result of the reaction using a ckA-pt a operon strain and the poxB gene as the cell matrix was 323 bp. and 255 bp. As a result of subsequent treatment of the clones from the plasmid helper pMWts-Int / Xis, carried out by re-sieving to individual colonies on plates with LB medium and selection of Cm ^S Ap ^S variants, E. coli strain MG1655 was obtained (Δ a ckA-pt a , ΔroxB) with the a ckA-pt a and roxB genes deleted.

The E. coli strain with deleted a ckA-pt a , poxB and ldhA genes was obtained by transferring the chromosome fragment to the chromosome of E. coli strain MG1655 (Δ a ckA-pt a , ΔpoxB), with the ldhA gene replaced by the c a t gene flanked by attachment lambda phage sites using a transducing phage preparation carrying the targeted labeled genetic modification as described above. Purification of the resulting transductants from residual phage particles was carried out by sieving the clones to individual colonies on an agarized LB medium, followed by selection of variants resistant to chloramphenicol Cm ^R. In the chromosomes of the clones that form the fastest growing colonies of the correct form, the ldhA gene was replaced by the c a t gene, flanked by the lambda phage sites, by PCR analysis using locus-specific primers P11 (SEQ ID NO: 45) and P12 (SEQ ID NO: 46) ) Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. Used Taq DNA polymerase, the appropriate buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania). The length of PCR products resulting from reactions using the parental strain MG1655 (Δ a ckA-pt a , ΔroxB) as a matrix of cells was 1299 bp for the intact ldhA gene. The length of PCR products resulting from the reaction using the obtained transductants as a matrix of cells was 2008 bp. for the ldhA gene, replaced by the c a t gene, flanked by attachment sites of the lambda phage. In order to remove the antibiotic resistance marker from the chromosomes of transductants, the selected clones were transformed with the plasmid pMWts-Int / Xis (WO 2007013638; RU 2005123423). The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The c a t gene was removed from transformed clones by growing separate strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removal of the c a t gene from transductant chromosomes and deletion of the ckA-pt a operon a, roxB gene, and ldhA gene in them was confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 41) and P4 (SEQ ID NO: 42), P7 (SEQ ID NO: 43) and P8 (SEQ ID NO: 44), P11 (SEQ ID NO: 45) and Ρ12 (SEQ ID NO: 46). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. The length of the PCR products obtained as a result of reactions using the original strain MG1655 as a matrix of cells was 3351 bp for the intact operon a ckA-pt a , 1872 bp for the intact RohB gene and 1299 bp for the intact ldhA gene. The length of the respective PCR products obtained from the reaction using as template cells from strain operon deleted a ckA-pt a, gene rohV and ldhA gene was 323 bp, 255 bp and 411 bp By curing the clones from the pMWts-Int / Xis helper plasmid by re-sieving to individual colonies on plates with LB medium and selection of Cm ^S Ap ^S variants, E. coli strain MG1655 (Δ a ckA-pt a , ΔroxB, ΔldhA) with the a ckA-pt a , roxB and ldhA genes deleted.

The E. coli strain with deleted a ckA-pt a , poxB, ldhA and a dhE genes was obtained by transferring the chromosome fragment with the a dhE gene replaced by the dhE gene to the chromosome of E. coli strain MG1655 (Δ a ckA-pt a , ΔroxB, ΔldhA) c a t flanked by attachment sites of the lambda phage using a transducing phage preparation carrying the targeted labeled genetic modification as described above. Purification of the resulting transductants from residual phage particles was carried out by sieving the clones to individual colonies on an agarized LB medium, followed by selection of variants resistant to chloramphenicol Cm ^R. In the chromosomes of the clones forming the fastest growing colonies of the correct form, the replacement of the a dhE gene with the c a t gene, flanked by attachment sites of the lambda phage, by PCR analysis using locus-specific primers P15 (SEQ ID NO: 47) and PI6 (SEQ ID NO: 48). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. Used Taq DNA polymerase, the appropriate buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania). The length of PCR products resulting from reactions using the parental strain MG1655 (Δ a ckA-pt a , ΔroxB, ΔldhA) as a matrix of cells was 2903 bp for the intact gene a dhE. The length of the PCR products obtained as a result of the reaction using the obtained transductants as a matrix of cells was 1926 bp for the a dhE gene replaced by the c a t gene flanked by attachment sites of the lambda phage. In order to remove the antibiotic resistance marker from the chromosomes of transductants, the selected clones were transformed with the plasmid pMWts-Int / Xis (WO 2007 013638; RU 2005123423). The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The c a t gene was removed from transformed clones by growing separate strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removing the c a t gene from the transductant chromosomes and dividing the ckA-pt a operon a, poxB gene, ldhA gene, and dhE gene a in them was confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 41) and P4 ( SEQ ID NO: 42), P7 (SEQ ID NO: 43) and P8 (SEQ ID NO: 44), P11 (SEQ ID NO: 45) and P12 (SEQ ID NO: 46), P15 (SEQ ID NO: 47 ) and PI6 (SEQ ID NO: 48). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. The length of the PCR products obtained as a result of reactions using the original strain MG1655 as a matrix of cells was 3351 bp for the intact operon a ckA-pt a , 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene and 2903 bp for the intact gene a dhE. The length of the respective PCR products obtained from the reaction using as template cells from strain operon deleted a ckA-pt a, rohV gene, ldhA gene and adhE gene was 323 bp, 255 bp, 411 bp . and 329 bp. As a result of curing the clones from the helper plasmid pMWts-Int / Xis, carried out by re-sieving to individual colonies on plates with LB medium and selection of Cm ^S Ap ^S variants, E. coli strain MG1655 was obtained ( Δ a ckA-pt a , ΔroxB, ΔldhA, Δ a dhE) with inactivated genes a ckA-pt a , roxB, ldhA and a dhE.

E. coli strain deleted of the genes with a ckA-pt a, rohV, ldhA, a dhE ptsG and obtained transfer into the chromosome of E. coli MG1655 strain (Δ a ckA-pt a, ΔrohV, ΔldhA, ΔadhE) chromosome fragment with a gene ptsG replaced by the c a t gene, flanked by the attachment sites of the lambda phage, using a transducing phage preparation carrying the targeted labeled genetic modification as described above. Purification of the resulting transductants from residual phage particles was carried out by sieving the clones to individual colonies on an agarized LB medium, followed by selection of variants resistant to chloramphenicol Cm ^R. In the chromosomes of the clones that form the fastest growing colonies of the correct form, the replacement of the ptsG gene with the c a t gene, flanked attachment sites of the lambda phage, PCR analysis using locus-specific primers P19 (SEQ ID NO: 49) and P20 (SEQ ID NO: 50) was confirmed ) Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. Used Taq DNA polymerase, the appropriate buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania). The length of PCR products resulting from reactions using the parental strain MG1655 (Δ a ckA-pt a , ΔroxB, ΔldhA, Δ a dhE) as a matrix of cells was 1494 bp for the intact ptsG gene. The length of PCR products resulting from the reaction using the obtained transductants as a matrix of cells was 1759 bp. for the ptsG gene replaced by the cat gene, flanked by attachment sites of the lambda phage. In order to remove the antibiotic resistance marker from the chromosomes of transductants, the selected clones were transformed with the plasmid pMWts-Int / Xis (WO 2007 013638; RU 2005123423). The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The c a t gene was removed from transformed clones by growing separate strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removal of the c a t gene from transductant chromosomes and deletion of the ckA-pt a operon a gene, the poxB gene, the ldhA gene, the dhE gene a , and the ptsG gene in them were confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 41) and P4 (SEQ ID NO: 42), P7 (SEQ ID NO: 43) and P8 (SEQ ID NO: 44), P11 (SEQ ID NO: 45) and P12 (SEQ ID NO: 46), P15 (SEQ ID NO: 47) and P16 (SEQ ID NO: 48), P19 (SEQ ID NO: 49) and P20 (SEQ ID NO: 50). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. The length of the PCR products obtained as a result of reactions using the original strain MG1655 as a matrix of cells was 3351 bp for the intact operon a ckA-pt a , 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact a dhE gene and 1494 bp for the intact ptsG gene. The length of the corresponding PCR products obtained as a result of the reaction using a ckA-pt a operon strain, the poxB gene, the ldhA gene, the dhE gene and the ptsG gene as a matrix of cells was 323 bp, 255 bp, 411 bp, 329 bp and 162 bp. As a result of curing the clones from the helper plasmid pMWts-Int / Xis, carried out by re-sieving to individual colonies on plates with LB medium and selection of Cm ^S Ap ^S variants, E. coli strain MG1655 was obtained ( Δ a ckA-pt a , ΔpohB, ΔldhA, Δ a dhE, ΔptsG) with inactivated genes a ckA-pt a , pohB, IdhA, a dhE and ptsG, named MSG0.1.

1.2. Construction of the strain MSG0.2.

Strain MSG0.2 (E. coli MG1655 Δ a skA, Δpt a , Δpth, Δpoh, ΔldhA, Δ a dhE, ΔptsG, P _tac -galP) was obtained on the basis of strain MSG0.1 (E. coli MG1655 Δ a skA, Δpt a , ΔroxB, ΔldhA, Δ a dhE, ΔptsG) as a result of replacing the native regulatory region of the galP gene in it with the P _tac promoter.

Initially, the target chromosome modification was obtained individually using strain E. coli MG1655 as a recipient. To enhance the expression of the galP gene, a shortened version of the P _tac promoter, which provides constitutive transcription in LacI positive strains, was integrated into the chromosome of the recipient strain in front of the coding region of the galP gene. Based on the obtained strain bearing the marked target chromosome modification, a preparation of the corresponding P1 transducing phage was obtained. Then, the Ρ1 transduction obtained modification was introduced into the chromosome of the target strain. After transductions, the antibiotic resistance marker flanked by the attachment sites of the lambda phage was removed from the chromosome of the obtained strain using the plasmid pMWts-Int / Xis (WO 2007 013638; RU 2005123423), including the Int / Xis genes of the dependent site-specific recombination system of the lambda phage .

A DNA fragment was originally obtained to replace the native regulatory region of the galP gene.

Obtaining the aforementioned artificial DNA fragment, for integration into the corresponding section of the bacterial chromosome, was carried out in several stages. In the first step, PCR obtained DNA fragment containing the early recognition site of restriction endonuclease BglII, promoter is P _tac, 18 nucleotides complementary to the coding region of the previous portion galP gene and 18 nucleotides complementary to the 5'-terminal region of the galP gene coding portion. PCR was performed using primers P21 (SEQ ID NO: 51) and P22 (SEQ ID NO: 52) and plasmid pDR540 (sequence with accession number U13847 in the GenBank database; gi: 595699) as a template. Primer P21 contains a BglII recognition site and a region homologous to the 5 ′ end of the P _tac promoter. Primer P22 contains 18 nucleotides complementary to the region of the preceding coding part of the galP gene, 18 nucleotides complementary to the 5′-terminal region of the coding part of the galΡ gene, and a region complementary to the 3 ′ end of the P _tac promoter. Temperature profile for PCR: 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 deoxyribonucleoside triphosphates manufactured by Fermentas (Lithuania).

In parallel, the second stage of constructing the DNA fragment was carried out. A DNA fragment containing the Cm ^R marker encoded by the c a t gene was obtained by PCR using primers P23 (SEQ ID NO: 53) and P24 (SEQ ID NO: 54) and plasmid pMW118-attL-Cm-attR (Katashkina Zh . 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 P23 contains 36 nucleotides homologous to the DNA site located at 174 nucleotides of the previously coding region of the galP gene and a 28-nucleotide DNA site complementary to the DNA site located at the 3 ′ end of the a ttR region. Primer P24 contains a BglII recognition site and a 28-nucleotide-sized DNA region homologous to the DNA region located at the 5 ′ end of the a ttL region. Temperature profile for PCR: 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 according to the standard procedure (Maniatis T., Fritsch Ε. F., 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 P22 (SEQ ID NO: 52) and P23 (SEQ ID NO: 53). Temperature profile for PCR: 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 1756 bp PCR product was purified by agarose gel electrophoresis and isolated using the Qiagen QIAquick Gel Extraction Kit (Qiagen, United States) according to the manufacturer's recommendations. The resulting fragment contained the P _tac promoter, the chloramphenicol resistance gene (c a t gene encoding chloramphenicol acetyl transferase) flanked by the a ttL and a ttR sites of the lambda phage, as well as 36-unit regions of flank homology with a DNA region preceding the galP gene coding region and a DNA region including the 5′-region of the coding region of the galP gene.

Integration of the obtained linear DNA fragment into the chromosome of E. coli strain MG1655 was carried out using the system of Red-dependent homologous recombination of phage λ.

For this, recombinant DNA was introduced by electrotransformation into cells of the E. coli strain MG1655 containing the plasmid pKD46 (Datsenko, K.A. and Wanner, BL, 2000, Proc. Natl. Acad. Sci. USA, 97 (12): 6640-6645) .

Electrocompetent cells of strain Ε. coli MG1655 containing the plasmid pKD46 was obtained as described above. Electroporation was performed using 70 μl of cells and ~ 300 ng of an individual PCR product. After electroporation, 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 plates with L-agar containing 30 μg / ml chloramphenicol and grown at 37 ° C to select Cm ^R recombinants. The pKD46 helper plasmid was removed by sieving the clones to individual colonies on LB agar medium with selection of chloramphenicol-resistant and ampicillin-sensitive Cm ^R Ap ^S variants.

The fact that the proposed and experimentally obtained chromosome structures correspond to the corresponding Cm ^R Ap ^S colonies was confirmed by PCR analysis using locus-specific primers P25 (SEQ ID NO: 55) and P25 (SEQ ID NO: 56). The length of PCR products obtained by reactions using the parental strain MG1655 and primers P25 and P26 as a matrix of cells was 317 bp. for the intact regulatory region of the g a lP gene. The length of the PCR product obtained by the reaction using the mutant strain and primers P25 and P26 as a matrix of cells was 1845 bp. for the modified regulatory region of the gene g a lP. Also, in the chromosomes of the obtained clones, the correspondence between the planned and experimentally obtained nucleotide sequences of the new regulatory element introduced before the coding region of the galP gene was confirmed by sequencing.

Based on the selected Cm ^R Ap ^S variant strain in which the native regulatory region of the galP gene is replaced by the P _tac promoter, conjugated to the c a t gene flanked by attachment sites of the lambda phage, a preparation of the corresponding P1 transducing phage was obtained.

Strain Ε. coli with the deleted genes a ckA-pt a , poxB, ldhA, a dhE and ptsG, and enhanced expression of the galP gene, obtained by transfer to the chromosome of strain MSG0.1 (E. coli MG1655 Δ a ckA-pt a , ΔroxB, ΔldhA, Δ a dhE, ΔptsG) fragment of the chromosome in which the native regulatory region of the galP gene is replaced by the promoter P _tac , conjugated to the cat gene, flanked by attachment sites of the lambda phage, using a transducing phage preparation carrying the targeted labeled genetic modification, as described above. Purification of the resulting transductants from residual phage particles was carried out by sieving the clones to individual colonies on an agarized LB medium, followed by selection of variants resistant to chloramphenicol Cm ^R. In the chromosomes of the clones that form the fastest growing colonies of the correct shape, the replacement of the native regulatory region of the galP gene with the P _tac promoter, coupled to the c a t gene flanked by attachment sites of the lambda phage, was confirmed by PCR analysis using locus-specific primers P25 (SEQ ID NO: 55 ) and P26 (SEQ ID NO: 56). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. Used Taq DNA polymerase, the appropriate buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania). The length of PCR products resulting from reactions using the parental strain MSG0.1 as a matrix of cells was 317 bp for the intact regulatory region of the galP gene. The length of the PCR products obtained by the reaction using the obtained transductants as a matrix of cells was 1845 bp. bp for the regulatory region of the galP gene, replaced by the P _tac promoter, conjugated to the c a t gene, flanked by attachment sites of the lambda phage. In order to remove the antibiotic resistance marker from the chromosomes of transductants, the selected clones were transformed with the plasmid pMWts-Int / Xis. The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The c a t gene was removed from transformed clones by growing separate strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removal of the c a t gene from transductant chromosomes and deletion of the ckA-pt a operon a gene, the poxB gene, the ldhA gene, the dhE gene a , and the ptsG gene, as well as the presence of the P _tac promoter in front of the galP gene, were confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 41) and P4 (SEQ ID NO: 42), P7 (SEQ ID NO: 43) and P8 (SEQ ID NO: 44), PI 1 (SEQ ID NO: 45 ) and P12 (SEQ ID NO: 46), P15 (SEQ ID NO: 47) and PI6 (SEQ ID NO: 48), P19 (SEQ ID NO: 49) and P20 (SEQ ID NO: 50), P25 (SEQ ID NO: 55) and P26 (SEQ ID NO: 56). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. The length of the PCR products obtained as a result of reactions using the original strain MG1655 as a matrix of cells was 3351 bp for the intact operon a ckA-pt a , 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact gene a dhE, 1494 bp for the intact ptsG gene and 317 bp for the intact regulatory region of the galP gene. The length of the respective PCR products obtained from the reaction using as template cells from strain operon deleted a ckA-pt a, rohV gene, gene ldhA, genome a dhE, ptsG gene and containing a P _tac promoter in front of galP gene was 323 bp ., 255 bp, 411 bp, 329 bp, 162 bp and 248 bp. As a result of curing the clones from the helper plasmid pMWts-Int / Xis, carried out by re-sieving to individual colonies on plates with LB medium and selection of Cm ^S Ap ^S variants, E. coli strain MG1655 was obtained ( Δ a ckA-pt a , ΔpohB, ΔldhA, Δ a dhE, ΔptsG, P _tac -galP) with inactivated a ckA-pt a , pohB, ldhA, a dhE and ptsG genes, and enhanced galP gene expression named MSG0.2 .

1.3. Construction of the strain MSG 1.0.

The strain MSG1.0 (E. coli MG1655 Δ a skA, Δpt a , ΔroxB, ΔldhA, Δ a dhE, ΔptsG, P _tac -galP, P _L -glk) was obtained on the basis of strain MSG0.2 (Ε. Coli MG1655 Δ a ckA-pt a , ΔroxB, ΔldhA, Δ a dhE, ΔptsG, P _tac -galP) by replacing the native regulatory region of the glk gene in it with the P _L lambda phage promoter.

Initially, the target chromosome modification was obtained individually using strain E. coli MG1655 as a recipient. To enhance expression of the glk gene, the P _L lambda phage promoter was integrated into the chromosome of the recipient strain in front of the coding region of the glk gene. Based on the obtained strain bearing the marked target chromosome modification, a preparation of the corresponding P1 transducing phage was obtained. Next, P1 transduction, the resulting modification was introduced into the chromosome of the target strain. After transduction, the antibiotic resistance marker flanked by the attachment sites of the lambda phage was removed from the chromosome of the obtained strain using the plasmid pMWts-Int / Xis (WO 2007 013638; RU 2005123423), including the Int / Xis genes of the dependent site-specific recombination system of the lambda phage .

A DNA fragment was originally obtained to replace the native regulatory region of the glk gene.

Obtaining the aforementioned artificial DNA fragment, for integration 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 recognition site for the restriction endonuclease BglII, P _L lambda phage and 35 nucleotides complementary to the region of the previous coding part of the glk gene. PCR was performed using primers P27 (SEQ ID NO: 57) and P28 (SEQ ID NO: 58) and lambda phage genomic DNA (sequence with accession number NC_001416.1 in the GenBank database, gi: 9626243) as a template. Primer P27 contains a Bglll recognition region and a region homologous to the 5′-end of the P _L promoter. Primer P28 contains 35 nucleotides complementary to the region of the preceding coding portion of the glk gene and a region complementary to the 3 ′ end of the P _L promoter. Temperature profile for PCR: 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 deoxyribonucleoside triphosphates manufactured by Fermentas (Lithuania).

In parallel, the second stage of constructing the DNA fragment was carried out. A DNA fragment containing the Cm ^R marker encoded by the c a t gene was obtained by PCR using primers P29 (SEQ ID NO: 59) and P24 (SEQ ID NO: 54) and plasmid pMW118-attL-Cm-attR (Katashkina Zh . 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 P29 contains 36 nucleotides homologous to the DNA region located 135 nucleotides of the previously coding region of the glk gene and a DNA region 28 nucleotides in length complementary to the DNA region located at the 3 ′ end of the a ttR region. Primer P24 contains a BglII recognition site and a 28-nucleotide-sized DNA region homologous to the DNA region located at the 5 ′ end of the a ttL region. Temperature profile for PCR: 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 according to the standard procedure (Maniatis T., Fritsch Ε. F., 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 P28 (SEQ ID NO: 58) and P29 (SEQ ID NO: 59). Temperature profile for PCR: 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 1955 bp PCR product was purified by agarose gel electrophoresis and isolated using the Qiagen QIAquick Gel Extraction Kit (Qiagen, United States) according to the manufacturer's recommendations. The resulting fragment contained the P _L lambda phage promoter, the chloramphenicol resistance gene (c a t gene encoding chloramphenicol acetyl transferase) flanked by the a ttL and a ttR phages of the lambda phage, as well as the flank homology region with the DNA region preceding the coding region of the glk gene for 135 nucleotides the DNA region immediately preceding the coding region of the glk gene.

Integration of the obtained linear DNA fragment into the chromosome of E. coli strain MG1655 was carried out using the system of Red-dependent homologous recombination of phage λ.

For this, recombinant DNA was introduced by electrotransformation into cells of E. coli strain MG1655 containing plasmid pKD46 (Datsenko, K.A. and Wanner, BL, 2000, Proc. Natl. Acad. Sci. USA, 97 (12): 6640-6645) .

Electrocompetent cells of strain Ε. coli MG1655 containing the plasmid pKD46 was obtained as described above. Electroporation was performed using 70 μl of cells and ~ 300 ng of an individual PCR product. After electroporation, 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 plates with L-agar containing 30 μg / ml chloramphenicol and grown at 37 ° C to select Cm ^R recombinants. The pKD46 helper plasmid was removed by sieving the clones to individual colonies on LB agar medium with selection of chloramphenicol-resistant and ampicillin-sensitive Cm ^R Ap ^S variants.

The fact that the proposed and experimentally obtained chromosome structures correspond to the corresponding Cm ^R Ap ^S colonies was confirmed by PCR analysis using locus-specific primers P30 (SEQ ID NO: 60) and P31 (SEQ ID NO: 61). The length of PCR products resulting from reactions using the parental strain MG1655 and primers P30 and P31 as a matrix of cells was 234 bp. for the intact regulatory region of the glk gene. The length of the PCR product obtained by the reaction using the mutant strain and primers P30 and P31 as a matrix of cells was 2015 bp. for the modified regulatory region of the glk gene. Also, in the chromosomes of the obtained clones, the correspondence between the planned and experimentally obtained nucleotide sequences of the new regulatory element introduced before the coding region of the glk gene was confirmed by sequencing.

Based on the selected variant Cm ^R Ap ^S variant strain in which the native regulatory region of the galP gene is replaced by the P _L lambda phage promoter coupled to the c a t gene flanked by attachment sites of the lambda phage, a preparation of the corresponding P1 transducing phage was obtained.

The E. coli strain with deleted a ckA-pt a , poxB, ldhA, a dhE and ptsG genes, and enhanced expression of the galP and glk genes was obtained by transferring the strain MSG0.2 (E. coli MG1655 Δ a ckA-pt a , ΔroxB, ΔldhA, Δ a dhE, ΔptsG, P _tac -galP) of the chromosome fragment in which the native regulatory region of the glk gene is replaced by the promoter P _{L of the} lambda phage linked to the c a t gene flanked by the attachment sites of the lambda phage using the transducing preparation carrying the targeted labeled genetic modification as described above. Purification of the resulting transductants from residual phage particles was carried out by sieving the clones to individual colonies on an agarized LB medium, followed by selection of variants resistant to chloramphenicol Cm ^R. In the chromosomes of the clones that form the fastest growing colonies of the correct form, the replacement of the native regulatory region of the glk gene with the P _L phage lambda promoter coupled to the c a t gene flanked by the lambda phage sites was confirmed by PCR analysis using locus-specific P30 primers (SEQ ID NO : 60) and P31 (SEQ ID NO: 61). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. Used Taq DNA polymerase, the appropriate buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania). The length of PCR products resulting from reactions using the parental strain MSG0.2 as a matrix of cells was 234 bp for the intact regulatory region of the glk gene. The length of the PCR products obtained as a result of the reaction using the obtained transductants as a matrix of cells was 2015 bp. bp for the regulatory region of the glk gene, replaced by the P _L lambda phage promoter, coupled to the c a t gene, flanked by the attachment sites of the lambda phage. In order to remove the antibiotic resistance marker from the chromosomes of transductants, the selected clones were transformed with the plasmid pMWts-Int / Xis. The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The c a t gene was removed from transformed clones by growing separate strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removal of the c a t gene from the transductant chromosomes and deletion of the ckA-pt a operon a gene, the poxB gene, the ldhA gene, the dhE gene a , and the ptsG gene, as well as the presence of the P _tac promoter in front of the galP gene and the P _L promoter lambda phage in front of the glk gene were confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 41) and P4 (SEQ ID NO: 42), P7 (SEQ ID NO: 43) and P8 (SEQ ID NO: 44) , P11 (SEQ ID NO: 45) and P12 (SEQ ID NO: 46), P15 (SEQ ID NO: 47) and P16 (SEQ ID NO: 48), P19 (SEQ ID NO: 49) and P20 (SEQ ID NO: 50), P25 (SEQ ID NO: 55) and P26 (SEQ ID NO: 56), P30 (SEQ ID NO: 60) and P3 1 (SEQ ID NO: 61). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. The length of the PCR products obtained by reactions using the original MG1655 strain as a matrix of cells was 3351 bp; for the intact operon a ckA-pt a , 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact gene a dhE, 1494 bp for the intact ptsG gene, 317 bp for the intact regulatory region of the galP gene and 234 bp for the intact regulatory region of the glk gene. The length of the respective PCR products obtained from the reaction using as template cells of strain with deleted operon and ska-pt a, gene rohV genome ldhA, genome a dhE, gene ptsG and containing promoter P _tac before gene galP promoter and P _L of phage the lambda in front of the glk gene was 323 bp, 255 bp, 411 bp, 329 bp, 162 bp, 248 bp and 418 bp. As a result of treatment of the clonfv from the plasmid helper pMWts-Int / Xis, carried out by re-sieving to individual colonies on plates with LB medium and selection of Cm ^S Ap ^S variants, E. coli strain MG1655 was obtained ( Δ a ckA, Δpt a , ΔroxB, ΔldhA, Δ a dhE, ΔptsG, P _tac -g a lP, P _L -glk) with inactivated a ckA-pt a , pohB, ldhA, a dhE and ptsG genes, and enhanced expression genes g a lP and glk named MSG1.0.

1.4. The construction of the inventive strain FUM1.0.

Strain FUM1.0 (Ε. Coli MG1655 Δ and SKA, Δpt a, ΔrohV, ΔldhA, Δ a dhE, ΔpflB , ΔfrdA, ΔfrdB, ΔsdhA, ΔsdhB, Δ a ceA, Δ a ceB, ΔptsG, P tac -galP, P _L- glk) was obtained on the basis of strain MSG1.0 (Ε. Coli MG1655 Δ a ckA, Δpta, ΔroxB, ΔldhA, Δ a dhE, ΔptsG, P _tac -galP, P _L- glk) as a result of inactivation of pflB genes in it , frdA, frdB, sdhA, sdhB, and ceA and a ceB.

Initially, all chromosomal modifications were obtained individually using E. coli strain MG1655 as a recipient. Based on selected individual colonies of strains carrying marked target chromosome modifications, preparations of the corresponding P1 transducing phages were obtained. Next, a series of sequential transductions of the resulting modifications are combined on the chromosome of the target strain. After each round of transductions, the antibiotic resistance marker flanked by the lambda phage sites was removed from the chromosome of the obtained strains using the plasmid pMWts-Int / Xis (WO 2007 013638; RU 2005123423), including the Int / Xis genes of the dependent site-specific recombination system phage lambda.

DNA fragments were originally obtained for inactivation of the pflB, frdA and frdB, sdhA and sdhB, and ceA and a ceB genes.

Linear DNA fragments for fission of target genes were obtained by PCR using primer pairs P32 (SEQ ID NO: 62) and P33 (SEQ ID NO: 63), P36 (SEQ ID NO: 64) and P37 (SEQ ID NO: 65) , P40 (SEQ ID NO: 66) and P41 (SEQ ID NO: 67), P44 (SEQ ID NO: 68) and P45 (SEQ ID NO: 69) and plasmids pMWl 18-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.

Primers P32, P36, P40, and P44 contain 36 nucleotide DNA regions homologous to the DNA regions located at the 5 ′ ends of the pflB, frdA, sdhA, and a ceB genes, and 28 nucleotide DNA regions complementary to the DNA region located on 3′-end of the region a ttR. Primers P33, P37, P41, and P45 contain 36 nucleotide DNA regions complementary to the DNA regions located at the 3′-ends of the pflB, frdB, sdhB and a ceA genes, as well as 28 nucleotide DNA regions homologous to the DNA region located on 5′-end of the region a ttL. Temperature profile for PCR: 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 deoxyribonucleoside triphosphates manufactured by Fermentas (Lithuania).

Received PCR products with a length of 1700 bp To inactivate the genes pflB, frdA and frdB, sdhA and sdhB, and ceA and a ceB, were individually purified by agarose gel electrophoresis and isolated using the Qiagen QIAquick Gel Extraction Kit (Qiagen, United States) according to the manufacturer's recommendations. The resulting fragments contain the chloramphenicol resistance gene (c a t gene encoding chloramphenicol acetyl transferase) flanked by the a ttL and a ttR sites of the lambda phage, as well as 36 linking regions of flank homology with coding regions of the target genes.

Integration of the obtained linear DNA fragments into the chromosome of E. coli strain MG1655 was carried out using the system of Red-dependent homologous recombination of phage λ.

For this, recombinant DNA was introduced by electrotransformation into individual clones of E. coli strain MG1655 containing the plasmid pKD46 (Datsenko, K.A. and Wanner, BL, 2000, Proc. Natl. Acad. Sci. USA, 97 (12): 6640-6645 )

Electrocompetent cells of strain Ε. coli MG1655 containing the plasmid pKD46 was obtained as described above. Electroporation was performed using 70 μl of cells and ~ 300 ng of an individual PCR product. After electroporation, 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 plates with L-agar containing 30 m kg / ml chloramphenicol and grown at 37 ° C to select Cm ^R recombinants. The pKD46 helper plasmid was removed by sieving the clones to individual colonies on LB agar medium with selection of chloramphenicol-resistant and ampicillin-sensitive Cm ^R Ap ^S variants.

Correspondence of the putative and experimentally obtained chromosome structures of the selected Cm ^R Ap ^S colonies was confirmed by PCR analysis using pairs of locus-specific primers P34 (SEQ ID NO: 70) and P35 (SEQ ID NO: 71), P38 (SEQ ID NO: 72) and P39 (SEQ ID NO: 73), P42 (SEQ ID NO: 74) and P43 (SEQ ID NO: 75), P46 (SEQ ID NO: 76) and P47 (SEQ ID NO: 77), for the pflB gene, aunt frdA and frdB, sdhA and sdhB genes, and a ceA and a ceB genes, respectively. Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C.

The length of PCR products resulting from reactions using the parental strain MG1655 as a matrix of cells was 2566 bp for the intact pfl gene, 2676 bp for intact genes frdA and frdB, 2731 bp for intact sdhA and sdhB genes, and 3267 bp for the genes a ceA and a ceB. The length of PCR products obtained as a result of the reaction using mutant strains as a matrix of cells was 1983 bp. for the inactivated pflB gene, 1839 bp for inactivated frdA and frdB genes, 1931 bp for inactivated sdhA and sdhB genes, and 2030 bp for inactivated genes a ceA and a ceB.

On the basis of the selected individual Cm ^R Ap ^S colonies of strains replaced gene c a t genome pflB, and genes frdA and frdB, genes sdhA and sdhB, and genes and CEA and a sowing by standard methods (Sambrook et al, "Molecular Cloning .: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989) prepared preparations of the corresponding Ρ1-transducing phages.

Strain Ε. coli with the deleted genes a ckA-pt a , poxB, ldhA, a dhE, pflB and ptsG, and enhanced expression of the galP and glk genes, obtained by transferring the strain MSG1.0 (Ε. coli MG1655 Δ a ckA, Δpt a , ΔroxB , ΔldhA, Δ a dhE, ΔptsG, p _tac -galP, P _L -glk) of the chromosome fragment with the pflB gene replaced by the cat gene flanked by attachment sites of the lambda phage using a transducing phage preparation carrying the target tagged genetic modification as described above . Purification of the resulting transductants from residual phage particles was carried out by sieving the clones to individual colonies on an agarized LB medium, followed by selection of variants resistant to chloramphenicol Cm ^R. In the chromosomes of the clones that form the fastest growing colonies of the correct shape, the replacement of the pflB gene with the c a t gene, flanked attachment sites of the lambda phage, PCR analysis using locus-specific primers P34 (SEQ ID NO: 70) and P35 (SEQ ID NO: 71) was confirmed ) Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. Used Taq DNA polymerase, the appropriate buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania). The length of PCR products resulting from reactions using the parental strain MSG1.0 as a matrix of cells was 2566 bp for the intact pflB gene. The length of PCR products resulting from the reaction using the obtained transductants as a matrix of cells was 1983 bp. for the pflB gene, replaced by the c a t gene, flanked by attachment sites of the lambda phage. In order to remove the antibiotic resistance marker from the chromosomes of transductants, the selected clones were transformed with the plasmid pMWts-Int / Xis (WO 2007 013638; RU 2005123423). The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The c a t gene was removed from transformed clones by growing separate strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removing the c a t gene from transductant chromosomes and dividing the ckA-pt a operon a, poxB gene, ldhA gene, a dhE gene, pflB gene, and ptsG gene, as well as the presence of the P _tac promoter, in front of the galP gene, and the promoter P _L phage lambda in front of the glk gene was confirmed by PCR using the appropriate pairs of locus-specific primers P3 (SEQ ID NO: 41) and P4 (SEQ ID NO: 42), P7 (SEQ ID NO: 43) and P8 (SEQ ID NO: 44), P11 (SEQ ID NO: 45) and P12 (SEQ ID NO: 46), P15 (SEQ ID NO: 47) and P16 (SEQ ID NO: 48), P34 (SEQ ID NO: 70) and P35 ( SEQ ID NO: 71), PI9 (SEQ ID NO: 49) and P20 (SEQ ID NO: 50), P25 (SEQ ID NO: 55) and P26 (SEQ ID NO: 56), P30 (SEQ ID NO: 60 ) and P31 (SEQ ID NO: 61). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. The length of the PCR products obtained as a result of reactions using the original strain MG1655 as a matrix of cells was 3351 bp for the intact operon a ckA-pt a , 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact a dhE gene, 2566 bp for the intact pflB gene, 1494 bp for the intact ptsG gene, 317 bp for the intact regulatory region of the galP gene and 234 bp for the intact regulatory region of the glk gene. The length of the respective PCR products obtained from the reaction using as template cells of strain with deleted operon a ckA-pt a, gene rohV genome ldhA, genome a dhE, gene pflB, gene ptsG and containing promoter P _tac before gene galP promoter and The P _L phage of the lambda in front of the glk gene was 323 bp, 255 bp, 411 bp, 329 bp, 386 bp, 162 bp, 248 bp and 418 bp. As a result of curing the clones from the helper plasmid pMWts-Int / Xis, carried out by re-sieving to individual colonies on plates with LB medium and selection of Cm ^S Ap ^S variants, E. coli strain MG1655 was obtained ( Δ a cKA, Δpta, ΔpohB, ΔldhA, Δ a dhE, ΔpflB, ΔptsG, P _tac -galP, P _L -glk) with inactivated a ckA-pt a , poxB, ldhA, a dhE, pflB and ptsG genes, and amplified gene expression of galP and glk.

Strain Ε. coli genes deleted from a ckA-pt a, poxB, ldhA, a dhE, pflB, frdA, frdB and ptsG, and enhanced expression of genes galP and glk, obtained by transfer into the chromosome of E. coli MG1655 (Δ as SKA, Δpt a, ΔroxB, ΔldhA, Δ a dhE, ΔpflB, ΔptsG, P _tac -galP, P _L -glk) of the chromosome fragment with the frdA and frdB genes replaced by the cat gene flanked by attachment sites of the lambda phage using a preparation of a transducing phage that carries the target phage carrying the modification as described above. Purification of the resulting transductants from residual phage particles was carried out by sieving the clones to individual colonies on an agarized LB medium, followed by selection of variants resistant to chloramphenicol Cm ^R. In the chromosomes of the clones that form the fastest growing colonies of the correct form, the replacement of the frdA and frdB genes with the c a t gene, flanked attachment sites of the lambda phage, PCR analysis using locus-specific primers P38 (SEQ ID NO: 72) and P39 (SEQ ID NO : 73). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. Used Taq DNA polymerase, the appropriate buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania). The length of PCR products resulting from reactions using the parental strain MG1655 as a matrix of cells (Δ a skA, Δpt a , ΔpohB, ΔldhA, Δ a dhE, ΔpflB, ΔptsG, P _tac- galP, P _L -glk) was 2676 bp for intact frdA and frdB genes. The length of PCR products resulting from the reaction using the obtained transductants as a matrix of cells was 1839 bp. for the frdA and frdB genes replaced by the cat gene, flanked by attachment sites of the lambda phage. In order to remove the antibiotic resistance marker from the chromosomes of transductants, the selected clones were transformed with the plasmid pMWts-Int / Xis (WO 2007 013638; RU 2005123423). The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The c a t gene was removed from transformed clones by growing separate strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Deleting gene c a t chromosome transductants and deletion in these genes operon a ckA-pt a, gene rohV, ldhA gene, a dhE, pflB gene, genes frdA and frdB, and ptsG gene as well as the fact of promoter P _tac, before the galP gene, and the Pl promoter of the lambda phage before the glk gene, PCR was confirmed using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 41) and P4 (SEQ ID NO: 42), P7 (SEQ ID NO: 43) and P8 (SEQ ID NO: 44), P11 (SEQ ID NO: 45) and Ρ12 (SEQ ID NO: 46), P15 (SEQ ID NO: 47) and Ρ16 (SEQ ID NO: 48), P34 (SEQ ID NO: 70) and P35 (SEQ ID NO: 71), P38 (SEQ ID NO: 72) and P39 (SEQ ID NO: 73), P19 (SEQ ID NO: 49) and P20 (SEQ ID NO: 50), P25 ( SEQ ID NO: 55) and P26 (SEQ ID NO: 56), P30 (SEQ ID NO: 60) and P31 (SEQ ID NO: 61). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. The length of the PCR products obtained as a result of reactions using the original strain MG1655 as a matrix of cells was 3351 bp for the intact operon a ckA-pt a , 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact a dhE gene, 2566 bp for the intact pflB gene, 2676 bp for intact genes frdA and frdB, 1494 bp for the intact ptsG gene, 317 bp; for the intact regulatory region of the galP gene and 234 bp for the intact regulatory region of the glk gene. The length of the respective PCR products obtained from the reaction using as template cells of strain with deleted operon a ckA-pt a, gene rohV genome ldhA, genome a dhE, gene pflB, genes frdA and frdB, gene ptsG and containing promoter P _tac before the galP gene and the P _L promoter of the lambda phage before the glk gene was 323 bp, 255 bp, 411 bp, 329 bp, 386 bp, 242 bp, 162 bp, 248 bp and 418 bp. As a result of curing the clones from the helper plasmid pMWts-Int / Xis, carried out by re-sieving to individual colonies on plates with LB medium and selection of Cm ^S Ap ^S variants, E. coli strain MG1655 was obtained ( Δ a ckA, Δpt a , ΔpohB, ΔldhA, ΔadhE, ΔpflB, ΔfrdA, ΔfrdB, ΔptsG, P _tac -galP, P _L -glk) with inactivated genes a ckA-pt a , pohB, ldhA, a dhEfl, , frdB and ptsG, and enhanced gene expression of galP and glk.

Strain Ε. coli genes deleted from a ckA-pt a, rohV, ldhA, a dhE, pflB, frdA, frdB, sdhA, sdhB and ptsG, and enhanced expression of genes galP and glk, obtained by transfer into the chromosome of E. coli MG1655 (Δ a ckA , Δpt a , ΔpohB, ΔldhA, Δ a dhE, ΔpflB, ΔfrdA, ΔfrdB, ΔptsG, P _tac -galP, P _L -glk) of the chromosome fragment with sdhA and sdhB genes replaced by the cat gene, flanked attachment with phage sites a phage transducing preparation carrying a targeted labeled genetic modification as described above. Purification of the resulting transductants from residual phage particles was carried out by sieving the clones to individual colonies on an agarized LB medium, followed by selection of variants resistant to chloramphenicol Cm ^R. In the chromosomes of the clones that form the fastest growing colonies of the correct shape, the replacement of the sdhA and sdhB genes by the cat gene, flanked attachment sites of the lambda phage, PCR analysis using locus-specific primers P42 (SEQ ID NO: 74) and P43 (SEQ ID NO: 75) was confirmed ) Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. Used Taq DNA polymerase, the appropriate buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania). The length of PCR products resulting from reactions using the parental strain MG1655 as a matrix of cells (Δ a ckA, Δpt a , ΔroxB, ΔldhA, Δ a dhE, ΔpflB, ΔfrdA, ΔfrdB, ΔptsG, P _tac- galP, P _L- glk ), amounted to 2731 bp for intact sdhA and sdhB genes. The length of the PCR products obtained by the reaction using the obtained transductants as a matrix of cells was 1931 bp for the sdhA and sdhB genes replaced by the c a t gene flanked by attachment sites of the lambda phage. In order to remove the antibiotic resistance marker from the chromosomes of transductants, the selected clones were transformed with the plasmid pMWts-Int / Xis (WO 2007 013638; RU 2005123423). The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The c a t gene was removed from transformed clones by growing separate strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Deleting gene c a t chromosome transductants and deletion in these genes operon a ckA-pt a, rohV gene, ldhA gene, a dhE, pflB gene, genes frdA and frdB, genes sdhA and sdhB and ptsG gene as well as the fact of the promoter P _tac , before the galP gene, and the promoter P _L phage lambda before the glk gene was confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 41) and P4 (SEQ ID NO: 42), P7 (SEQ ID NO : 43) and P8 (SEQ ID NO: 44), PI 1 (SEQ ID NO: 45) and P12 (SEQ ID NO: 46), P15 (SEQ ID NO: 47) and P16 (SEQ ID NO: 48), P34 (SEQ ID NO: 70) and P35 (SEQ ID NO: 71), P38 (SEQ ID NO: 72) and P39 (SEQ ID NO: 73), P42 (SEQ ID NO: 74) and P43 (SEQ ID NO : 75), P19 (SEQ ID NO: 49) and P20 (SEQ ID NO: 50), P25 (SEQ ID NO: 55) and P26 (SEQ ID NO: 56), P30 (SEQ ID NO: : 60) and P31 (SEQ ID NO: 61). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. The length of the PCR products obtained as a result of reactions using the original strain MG1655 as a matrix of cells was 3351 bp for the intact operon a ckA-pt a , 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact a dhE gene, 2566 bp for the intact pflB gene, 2676 bp for intact genes frdA and frdB, 2731 bp for intact sdhA and sdhB genes, 1494 bp for the intact ptsG gene, 317 bp for the intact regulatory region of the galP gene and 234 bp for the intact regulatory region of the glk gene. The length of the corresponding PCR products obtained as a result of the reaction using a ckA-pta operon strain, the poxB gene, the ldhA gene, the a dhE gene, the pflB gene, the frdA and frdB genes, the sdhA and sdhB genes, the ptsG and containing the P _tac promoter in front of the galP gene and the P _L promoter of the lambda phage in front of the glk gene was 323 bp, 255 bp, 411 bp, 329 bp, 386 bp, 242 bp n., 334 bp, 162 bp, 248 bp and 418 bp. As a result of curing the clones from the helper plasmid pMWts-Int / Xis, carried out by re-sieving to individual colonies on plates with LB medium and selection of Cm ^S Ap ^S variants, E. coli strain MG1655 was obtained ( Δ a clA, Δpta, ΔrohV, ΔldhA, Δ a dhE, ΔpflB , ΔfrdA, ΔfrdB, ΔsdhA, ΔsdhB, ΔptsG, P tac -galP, P L -glk) inactivated genes a ckA-pt a, rohV, ldhA, a dhE, pflB, frdA, frdB, sdhA, sdhB and ptsG, and enhanced expression of the galP and glk genes.

Strain Ε. coli with deleted genes a ckA-pt a , poxB, ldhA, a dhE, pflB, frdA, frdB, sdhA, sdhB, a ceA, and ceB and ptsG, and enhanced expression of the galP and glk genes, obtained by transfer to strain E chromosome . coli MG1655 (Δ as SKA, Δpt a, ΔrohV, ΔldhA, Δ a dhE, ΔpflB , ΔfrdA, ΔfrdB, ΔsdhA, ΔsdhB, ΔptsG, P tac -galP, P L -glk) chromosome fragment genes as CEA and CEB and replaced by the cat gene, flanked by attachment sites of the lambda phage, using a transducing phage preparation carrying the targeted labeled genetic modification as described above. Purification of the resulting transductants from residual phage particles was carried out by sieving the clones to individual colonies on an agarized LB medium, followed by selection of variants resistant to chloramphenicol Cm ^R. In the chromosomes of the clones that form the fastest growing colonies of the correct form, the replacement of a ceA and a ceB genes with c a t, flanked attachment sites of the phage lambda, PCR analysis using locus-specific primers P46 (SEQ ID NO: 76) and P47 (SEQ ID NO: 77). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. Used Taq DNA polymerase, the appropriate buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania). The length of PCR products resulting from reactions using the parental strain MG1655 as a matrix of cells (Δ a cKA, Δpt a , ΔpohB, ΔldhA, Δ a dhE, ΔpflB, ΔfrdA, ΔfrdB, ΔsdhA, ΔsdhB, ΔptsG, P _tac -gg P _L -glk) was 3267 bp for the intact ACEA and ACEB genes. The length of the PCR products obtained by the reaction using the obtained transductants as a matrix of cells was 2030 bp for the genes a ceA and a ceB, replaced by the c a t gene flanked by attachment sites of the lambda phage. In order to remove the antibiotic resistance marker from the chromosomes of transductants, the selected clones were transformed with the plasmid pMWts-Int / Xis (WO 2007 013638; RU 2005123423). The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The c a t gene was removed from transformed clones by growing separate strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Deleting gene c a t chromosome transductants and deletion in these genes operon a ckA-pt a, gene rohV, ldhA gene, a dhE, pflB gene, genes frdA and frdB, genes sdhA and sdhB, genes and CEA and a sowing, and the ptsG gene, as well as the fact of the presence of the P _tac promoter, before the galP gene, and the P _L promoter of the lambda phage before the glk gene, were confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 41) and P4 (SEQ ID NO: 42), P7 (SEQ ID NO: 43) and P8 (SEQ ID NO: 44), P11 (SEQ ID NO: 45) and P12 (SEQ ID NO: 46), P15 (SEQ ID NO: 47) and P16 ( SEQ ID NO: 48), P34 (SEQ ID NO: 70) and P35 (SEQ ID NO: 71), P38 (SEQ ID NO: 72) and P39 (SEQ ID NO: 73), P42 (SEQ ID NO: 74 ) and P43 (SEQ ID NO: 75), P46 (SEQ ID NO: 76) and P47 (SEQ ID NO: 77), P19 (SEQ ID NO: 49) and P20 (SEQ ID NO: 50), P25 (SEQ ID NO: 55) and P26 (SEQ ID NO: 56), P30 (SEQ ID NO: 60) and P31 (SEQ ID NO: 61). Temperature profile for PCR testing: denaturation at 95 ° C for 10 min; profile for 30 cycles: 30 sec at 95 ° C, 30 sec at 50 ° C, 2 min at 72 ° C; final step: 7 min at 72 ° C. The length of the PCR products obtained as a result of reactions using the original strain MG1655 as a matrix of cells was 3351 bp for the intact operon a ckA-pt a , 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact a dhE gene, 2566 bp for the intact pflB gene, 2676 bp for intact genes frdA and frdB, 2731 bp for intact sdhA and sdhB genes, 3267 bp for intact genes a ceA and a ceB, 1494 bp for the intact ptsG gene, 317 bp for the intact regulatory region of the galP gene and 234 bp for the intact regulatory region of the glk gene. The length of the respective PCR products obtained from the reaction using as template cells of strain with deleted operon a ckA-pt a, gene rohV genome ldhA, genome a dhE, gene pflB, genes frdA and frdB, genes sdhA and sdhB, genes and ceA and a ceB, the ptsG gene and containing the P _tac promoter before the galP gene and the P _L promoter lambda phage before the glk gene were 323 bp, 255 bp, 411 bp, 329 bp, 386 bp, 242 bp, 334 bp, 433 bp, 162 bp, 248 bp and 418 bp. As a result of curing the clones from the helper plasmid pMWts-Int / Xis, carried out by re-sieving to individual colonies on plates with LB medium and selection of Cm ^S Ap ^S variants, E. coli strain MG1655 was obtained ( Δ a ckA, Δpta, ΔrohV, ΔldhA, Δ a dhE, ΔpflB , ΔfrdA, ΔfrdB, ΔsdhA, ΔsdhB, Δ a ceA, Δ a ceB, ΔptsG, P tac -galP, P L -glk) inactivated genes a ckA- pt a , poxB, ldhA, ΔdhE, pflB, frdA, frdB, sdhA, sdhB, a ceA, and ceB and ptsG, and enhanced expression of the galP and glk genes, called Escherichia coli FUM1.0.

Example 2. The production of fumaric acid of the inventive strain of bacteria Escherichia coli FUM1.0.

Cells of FUM1.0 strain or control starting MG1655 strain are grown overnight in M9 medium (Sambrook, J., Fritsch, F. F., and Maniatis, T., "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) containing 2 g / l glucose at 37 ° C. 5 ml of the resulting overnight cultures are diluted 10 times, 45 ml of M9 medium containing 5 g / l tryptone and 10 g / l glucose are added. The resulting cultures were grown in 750 ml flasks closed with cotton plugs at 37 ° C on a rotary shaker at 250 rpm for 20 hours. The resulting cell suspensions are centrifuged for 15 minutes at 4000 rpm at 4 ° C. Precipitation was resuspended in 15 ml of M9 minimal saline containing 4.5 g / L glucose and 4.5 g / L NaHCO ₃ . Then, the resulting cell cultures are incubated for 24 hours in 15 ml tubes closed with unventilated tubes at 37 ° C on a rotary shaker at 250 rpm. The concentrations of organic acids and residual glucose in the culture fluid freed from the biomass by centrifugation were determined by high performance liquid chromatography using a Waters HPLC system (Waters, Milford, MA, USA). To measure the concentration of organic acids, a ReproSil-Pur C18-AQ reverse phase column (4 × 250 mm, 5 μm, Dr. Maisch, Ammerbuch, Germany) was used. Detection is 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 is used as the mobile phase. To measure glucose concentration, the Waters HPLC system is equipped with a Waters 2414 refractive 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 is used as the mobile phase. Substances are identified by comparing their retention times with the retention times of their respective standards.

The ethanol concentration in the culture fluid is determined by gas chromatography with a flame ionization detector on an OmegaWax column (30 m, 0.25 mm east, 0.25 μm film thickness) (Supelco, USA). A GC-17A "Shimadzu" chromatograph (Japan) equipped with an AOC-20i autosampler is used. Helium with a constant flow rate of 1.5 ml / min is used as carrier gas. The temperature of the column thermostat is 4 minutes 40 ° C, followed by a rise to 200 ° C at a speed of 30 ° C / min, holding for 1 min and returning to the initial temperature. Injector temperature = 150 ° C. Detector temperature = 240 ° C.

The molar concentrations of glucose consumed by the strains, accumulated metabolites and the molar yield of fumaric acid as a result of the corresponding tube fermentations are presented in Table 1, from which it follows that the conversion rate of glucose to fumaric acid demonstrated by the inventive bacterial strain Escherichia coli FUM1.0 in the anaerobic productive phase is 1, 31 mol / mol, reaching 65.5% of the theoretically possible maximum, and significantly exceeds the performance of all currently known bacterial strains of producers fumaric acid.

Table 1 Strain Glucose consumed Lactic acid Acetic acid Ethanol succinic acid Fumaric acid Molar yield of fumaric acid mm mm mm mm mm mm mol / mol MG1655 25.0 12.8 ± 0.3 10.2 ± 0.5 12.3 ± 0.4 3.7 ± 0.1 - - FUM1.0 25.0 - 3.0 ± 0.2 14.9 ± 0.3 - 32.8 ± 0.5 1.31 ± 0.02

Claims (3)

1. The bacterial strain Escherichia coli FUM1.0 is a fumaric acid producer obtained by inactivation of the ackA, pta, poxB, ldhA, adhE, pflB, frdA, frdB, sdhA, sdhB, aceA, aceB and ptsG genes, and enhancing the expression of galP and glk in the strain Escherichia coli K-12 MG1655. 2. The bacterial strain Escherichia coli according to claim 1, characterized in that the expression of the galP and glk genes is enhanced by replacing the nucleotide sequences of the natural promoters controlling the expression of the galP and glk genes on the chromosome with stronger promoters. 3. A method of producing fumaric acid by culturing a strain according to claim 1 under suitable conditions, followed by isolation of fumaric acid from the culture fluid.

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