RU2603004C1 - Strain of bacteria escherichia coli-succinic acid producer (versions) and method for preparing succinic acid using this strain - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed versions of the bacteria Escherichia coli, which are succinic acid producers. Escherichia coli bacterium is modified so that the genes ackA, pta, poxB, ldhA, adhE, ptsG are inactivated in it, expression of genes galP and glk is strengthened, and bacterium possessing activity of pyruvate carboxylase. Also disclosed a version of bacteria Escherichia coli, having inactivated genes ackA, pta, poxB, ldhA, adhE, ptsG, icIR reinforced by expression of genes galP and glk, and activity of pyruvate carboxylase. Also disclosed a version of bacteria Escherichia coli, having inactivated genes ackA, pta, poxB, ldhA, adhE, ptsG, enhanced by expression of genes galP and glk, enhanced by expression of genes aceE, aceF and lpdA, and activity of pyruvate carboxylase. Also disclosed a version of bacteria Escherichia coli, having inactivated genes ackA, pta, poxB, ldhA, adhE, ptsG, icIR, enhanced by expression of genes galP and glk, enhanced by expression of genes aceE, aceF and lpdA, and activity of pyruvate carboxylase. Also disclosed a version of bacteria Escherichia coli, having inactivated genes ackA, pta, poxB, IdhA, adhE, ptsG, pflB, enhanced by expression of genes galP and glk, enhanced by expression of genes aceE, aceF and lpdA, and activity of pyruvate carboxylase. Also disclosed a version of bacteria Escherichia coli, having inactivated genes ackA, pta, poxB, ldhA, adhE, ptsG, pflB, icIR, enhanced by expression of genes galP and glk, enhanced by expression of genes aceE, aceF and lpdA, and activity of pyruvate carboxylase. Disclosed a method for preparing succinic acid with using the above versions.

EFFECT: group of inventions provides higher yield of succinic acid.

19 cl, 2 tbl, 2 ex

Description

The present invention relates to biotechnology, in particular to strains of bacteria Escherichia coli - producers of succinic acid capable of efficient production of succinic acid from glucose, as well as a method for producing succinic acid using these strains, with improved rates of conversion of the substrate to the target product close to the maximum theoretically possible values.

Succinic acid is considered as one of the most important “building blocks” for the production of a wide range of high value-added chemicals such as organic solvents, plasticizers, biodegradable plastics, etc. (Cukalovic A., Stevens CV, Feasibility of production methods for succinic acid derivatives : a marriage of renewable resources and chemical technology. Biofuels, Bioprod Bioref., 2008, 2 (6): 505-529). The potential market for succinic acid and its derivatives is estimated at 6 million tons / year.

Traditionally, succinic acid is obtained by petrochemical synthesis from maleic anhydride, however, in recent years significant progress has been made in commercializing and industrializing the microbiological synthesis of succinic acid from renewable raw materials (Jansen ML, van Gulik WM Towards large scale fermentative production of succinic acid. Curr Opin Biotechnol., 2014, 30: 190-197). The basis of these processes is the fermentation of carbohydrate-containing substrates by highly effective microbial strains obtained using directed metabolic engineering. Due to the relative cheapness and significant production volumes, the preferred substrate for the microbiological production of succinic acid is glucose, the most common carbohydrate in plant biomass (Song N. Lee SY, Production of succinic acid by bacterial fermentation. Enz. Microbial Technol., 2006, 39 (3): 352-361). The best indicators of glucose to succinic acid conversion are demonstrated by producer strains based on Escherichia coli bacteria (Cheng K.K., et al. Improved succinate production by metabolic engineering. 2013 Biomed Res Int. Doi: 10.1155 / 2013/538790).

The optional anaerobic bacterium Escherichia coli is capable of synthesizing succinic acid in a limited set of biochemical reactions. These include reactions of the oxidative cycle of tricarboxylic acids, reactions of one of the branches of mixed acid fermentation, known as the reducing branch of the cycle of tricarboxylic acids, and the reaction of the glyoxylate shunt.

The most effective microbiological process for obtaining succinic acid is anaerobic fermentation of glucose, with the formation of the target substance in the reducing branch of the tricarboxylic acid cycle. In this process, the maximum possible conversion rate of the substrate into the product based on carbon is 2 mol / mol. This is due to the fixation of CO ₂ at the stage of formation from glycolytic intermediates of oxaloacetate, a precursor in the anaerobic pathway of glucose fermentation into succinic acid. However, the feasibility of this process is hindered by its redox imbalance. The conversion of each of the two oxaloacetate molecules obtained from glycolytic glucose utilization intermediates to succinic acid requires 2 reduced equivalents (NADH), while only half of the required equivalents are formed during glycolysis (1 NADH per oxaloacetate molecule). This leads to the fact that the best indicators of the production of succinic acid by specially obtained mutant strains are achieved in biosynthesis processes, which are a combination of two biochemical pathways - the reducing branch of the tricarboxylic acid cycle and glyoxylate shunt. With the participation of the glyoxylate shunt, two succinic acid molecules can be synthesized from one oxaloacetate molecule, two acetyl-CoA molecules, and one NADH. The glyoxylate shunt consuming less NADH for each synthesized succinic acid molecule acts in this case as a donor of “residual” glycolytic NADH for highly productive formation of the target substance in the reduction part of the tricarboxylic acid cycle. However, since 2 Acetyl-CoA molecules formed from a glycolytic precursor with CO ₂ loss are involved in the glyoxylate shunt, along with oxaloacetate, the contribution of this process to the total biosynthesis of succinic acid leads to a decrease in the total yield of the product. The final yield of the target product is thus determined by the ratio of the contributions of each of the pathways to the biosynthesis of succinic acid. While the key biosynthesis reactions of oxaloacetate from tri-carbon glycolytic precursors are NADH independent, the formation of Acetyl-CoA from glycolytically formed pyruvic acid may be accompanied by additional generation of NADH. In the cells Ε. coli oxidation of pyruvate to Acetyl-CoA, depending on aeration conditions, is catalyzed by either pyruvate dehydrogenase or pyruvate format lyase. The conversion of pyruvic acid to Acetyl-CoA under the action of pyruvate format lyase is not accompanied by the formation of NADH, while the action of pyruvate dehydrogenase leads to the formation of an additional NADH molecule on each formed Acetyl-CoA. Depending on the participation of specific enzymes in the formation of Acetyl-CoA, the biosynthesis of succinic acid from glucose under the combined action of the reducing branch of the tricarboxylic acid cycle and glyoxylate shunt can be carried out in three redox balanced modes, characterized by different values of the maximum theoretically possible yield of the product.

When Acetyl-CoA is formed under the action of pyruvate, the lyase format, redox balanced anaerobic biosynthesis of succinic acid from glucose is possible as a result of the combined action of the reducing branch of the tricarboxylic acid cycle and the glyoxylate shunt with a carbon flux distribution between the two pathways at a ratio of 60:40, which determines the maximum possible the conversion ratio of the substrate into the target product is 1.6 mol / mol. When Acetyl-CoA is formed under the influence of both pyruvate lyase format and pyruvate dehydrogenase, the anaerobic biosynthesis of succinic acid from glucose can be redox-balanced when the carbon flux is distributed between the reducing branch of the tricarboxylic acid cycle and the glyoxylate shunt in a ratio of 50:50, which determines the maximum the possible conversion rate of the substrate into the target product is 1.6 (6) mol / mol. In the case of the formation of acetyl-CoA under the action of pyruvate dehydrogenase exclusively, the distribution of the carbon flux between the reducing branch of the tricarboxylic acid cycle and the glyoxylate shunt in the ratio of 57.14: 42.86 provides redox balanced anaerobic biosynthesis of succinic acid from glucose and determines the maximum possible conversion coefficient the substrate in the target product is 1.71 mol / mol.

It should be noted that in E. coli, the conversion of pyruvic acid to Acetyl-CoA under anaerobic conditions is effected by the pyruvate lyase format, while the expression of the pyruvate dehydrogenase complex genes during anaerobiosis is repressed (see, for example, Soini J. et al., Norvaline is accumulated after a down-shift of oxygen in Escherichia coli W3110. Microb Cell Fact., 2008, 7:30, doi: 10.1186 / 1475-2859-7-30). Thus, the involvement of pyruvate dehydrogenase in the anaerobic biosynthesis of acetyl-CoA requires a corresponding increase in the expression of genes encoding the components of this enzymatic complex.

Among E. coli strains of succinic acid producers obtained by directed metabolic engineering without the use of mutagenesis and selection methods, representative strains characterized by the highest possible conversion rate of glucose to succinic acid 1.6 mol / mol are SBS550MG [pHL413] (Sánchez AM et al. , Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity. Metab Eng. 2005, 7 (3), 229-239; Cox SJ et al., Development of a metabolic network design and optimization framework incorporating implementation constraints: A succinate production case study. Metab Eng., 2006, 8: 46-57), SGM1.0 [pPYC] and SGM1.1 [pPYC] (RU 25280 56). Strains Ε. coli succinic acid producers characterized by the highest possible conversion of glucose to succinic acid 1.6 (6) mol / mol are SGM2.0 [pPYC] and SGM2.1 [pPYC] (RU 2528056). The producing strains with the highest possible conversion rate of glucose to succinic acid 1.71 mol / mol correspond to NZ-041 (Zhu X. et al. Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli. Metab Eng ., 2014, doi: 10.1016 / j.ymben.2014.05.003), SGM3.0 [pPYC] and SGM3.1 [pPYC] (RU 2528056).

Strains Ε. coli SBS550MG [pHL413], SGM1.0 [pPYC], SGM1.1 [pPYC]; We consider SGM2.0 [pPYC] and SGM2.1 [pPYC], as well as NZ-041, SGM3.0 [pPYC] and SGM3.1 [pPYC] as the closest analogues of the induced strains of succinic acid producers.

It should be noted that directed genetic modifications leading to obtaining strains of E. coli of producers of succinic acid, capable of synthesizing the target substance with yields close to the corresponding theoretical values, can differ in their set. A rational choice of target genes, based only on the analysis of available information on the functioning of target biochemical pathways, cannot reliably determine success in improving the production of succinic acid 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. At the same time, the actually achieved indicators of the conversion rates of glucose to succinic acid for strains characterized by various maximum possible output values vary depending on the set of specific modifications introduced into the corresponding strains.

In the SBS550MG strain [pHL413], the genes aca, pta, ldhA, adhE, iclR, encoding acetate kinase, phosphotransacetylase, lactate dehydrogenase, aldehyde / alcohol dehydrogenase, and transcriptional repressor gene expressing coding for lactic enzyme pyruvate carboxylase. However, this strain contains an intact rohB gene encoding pyruvate oxidase, which competes with succinic acid biosynthesis for the metabolite precursor pyruvic acid. The conversion of pyruvic acid to Acetyl-CoA in strain SBS550MG [pHL413] is effected by the pyruvate lyase format, which determines the maximum possible conversion rate of glucose to succinic acid - 1.6 mol / mol (Cox SJ et al., Development of a metabolic network design and optimization framework incorporating implementation constraints: a succinate production case study. (Metab. Eng., 2006, 8: 46-57). In rich complex environments, the SBS550MG strain [pHL413] synthesizes succinic acid from glucose with a conversion factor of 1.59 mol / mol (Sánchez AM et al., Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity. Metab Eng. 2005, 7 (3): 229-239), 1.4 mol / mol (PCT / FR 2010/050230) and 1.25 mol / mol (Martínez I. et al., Metabolic impact of the level of aeration during cell growth on anaerobic succinate production by an engineered Escherichia coli strain. Metab Eng., 2010, 12: 499-509) depending on cultivation conditions. These conversion ratios were achieved with succinic acid production in LB medium containing a significant amount of amino acids. Under conditions of anaerobiosis, the oxidative deamination of amino acids, accompanied by the generation of NADH, can provide additional reduced glycolytic equivalents necessary for the formation of succinic acid in the reducing branch of the tricarboxylic acid cycle. When amino acids are exhausted in the medium, the strain loses an additional source of reduced equivalents for the effective biosynthesis of succinic acid. Indeed, when carrying out the process of anaerobic biosynthesis of succinic acid from glucose with the SBS550MG strain [pHL413] in a minimal salt medium with glucose, the conversion coefficient of the substrate into the target product is 1.3 mol / mol (WO 2009/083756).

In the strains SGM1.0 [pPYC] and SGM1.1 [pPYC], the genes acA, pta, poxB, ldhA, adhE, encoding acetate kinase, phosphotransacetylase, pyruvate oxidase, lactate dehydrogenase, aldehyde / alcohol dehydrogenase B, and coding pyruvate carboxylase. In the SGM1.1 [pPYC] strain, the iclR gene encoding a protein is also a transcriptional repressor of the expression of genes encoding glyoxylate shunt enzymes. The conversion of pyruvic acid to Acetyl-CoA in strains SGM1.0 [pPYC] and SGM1.1 [pPYC] is carried out under the action of the pyruvate lyase format, which determines the maximum possible conversion rate of glucose to succinic acid - 1.6 mol / mol. In a minimal salt medium, strains SGM1.0 [pPYC] and SGM1.1 [pPYC] synthesize succinic acid from glucose with conversion coefficients of 1.31 mol / mol and 1.32 mol / mol, respectively (RU 2528056).

In the SGM2.0 [pPYC] and SGM2.1 [pPYC] strains, the ackA, pta, poxB, ldhA, adhE genes encoding acetate kinase, phosphotransacetylase, pyruvate oxidase, lactate dehydrogenase, aldehyde / alcohol dehydrogenase, and aldehyde / alcohol dehydrogenase are inactivated. and lpdA encoding pyruvate dehydrogenase components, and the Bacillus subtilis Rus A gene encoding pyruvate carboxylase is expressed. In the SGM2.1 strain [pPYC], the iclR gene encoding a protein is also a transcriptional repressor of the expression of genes encoding glyoxylate shunt enzymes. The conversion of pyruvic acid into Acetyl-CoA in strains SGM2.0 [pPYC] and SGM2.1 [pPYC] is carried out under the action of both pyruvate lyase format and pyruvate dehydrogenase, which determines the maximum possible conversion rate of glucose to succinic acid - 1.6 ( 6) mol / mol. In a minimal salt medium, strains SGM2.0 [pPYC] and SGM2.1 [pPYC] synthesize succinic acid from glucose with conversion factors of 1.42 mol / mol and 1.49 mol / mol, respectively (RU 2528056).

In strain NZ-041, inactivated acA, pta, ldhA, pflB, ptsl genes encoding acetate kinase, phosphotransacetylase, lactate dehydrogenase, pyruvate format lyase and enzyme I phosphoenolpyruvate dependent sugar transport system, as well as increased expression of ACE, Ape, ace components of pyruvate dehydrogenase, the pck gene encoding phosphoenolpyruvate carboxykinase, and the galP gene encoding the galactose H ⁺ simporter. However, this strain contains intact genes poxB and adhE, encoding pyruvate oxidase and aldehyde / alcohol dehydrogenase, competing with succinic acid biosynthesis for the precursor metabolites pyruvic acid and acetyl-CoA, respectively. The conversion of pyruvic acid to Acetyl-CoA in strain NZ-041 is carried out under the action of pyruvate dehydrogenase, which determines the maximum possible conversion rate of glucose to succinic acid - 1.71 mol / mol. In a minimal salt medium, strain NZ-041 synthesizes succinic acid from glucose with a conversion coefficient of 1.31 mol / mol (Zhu X. et al. Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli. Metab Eng ., 2014, doi: 10.1016 / j.ymben.2014.05.003).

In the strains SGM3.0 [pPYC] and SGM3.1 [pPYC], the ackA, pta, poxB, ldhA, adhE, pflB genes encoding acetate kinase, phosphotransacetylase, pyruvate oxidase, lactate dehydrogenase, aldehyde / alcoholase dehydrogenase, dehydrogenase, dehydrogenase enhanced expression of ACEE, aceF and lpdA genes encoding pyruvate dehydrogenase components, and Bacillus subtilis RusA gene encoding pyruvate carboxylase was expressed. In the SGM3.1 strain [pPYC], the iclR gene encoding a protein is also a transcriptional repressor of the expression of genes encoding glyoxylate shunt enzymes. The conversion of pyruvic acid to Acetyl-CoA in strains SGM3.0 [pPYC] and SGM3.1 [pPYC] is carried out under the action of pyruvate dehydrogenase, which determines the maximum possible conversion rate of glucose to succinic acid - 1.71 mol / mol. In a minimal salt medium, the strains SGM3.0 [pPYC] and SGM3.1 [pPYC] synthesize succinic acid from glucose with conversion ratios of 1.46 mol / mol and 1.66 mol / mol, respectively (RU 2528056).

The objective of the invention is to expand the range of strains of bacteria Escherichia coli - producers of succinic acid, capable of synthesizing the target substance with increased efficiency, and methods for producing succinic acid using these strains.

The problem is solved by

- construction of strains of Escherichia coli - producers of succinic acid:

1. MSG1.0 [pPYC], which has the inactivated ackA, pta, poxB, ldhA, adhE, ptsG genes encoding acetate kinase, phosphotransacetylase, pyruvate oxidase, lactate dehydrogenase, aldehyde / alcohol dehydrogenase, and genomegase glucose, enhanced glucose, and glk, encoding the N ⁺ -simporter galactose and glucokinase, and pyruvate carboxylase activity;

2. MSG1.1 [pPYC], which has the inactivated ackA, pta, poxB, ldhA, adhE, ptsG, iclR genes encoding acetate kinase, phosphotransacetylase, pyruvate oxidase, lactate dehydrogenase, aldehyde / alcohol dehydrogenase, permerase transgenase, permease glucose repressase, permerase encoding glyoxylate shunt enzymes, respectively, by enhanced expression of the galP and glk genes encoding the galactose H ⁺ simporter and glucokinase, as well as pyruvate carboxylase activity;

3. MSG2.0 [pPYC], which has inactivated ackA, pta, poxB, ldhA, adhE, ptsG genes encoding acetate kinase, phosphotransacetylase, pyruvate oxidase, lactate dehydrogenase, aldehyde / alcohol dehydrogenase, and genomegase glucose, permease glucose, and glk encoding the H ⁺ -symptor of galactose and glucokinase, enhanced expression of the ACEE, aceF and lpdA genes encoding the components of pyruvate dehydrogenase, as well as the activity of pyruvate carboxylase;

4. MSG2.1 [pPYC], which has the inactivated genes of acA, pta, poxB, ldhA, adhE, ptsG, iclR, encoding acetate kinase, phosphotransacetylase, pyruvate oxidase, lactate dehydrogenase, aldehyde / alcohol dehydrogenase, permerase transgenase, permerase transgenase, permerase, dehydrogenase, permease encoding glyoxylate shunt enzymes, respectively, by enhanced expression of the galP and glk genes encoding the H ⁺ -symptor of galactose and glucokinase, enhanced expression of the aceEE, aceF and lpdA genes, encoding pyruvate dehydrogenase components, as well as pyruvate carboxylase activity;

5. MSG3.0 [pPYC], which has the inactivated genes akA, pta, pohB, ldhA, adhE, ptsG, pflB, encoding acetate kinase, phosphotransacetylase, pyruvate oxidase, lactate dehydrogenase, aldehyde / alcohol dehydrogenase, permease lyase, permease, permease, permease, respectively, enhanced expression of the galP and glk genes encoding the H ⁺ -symptor of galactose and glucokinase, enhanced expression of the aceeE, aceF and lpdA genes encoding the components of pyruvate dehydrogenase, as well as the activity of pyruvate carboxylase;

6. MSG3.1 [pPYC], which has the inactivated genes acA, pta, poxB, ldhA, adhE, ptsG, pflB, iclR, encoding acetate kinase, phosphotransacetylase, pyruvate oxidase, lactate dehydrogenase, aldehyde / alcohol glucose dehydrogenase, dehydrogenase, dehydrogenase, dehydrogenase lyase and transcriptional repressor genes encoding enzymes of the glyoxylate shunt, respectively, enhanced expression of genes galP and glk, encoding the N ⁺ -simporter galactose and glucokinase, enhanced expression of genes aseE, aceF and lpdA, coding for pyruvate dehydrogenase component and the activity of pyruvate carbo silazy;

- development of a method for producing succinic acid using the claimed strains.

The conversion of pyruvic acid to Acetyl-CoA is carried out in the claimed strains either under the influence of pyruvate format lyase, or under the influence of pyruvate format lyase and pyruvate dehydrogenase, or under the action of exclusively pyruvate dehydrogenase which determines the maximum possible conversion rates of glucose to succinic acid - 1.6 mol / mol, 1.6 (6) mol / mol, 1.71 mol / mol. The inventive strains are capable of anaerobically synthesizing succinic acid from glucose with conversion coefficients close to the maximum theoretically possible values.

The object of the present invention are bacterial strains of the species Escherichia coli, with the ability to efficiently convert glucose to succinic acid.

Also an object of the present invention is a bacterial strain Escherichia coli with inactivated genes acA, pta, poxB, ldhA, adhE, ptsG, enhanced expression of the galP and glk genes, and having pyruvate carboxylase activity - producing succinic acid with the highest possible conversion of glucose to succinic acid - 1.6 mol / mol;

Also an object of the present invention is a bacterial strain Escherichia coli with inactivated genes asA, pta, poxB, ldhA, adhE, ptsG, iclR, enhanced by the expression of galP and glk genes, and having pyruvate carboxylase activity - producing succinic acid with the highest possible glucose to succinic conversion rate acid - 1.6 mol / mol;

Also an object of the present invention is a bacterial strain Escherichia coli with inactivated genes of acA, pta, poxB, ldhA, adhE, ptsG, enhanced expression of the galP and glk genes, enhanced expression of the ACEE, ACE F and lpdA genes, and having pyruvate carboxylase activity, a producer of succinic acid with the highest possible conversion rate of glucose to succinic acid - 1.6 (6) mol / mol;

Also an object of the present invention is a bacterial strain Escherichia coli with inactivated genes askA, pta, poxB, ldhA, adhE, ptsG, iclR, enhanced expression of the galP and glk genes, enhanced expression of the ACEE, aceF and lpdA genes, and with the activity of amber pyruvate carboxylase acids with the highest possible conversion rate of glucose to succinic acid - 1.6 (6) mol / mol;

Also an object of the present invention is a bacterial strain Escherichia coli with inactivated genes askA, pta, poxB, ldhA, adhE, ptsG, pflB, enhanced expression of the galP and glk genes, enhanced expression of the ACEE, aceF and lpdA genes, and with the activity of amber pyruvate carboxylase acids with the highest possible conversion rate of glucose to succinic acid - 1.71 mol / mol;

Also an object of the present invention is a bacterial strain Escherichia coli with inactivated genes asA, pta, poxB, ldhA, adhE, ptsG, pflB, iclR, enhanced expression of the galP and glk genes, enhanced expression of the acee, aceF and lpdA genes, and having pyruvate carboxylase activity - succinic acid producer with the highest possible conversion rate of glucose to succinic acid - 1.71 mol / mol;

Also the subject of the present invention are Escherichia coli bacterial strains described above, wherein pyruvate carboxylase activity is provided by introducing into the bacterium a DNA molecule containing a gene encoding an enzyme having an activity classified as K.F. 6.4.1.1.

Also an object of the present invention are Escherichia coli bacterial strains described above, in which the expression of galP and glk genes is enhanced by replacing the nucleotide sequences of natural promoters controlling the expression of galP and glk genes on chromosomes with stronger promoters.

Also an object of the present invention are recombinant strains of Escherichia coli bacteria in which the expression of the ACEE, aceF and lpdA genes is enhanced by replacing the nucleotide sequence of the natural promoter controlling the expression of the aceEF-lpdA gene of the operon on the chromosome with a stronger promoter.

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

Another object of the present invention is the method described above, in which the process of culturing recombinant strains includes an aerobic stage of biomass accumulation and an anaerobic stage of production of succinic 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 “bacterial strain Escherichia coli with inactivated genes asA, pta, poxB, ldhA, adhE, ptsG means that the bacterium has been modified so that as a result of the modification such a bacterium contains a reduced amount of proteins AckA, Pta, PoxB, LdhA, AdhE, PtsG compared with an unmodified bacterium, or the specified bacterium is not able to synthesize AckA, Pta, PoxB, LdhA, AdhE, PtsG proteins.

The term "bacterial strain Escherichia coli with inactivated genes asA, pta, poxB, ldhA, adhE, ptsG, iclR" means that the bacterium has been modified so that as a result of the modification this bacterium contains a reduced amount of proteins AckA, Pta, PoxB, ldhA , AdhE, PtsG and lclR compared to an unmodified bacterium, or the indicated bacterium is not able to synthesize AckA, Pta, PoxB, LdhA, AdhE, PtsG and IclR proteins.

The term "bacterial strain Escherichia coli with inactivated genes asA, pta, poxB, ldhA, adhE, ptsG, pflB" means that the bacterium has been modified so that as a result of the modification this bacterium contains a reduced amount of proteins AckA, Pta, PoxB, LdhA , AdhE, PtsG and PflB compared with an unmodified bacterium, or the indicated bacterium is not able to synthesize AckA, Pta, PoxB, LdhA, AdhE, PtsG and PflB proteins.

The term "bacterial strain Escherichia coli with inactivated genes asA, pta, poxB, ldhA, adhE, ptsG, pflB, iclR" means that the bacterium has been modified so that as a result of the modification this bacterium contains a reduced amount of AckA, Pta, PoxB proteins , LdhA, AdhE, PtsG, PflB and IclR compared with an unmodified bacterium, or the specified bacterium is not able to synthesize AckA, Pta, PoxB, LdhA, AdhE, PtsG, PflB and IclR proteins.

The term "inactivation of the genes akA, pta, pohB, ldhA, adhE, ptsG" means that the natural expression of modified DNA regions is impossible due to deletions of these genes or their parts or modification of regions adjacent to genes that include sequences that control the expression of the corresponding genes, such as promoters, enhancers, attenuators, etc.

The term “iclR gene inactivation” means that natural expression of a modified DNA region is not possible due to deletions of a given gene or part of it or modification of regions adjacent to a gene that include sequences that control gene expression, such as promoters, enhancers, attenuators, etc. d.

The term "inactivation of the pflB gene" means that the natural expression of a modified portion of DNA is impossible due to deletions of a given gene or part of it or modification of regions adjacent to a gene that include sequences that control gene expression, such as promoters, enhancers, attenuators, etc. d.

The presence or absence of acA, pta, poxB, ldhA, adhE, ptsG, and iclR and / or pflB genes on the chromosome of a bacterium can be determined by known methods, including PCR, Southern blotting, 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-PAGE electrophoresis followed by PCR immunoblotting (Western blotting) and the like.

AskA gene (synonyms: ESK2290, b2296) encodes an AskA protein (synonym: B2296), which exhibits acetate kinase activity, classified as K.F. 2.7.2.1. The acA gene (nucleotides from 2,411,492 to 2,412,694 in the sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the open reading frame yfbV and the pta gene on the chromosome of E. coli K-12 strain. The nucleotide sequence of the ackA gene and the amino acid sequence of AckA encoded by the asKA gene are shown in the Sequence Listing Numbers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2), respectively. The pta gene (synonyms: ESK2291, b2297) encodes a Pta protein (synonym: B2297), which exhibits phosphotransacetylase activity, classified as K.F. 2.3.1.8. The pta 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 askA gene and the open yfcC reading frame on the chromosome of E. coli Κ12 strain. The nucleotide sequence of the pta gene and the amino acid sequence of Pta encoded by the pta gene are shown in the Sequence Listing under the numbers 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4), respectively. The poxB gene (synonyms: ESK0862, b0871) encodes the PohB protein (synonym: B0871), exhibiting 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 PohB encoded by the PohB gene are shown in the Sequence Listing under the numbers 5 (SEQ ID NO: 5) and 6 (SEQ ID NO: 6), respectively. The ldhA gene (synonyms: ESK1377, 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 LdhA encoded by the ldhA gene are shown in the Sequence Listing under the numbers 7 (SEQ ID NO: 7) and 8 (SEQ ID NO: 8), respectively. The adhE gene (synonyms: ESK1235, b1241) encodes an AdhE protein (synonym: B1241), which shows the activity of alcohol dehydrogenase and aldehyde dehydrogenase, classified as K.F. 1.1.1.1. and K.F. 1.2.1.3. The adhE 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 adhE gene and the amino acid sequence of AdhE encoded by the adhE gene are shown in the sequence Listing under the numbers 9 (SEQ ID NO: 9) and 10 (SEQ ID NO: 10), respectively. The ptsG gene (synonyms: ESK1087, 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 numbers 11 (SEQ ID NO: 11) and 12 (SEQ ID NO: 12), respectively. The iclR gene (synonyms: ESK4010, b4018) encodes the IclR protein (synonym: B4018), which is a transcriptional repressor of the aseBAC operon encoding glyoxylate shunt enzymes. The iclR gene (nucleotides complementary to nucleotides 4,220,827 to 4,221,651 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the arpA gene and metH gene on the chromosome of E. coli K-12 strain. The nucleotide sequence of the iclR gene and the amino acid sequence of the IclR encoded by the iclR gene are shown in the Sequence Listing Nos. 13 (SEQ ID NO: 13) and 14 (SEQ ID NO: 14), respectively. The pflΒ gene (synonyms: ESK0894, b0903) encodes a PflB protein (synonym: B0903), which exhibits 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 encoded by the pfl gene are shown in the Sequence Listing Nos. 15 (SEQ ID NO: 15) and 16 (SEQ ID NO: 16), respectively.

Since representatives of various strains of the Escherichia coli species may experience some variations in the nucleotide sequences, the concept of an inactivated gene is not limited to the 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 ), but may 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 encoding variants of the AckA, Pta, PoxB, LdhA, AdhE, PtsG, IclR, and PflB proteins. 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 deletion, 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 its type. 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 and SEQ ID NO: 16. 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 replacement 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 Gln, His or Lys, replacing Asn with Glu, Gln, Lys, His or Asp, replacing Asp with Asn, Glu or Gln, replacing Cys with Ser or Ala, replacing Gln with Asn, Glu, Lys, His, Asp or Arg, replacing Glu with Asn, Gln, Lys or Asp, replacing Gly with Pro, replacing His with Asn, Lys, Gln, Arg or Tyr, replacing Ile with Leu, Met, Val or Phe, replace Leu with Ile, Met, Val or Phe, replace Lys with Asn, Glu, Gln, His or Arg, replace Met with Ile, Leu, Val or Phe, replace Phe with Trp, Tyr, Met, Ile or Leu, replacing Ser with Thr or Ala, replacing Thr with Ser or Ala, replacing Trp with Phe or Tyr, replacing Tyr with His, Phe or Trp, and replacing Val with Met, Ile or Leu. The replacements, deletions, insertions, additions, permutations, etc. described above one or more amino acid residues include naturally occurring mutations (mutant or variant) depending on species differences or individual differences in microorganisms containing the genes asA, pta, poxB, ldhA, adhE, ptsG, iclR and pflB. 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 and SEQ ID NO: 15 using, for example, site-specific mutagenesis, such that the site-specific amino acid residue in the corresponding encoded protein includes substitutions, deletions, insertions or additions.

Therefore, variants of the proteins encoded by the genes akA, pta, poxB, ldhA, adhE, ptsG, iclR and pflB can have a homology of at least 80%, preferably at least 90%, and most preferably at least 95%, relative to the total amino acid the sequence shown in the List of sequences numbered 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, and SEQ ID NO: 16, respectively.

In this regard, the genes akA, pta, poxB, ldhA, adhE, ptsG, iclR and pflB can be variants that hybridize under stringent conditions with the nucleotide sequences shown in the Sequence Listing No. 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), or with a probe that can be synthesized based on the indicated nucleotide sequence. "Stringent conditions" include those conditions under which specific hybrids, for example hybrids with a homology of at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95 % are formed, and non-specific hybrids, 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, is 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.

The expression of the genes akA, pta, poxB, ldhA, adhE, ptsG, as well as iclR and / or pflB can be weakened by introducing a mutation into the corresponding gene on the chromosome, in which the intracellular activity of the protein encoded by the gene is reduced or absent compared to the 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., Biol. Chem., 1997, 272: 8611-8617; Kwon, DH et al., J. Antimicrob. Chemother., 2000, 46: 793-796). Expression of the genes akA, pta, poxB, ldhA, adhE, ptsG, as well as iclR and / or pflB can also be attenuated by modification of regulatory sequences, such as the promoter or Shine-Dalgarno (SD) sequence (PCT application WO 95/34672; Carrier, T.A. and Keasling, JD, Biotechnol Prog., 1999, 15: 58-64).

For example, 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 One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA, 2000, 97 (12): 6640-6645) or a method using a plasmid whose temperature-sensitive replication is (US patent 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 disruption using homologous recombination, and / or mutagenesis insertion deletion (Yu D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 5978-5983; Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97 (12): 6640-6645) 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 based on the gene sequence, in situ fluorescence hybridization (FISH), and the like. The level of gene expression can be determined by various known methods, including Northern blotting, quantitative RT-PCR, etc. In addition, strains that can be used as a control include, for example, Escherichia coli K-12.

The galP gene (synonyms: ECK2938, b2943) 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 reading frame yggI 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 under numbers 17 (SEQ ID NO: 17) and 18 (SEQ ID NO: 18), respectively. The glk gene (synonyms: ECK2384, 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 reading frame yfeO 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 Numbers 19 (SEQ ID NO: 19) and 20 (SEQ ID NO: 20), respectively.

Genes aseE, aceF and lpdA (synonyms: b0114, ESK0113; b0115, ESK0114; b0116, ESK0115) encode proteins AseE, AceF and LpdA - enzymatic complex components regenerate NAD ⁺ pyruvate dehydrogenase. The aceEF-lpdA genes of the operon (nucleotides homologous to nucleotides 123017 through 129336; in sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990) are located between the pdhR and ucH genes on the E. coli K-12 chromosome. The nucleotide sequences of the ACEE, aceF and lpdA genes and the amino acid sequences of the ACEE, AceF and LpdA proteins encoded by the ACEE, aceF and lpdA genes are shown in SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 22 , SEQ ID NO: 24, SEQ ID NO: 26, respectively.

The term “protein variant” is also applicable to the GalP and Glk proteins encoding glucokinase and the H ⁺ galactose simulator, and the AcEE, AceF, and LpdA proteins, which are components of the NAD ⁺ pyruvate dehydrogenase enzymatic complex. The term "protein variant" is interpreted in the same way as described above.

In this regard, the galP, glk genes and the ACEE, aceF and lpdA genes can also exist as variants that hybridize under stringent conditions with the nucleotide sequences shown in SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or with probes that can be synthesized based on the indicated nucleotide sequences, provided that they encode the functional proteins GalP, Glk and AcEE, AceF and LpdA. 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 target sequences present on the chromosome in multiple copies. Multiple copy sequences in chromosomal DNA include, but are not limited to, repetitive DNA or inverted repeats at the ends of transposed elements. It is also possible to incorporate the gene into the transposon and 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, 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 L. et al., Annu. Rev. Microbiol., 1981, 35: 365-403; Hui A. et al., EMBO J., 1984.3: 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.

The bacterial strains of Escherichia coli according to the present invention are also the strains described above, modified in such a way that they acquire pyruvate carboxylase activity. Pyruvate carboxylase activity can be achieved, in particular, by the presence in the bacterium of a DNA molecule containing a gene encoding pyruvate carboxylase.

Pyruvate carboxylase is an enzyme capable of catalyzing the carboxylation reaction of pyruvic acid to form oxalacetic acid: ATP + pyruvate + HCO ₃ ^- = ADP + phosphate + oxaloacetate. The indicated activity is classified as K.F. 6.4.1.1. The presence of pyruvate carboxylase activity can be ascertained, for example, using the method described by Peters-Wendisch PG et al. (Pyruvate carboxylase from Corynebacterium glutamicum: characterization, expression and inactivation of the rus gene. Microbiology, 1998, 144: 915-927). An example of an enzyme having pyruvate carboxylase activity is RusA protein from Bacillus subtilis (sequence with accession number NP_389369.1 in the GenBank database, gi: 16078550) encoded by the RusA gene (nucleotides 1554185 to 1557631 in sequence with accession number NC_000964.3 in the GenBank database, gi: 255767013). The nucleotide sequence of the RusA gene and the amino acid sequence of the RusA protein encoded by it are shown in SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

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

In this regard, the rusA gene can also exist in the form of variants that hybridize under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 27, or with a probe that can be synthesized based on the specified nucleotide sequence, provided that they encode functional protein RusA. The term "stringent conditions" is interpreted in the same way as described above.

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

The Escherichia coli bacterial strains of the present invention are the strains described above, modified in such a way that they acquire pyruvate carboxylase activity by introducing into the bacterium a DNA molecule containing the gene encoding the pyruvate carboxylase in the plasmid vector.

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

A method of obtaining succinic acid in General.

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

According to the claimed method, the cultivation, isolation and purification of succinic acid or its salt from a culture or similar liquid is carried out in a manner similar to traditional fermentation methods in which succinic 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 introduced into the medium by means of 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. Usually, culturing for 1 to 5 days leads to the accumulation of succinic 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 succinic acid or its salt can be isolated and purified by electrodialysis, ion exchange chromatography, reverse phase chromatography, concentration and / or crystallization.

Example 1. Construction of strains of bacteria Escherichia coli.

The following strains and plasmids were designed and used -

The wild-type strain Escherichia coli K-12 MG1655 (VKPM B-6195) was used as the base for the construction of all recombinant strains. The plasmid pPYC containing the Rus A gene encoding the functionally active pyruvate carboxylase from Bacillus subtilis is known (RU 2466186).

1.1. Construction of the strain MSG0.1.

The strain MSG0.1 (E. coli MG1655 ΔackA, Δpta, ΔroxB, ΔldhA, ΔadhE, ΔptsG) was obtained on the basis of strain E. coli MG1655 as a result of inactivation of the genes asA, pta, roxB, ldhA, adhE and ptsG by recombinant on a method using λRed-dependent homologous recombination. The principle of the method is described in detail, for example, in (Sawitzke J. et al., Recombineering: In Vivo Genetic Engineering in E. coli, S. enterica, and Beyond. Methods in Enzymology., 2007, 421: 171-199), and examples its 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 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.

First, DNA fragments were obtained for inactivation of the genes akA, pta, poxB, ldhA, adhE and ptsG.

Linear DNA fragments for deletions of the target genes were obtained by PCR using primer pairs P1 (SEQ ID NO: 29) and P2 (SEQ ID NO: 30), P5 (SEQ ID NO: 31) and P6 (SEQ ID NO: 32) , P9 (SEQ ID NO: 33) and P10 (SEQ ID NO: 34), P13 (SEQ ID NO: 35) and P14 (SEQ ID NO: 36), P17 (SEQ ID NO: 37) and P18 (SEQ ID NO: 38) and plasmids pMW118-attL-Cm-attR (Katashkina J.I. et al., Directional change in the level of gene expression in the bacterial chromosome. Mol. Biol. 2005, 39 (5): 823-831) as matrices.

Primers P1, P5, P9, P13, and P17 contain 36 nucleotide DNA regions homologous to the DNA regions located at the 5'-ends of the acA, poxB, ldhA, adhE, and ptsG genes, and 28 nucleotide DNA regions complementary to the DNA region, located at the 3'-end of the attR region. Primers P2, P6, P10, P14, and P18 contain 36 nucleotide DNA regions complementary to the DNA regions located at the 3'-ends of the pta, poxB, ldhA, adhE and ptsG genes, as well as 28 nucleotide DNA regions homologous to the DNA region located at the 5'-end of the attL. 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 for inactivation of the genes akA and pta, poxB, ldhA, adhE and ptsG, 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 (the cat gene encoding chloramphenicolacetyltransferase) flanked by the attL and attR 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., Wanner BL One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA, 2000, 97 (12): 6640-6645) contains a phage DNA fragment λ 2154 bp long (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 (genes γ, β, exo) under the control of the promoter P _ar-B induced by arabinose. 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 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: 39) and P4 (SEQ ID NO: 40), P7 (SEQ ID NO: 41) and P8 (SEQ ID NO: 42), P11 (SEQ ID NO: 43) and P12 (SEQ ID NO: 44), P15 (SEQ ID NO: 45) and P16 (SEQ ID NO: 46), P19 (SEQ ID NO: 47) and P20 (SEQ ID NO: 48), for the ackA-pta operon and the poxB, ldhA, adhE, and ptsG 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 3351 bp for the intact operon ackA-pta, 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact adhE 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 ackA-pta, 1852 bp for inactivated RohB gene, 2008 bp for the inactivated ldhA gene, 1926 bp for the inactivated adhE gene and 1759 bp for the inactivated ptsG gene.

Based on selected individual Cm ^R Ap ^S colony of strains with the cat gene replaced by the ackA-pta operon and the poxB, ldhA, adhE and ptsG genes according to the standard method (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989), preparations of the corresponding P1 transducing phages were obtained.

Strain Ε. coli with deleted akA, pta genes was obtained by transferring a chromosome fragment to the chromosome of E. coli strain MG1655, with the ackA-pta operon replaced by the cat gene, flanked by attachment sites of the lambda phage, using a transducing phage preparation that carries the targeted marked genetic modification. For this, E. coli strain 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 transducing phage 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 ackA-pta operon genes with the cat gene, flanked attachment sites of the lambda phage, PCR analysis using locus-specific primers P3 (SEQ ID NO: 39) and P4 (SEQ ID NO: 40). 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 ackA-pta operon. 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 ackA-pta operon 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 cat gene was removed from transformed clones by growing individual strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removal of the cat gene from transductant chromosomes and deletion of the ackA-pta operon genes in them was confirmed by PCR using the corresponding 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 ackA-pta operon. The length of the PCR products obtained by the reaction using the ackA-pta operon strain as a matrix of cells was 323 bp. As a result of subsequent curing of the clones from the pMWts-Int / Xis helper plasmid, re-screened to of individual colonies on plates with LB medium and selection of Cm ^S Ap ^S variants, E. coli strain MG1655 (ΔackA, Δpta) with deleted akA, pta genes was obtained.

The E. coli strain with deleted akA, pta, and poxB genes was obtained by transferring a chromosome fragment to the chromosome of E. coli strain MG1655 (ΔackA-pta) with the poxB gene replaced by the cat gene flanked by attachment sites of the lambda phage using a preparation of a transducing phage carrying 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 cat gene, flanked attachment sites of the lambda phage, PCR analysis using locus-specific primers P7 (SEQ ID NO: 41) and P8 (SEQ ID NO: 42) 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 (ΔackA, Δpta) 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 cat gene, flanked by attachment sites of the phage lambda. 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 cat gene was removed from transformed clones by growing individual strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removal of the cat gene from the transductant chromosomes and deletion of the ackA-pta operon and roxB genes in them was confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 39), P4 (SEQ ID NO: 40) and P7 (SEQ ID NO: 41), P8 (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. 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 ackA-pta operon and 1872 bp for the intact rohB gene. The length of the corresponding PCR products obtained as a result of the reaction using the ackA-pta operon strain and the poxB gene as a matrix of cells 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 (ΔackA, Δpta, ΔroxB) with the ackA-pta and roxB genes deleted.

The E. coli strain with deleted genes askA, pta, poxB and ldhA was obtained by transferring the chromosome fragment to the chromosome of E. coli strain MG1655 (ΔackA, Δpta, ΔpoxB), with the ldhA gene replaced by the cat gene flanked by attachment sites using phage lambda a transducing phage carrying the targeted 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 ldhA gene was replaced by the cat gene, flanked by the phage lambda sites, by PCR analysis using locus-specific primers Ρ11 (SEQ ID NO: 43) and P12 (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. 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 (ΔackA-pta, 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 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 cat gene was removed from transformed clones by growing individual strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removal of the cat gene from transductant chromosomes and deletion of the ackA-pta operon gene, the poxB gene, and the ldhA gene in them was confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 39) and P4 (SEQ ID NO: 40), P7 (SEQ ID NO: 41) and P8 (SEQ ID NO: 42), P11 (SEQ ID NO: 43) and P12 (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 ackA-pta, 1872 bp for the intact RohB gene and 1299 bp for the intact ldhA gene. The length of the corresponding PCR products obtained as a result of the reaction using the ackA-pta operon strain, the poxB gene and the ldhA gene as a cell matrix was 323 bp, 255 bp and 411 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 ( ΔackA, Δpta, ΔroxB, ΔldhA) with the deleted genes acA, pta, poxB and ldhA.

The strain E. coli with deleted genes aslA, pta, poxB, ldhA and adhE was obtained by transferring the chromosome fragment to the chromosome of E. coli strain MG1655 (ΔackA, Δpta, ΔroxB, ΔldhA) with the adhE gene replaced by the cat gene, flanked attachment site using a phage-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 shape, the replacement of the adhE gene with the cat gene, flanked attachment sites of the lambda phage, PCR analysis using locus-specific primers P15 (SEQ ID NO: 45) and PI6 (SEQ ID NO: 46) 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. Taq DNA polymerase, the corresponding buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania) were used. The length of PCR products resulting from reactions using the parental strain MG1655 (ΔackA, Δpta, ΔpoxB, ΔldhA) as a matrix of cells was 2903 bp for the intact adhE gene. 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 adhE 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 cat gene was removed from transformed clones by growing individual strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removal of the cat gene from the transductant chromosomes and the division of the ackA-pta operon, roxB gene, ldhA gene, and adhE gene into them was confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 39) and P4 (SEQ ID NO: 40 ), P7 (SEQ ID NO: 41) and P8 (SEQ ID NO: 42), P11 (SEQ ID NO: 43) and P12 (SEQ ID NO: 44), P15 (SEQ ID NO: 45) and P16 (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 ackA-pta, 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene and 2903 bp for the intact adhE gene. The length of the corresponding PCR products obtained by the reaction using the ackA-pta operon strain, the poxB gene, the ldhA gene and the adhE gene as a cell matrix 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 ( ΔackA, Δpta, ΔpoxB, ΔldhA, ΔadhE) with inactivated genes akA, pta, pohB, ldhA and adhE.

Strain Ε. coli with delegated genes ackA-pta, poxB, ldhA, adhE and ptsG was obtained by transferring the E. coli strain MG1655 (ΔackA, Δpta, ΔroxB, ΔldhA, ΔadhE) chromosome fragment to the chromosome, with the ptsG gene replaced by the at lambda, 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. On the chromosomes of the clones that form the fastest growing colonies of the correct shape, the replacement of the ptsG gene with the cat gene, flanked attachment sites of the lambda phage, PCR analysis using locus-specific primers P19 (SEQ ID NO: 47) and P20 (SEQ ID NO: 48) 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 (ΔackA, Δpta, ΔroxB, ΔldhA, ΔadhE) 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 cat gene was removed from transformed clones by growing individual strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removal of the cat gene from the transductant chromosomes and deletion of the ackA-pta operon gene, poxB gene, ldhA gene, adhE gene and ptsG gene in them was confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 39) and P4 (SEQ ID NO: 40), P7 (SEQ ID NO: 41) and P8 (SEQ ID NO: 42), P11 (SEQ ID NO: 43) and P12 (SEQ ID NO: 44), P15 (SEQ ID NO: 45) and P16 (SEQ ID NO: 46), P19 (SEQ ID NO: 47) and P20 (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 ackA-pta, 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact adhE gene and 1494 bp for the intact ptsG gene. The length of the corresponding PCR products obtained as a result of the reaction using the ackA-pta operon strain, the poxB gene, the ldhA gene, the adhE gene, and the ptsG gene as a cell matrix was 323 bp, 255 bp, 411 bp n., 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 ( ΔackA, Δpta, ΔpohB, ΔldhA, ΔadhE, ΔptsG) with inactivated genes askA, pta, pohB, ldhA, adhE and ptsG, named MSG0.1.

1.2. Construction of the strain MSG0.2.

Strain MSG0.2 (E. coli MG1655 ΔaskA, Δpta, ΔrohV, ΔldhA, ΔadhE, ΔptsG, P tac -galP) obtained based on the strain MSG0.1 (E. coli MG1655 ΔaskA, Δpta, ΔrohV, ΔldhA, ΔadhE, 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 expression of the galP gene, a truncated variant 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 carrying the marked target chromosome modification, the preparation of the corresponding Ρ1-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 .

First, a DNA fragment was 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. At the first stage, PCR was used to obtain a DNA fragment containing the BglII restriction endonuclease recognition site, the _Ptac promoter, 18 nucleotides complementary to the region of the preceding coding part of the galP gene, and 18 nucleotides complementary to the 5'-terminal region of the coding part of the gal R gene. PCR was performed. using primers P21 (SEQ ID NO: 49) and P22 (SEQ ID NO: 50) and plasmid pDR540 (sequence with accession number U13847 in the GenBank database; gi: 595699) as a matrix. Primer P21 contains a BglII recognition site and a region homologous to the 5'-end of the promoter P _tac . Primer P22 contains 18 nucleotides complementary to the region of the preceding coding part of the galP gene, 18 nucleotides complementary to the 5 ′ end region of the coding part of the galP 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. The DNA fragment containing the Cm ^R marker encoded by the cat gene was obtained by PCR using primers P23 (SEQ ID NO: 51) and P24 (SEQ ID NO: 52) and plasmid pMW118-attL-Cm-attR (Katashkina J.I. . et al., Directional change in the level of gene expression in the bacterial chromosome, Mol. Biol., 2005, 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 attR 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 attL 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 EF, Sambrook, J .: Molecular Cloning: A Laboratory Manual. 2 ^nd edn. Cold Spring Harbor , NY: Cold Spring Harbor Laboratory Press, 1989).

The ligation product was amplified by PCR using primers P22 (SEQ ID NO: 50) and P23 (SEQ ID NO: 51). 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 (cat gene encoding chloramphenicol acetyl transferase) flanked by the attL and attR sites of the lambda phage, as well as 36 flank homology regions with a DNA region preceding the galP gene coding region and a DNA region containing 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 E. coli strain MG1655 containing plasmid pKD46 (Datsenko K.A., Wanner BL One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA, 2000 97 (12): 6640-6645).

Electrocompetent cells of E. coli strain MG1655 containing plasmid pKD46 were obtained as described above. Electroporation was performed using 70 μl of cells and ~ 300 ng of 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 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: 53) and P26 (SEQ ID NO: 54). 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 galP 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 galP 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 galP gene was confirmed by sequencing.

Based on the selected Cm ^R Ap ^S variant of the strain in which the native regulatory region of the galP gene is replaced by the P _tac promoter, conjugated to the cat 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 genes acA, pta, poxB, ldhA, adhE, ptsG, and enhanced expression of the galP gene was obtained by transferring the strain MSG0.1 (E. coli MG1655 ΔackA, Δpta, ΔroxB, ΔldhA, ΔadhE, to the chromosome) a fragment of the chromosome in which the native regulatory region of the galP gene is replaced by the P _tac promoter, 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. The chromosomes clones forming most rapidly growing colonies of the correct form, replacing the native regulatory region of the gene galP promoter P _tac, conjugate with the gene cat, flanked attechment sites of lambda phage, was confirmed by PCR analysis using locus specific primers P25 (SEQ ID NO: 53) and P26 (SEQ ID NO: 54). 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 regulatory region for galP, the replaced gene promoter P _tac, conjugate with the gene cat, flanked attechment sites of 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 cat gene was removed from transformed clones by growing individual strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removal of the cat gene from transductant chromosomes and deletion of the ackA-pta operon gene, the poxB gene, the ldhA gene, the adhE gene, and the ptsG gene in them, as well as the presence of the P _tac promoter in front of the galP gene, was confirmed by PCR using the corresponding locus-specific pairs primers P3 (SEQ ID NO: 39) and P4 (SEQ ID NO: 40), P7 (SEQ ID NO: 41) and P8 (SEQ ID NO: 42), P11 (SEQ ID NO: 43) and P12 (SEQ ID NO: 44), P15 (SEQ ID NO: 45) and P16 (SEQ ID NO: 46), P19 (SEQ ID NO: 47) and P20 (SEQ ID NO: 48), P25 (SEQ ID NO: 53) and P26 (SEQ ID NO: 54). 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 ackA-pta, 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact adhE gene, 1494 bp for the intact ptsG gene and 317 bp for the intact regulatory region of the galP gene. The length of the corresponding PCR products obtained as a result of the reaction using the ackA-pta operon strain, the poxB gene, the ldhA gene, the adhE gene, and the ptsG gene and containing the P _tac promoter in front of the 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 ( ΔackA, Δpta, ΔroxB, ΔldhA, ΔadhE, ΔptsG, P _tac -galP) with inactivated genes askA, pta, poxΒ, ldhA, adhE and ptsG, and enhanced expression of the galP gene, called MSG0.2.

1.3. Construction of the strain MSG1.0.

The strain MSG1.0 (E. coli MG1655 ΔackA, Δpta, ΔroxB, ΔldhA, ΔadhE, ΔptsG, P _tac -galP, P _L -glk) was obtained on the basis of the strain MSG0.2 (Ε. Coli MG1655 ΔackA, Δpta, Δhax, Δhax , ΔadhE, ΔptsG, P _tac -galP) as a result of replacing the native regulatory region of the glk gene in it with the promoter P _L phage lambda.

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 .

First, a DNA fragment was 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: 55) and P28 (SEQ ID NO: 56) and the lambda phage genomic DNA (sequence with accession number NC_001416.1 in the GenBank database, gi: 9626243) as a template. Primer P27 contains a BglII recognition site and a region homologous to the 5'-end of the P _L promoter. Primer P28 contains region complementary to the 35 preceding the coding portion glk gene and a region complementary to the 3'-end of the promoter P _L nucleotides. 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 cat gene was obtained by PCR using primers P29 (SEQ ID NO: 57) and P24 (SEQ ID NO: 52) and plasmid pMW118-attL-Cm-attR (Katashkina J.I. . et al., Directional change in the level of gene expression in the bacterial chromosome, Mol. Biol., 2005, 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 attR 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 attL 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 EF, Sambrook, J .: Molecular Cloning: A Laboratory Manual. 2 ^nd edn. Cold Spring Harbor , NY: Cold Spring Harbor Laboratory Press, 1989).

The ligation product was amplified by PCR using primers P28 (SEQ ID NO: 56) and P29 (SEQ ID NO: 57). 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 (cat gene encoding chloramphenicol acetyltransferase) flanked by the attL and attR sites of the lambda phage, as well as the flank homology region with the DNA region preceding the 135 coding region of the glk gene and the DNA region itself 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 strain E, coli MG1655 containing plasmid pKD46 (Datsenko K.A., Wanner BL One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA, 2000 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 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: 58) and P31 (SEQ ID NO: 59). 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 Cm ^R Ap ^S variant of the strain in which the native regulatory region of the glk gene was replaced by the P _L phage lambda promoter, coupled to the cat gene, flanked by attachment sites of the lambda phage, a preparation of the corresponding Ρ1-transducing phage was obtained.

The E. coli strain with deleted genes askA, pta, poxB, ldhA, adhE, ptsG, and enhanced expression of the galP and glk genes was obtained by transferring the strain MSG0.2 to the chromosome. (E. coli MG1655 ΔackA, Δpta, ΔroxB, ΔldhA, ΔadhE, ΔptsG, P _tac -galP) of the chromosome fragment in which the native regulatory region of the glk gene is replaced by the P _L lambda phage promoter, conjugated to the cat gene, flanked by the attachment 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 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 cat gene, flanked by attachment sites of the lambda phage, was confirmed by PCR analysis using locus-specific primers P30 (SEQ ID NO: 58 ) and P31 (SEQ ID NO: 59). 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 cat 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 cat gene was removed from transformed clones by growing individual strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removal of the cat gene from transductant chromosomes and deletion of the ackA-pta operon gene, poxB gene, ldhA gene, adhE gene, and ptsG gene in them, as well as the presence of the P _tac promoter in front of the galP gene and the P _L lambda phage promoter in front of the glk gene PCR was confirmed using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 39) and P4 (SEQ ID NO: 40), P7 (SEQ ID NO: 41) and P8 (SEQ ID NO: 42), P11 (SEQ ID NO: 43) and P12 (SEQ ID NO: 44), P15 (SEQ ID NO: 45) and P16 (SEQ ID NO: 46), P19 (SEQ ID NO: 47) and P20 (SEQ ID NO: 48), P25 (SEQ ID NO: 53) and P26 (SEQ ID NO: 54), P30 (SEQ ID NO: 58) and P31 (SEQ ID NO: 59). 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 ackA-pta, 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact adhE 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 corresponding PCR products obtained as a result of the reaction using the ackA-pta operon strain, the poxB gene, the ldhA gene, the adhE gene, the ptsG gene and containing the P _tac promoter in front of the galP gene and the P _L promoter in front of the gene glk was 323 bp, 255 bp, 411 bp, 329 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 ( ΔaskA, Δpta, ΔrohV, ΔldhA, ΔadhE, ΔptsG, P tac -galP, P L -glk) inactivated genes askA, pta, rohV, ldhA, adhE, ptsG, and enhanced expression of genes galP and glk, named MSG1.0.

1.4. Construction of the strain MSG1.1.

The strain MSG1.1 (E. coli MG1655 ΔackA, Δpta, ΔroxB, ΔldhA, ΔadhE, ΔptsG, ΔiclR, P _tac- galP, P _L -glk) was obtained on the basis of strain MSG1.0 (Ε. Coli MG1655 ΔackA, Δpta, Δpt , ΔldhA, ΔadhE, ΔptsG, P _tac -galP, P _L -glk) as a result of inactivation of the iclR gene in it.

Initially, the target chromosome modification was obtained individually using strain E. coli MG1655 as a recipient. 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 introduced into the chromosome of the target recombinant strain. After transduction, the antibiotic resistance marker flanked by attachment to lambda phage sites was removed from the chromosome of the obtained strain using the plasmid pMWts-Int / Xis (WO 2007 013638; RU 2005 123423), including the Int / Xis genes of the dependent site-specific phage recombination system lambda.

A DNA fragment was first obtained to inactivate the iclR gene.

A linear DNA fragment for dividing the target gene was obtained by PCR using primers P32 (SEQ ID NO: 60) and P33 (SEQ ID NO: 61) and plasmid pMW118-attL-Cm-attR (Katashkina J.I. et al., Directional change in the level of gene expression in the bacterial chromosome (Mol. Biol. 2005, 39 (5): 823-831) as a matrix.

Primer P32 contains a 36 nucleotide DNA region homologous to the DNA region located at the 5 ′ end of the iclR gene and a 28 nucleotide DNA region complementary to the DNA region located at the 3 ′ end of the attR region. Primer P33 contains a 36-nucleotide DNA region complementary to the DNA region located at the 3 ′ end of the iclR gene, as well as a 28 nucleotide DNA region homologous to the DNA region located at the 5 ′ end of the attL 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).

The resulting PCR product with a length of 1700 bp to inactivate the iclR gene, they were purified by agarose gel electrophoresis and isolated, according to the manufacturer's recommendations, using the Qiagen QIAquick Gel Extraction Kit (Qiagen, USA). The resulting fragment contains the chloramphenicol resistance gene (the cat gene encoding chloramphenicolacetyltransferase) flanked by the attL and attR sites of the lambda phage, as well as 36 linking regions of flank homology with coding regions of the target 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., Wanner BL One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA, 2000 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 L plates. 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 putative and predicted chromosome structures corresponded to the selected Cm ^R Ap ^S colonies was confirmed by PCR analysis using locus-specific primers P34 (SEQ ID NO: 62) and P35 (SEQ ID NO: 63). 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 product obtained by reactions using the parental strain MG1655 as a matrix of cells was 946 bp for the intact iclR gene. The length of the PCR product obtained by the reaction using a mutant strain as a matrix of cells was 1781 bp for the inactivated iclR gene.

Based on the selected Cm ^R Ap ^S strain variant with the cat gene replaced by the iclR gene, a preparation of the corresponding Ρ1-transducing phage was obtained.

The E. coli strain with deleted genes acA, pta, poxB, ldhA, adhE, ptsG, iclR, and enhanced expression of the galP and glk genes was obtained by transferring to the chromosome of strain MSG 1.0 (MG. Coli MG1655 ΔackA, Δpta, ΔroxB, ΔhdA, E _{, ΔptsG, P tac -galP, P} L -glk) chromosome fragment with the gene iclR, gene replaced cat, flanked attechment sites of lambda phage, using the preparation transducing phage carrying the labeled target 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. On the chromosomes of the clones that form the fastest growing colonies of the correct shape, the iclR gene was replaced by the cat gene, flanked by the phage lambda sites, by PCR analysis using locus-specific primers P34 (SEQ ID NO: 62) and P35 (SEQ ID NO: 63). 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 946 bp for the intact iclR gene. The length of the PCR products obtained as a result of the reaction using the obtained transductants as a matrix of cells was 1781 bp for the iclR 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. The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The cat gene was removed from transformed clones by growing individual strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Deleting gene cat chromosome transductants and deletion in these genes operon ackA-pta, rohV gene, ldhA gene, adhE gene, ptsG gene and iclR gene as well as the fact of promoter P _tac, before genome galP, and a promoter P _L of phage lambda PCR was confirmed before the glk gene using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 39) and P4 (SEQ ID NO: 40), P7 (SEQ ID NO: 41) and P8 (SEQ ID NO: 42), P11 (SEQ ID NO: 43) and P12 (SEQ ID NO: 44), P15 (SEQ ID NO: 45) and P16 (SEQ ID NO: 46), P19 (SEQ ID NO: 47) and P20 (SEQ ID NO: 48), P34 (SEQ ID NO: 62) and P35 (SEQ ID NO: 63), P25 (SEQ ID NO: 53) and P26 (SEQ ID NO: 54), P30 (SEQ ID NO: 58) and P31 ( SEQ ID NO: 59). 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 ackA-pta, 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact adhE gene, 1494 bp for the intact ptsG gene, 946 bp for the intact iclR 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 by the reaction using the ackA-pta operon strain, roxB gene, ldhA gene, adhE gene, ptsG gene, iclR gene and containing the P _tac promoter in front of the galP gene and the P _L phage promoter as a cell matrix the lambda in front of the glk gene was 323 bp, 255 bp, 411 bp, 329 bp, 162 bp, 184 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 ( ΔackA, Δpta, ΔpohB, ΔldhA, ΔadhE, ΔptsG, ΔiclR, P _tac -galP, P _L -glk) with inactivated acA, pta, pohB, ldhA, adhE, ptsG, iclR genes, and enhanced gene expression gal, and galP MSG1.1.

1.5. Construction of the strain MSG2.0.

The strain MSG2.0 (E. coli MG1655 ΔackA, Δpta, ΔroxB, ΔldhA, ΔadhE, ΔptsG, P _tac -galP, P _L- glk, P _L -aceEF-lpdA) was obtained on the basis of strain MSG 1.0 (Ε. Coli MG1655 ΔackA , Δpta, ΔpoxB, ΔldhA, ΔadhE, ΔptsG, P _tac -galP, P _L -glk) by replacing the native regulatory region of the aceEF-lpdA operon in it with the P _L phage lambda promoter.

Initially, the target chromosome modification was obtained individually using strain E. coli MG1655 as a recipient. To enhance the expression of the ACEE, aceF, and IpdA constituting the aceEF-lpdA operon, the PL promoter of the lambda phage linked to the naturally occurring Shine-Dalgarno (SD sequence) ACEE gene was integrated upstream of the coding region of the ACEE gene, which is the first in the corresponding operon, in chromosome of the recipient strain. Based on the obtained strain bearing the marked target chromosome modification, a preparation of the corresponding Ρ1-transducing phage was obtained. Next, P1 transduction, the resulting modification was introduced into the chromosome of the target recombinant 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 first obtained to replace the native regulatory region of the aceEF-lpdA operon

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 P _L, SD sequence of the gene aseE and finally 36 nucleotides complementary to the 5'-end of the coding region of the gene aseE. The fragment was obtained in two stages. At the first stage, using the lambda phage genomic DNA as a template (sequence with accession number NC_001416.1 in the GenBank database, gi: 9626243), a DNA fragment containing the BglII recognition site, the P _L promoter, and the ACeE gene SD sequence was obtained at the beginning . PCR was performed using primers P27 (SEQ ID NO: 55) and P36 (SEQ ID NO: 64). Primer P27 contains a BglII recognition site and a region homologous to the 5'-end of the P _L promoter. Primer P36 contains the ACEE gene SD sequence 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).

The resulting PCR product, purified on an agarose gel and isolated according to the manufacturer's recommendations using the Qiagen QIAquick Gel Extraction Kit, was used as a template in the next round of PCR. Primers P27 (SEQ ID NO: 55) and P37 (SEQ ID NO: 65) were used. Primer P37 contains a region complementary to the 3 ′ end of the P _L promoter, an ACEE gene SD sequence, and 36 nucleotides from the ACEE gene reading frame. Temperature profile for PCR: denaturation at 95 ° C for 5 min; subsequent 25 cycles: 30 sec at 95 ° C, 30 sec at 53 ° C, 60 sec at 72 ° C; and final polymerization: 7 min at 72 ° C. Used Pfu DNA polymerase, the appropriate buffer and deoxynucleoside triphosphates manufactured by Fermentas (Lithuania).

In parallel, the second stage of constructing the DNA fragment was carried out. A DNA fragment containing the Cm ^R marker encoded by the cat gene was obtained by PCR using primers P38 (SEQ ID NO: 66) and P24 (SEQ ID NO: 52) and plasmid pMW118-attL-Cm-attR (Katashkina J.I. . et al., Directional change in the level of gene expression in the bacterial chromosome, Mol. Biol., 2005, 39 (5): 823-831) as a matrix. Primer P38 contains 36 nucleotides homologous to the DNA region preceding the directly coding region of the ACEE gene and a DNA region of 28 nucleotides in size complementary to the DNA region located at the 3'-end of the attR 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 attL 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 EF, Sambrook, J .: Molecular Cloning: A Laboratory Manual. 2 ^nd edn. Cold Spring Harbor , NY: Cold Spring Harbor Laboratory Press, 1989).

The ligation product was amplified by PCR using primers P37 (SEQ ID NO: 65) and P38 (SEQ ID NO: 66). 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 1970 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 promoter linked to the natural Shine-Dalgarno sequence (SD sequence) of the ACEE gene, the chloramphenicol resistance gene (cat gene encoding chloramphenicol acetyl transferase) flanked by the attL and attR sites of the lambda phage, as well as 36-unit regions of flank homology with the DNA region immediately preceding the coding region of the ACEE gene and the 5'-region of the coding region of the ACEE 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.Α., Wanner BL One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA, 2000 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 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 pairs of locus-specific primers P39 (SEQ ID NO: 67) and P40 (SEQ ID NO: 68), P27 (SEQ ID NO: 55) and P40 (SEQ ID NO: 68). The length of the PCR product obtained by the reaction using the parental strain MG1655 and primers P39 and P40 as a matrix of cells was 201 bp for the intact regulatory region of the aceEF-lpdA operon. The length of the PCR product obtained by the reaction using the mutant strain and primers P39 and P40 as a matrix of cells was 2099 bp. for the modified regulatory region of the aceEF-lpdA operon. When using a pair of primers P27 and P40 and the chromosome of the parent strain as a template, specific fragments were not observed among PCR products due to the absence of the wild-type promoter P _L lambda phage in the chromosome of E. coli strain. The length of the PCR product obtained by the reaction using the mutant strain and primers P27 and P40 as a matrix of cells was 377 bp. 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 ACEE gene was confirmed by sequencing.

Based on the selected Cm ^R Ap ^S variant strain in which the native regulatory region of the aceEF-lpdA operon is replaced by the P _L promoter linked to the ACEE SD gene sequence and linked to the cat 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 genes askA, pta, poxB, ldhA, adhE, ptsG, enhanced expression of the galP and glk genes, and enhanced expression of the acee, aceF, and lpdA genes was obtained by transfer to the chromosome of strain MSG1.0 (E. coli MG1655 ΔackA, Δpta, ΔroxB, ΔldhA, ΔadhE, ΔptsG, P _tac -galP, P _L -glk) of the chromosome fragment in which the native regulatory region of the aceEF-lpdA operon is replaced by the P _L promoter linked to the ACeE gene SD sequence and linked to the cat gene, flanks attachment to lambda phage sites 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 native regulatory region of the aceEF-lpdA operon by an artificial regulatory element conjugated to the cat gene, flanked by attachment sites of the lambda phage, was confirmed by PCR analysis using locus-specific primers P39 (SEQ ID NO: 67 ) and P40 (SEQ ID NO: 68). 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 MSG 1.0 as a matrix of cells was 201 bp. for the intact regulatory region of the aceEF-lpdA operon. The length of PCR products resulting from the reaction using the obtained transductants as a matrix of cells was 2099 bp. for the regulatory region of the aceEF-lpdA operon, replaced by an artificial regulatory element conjugated to 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. The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The cat gene was removed from transformed clones by growing individual strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removing the cat gene from the transductant chromosomes and deleting the ackA-pta operon gene, the poxB gene, the ldhA gene, the adhE gene, the ptsG gene, and the presence of the P _tac promoter in front of the galP gene, the P _L lambda phage promoter in front of the glk gene, and promoter P _L phage lambda in front of the aceEF-lpdA operon genes was confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 39) and P4 (SEQ ID NO: 40), P7 (SEQ ID NO: 41) and P8 ( SEQ ID NO: 42), P11 (SEQ ID NO: 43) and P12 (SEQ ID NO: 44), P15 (SEQ ID NO: 45) and P16 (SEQ ID NO: 46), P19 (SEQ ID NO: 47 ) and P20 (SEQ ID NO: 48), P25 (SEQ ID NO: 53) and P26 (SEQ ID NO: 54), P30 (SEQ ID NO: 58) and P31 (SEQ ID NO: 59), P39 (SEQ ID NO: 67) and P40 (SEQ ID NO: 68). 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 ackA-pta, 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact adhE gene, 1494 bp for the intact ptsG gene, 317 bp for the intact regulatory region of the galP gene, 234 bp for the intact regulatory region of the glk gene and 201 bp for the intact regulatory region of the aceEF-lpdA operon. The length of the corresponding PCR products obtained as a result of the reaction using the ackA-pta operon strain, the poxB gene, the ldhA gene, the adhE gene, the ptsG gene, and containing the P _tac promoter in front of the galP gene, the P _L promoter of the lambda phage the glk genome and the P _L promoter of the lambda phage in front of the aceEF-lpdA operon genes were 323 bp, 255 bp, 411 bp, 329 bp, 162 bp, 248 bp , 418 bp and 502 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 ( ΔackA, Δpta, ApoxB, ΔldhA, ΔadhE, ΔptsG, P _tac -galP, P _L -glk, P _L -aceEF-lpdA) with inactivated genes akA, pta, pohB, ldhA, adhE, ptsG, enhanced by the expression of galP and glk genes , and enhanced expression of the ACEE, aceF, and lpdA genes, named MSG2.0.

1.6. Construction of the strain MSG2.1.

The strain MSG2.1 (E. coli MG1655 ΔackA, Δpta, ΔpoxB, ΔldhA, ΔadhE, ΔptsG, ΔiclR, P _tac -galP, P _L- glk, P _L -aceEF-lpdA) was obtained on the basis of strain MSG2.0 (E. coli MG1655 ΔackA, Δpta, ΔpoxB, ΔldhA, ΔadhE, ΔptsG, P _tac -galP, P _L- glk, P _L -aceEF-lpdA) as a result of inactivation of the iclR gene in it.

The E. coli strain with deleted genes aca, pta, poxB, ldhA, adhE, ptsG, iclR, enhanced expression of the galP and glk genes, and enhanced expression of the aceE, aceF and IpdA genes was obtained by transfer of the MSG2.0 strain (E. coli MG1655 to the chromosome ΔackA, Δpta, ΔpoxB, ΔldhA, ΔadhE, ΔptsG, P _tac -galP, P _L -glk, P _L -aceEF-lpdA) fragment of the chromosome, with the iclR gene replaced by the cat gene, flanked by attachment sites of the lambda phage using the preparation transducing phage obtained previously (Example 1.4). P1 transduction was carried out 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. On the chromosomes of the clones that form the fastest growing colonies of the correct shape, the iclR gene was replaced by the cat gene, flanked by the phage lambda sites, by PCR analysis using locus-specific primers P34 (SEQ ID NO: 62) and P35 (SEQ ID NO: 63). 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 MSG2.0 as a matrix of cells was 946 bp for the intact iclR gene. The length of the PCR products obtained as a result of the reaction using the obtained transductants as a matrix of cells was 1781 bp for the iclR 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. The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The cat gene was removed from transformed clones by growing individual strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removing the cat gene from the transductant chromosomes and dividing the ackA-pta operon, roxB gene, ldhA gene, adhE gene, ptsG gene, and iclR gene in them, as well as the presence of the P _tac promoter, in front of the galP gene, the P _L lambda phage promoter the glk gene and the P _L lambda phage promoter in front of the aceEF-lpdA operon genes were confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 39) and P4 (SEQ ID NO: 40), P7 (SEQ ID NO: 41) and P8 (SEQ ID NO: 42), P11 (SEQ ID NO: 43) and P12 (SEQ ID NO: 44), P15 (SEQ ID NO: 45) and P16 (SEQ ID NO: 46), P19 (SEQ ID NO: 47) and P20 (SEQ ID NO: 48), P34 (SEQ ID NO: 62) and P35 (SEQ ID NO: 63), P25 (SEQ ID NO: 53) and P26 (SEQ ID NO: 54), P30 (SEQ ID NO: 58) and P31 (SEQ ID NO: 59), P39 (SEQ ID NO: 67) and P40 (SEQ ID NO: 68). 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 ackA-pta, 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact adhE gene, 1494 bp for the intact ptsG gene, 946 bp for the intact iclR gene, 317 bp for the intact regulatory region of the galP gene, 234 bp for the intact regulatory region of the glk gene and 201 bp for the intact regulatory region of the aceEF-lpdA operon. The length of the corresponding PCR products obtained as a result of the reaction using the ackA-pta operon strain, roxB gene, ldhA gene, adhE gene, ptsG gene, iclR gene and containing the P _tac promoter in front of the galP gene, P _L phage promoter, as a cell matrix the lambda in front of the glk gene and the promoter P _L phage of the lambda in front of the aceEF-lpdA operon genes was 323 bp, 255 bp, 411 bp, 329 bp, 162 bp, 184 bp n., 248 bp, 418 bp and 502 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 (ΔackA, Δpta, ΔroxB, ΔldhA , ΔadhE, ΔptsG, ΔiclR, P _tac -galP, P _L- glk, P _L -aceEF-lpdA) with inactivated genes akA, pta, pohV, ldhA, adhE, ptsG, iclR, enhanced by the expression of galP and glk genes, and enhanced expression of ACEE, aceF and lpdA genes, named MSG2.1.

1.7. Construction of the strain MSG3.0.

The strain MSG3.0 (Ε. Coli MG1655 ΔackA, Δpta, ΔroxB, ΔldhA, ΔadhE, ΔptsG, ΔpflB, P _tac -galP, P _L -glk, P _L -aceEF-lpdA) was obtained on the basis of strain MSG2.0 (Ε. coli MG1655 ΔackA, Δpta, ΔroxB, ΔldhA, ΔadhE, ΔptsG, P _tac -galP, P _L -glk, P _L -aceEF-lpdA) as a result of inactivation of the pflB gene in it.

Initially, the target chromosome modification was obtained individually using strain E. coli MG1655 as a recipient. 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 recombinant 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 first obtained to inactivate the pflB gene.

A linear DNA fragment for deletion of the target gene was obtained by PCR using primers P41 (SEQ ID NO: 69) and P42 (SEQ ID NO: 70) and plasmid pMW118-attL-Cm-attR (Katashkina J.I. et al., Directional change in the level of gene expression in the bacterial chromosome (Mol. Biol. 2005, 39 (5): 823-831) as a matrix.

Primer P41 contains a 36 nucleotide DNA region homologous to the DNA region located at the 5 ′ end of the pflB gene and a 28 nucleotide DNA region complementary to the DNA region located at the 3 ′ end of the attR region. Primer P42 contains a 36 nucleotide DNA region complementary to the DNA region located at the 3 ′ end of the pflB gene, and a 28 nucleotide DNA region homologous to the DNA region located at the 5 ′ end of the attL 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).

The resulting PCR product with a length of 1700 bp to inactivate the pflB gene, it was purified by agarose gel electrophoresis and isolated, according to the manufacturer's recommendations, using the Qiagen QIAquick Gel Extraction Kit (Qiagen, USA). The resulting fragment contained the chloramphenicol resistance gene (the cat gene encoding chloramphenicolacetyltransferase) flanked by the attL and attR sites of the lambda phage, as well as 36-link flank homology regions with coding regions of the target 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., Wanner BL One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA, 2000 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 L plates. 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 corresponded to the selected Cm ^R Ap ^S colonies was confirmed by PCR analysis using locus-specific primers P43 (SEQ ID NO: 71) and P44 (SEQ ID NO: 72). 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 product obtained by reactions using the parental strain MG1655 as a matrix of cells was 2566 bp for the intact pflB gene. The length of the PCR product obtained by the reaction using a mutant strain as a matrix of cells was 1983 bp. for the inactivated iclR gene.

Based on the selected Cm ^R Ap ^S variant of the strain with the cat gene replaced by the pflB gene, a preparation of the corresponding P1 transducing phage was obtained.

The E. coli strain with delegated genes acA, pta, poxB, ldhA, adhE, ptsG, pflB, enhanced expression of the galP and glk genes, and enhanced expression of the aceE, aceF and lpdA genes was obtained by transfer of the MSG2.0 strain to the chromosome (E. coli MG1655 ΔackA, Δpta, ΔpohB, ΔldhA, ΔadhE, ΔptsG, P _tac -galP, Ρ _L- glk, P _L -aceEF-lpdA) of the chromosome fragment, with the pflB gene replaced by the cat gene, flanked by the lambda phage sites using the lambda phage with phage carrying the targeted 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. On the chromosomes of the clones that form the fastest growing colonies of the correct shape, the replacement of the pflB gene with the cat gene, flanked attachment sites of the lambda phage, PCR analysis using locus-specific primers P43 (SEQ ID NO: 71) and P44 (SEQ ID NO: 72) 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 MSG2.0 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 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 cat gene was removed from transformed clones by growing individual strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removal of the cat gene from transductant chromosomes and deletion of the ackA-pta operon gene, poxB gene, ldhA gene, adhE gene, ptsG gene, and pflB gene in them, as well as the presence of the P _tac promoter in front of the galP gene, P _L lambda phage promoter before the glk gene and the P _L lambda phage promoter in front of the aceEF-lpdA operon genes were confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 39) and P4 (SEQ ID NO: 40), P7 (SEQ ID NO: 41) and P8 (SEQ ID NO: 42), P11 (SEQ ID NO: 43) and P12 (SEQ ID NO: 44), P15 (SEQ ID NO: 45) and P16 (SEQ ID NO: 46), P19 (SEQ ID NO: 47) and P20 (SEQ ID NO: 48), P43 (SEQ ID NO: 71) and P44 (SEQ ID NO: 72), P25 (SEQ ID NO: 53) and P26 (SEQ ID NO: 54), P30 (SEQ ID NO: 58) and P31 (SEQ ID NO: 59), P39 (SEQ ID NO: 67) and P40 (SEQ ID NO: 68). 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 ackA-pta, 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact adhE gene, 1494 bp for the intact ptsG gene, 2566 bp for the intact pflB gene, 317 bp for the intact regulatory region of the galP gene, 234 bp for the intact regulatory region of the glk gene and 201 bp for the intact regulatory region of the aceEF-lpdA operon. The length of the corresponding PCR products obtained as a result of the reaction using the ackA-pta operon strain, roxB gene, ldhA gene, adhE gene, ptsG gene, pflB gene and containing the P _tac promoter in front of the galP gene, P _L phage promoter, as a cell matrix the lambda in front of the glk gene and the promoter P _L phage of the lambda in front of the aceEF-lpdA operon genes was 323 bp, 255 bp, 411 bp, 329 bp, 162 bp, 386 bp n., 248 bp, 418 bp and 502 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 ( ΔackA, Δpta, ΔrohB, ΔldhA, ΔadhE, ΔptsG, ΔpflB, P _tac -galP, P _L -glk, P _L -aceEF-lpdA) with inactivated genes askA, pta, pohB, ldhA, adhE, ptsG, galP and glk genes, and enhanced expression of the ACEE, aceF, and lpdA genes, called MSG3.0.

1.8. Construction of the strain MSG3.1.

Strain MSG3.1 (E. coli MG1655 ΔackA, Δpta, ΔroxB, ΔldhA, ΔadhE, ΔptsG, ΔpflB, ΔiclR, P _tac -galP, P _L -glk, P _L -aceEF-lpdA) was obtained on the basis of strain MSG3.0 ( Col. Coli MG1655 ΔackA, Δpta, ΔroxB, ΔldhA, ΔadhE, ΔptsG, ΔpflB, P _tac -galP, P _L -glk, P _L -aceEF-lpdA) as a result of inactivation of the iclR gene in it.

The E. coli strain with deleted genes askA, pta, poxB, ldhA, adhE, ptsG, pflB, iclR, enhanced expression of the galP and glk genes, and enhanced expression of the acee, aceF and lpdA genes was obtained by transferring the strain MSG3.0 to the chromosome (E. coli MG1655 ΔackA, Δpta, ΔroxB, ΔldhA, ΔadhE, ΔptsG, ΔpflB, P _tac -galP, P _L -glk, P _L -aceEF-lpdA) chromosome fragment, with iclR gene replaced by cat gene, flanked attachment site using the preparation of the corresponding transducing phage obtained previously (Example 1.4). P1 transduction was carried out 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. On the chromosomes of the clones that form the fastest growing colonies of the correct shape, the iclR gene was replaced by the cat gene, flanked by the phage lambda sites, by PCR analysis using locus-specific primers P34 (SEQ ID NO: 62) and P35 (SEQ ID NO: 63). 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 MSG3.0 as a matrix of cells was 946 bp for the intact iclR gene. The length of the PCR products obtained as a result of the reaction using the obtained transductants as a matrix of cells was 1781 bp for the iclR 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. The selection of transformed clones was performed using LB medium containing ampicillin (100 μg / ml). The cat gene was removed from transformed clones by growing individual strain colonies on plates with LB medium with selection of chloramphenicol-sensitive and ampicillin-resistant Cm ^S Ap ^R variants. Removal of the cat gene from transductant chromosomes and deletion of the ackA-pta operon gene, poxB gene, ldhA gene, adhE gene, ptsG gene, pflΒ gene, and iclR gene, as well as the presence of the P _tac promoter, in front of the galP gene, P _L promoter lambda phage in front of the glk gene and the P _L promoter phage lambda in front of the aceEF-lpdA operon genes were confirmed by PCR using the corresponding pairs of locus-specific primers P3 (SEQ ID NO: 39) and P4 (SEQ ID NO: 40), P7 (SEQ ID NO : 41) and P8 (SEQ ID NO: 42), P11 (SEQ ID NO: 43) and P12 (SEQ ID NO: 44), P15 (SEQ ID NO: 45) and P16 (SEQ ID NO: 46), P19 (SEQ ID NO: 47) and P20 (SEQ ID NO: 48), P43 (SEQ ID NO: 71) and P44 (SEQ ID NO: 72), P34 (SEQ ID NO: 62) and P35 (SEQ ID NO: 63), P25 (SEQ ID NO: 53) and P26 (SEQ ID NO: 54), P30 (SEQ ID NO: 58) and P31 (SEQ ID NO: 59), P39 (SEQ ID NO: 67) and P40 (SEQ ID NO: 68). 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 ackA-pta, 1872 bp for the intact RohB gene, 1299 bp for the intact ldhA gene, 2903 bp for the intact adhE gene, 1494 bp for the intact ptsG gene, 2566 bp for the intact pflB gene, 946 bp for the intact iclR gene, 317 bp for the intact regulatory region of the galP gene, 234 bp for the intact regulatory region of the glk gene and 201 bp for the intact regulatory region of the aceEF-lpdA operon. The length of the corresponding PCR products obtained as a result of the reaction using the ackA-pta operon strain, roxB gene, ldhA gene, adhE gene, ptsG gene, pflB gene, iclR gene and containing the P _tac promoter in front of the galP gene, the promoter The P _L phage lambda in front of the glk gene and the promoter P _L phage lambda in front of the aceEF-lpdA operon genes was 323 bp, 255 bp, 411 bp, 329 bp, 162 bp, 386 bp, 184 bp, 248 bp, 418 bp and 502 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 ( ΔackA, Δpta, ΔpoxB, ΔldhA, ΔadhE, ΔptsG, ΔpflB, ΔiclR, P _tac -galP, P _L- glk, P _L -aceEF-lpdA) with inactivated genes as AkA, pta, pokhB, ldhA, adhE, adhF, iclR, enhanced expression of the galP and glk genes, and enhanced expression of the acEE genes, aceFn lpdA, named MSG3.1.

1.9. Obtaining strains MSG1.0 [pPYC], MSG1.1 [pPYC], MSG2.0 [pPYC], MSG2.1 [pPYC], MSG3.0 [pPYC], MSG3.1 [pPYC].

Bacterial strains Escherichia coli MSG1.0 [pPYC], MSG1.1 [pPYC], MSG2.0 [pPYC], MSG2.1 [pPYC], MSG3.0 [pPYC], MSG3.1 [pPYC] - succinic acid producers - obtained by transformation of strains MSG1.0, MSG1.1, MSG2.0, MSG2.1, MSG3.0, MSG3.1 with the pPYC plasmid containing the RusA gene encoding the functionally active pyruvate carboxylase from Bacillus subtilis (RU 2466186), according to standard technique (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989).

Example 2. Production of succinic acid by the claimed strains of bacteria Escherichia coli.

2.1. Characterization of the conversion rates of glucose to succinic acid by the claimed bacterial strains of Escherichia coli.

Cells of strain MSG1.0 [pPYC] or MSG1.1 [pPYC] or MSG2.0 [pPYC] or MSG2.1 [pPYC] or MSG3.0 [pPYC] or MSG3.1 [pPYC] are grown overnight in M9 medium (Sambrook, J., Fritsch, EF, and Maniatis, T., "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) containing 2 g / l glucose and 1 g / l tryptone, 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 7 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 salt medium containing 5 g / l glucose and 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. To maintain the pPYC plasmid, all media contain an additional 100 mg / L ampicillin. 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 organic acid concentrations, a Rezex ROA-Organic Acid H + ion-exclusion column (8%) (300 × 7.8 mm, 8 μm, Phenomenex, USA) was used. Detection is carried out at a wavelength of 210 nm. As the mobile phase, an aqueous solution of sulfuric acid (2.5 mmol) is used with a flow rate of 0.5 ml / min. To measure glucose concentration, the Waters HPLC system is equipped with a Waters 2414 refractory detector and a Spherisorb-NH2 column (4.6 × 250 mm, 5 μm, Waters, USA). A mixture of acetonitrile / ethyl acetate / water (volume ratio 76/4/20) with a flow rate of 1 ml / min is used as the mobile phase. Substances are identified by comparing their retention times with the retention times of their respective standards.

The molar concentrations of glucose consumed by the strains, accumulated metabolites and molar yields of succinic acid as a result of the corresponding in vitro fermentation are presented in Table 1.

The conversion rates of glucose to succinic acid demonstrated by the claimed bacterial strains of Escherichia coli MSG 1.0 [pPYC] and MSG 1.1 [pPYC] are 1.56 mol / mol and 1.59 mol / mol and are superior to those of the strains of the closest analogues SBS550MG [pHL413], SGM1 .0 [pPYC] and SGM1.1 [pPYC] (1.3 mol / mol, 1.31 mol / mol and 1.32 mol / mol), reaching 97% and 99% of the corresponding theoretical maximum of -1.6 mol / mol.

The conversion rates of glucose to succinic acid demonstrated by the claimed bacterial strains of Escherichia coli MSG2.0 [pPYC] and MSG2.1 [pPYC] are 1.64 mol / mol and 1.65 mol / mol and are superior to similar strains of the closest analogs of SGM2.0 [ pPYC] and SGM2.1 [pPYC] (1.42 mol / mol and 1.49 mol / mol), reaching 98% and 99% of the corresponding theoretical maximum - 1.6 (6) mol / mol.

The conversion rate of glucose to succinic acid demonstrated by the claimed bacterial strains of Escherichia coli MSG3.0 [pPYC] and MSG3.1 [pPYC] is 1.7 mol / mol and exceeds the similar rates of strains of the closest analogues NZ-041, SGM3.0 [pPYC] and SGM3.1 [pPYC] (1.31 mol / mol, 1.46 mol / mol and 1.66 mol / mol), reaching 99% of the corresponding theoretical maximum of 1.71 mol / mol.

2.2. Characterization of succinic acid accumulation by the claimed bacterial strains of Escherichia coli MSG3.0 [pPYC] and MSG3.1 [pPYC].

Cells of strain MSG3.0 [pPYC] or MSG3.1 [pPYC] are grown overnight in M9 medium (Sambrook, J., Fritsch, EF, and Maniatis, T., "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) containing 2 g / l glucose and 1 g / l tryptone at 37 ° C. 5 ml of the resulting overnight cultures are diluted 10 times, 45 ml of M9 medium containing 10 g / l glucose and 4 ml of a solution of wheat gluten neutralized acid hydrolyzate to pH 7.0 (Cargill, Efremovo, Russia) are added with dissolved solids 115 g / l The resulting cultures were grown in 750 ml flasks closed with cotton plugs at 37 ° C on a rotary shaker at 250 rpm for 7 hours. Then, cell suspensions are seeded in a fermentation apparatus (1L Microbial BioBundle System, Applikon Biotechnology, Netherlands) containing 450 ml of neutralized combination medium (3 g / L KH ₂ PO ₄ , 6.8 g / L K ₂ HPO ₄ × 3H ₂ O ; 1 g / l NaCl; 5 g / l (NH ₄ ) ₂ HPO ₄ ; 0.25 g / l MgSO ₄ × 7H ₂ O; 0.01 g / l CaCl ₂ × 2H ₂ O; 5 mg / l thiamine and 40 ml / l of wheat gluten acid hydrolyzate, 100 mg / l of ampicillin, 2 g / l of glucose). Aerobic biomass accumulation is carried out at 37 ° C for 7 hours at a stirrer speed of 800 rpm and an air flow of 0.6 l / l / min. An anaerobic productive stage is initiated by adding 100 ml of a 50% glucose solution to a final concentration of 83.3 (3) g / l, reducing the stirrer speed to 250 rpm and replacing the incoming gas with CO ₂ at a flow rate of 0.5 l / min. During the anaerobic phase, the pH of the medium is maintained at a value of 7.0 by automatically adding 40% NaOH solution (total volume ~ 110 ml). The concentration of succinic acid in the culture fluid is determined by high performance liquid chromatography as described above (Example 2.1).

Representative concentrations of succinic acid accumulated as a result of the corresponding fermentations using Escherichia coli bacterial strains MSG3.0 [pPYC] and MSG3.1 [pPYC] are presented in Table 2.

Thus, the claimed strains of bacteria Eshcerichia coli are highly effective and promising for use in industry by producers, allowing to obtain succinic acid from glucose with high conversion ratios, close to the maximum possible values.

Claims (19)

1. The bacterium Escherichia coli is a succinic acid producer modified in such a way that the genes ackA, pta, pohB, ldhA, adhE, ptsG are inactivated in it, the expression of the galP and glk genes is enhanced, and the bacterium has pyruvate carboxylase activity. 2. The bacterium according to claim 1, characterized in that the activity of pyruvate carboxylase is ensured by introducing into the bacterium a DNA molecule containing a gene encoding an enzyme having an activity classified as K.F. 6.4.1.1. 3. The bacterium 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. 4. The bacterium Escherichia coli is a succinic acid producer modified in such a way that the genes ackA, pta, poxB, IdhA, adhE, ptsG, iclR are inactivated in it, the expression of the galP and glk genes is enhanced, and the bacterium has pyruvate carboxylase activity. 5. The bacterium according to claim 4, characterized in that the activity of pyruvate carboxylase is provided by introducing into the bacterium a DNA molecule containing a gene encoding an enzyme having an activity classified as K.F. 6.4.1.1. 6. The bacterium according to claim 4, 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. 7. The bacterium Escherichia coli is a producer of succinic acid, modified in such a way that the genes ACA, pta, poXB, ldhA, adhE, ptsG are inactivated in it, the expression of the galP and glk genes is enhanced, the expression of the ACEE, aceF and lpdA genes is enhanced, and the bacterium possesses pyruvate carboxylase activity. 8. The bacterium according to claim 7, characterized in that the activity of pyruvate carboxylase is ensured by introducing into the bacterium a DNA molecule containing a gene encoding an enzyme having an activity classified as K.F. 6.4.1.1. 9. The bacterium according to claim 7, characterized in that the expression of the galP and glk genes, as well as the ACEE, aceF and lpdA genes, is enhanced by replacing the bacterial chromosome of the nucleotide sequences of natural promoters that control the expression of galP and glk genes, as well as aceEF operon genes -lpdA, to stronger promoters. 10. The bacterium Escherichia coli is a succinic acid producer modified in such a way that the ackA, pta, poxB, ldhA, adhE, ptsG, iclR genes are inactivated, expression of the galP and glk genes is enhanced, expression of the aceE, aceF and lpdA genes is enhanced, and the bacterium has pyruvate carboxylase activity. 11. The bacterium according to claim 10, characterized in that the activity of pyruvate carboxylase is ensured by introducing into the bacterial cells a DNA molecule containing a gene encoding an enzyme having an activity classified as K.F. 6.4.1.1. 12. The bacterium according to claim 10, characterized in that the expression of the galP and glk genes, as well as the ACEE, aceF and lpdA genes, is enhanced by replacing the bacterial chromosome of the nucleotide sequences of natural promoters that control the expression of galP and glk genes, as well as aceEF operon genes -lpdA, to stronger promoters. 13. The bacterium Escherichia coli is a succinic acid producer modified in such a way that the ackA, pta, poxB, ldhA, adhE, ptsG, pflB genes are inactivated, the expression of the galP and glk genes is enhanced, the expression of the aceE, aceF and lpdA genes is enhanced, and the bacterium has pyruvate carboxylase activity. 14. The bacterium according to claim 13, characterized in that the activity of pyruvate carboxylase is ensured by introducing into the bacterium a DNA molecule containing a gene encoding an enzyme having an activity classified as K.F. 6.4.1.1. 15. The bacterium according to claim 13, characterized in that the expression of the galP and glk genes, as well as the ACEE, aceF and lpdA genes, is enhanced by replacing the bacterial chromosome of the nucleotide sequences of natural promoters that control the expression of galP and glk genes, as well as aceEF operon genes -lpdA, to stronger promoters. 16. The bacterium Escherichia coli is a producer of succinic acid, modified in such a way that the genes ackA, pta, poxB, ldhA, adhE, ptsG, pflB, iclR are inactivated in it, the expression of the galP and glk genes is enhanced, the expression of the ACEE, aceF and lpdA genes is enhanced , and the bacterium has pyruvate carboxylase activity. 17. The bacterium according to claim 16, characterized in that the activity of pyruvate carboxylase is provided by introducing into the bacterium a DNA molecule containing a gene encoding an enzyme having an activity classified as K.F. 6.4.1.1. 18. The bacterium according to claim 16, characterized in that the expression of the galP and glk genes, as well as the ACEE, aceF and lpdA genes, is enhanced by replacing the bacterial chromosome of the nucleotide sequences of natural promoters that control the expression of galP and glk genes, as well as aceEF operon genes -lpdA, to stronger promoters. 19. A method for producing succinic acid by culturing a bacterium according to claim 1, or 4, or 7, or 10, or 13, or 16 in a nutrient medium and isolating succinic acid from the culture fluid.

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