RU2288264C2 - Bacterium belonging to escherichia genus as producer of l-threonine and method for preparing l-threonine - Google Patents

Abstract

FIELD: biotechnology, amino acids, microbiology.

SUBSTANCE: invention represents a method for preparing amino acid L-threonine by using bacterium belonging to Escherichia genus. This bacterium shows ability for production of L-threonine and modified by so manner that expression of gene chosen from the group glk, pgi, pfkA, tpiA, gapA, pgk, eno and pykA encoding glycogenolysis enzyme is enhanced. The claimed invention provides preparing L-threonine with the high degree of effectiveness.

EFFECT: improved preparing method.

7 cl, 1 dwg, 12 tbl, 9 ex

Description

Technical field

The present invention relates to biotechnology, specifically to a method for producing L-amino acids by fermentation, and more specifically to genes derived from Escherichia coli bacteria. The use of these genes increases the production of L-amino acids, for example, the production of L-threonine.

Brief Description of the Related Art

Traditionally, L-amino acids on an industrial scale can be obtained by fermentation using strains of microorganisms obtained from natural sources, or their mutants, specially modified in order to increase the production of L-amino acids.

Many methods have been described to increase the production of L-amino acids, for example, by transforming a microorganism of recombinant DNA (see, for example, US patent 4,278,765). These methods are based on increasing the activity of enzymes involved in the biosynthesis of amino acids and / or reducing the sensitivity of the target enzyme to reverse inhibition produced by the L-amino acid or by-products of the reaction (see, for example, US patents 4,346,170, 5,661,012 and 6,040,160).

Various strains are known to be used for the production of L-threonine by fermentation. These strains with increased activity of enzymes involved in the biosynthesis of L-threonine (US patents 5,175,107; 5,661,012; 5,705,371; 5,939,307; European patent 219027), strains resistant to certain chemicals, such as L-threonine and its analogues (PCT application WO 0114525 A1, European patent application 301572 A2, US patent 5,376,538), strains in which the sensitivity of the target enzyme to inhibition by the produced amino acid or its by-products is eliminated by feedback type (US patents 5,175,107 and 5,661,012), strains with inactivated enzymes of the degeneration system radiations of threonine (US patents 5,939,307 and 6,297,031).

The strain VKPM B-3996 (US patents 5,175,107 and 5,705,371) is by far the best known producer strain of threonine. To create a strain VKPM B-3996, several mutations and the plasmid described below were introduced into the parent strain E. coli K-12 (VKPM B-7). The mutant thrA gene (thrA442 mutation) encodes an aspartokinase homoserine dehydrogenase I protein, which is resistant to feedback threonine inhibition. The mutant UvA gene (ilvA442 mutation) encodes a threonine deaminase protein that has a reduced activity, which is expressed in a reduced level of isoleucine biosynthesis and in the leaky-type isoleucine deficient phenotype. In bacteria with the ilvA442 mutation, transcription of the thrABC operon is not repressed by isoleucine, which is very effective in the production of threonine. Inactivation of the tdh gene prevents the degradation of threonine. The genetic determinant of sucrose assimilation (scrKYABR genes) was introduced into this strain. To increase the expression of genes that control threonine biosynthesis, plasmid pVIC40 containing the thrA442BC mutant threonine operon was introduced into the intermediate strain TDH6. The amount of threonine accumulated during the fermentation of this strain reached 85 g / l.

Previously, the authors of the present invention obtained, starting from E. coli K 12, a mutant containing a thrR mutation (referred to hereinafter as rhtA23), which confers resistance to high concentrations of threonine and homoserine in a minimal nutrient medium (Astaurova, OB et al., Appl. Bioch. And Microbiol., 21, 611-616 (1985)). Phenotypically, a mutation is expressed in an increase in the production of L-threonine (USSR patent No. 974817), homoserine, and glutamate (Astaurova, OB et al., Appl. Bioch. And Microbiol., 27, 556-561, 1991, European patent application 1013765A) corresponding E. coli producer strain, such as VKPM-3996 strain. Moreover, the authors of the present invention showed that the rhtA gene is located at the 18th minute of the E. coli chromosome, next to the glnHPQ operon encoding the components of the glutamine transport system, and that the rhtA gene is identical to the open reading frame of ORF1 (ybiF gene, nucleotide numbers 764 to 1651 in sequence with accession number AAA2218541 in GenBank, gi: 440181), located between the genes pXB and oprX. The region expressing the protein encoded by ORF1 was called the rhtA gene (rht: resistance to homoserine and threonine - resistance to homoserin and threonine).

In addition, the authors of the present invention found that the rhtA23 mutation is a substitution of A for G position-1 relative to the start of the ATG codon (ABSTRACTS of 17 ^th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457; European Patent Application 1013765).

Under conditions when the main pathway for threonine biosynthesis has been studied and largely optimized, further improvement of the producer strain of threonine can be comprehended by increasing the efficiency of central metabolic pathways, such as glycolysis (Embden-Meyerhof pathway), which provides the cell with energy and various metabolic precursors.

The following enzymes participate in the glycolysis pathway: glucokinase (EC 2.7.1.2), encoded by the glk gene, phosphoglucose isomerase (EC 5.3.1.9), encoded by the pgi gene, phosphofructokinase-1 (fructose-6-P-1-kinase) (EC 2.7. 1.11), encoded by the pfkA gene, fructose-1,6-bisphosphataldolase (EC 4.1.2.13), encoded by the fbaA gene, triose phosphate isomerase (EC 5.3.1.1), encoded by the tpiA gene, glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), encoded by the gapA gene, phosphoglycerate kinase (EC 2.7.2.3) encoded by the pgk gene, phosphoglycerometase (EC 2.7.5.3) encoded by the gpmA gene, enolase (EC 4.2.1.11) encoded by the eno gene and and oenzimy pyruvate kinase (EC 2.7.1.40), encoded by the genes pykA and pykF (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F.C.Neidhardt, ASM Press, Washington D.C., 1996).

A process has been published for the production of L-lysine or other food additives containing L-lysine by fermentation of coryneform bacteria with increased expression of the allele of the chromosome glk gene under conditions conducive to the formation of a product of the indicated glk-glucokinase gene (PCT application WO 03054198 A1). A method has also been published for the preparation of shikimic acid and its derivatives by fermentation of E. coli bacteria transformed with recombinant DNA containing genes encoding a glucose facilitator protein and glucokinase from Zymononas mobilis, and thereby possessing the ability to synthesize shikimic acid from a carbon source of the growth medium (PCT application WO 0229078 A2). In addition, processes for producing intracellular intermediate metabolites using microorganisms, such as erythrose-4-phosphate, and alternative processes for producing compounds, for example, aromatic amino acids, such as L-phenylalanine using microorganisms, are described. Moreover, the activity of transaldolase in these producer microorganisms was increased (US patent 6,316,232). This' 232 patent, as the best mode for carrying out the invention, describes an additional increase in the activities of a transketolase or a transport protein carrying out PEP-independent import of sugar into the cell and / or glucokinase. The microorganisms claimed in the patent belong to the genera Escherichia, Seurratia, Bacillus, Corynebacterium or Breibacterium.

A method has been published for producing L-amino acids, in particular L-lysine, which includes culturing modified bacterial cells, in particular Corynebacterium glutamicum, containing an increased pool of NADPH as compared to unchanged bacterial cells, wherein said modified bacterial cell has an enhanced carbon flow through the oxidizing branch pentose phosphate cycle (PCT application WO 0107626 A2). This patent application, as the best mode for carrying out the invention, describes a modified bacterial cell with a weakened carbon flow through the glycolytic pathway due to the reduced catalytic activity of 6-phosphoglucose isomerase resulting from a mutation in resepgi. A similar method for producing L-lysine using coryneform bacteria with zero intracellular phosphoglucose isomerase (pgi) activity (US Pat. No. 6,586,214) is also published.

A method for producing L-lysine has been published, which includes culturing a coryneform bacterium producing L-lysine, in which the intracellular activity of 6-phosphofructokinase encoded by the pfkA gene is increased (European patent application EP 1195431 A1). But at the same time, the process of producing L-amino acids, in particular L-lysine, by fermentation was published, including the cultivation of coryneform bacteria to obtain the target L-amino acid, which has at least one gene encoding 6-phosphofructokinase (pfkA gene) and / or the gene encoding 1-phosphofructokinase (fruK gene) is attenuated (s) (PCT application WO 02074944 A1). In this publication, as the best practical implementation of the process of producing L-lysine using a coryneform bacterium, in addition to attenuating the pfK- and / or fruK gene / s, the expression of one or more genes from the group including the gene is also enhanced lysC encoding feedback inhibition resistant aspartate kinase, dapA gene encoding dihydrodipicolinate synthase, gap gene encoding glyceraldehyde phosphate dehydrogenase, rus encoding pyruvate carboxylase, mqo gene encoding malate chi it oxidoreductase gene zwal, coding for glucose 6-phosphate dehydrogenase, the gene lysE, encoding a lysine exporter, gene zwal, encoding a protein zwal, gene tpi, coding for triose phosphate isomerase and the gene pgk, encoding a 3-phosphoglycerate kinase.

A process is described for producing L-amino acids, such as L-lysine and L-threonine, by fermentation of coryneform bacteria in which at least the zwf gene is amplified (PCT application WO 0170995 A1). This publication describes, as the best method of carrying out the invention, a process for producing L-amino acids using coryneform bacteria, in which, in addition to amplification of the zwf gene, one or more genes additionally amplified or have increased expression simultaneously from the group consisting of: the dapA gene encoding dihydropicolinate synthase , lysC gene encoding feedback inhibition resistant aspartate kinase, gap gene encoding glycerolaldehyde-3-phosphate dehydrogenase, pyc gene encoding pyru atkarboksilazu gene tkt, coding for transketolase, a gene gnd, encoding the gluconate-6-phosphate, the gene lysE, encoding a lysine exporter, gene zwal, gene eno, encoding an enolase.

Describes coryneform bacteria producing L-amino acids, including L-threonine, containing from one copy on the chromosome at the natural locus and up to three additional copies of genes or open reading frames (ORF) selected from the group which, in addition to other genes, includes genes eno, gap, pgk and tpi (PCT applications WO 03014330 A2, WO 03040373 A2).

A process is described for producing L-glutamic acid by the fermentation of coryneform bacteria, in which the expression of the nucleotide sequence encoding D-alanine racemase (alr) is weakened, in particular, is absent (PCT application WO 0208437 A2). This publication describes, as the best embodiment, the process for producing L-glutamic acid using a coryneform bacterium, in which, in addition to the weakened expression of the nucleotide sequence encoding D-alanine racemase (alr), one or more genes from the group that are further enhanced or have increased expression among other genes, includes the gap and eno genes.

A method is described for the preparation of chemically pure compounds or metabolites, such as L-threonine, using microorganisms, in particular, coryneform bacteria or Escherichia coli, in which the ability to phosphorylate at least one protein was permanently increased due to mutation in at least one amino acid of the specified protein as a result, the yield of at least one pure compound synthesized by the microorganism was increased in comparison with the natural one (PCT application WO 03023016 A2). One example of such mutant proteins is enolase (enoS330E).

Among the genes encoding glycolysis enzymes, four genes were modified to increase the production of L-threonine by bacteria of the Enterobacteriaceae family, in particular Escherichia coli. These are the genes fbaA, gpmA (pgm), pykF and pfkB.

Thus, the process of producing L-amino acids, in particular L-threonine, was described by fermenting microorganisms of the Enterobacteriaceae family that produce the target L-amino acid, and in which the expression of the fba gene or nucleotide sequence that encodes the Fba protein is increased (PCT application WO 03004664 A2). At the same time, the same applicant filed PCT patent application WO 03004662 A2 for the enzymatic production of L-amino acids, in particular L-threonine, by fermentation of microorganisms of the Enterobacteriaceae family, producers of the target L-amino acid, in which the expression of one or more genes from the group including, among others, the fba gene, or the nucleotide sequence encoding the Fba protein, is weakened, in particular, is absent. This application does not contain practical examples.

A process was also announced for the production of L-amino acids, in particular L-threonine, by fermentation of microorganisms of the Enterobacteriaceae family, producers of the target amino acid, in which the expression of renapgm or the nucleotide sequence encoding the Pgm protein is increased (PCT application WO 03004598 A2). At the same time, the same applicant filed PCT patent application WO 03004662 A2 for the enzymatic production of L-amino acids, in particular L-threonine, by fermentation of microorganisms of the Enterobacteriaceae family, producers of the target L-amino acid, in which the expression of one or more genes from the group including, among others, the pgm gene, or the nucleotide sequence encoding the Pgm protein, is weakened, in particular, missing. This application does not contain practical examples.

A process has also been announced for the production of L-amino acids, in particular L-threonine, by fermentation of microorganisms of the Enterobacteriaceae family, producers of the target amino acid, in which expression of the pykF gene or the nucleotide sequence encoding the PykF protein is increased (PCT application WO 03008609 A2). At the same time, the same applicant filed PCT patent application WO 03008600 A2 for the enzymatic production of L-amino acids, in particular L-threonine, by fermentation of Enterobacteriaceae microorganisms, producers of the target L-amino acid, in which the expression of one or more genes from the group including, among others, the pykF gene, or the nucleotide sequence encoding the PykF protein, is weakened, in particular, missing. This application does not contain practical examples.

And finally, the production process of L-amino acids, in particular L-threonine, by fermentation of microorganisms of the Enterobacteriaceae family - producers of the target amino acid, in which the expression of the pfkB gene or the nucleotide sequence encoding the PfkB protein is increased (PCT application WO 03008610 A2). At the same time, the same applicant filed PCT patent application WO 03008600 A2 for the enzymatic production of L-amino acids, in particular L-threonine, by fermentation of Enterobacteriaceae microorganisms, producers of the target L-amino acid, in which the expression of one or more genes from the group including, among others, the pfkB gene, or the nucleotide sequence encoding the PfkB protein, is weakened, in particular, missing. This application does not contain practical examples.

To date, there is no evidence of the use of bacteria belonging to the genus Escherichia with increased expression of genes encoding glycolysis enzymes, such as glk, pgi, pfkA, tpiA, gapA, pgk, eno and pykA, for the production of L-threonine.

Description of the invention

The aim of the present invention is to increase the productivity of strains of producers of L-threonine and the provision of a method for producing L-threonine using these strains.

This goal was achieved by establishing the fact that genes such as glk, pgi, pfkA, tpiA, gapA, pgk, eno and pykA, encoding glycolysis enzymes cloned into a low copy vector, can increase the production of threonine. Thus, the present invention has been completed.

An object of the present invention is to provide a bacterium belonging to the genus Escherichia, a producer of L-threonine, wherein said bacterium is modified so that the activity of one or more glycolysis enzymes is increased.

It is also an object of the present invention to provide the above-described bacterium belonging to the genus Escherichia, a producer of L-threonine, wherein said bacterium is modified so that the expression of one or more genes selected from the group of genes glk, pgi, pfkA, tpiA, gapA, pgk , eno and pykA encoding glycolysis enzymes, or nucleotide sequences encoding the same enzymes, is amplified.

It is also an object of the present invention to provide a bacterium as described above, in which the expression of one or more of the indicated genes is enhanced by increasing the number of copies of this gene (s) or by modifying the sequence that controls the expression of the gene (s) such that the expression of the specified gene (s) amplified.

It is also an object of the present invention to provide a bacterium as described above in which the number of copies of a gene is increased by transforming the bacterium using a low copy vector containing the indicated gene (s).

It is also an object of the present invention to provide a bacterium as described above in which said genes are derived from a bacterium belonging to the genus Escherichia.

It is also an object of the present invention to provide a bacterium as described above, wherein said bacterium is modified so that said bacterium has enhanced expression of one or more genes selected from the group consisting of;

- mutant thrA gene encoding aspartokinase-homoserine dehydrogenase I, resistant to threonine inhibition by feedback type;

- thrB gene encoding homoserine kinase;

- thrC gene encoding threonine synthase; and

- rhtA gene encoding the putative transmembrane protein.

It is also an object of the present invention to provide a bacterium as described above, wherein said bacterium is modified so that the expression of said mutant thrA gene, said thrB gene, said thrC gene, and said rhtA gene is enhanced.

It is also an object of the present invention to provide a method for producing L-threonine, comprising growing the above-described bacteria in a nutrient and isolating L-threonine from the culture fluid.

The best way of carrying out the invention.

A bacterium according to the present invention is a bacterium belonging to the genus Escherichia, a producer of L-threonine, modified to increase the activity of one or more glycolysis enzymes. More specifically, the bacterium of the present invention is a bacterium belonging to the genus Escherichia, an L-threonine producer modified to enhance expression of one or more genes selected from the group of glk, pgi, pfkA, tpiA, gapA, pgk, eno and pykA encoding glycolysis enzymes, or nucleotide sequences that encode these enzymes.

According to the present invention, “a bacterium producing threonine” means a bacterium having the ability to accumulate L-threonine in a culture medium under conditions when the bacterium of the present invention is grown in said culture medium. The ability to produce L-threonine can be imparted or improved by selection. As used herein, the term “L-threonine-producing bacterium” also means a bacterium that is capable of producing L-threonine and causes the accumulation of L-threonine in a fermentation medium in quantities greater than the natural or parent E. coli strain, such as E. coli K-12.

The term "bacterium belonging to the genus Escherichia" means that the bacterium belongs to the genus Escherichia in accordance with the classification known to the person skilled in the field of microbiology. As an example of a microorganism belonging to the genus Escherichia used in the present invention, but not the only one in the specified genus, the bacterium Escherichia coli (E. coli) can be mentioned.

The range of bacteria belonging to the genus Escherichia that can be used in the present invention is not limited in any way, however, for example, the bacteria described in Neidhardt, F.C. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1) may be included in their number according to the present invention.

The term “modified in such a way that the expression of the gene / s is enhanced” means that the number of expressed copies of the gene / s is higher than that of an unmodified strain, for example, a natural strain. Examples of such modifications can be an increase in the number of expressed gene (s) per cell, an increase in the level of gene expression, and so on. The number of copies of an expressed gene can be measured, for example, by restriction of chromosomal DNA, followed by Southern hybridization, using a probe synthesized based on the nucleotide sequence of the gene under investigation, fluorescence in situ hybridization (FISH), and the like. The level of gene expression is determined using various methods, including Northern hybridization, quantitative RT-PCR, and the like. In addition, a strain of Escherichia coli K-12 may be mentioned as a natural strain that can serve as an object for comparison. As a result of increasing the expression level of the indicated gene (s), the effect of increasing the amount of accumulated L-threonine is observed.

The glycolysis enzymes according to the present invention are represented by glucokinase (EC 2.7.1.2) encoded by the glk gene, phosphoglucose isomerase (EC 5.3.1.9) encoded by the pgi gene, phosphofructokinase-1 (fructose-6-P 1 kinase) (EC 2.7.1.11 ), encoded by the pfkA gene, triose phosphate isomerase (EC 5.3.1.1), encoded by the tpiA gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12), encoded by the gapA gene, phosphoglycerate kinase (EC 2.7.2.3) p encoded enolase (EC 4.2.1.11) encoded by the eno gene and pyruvate kinase isoenzymes (EC 2.7.1.40) encoded by the pykA gene.

According to the present invention, the term "glucokinase" means an enzyme capable of catalyzing the reaction of ATP-dependent phosphorylation of glucose to form glucose-6-phosphate. The term "phosphoglucose isomerase" means an enzyme capable of catalyzing the conversion of glucose-6-phosphate to fructose-6-phosphate. The term "phosphofructokinase-1 (fructose-6-P 1-kinase)" means an enzyme capable of catalyzing the reaction of ATP-dependent phosphorylation of glucose-6-phosphate to form glucose-1,6-diphosphate. The term “triose phosphate isomerase” means an enzyme capable of catalysing the reaction of the interconversion of dioxaacetone phosphate to glyceraldehyde-3-phosphate and vice versa. The term "glyceraldehyde-3-phosphate dehydrogenase" means an enzyme capable of catalyzing the reaction of NAD ⁺ -dependent conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. The term "phosphoglycerate kinase" means an enzyme capable of catalyzing the dephosphorylation reaction of 1,3-diphosphoglycerate to form 3-phosphoglycerate and ATP. The term "enolase" means an enzyme capable of catalyzing the dehydration reaction of 2-phosphoglycerate to form phosphoenolpyruvate. The term "pyruvate kinase" means an enzyme capable of catalyzing the reaction of the formation of pyruvate from phosphoenolpyruvate with the release of ATP.

Any of these genes derived from bacteria belonging to the genus Escherichia and genes derived from other bacteria, such as coryneform bacteria, bacteria belonging to the genus Bacillus or the like, can be used as genes encoding these glycolysis enzymes. Among these genes, genes derived from bacteria belonging to the genus Escherichia are preferred.

As the gene encoding glucokinase from Escherichia coli, the glk gene is known (nucleotide numbers 2506481 to 2507446 in sequence with accession number NC_000913.1 in GenBank; gi: 16130320; SEQ ID NO: 1). The glk gene is located on the chromosome of E. coli K12 strain between open reading frames (ORFs) b2387 and b2389. As the gene encoding Escherichia coli phosphoglucose isomerase, the pgi gene is known (nucleotide numbers 4231337 to 4232986 in sequence with accession number NC_000913.1 in GenBank, gi: 16131851; SEQ ID NO: 3). The pgi gene is located on the chromosome of E. coli K12 strain between the lysC-ayjbE genes. As a gene encoding phosphofructokinase-1 from Escherichia coli, the pfkA gene is known (nucleotide numbers 4105132 to 4106094 in sequence with accession number NC_000913.1 in GenBank, gi: 16131754; SEQ ID NO: 5). FespfkA is located on the chromosome of E. coli K12 strain between the yiiP ORF reading frame and the sbp gene. As the gene encoding triosophosphatisomerase from Escherichia coli, the tpiA gene is known (nucleotide numbers 4108320 to 4109087 in sequence with accession number NC_000913.1 in GenBank, gi: 16131757; SEQ ID NO: 7). The tpiA gene is located on the chromosome of E. coli K12 strain between the cdh gene and the ORF reading frame. As the gene encoding glyceraldehyde-3-phosphate dehydrogenase from Escherichia coli, the gapA gene is known (nucleotide numbers from 1860795 to 1861790 in the sequence with accession number NC_000913.1 in GenBank, gi: 16129733;

SEQ ID NO: 9). The gapA gene is located on the chromosome of strain E. coli K12 between the reading frames of the уеАА ORF and уеАО ORF. As the gene encoding phosphoglycerate kinase from Escherichia coli, the pgk gene is known (nucleotide numbers 3069479 to 3070642 in sequence with accession number NC_000913.1 in GenBank, gi: 16130827; SEQ ID NO: 11). Ympgk is located on the chromosome of E. coli K12 strain between the fbaA and epd genes. As the gene encoding the enolase from Escherichia coli, the eno gene is known (nucleotide numbers 2904665 to 2905963 in sequence with accession number NC_000913.1 in GenBank, gi: 1613 0686;

SEQ ID NO: 13). The eno gene is located on the chromosome of E. coli K12 strain between the reading frame of b2778 ORF and the pyrG gene. The pykA gene is known as the gene encoding pyruvate kinase II from Escherichia coli (nucleotide numbers from 1935673 to 1937115 in sequence with accession number NC_000913.1 in GenBank, gi: 16129807; SEQ ID NO: 15). The pykA gene is located on the chromosome of E. coli K12 strain between the yebK ORF reading frame and the msbB gene. Therefore, the asd gene can be obtained by PCR (polymerase chain reaction; White, T.J. et al .. Trends Genet, 5.185 (1989)) using primers synthesized based on the nucleotide sequence of the gene.

An example of a glk gene from Escherichia coli is DNA encoding the following proteins (A) or (B):

(A) a protein that is represented by the amino acid sequence shown in the sequence listing under number 2 (SEQ ID NO: 2); or

(B) a protein that is represented by an amino acid sequence including deletions, substitutions, insertions, or the addition of one or more amino acids to the amino acid sequence listed in the sequence number 2 (SEQ ID NO: 2), and which has glucokinase activity.

An example of a pgi gene from Escherichia coli is DNA encoding the following proteins (C) or (D):

(C) a protein that is represented by the amino acid sequence shown in the sequence list under number 4 (SEQ ID NO: 4); or

(D) a protein that is represented by an amino acid sequence including deletions, substitutions, insertions, or the addition of one or more amino acids to the amino acid sequence listed in sequence number 4 (SEQ ID NO: 4), and which has phosphoglucose isomerase activity.

An example of a pfkA gene from Escherichia coli is DNA encoding the following proteins (E) or (F):

(E) a protein that is represented by the amino acid sequence shown in the sequence list under number 6 (SEQ ID NO: 6); or

(F) a protein that is represented by an amino acid sequence including deletions, substitutions, insertions, or the addition of one or more amino acids to the amino acid sequence listed in sequence number 6 (SEQ ID NO: 6), and which has phosphofructokinase-1 activity (fructose -6-P 1-kinase).

An example of a tpiA gene from Escherichia coli is DNA encoding the following proteins (G) or (H):

(G) a protein that is represented by the amino acid sequence shown in the sequence list under number 8 (SEQ ID NO: 8); or

(H) a protein that is represented by an amino acid sequence including deletions, substitutions, insertions, or the addition of one or more amino acids to the amino acid sequence listed in the sequence number 8 (SEQ ID NO: 8), and which has triosophosphatisomerase activity.

An example of a gapA gene from Escherichia coli is DNA encoding the following proteins (I) or (J):

(I) a protein that is represented by the amino acid sequence shown in the list of sequences at number 10 (SEQ ID NO: 10); or

(J) a protein that is represented by an amino acid sequence including deletions, substitutions, insertions, or addition of one or more amino acids to the amino acid sequence listed in sequence number 10 (SEQ ID NO: 10), and which has glyceraldehyde-3-phosphate dehydrogenase activity .

An example of a pgk gene from Escherichia coli is DNA encoding the following proteins (K) or (L):

(K) a protein that is represented by the amino acid sequence shown in the list of sequences at number 12 (SEQ ID NO: 12); or

(L) a protein that is represented by an amino acid sequence including deletions, substitutions, insertions, or the addition of one or more amino acids to the amino acid sequence listed in sequence number 12 (SEQ ID NO: 12), and which has phosphoglycerate kinase activity.

An example of an eno gene from Escherichia coli is DNA encoding the following proteins (M) or (N):

(M) a protein that is represented by the amino acid sequence shown in the sequence list under number 14 (SEQ ID NO: 14); or

(N) a protein that is represented by an amino acid sequence including deletions, substitutions, insertions, or the addition of one or more amino acids to the amino acid sequence listed in sequence number 14 (SEQ ID NO: 14), and which has enolase activity.

An example of a pykA gene from Escherichia coli is DNA encoding the following (O) or (P) proteins:

(O) a protein that is represented by the amino acid sequence shown in the list of sequences at number 16 (SEQ ID NO: 16); or

(P) a protein that is represented by an amino acid sequence including deletions, substitutions, insertions, or addition of one or more amino acids to the amino acid sequence listed in the sequence number 16 (SEQ ID NO: 16), and which has pyruvate kinase activity.

The number of “several” amino acids varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein. It can be from 2 to 30, preferably from 2 to 15, and more preferably from 2 to 5 for protein (A). This is due to the fact that some amino acids have high homology to each other and differences in such amino acids do not cause significant changes in the three-dimensional structure of the protein and its activity. Therefore, protein (B) may be a protein having a homology of not less than 30-50%, preferably 50-70%, more preferably 70-90% and most preferably more than 90% with respect to all amino acid residues constituting glucokinase and possessing glucokinase activity. The same approach applies to proteins (C), (E), (G), (I), (K), (M) and (O).

DNA encoding almost the same proteins as the glycolysis enzymes described above can be obtained, for example, by modifying the nucleotide sequence of the DNA encoding the enzyme, for example, using the site-directed mutagenesis method, so that one or more amino acid residues in certain locations contain deletions, substitutions, insertions or additives. DNA modified as described above can be obtained by conventional methods known in the art for the preparation of mutations. Such methods include treating the DNA encoding the protein of the present invention with hydroxylamine, or treating the bacterium containing said DNA with UV light or with a reagent such as N-methyl-N'-nitro-N-nitrosoguanidine or nitrous acid.

DNA encoding almost the same protein as glucokinase can be obtained by expressing DNA containing the above mutations in a suitable cell and studying the activity of any expressed product. DNA encoding practically the same protein as glucokinase can also be obtained by isolating DNA from genomic DNA encoding a glucokinase containing mutations, or from a cell containing it, and which hybridizes with a probe with a nucleotide sequence including, for example, a nucleotide sequence shown in Sequence Listing as sequence 1 (SEQ ID NO: 1), under stringent conditions, and which encodes a protein having glucokinase activity. The term "stringent conditions", mentioned here, means the conditions under which the so-called specific hybrids are formed, and non-specific ones are not formed. It is difficult to clearly describe such conditions in any numerical terms. However, for example, stringent conditions include conditions under which DNAs with a high degree of homology hybridize, for example, DNAs that have a homology of at least 50% relative to each other, and DNAs that have less homology do not hybridize with each other.

Several methods of calculation, such as BLAST search, FASTA search, and CrustalW, can be used to assess the degree of homology between DNA. BLAST (Basic Local Alignment Search Tool) is a self-learning search algorithm used by BLASTP, BLASTN, BLASTX, MEGABLAST, TBLASTN, and TBLASTX; these programs evaluate the significance of the results using statistical methods Karlin, Samuel and Stephen F. Altschul ("Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes". Proc. Natl. Acad. Sci. USA, 1990, 87 : 2264-68; "Applications and statistics for multiple high-scoring segments in molecular sequences". Proc. Natl. Acad. Sci. USA, 1993, 90: 5873-7). The FASTA search method is described by W. R. Pearson ("Rapid and Sensitive Sequence Comparison with FASTP and FASTA", Methods in Enzymology, 1990 183: 63-98). The ClustalW method is described by Thompson J.D., Higgins D.G. and Gibson T.J. ("CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice", Nucleic Acids Res. 1994, 22: 4673-4680).

On the other hand, an example of stringent conditions is the conditions under which DNA hybridizes with each other, at a salt concentration corresponding to standard washing conditions for Southern hybridization, for example, IxSSC, 0.1% SDS, preferably 0.1xSSC, 0.1% SDS at 60 ° C . The duration of washing depends on the type of filter used for blotting and, as a rule, on the recommendations of the manufacturer. For example, the recommended washing time for a Hybond ™ N + nylon filter (Amersham) under harsh conditions is 15 minutes.

As a probe, a portion of the nucleotide sequence numbered 1 (SEQ ID NO: 1) can be used. A probe of this kind can be obtained by PCR using as seeds the oligonucleotides obtained on the basis of the nucleotide sequence at number 1 and a DNA fragment containing the nucleotide sequence at number 1 as a matrix. When a DNA fragment with a length of about 300 base pairs is used as a probe, the washing conditions during hybridization correspond, for example, to 50 ° С, 2 × SSC, and 0.1% SDS.

Nucleotide deletions, substitutions, insertions or additions described above also include mutations that exist in nature (mutants or variants), for example, due to individual differences or species and genus differences in bacteria containing glucokinase.

DNA encoding substantially the same proteins as other glycolysis enzymes can be obtained by the method described above for glucokinase.

“Transformation of a bacterium using DNA encoding a protein” means the introduction of said DNA into a bacterial cell, for example, by conventional methods.

Transformation of the indicated DNA leads to an increase in the expression of the indicated gene encoding the protein of the present invention and an increase in the activity of the indicated protein in the bacterial cell. As a transformation method, any method described so far can be used. For example, a method of treating recipient cells with calcium chloride can be used to increase the permeability of DNA across the cell membrane described for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)).

Methods for increasing gene expression include methods for increasing the number of copies of a gene. The introduction of a gene into a vector capable of functioning in bacteria belonging to the genus Escherichia increases the number of copies of this gene. For this purpose, it is preferable to use low-copy vectors. Examples of low copy vectors are vectors pSC101, pMW118, pMW119 and the like. As used herein, the term “low copy vector” is used for vectors up to 5 copies per cell. As a transformation method, any method described so far can be used. For example, a method for treating recipient cells with calcium chloride can be used to increase DNA permeability across the cell membrane described for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53,159 (1970 )).

In addition, an increase in gene expression can be achieved by introducing plural copies of the specified gene into the bacterial chromosomes, for example, by homologous recombination, Mi integration, and the like. For example, one round of Mi integration allows you to embed up to three copies of an integrable gene in a bacterial chromosome.

Also, increased gene expression can be achieved by placing the DNA of the present invention under the control of a strong promoter. As strong promoters, for example, the lac promoter, trp promoter, trc promoter, pr or pl lambda phage promoters are known. The strength of the promoter is determined by the frequency of the act of starting RNA synthesis. Methods for evaluating promoter strength and examples of strong promoters are described by Deuschle, U., Kammerer, W., Gentz, R., Bujard, H. (Promoters in Escherichia coli: a hierarchy of in vivo strength indicates alternate structures. EMBO J., 5 , 2987-2994 (1986)).

Using a strong promoter can be combined with an increase in the number of copies of the gene.

In addition, the promoter can be enhanced, for example, by introducing a mutation in the specified promoter in order to increase the level of transcription of the gene located after the promoter. Further, it is known that the substitution of several nucleotides in the region between the ribosome binding site (RBS) and the start codon, and especially in the sequence immediately before the start codon, significantly affects the mRNA translatability. For example, a 20-fold change in expression level was found depending on the nature of the three nucleotides prior to the start codon (Gold et al., Annu. Rev. Microbiol., 35, 365-403, 1981; Hui et al., EMBO J. 3, 623-629, 1984). Previously, the present inventors have found that the rhtA23 mutation is a substitution of A for G position-1 relative to the ATG start codon (ABSTRACTS of 17 ^th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457). Therefore, it was suggested that the rhtA23 mutation enhances the expression of the rhtA gene and, as a result, increases the level of resistance to threonine, homoserine, and some other substances transported from the cell.

Moreover, it is possible to introduce nucleotide substitutions into the promoter region of one or more genes encoding glycolysis enzymes in the bacterial chromosome in such a way that said promoter becomes stronger. Changing the expression control sequence can be accomplished, for example, by the same method as replacing a gene using a temperature-sensitive plasmid, as described in international patent application WO / 18935 or Japanese patent application No. 1-215280.

An increase in the number of copies of one or more genes encoding glycolysis enzymes can be achieved by introducing plural copies of the gene into the chromosomal DNA of the bacterium. To introduce multiple copies of a gene into a bacterial chromosomal DNA, homologous recombination is used using the sequences present in the chromosomal DNA as targets. Repeating DNA fragments as well as inverted repeats present at the ends of mobile elements can be used as sequences present in several copies of chromosomal DNA. Also, as described in Japanese Patent Application No. 1-109985, it is possible to introduce the target gene into a transposon, for subsequent transformation with the introduction of multiple copies of the specified gene into the chromosomal DNA.

Methods for obtaining plasmid DNA, cutting and donating DNA, transformation, selection of oligonucleotides as seeds, and the like, can be conventional methods well known to those 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).

The bacterium according to the present invention can be obtained by introducing the above DNA into a bacterium already possessing the ability to produce L-threonine. On the other hand, a bacterium according to the present invention can be obtained by giving a bacterium already containing said DNA the ability to produce L-threonine.

Examples of parent strains related to the present invention, while not limiting the entire possible list of parent strains, are threonine producing strains belonging to the genus Escherichia, such as E. coli strain TDH-6 / pVIC40 (VKPM B-3996) ( US patents 5,175,107 and 5,705,371), E. coli strain NRRL-21593 (US patent 5,939,307), E. coli strain FERM BP-3756 (US patent 5,474,918), E. coli strains FERM BP-3519 and FERM BP-3520 (US patent 5,376,538), strain E, coli MG442 (Gusyatiner et al., Genetics, 14, 947-956 (1978)), E. coli strains VL643 and VL2055 (European patent application EP 1149911 A) and the like.

Strain TDH-6 is deficient in the thrC gene, is capable of assimilating glucose, and contains the leaky UvA gene. The specified strain contains a mutation in the rhtA gene, which causes resistance to high concentrations of threonine and homoserine. Strain B-3996 contains the plasmid pVIC40, which was obtained by introducing into the vector derived from the RSF1010 vector the thrA * BC operon including the thrA mutant gene encoding aspartokinase-homoserine dehydrogenase I, which has a significantly reduced feedback sensitivity to threonine inhibition. Strain B-3996 was deposited on November 19, 1987 at the All-Union Scientific Center for Antibiotics (RF, 113105, Moscow, Nagatinskaya St., 3-A) with inventory number RIA 1867. The strain was also deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) ( RF, 113545, Moscow, 1st Road passage, 1) with inventory number B-3996.

Preferably, the bacterium according to the present invention is further modified so that the specified bacterium is enhanced expression of one or more of the following genes in addition to one or more genes encoding glycolysis enzymes:

- mutant thrA gene encoding aspartokinase-homoserine dehydrogenase I, resistant to threonine inhibition by feedback type;

- thrB gene encoding homoserine kinase;

- thrC gene encoding threonine synthase.

Another preferred practical embodiment of the present invention is that said bacterium is modified by enhancing the expression of the rhtA gene encoding, as expected, a transmembrane protein. The most preferred embodiment of the present invention is a bacterium with enhanced expression of the gene (s) encoding / their glycolysis enzymes, mutant thrA gene, thrB, thrC and rhtA genes.

A method for producing L-threonine according to the present invention includes the steps of growing a bacterium according to the present invention in a culture medium in order to produce and accumulate L-threonine in a culture medium and isolate L-threonine from the culture fluid. According to the present invention, the cultivation, isolation and purification of L-threonine from a culture or similar liquid can be carried out in a manner similar to traditional fermentation methods in which the amino acid is produced using a microorganism.

The nutrient medium used for growing can be either synthetic or natural, provided that the medium contains sources of carbon, nitrogen, mineral additives and, if necessary, the appropriate amount of nutrient additives necessary for the growth of microorganisms. Carbon sources include various carbohydrates such as glucose and sucrose, as well as various organic acids. Depending on the nature of the assimilation of the microorganism used, alcohols such as ethanol and glycerin may be used. Various inorganic ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds, such as amines, natural nitrogen sources, such as peptone, soybean hydrolyzate, microorganism fermentolizate, can be used as a nitrogen source. As mineral additives, potassium phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium chloride and the like can be used. Thiamine and yeast extract can be used as vitamins.

Some nutritional supplements may, if necessary, be added to the culture medium. For example, if the microorganism needs isoleucine for growth (isoleucine auxotrophy), an appropriate amount of isoleucine can be added to the growth medium.

The cultivation is preferably carried out under aerobic conditions, such as mixing the culture fluid on a rocking chair, shaking with aeration, at a temperature in the range from 20 to 40 ° C, preferably in the range from 30 to 38 ° C. The pH of the medium is maintained in the range from 5 to 9, preferably from 6.5 to 7.2. The pH of the medium can be adjusted by ammonia, calcium carbonate, various acids, bases and buffer solutions. Typically, growing for 1 to 5 days leads to the accumulation of the target L-amino acid in the culture fluid.

After growth, solid residues, such as cells, can be removed from the culture fluid by centrifugation or filtration through a membrane, and then L-threonine can be isolated and purified by ion exchange chromatography, concentration and crystallization.

The drawing shows the structure of the synthetic mutant promoter P _A3m .

Examples.

In more detail, the present invention will be explained below with reference to the following Examples, not limiting the scope of the present invention. To assess the effect of amplification of genes encoding glycolysis enzymes on the production of L-threonine, E. coli strain VKPM B-3996 (US patent 5,175,107) was used as the parent strain. Plasmid pMW119 and its derivatives are compatible with plasmid pVIC40 (replicon pRSF1010), therefore both plasmids pVIC40 and plasmid derived from pMW119 containing the gene encoding the glycolysis enzyme can be simultaneously expressed in one bacterial cell. The tables below show the results of fermentation as the average of at least three independent experiments.

Example 1: Cloning of the glk gene from E. coli and the effect of increasing expression of the glk gene on L-threonine production.

The glk gene was obtained by PCR, chromosomal DNA from strain MG 1655 (VKPM B-6195) E. coli was used as a template, and primers PI (SEQ ID NO: 17) and P2 (SEQ ID NO: 18) were used as primers ) Primer PI contains a BamHI restriction enzyme recognition site at the 5 ′ end. Primer P2 contains a Sad restriction enzyme recognition site at the 5 ′ end. The obtained DNA fragment (968 bp), including the glk gene, was treated with restriction enzymes BamHI and Sad, and then cloned into the plasmid vector pMWl 19, previously modified by replacing the P _lac promoter with the pr promoter of the lambda phage, also treated with these restriction enzymes. Thus, the plasmid pMW-PR-glk containing the glk gene under the control of the pr promoter was obtained. Thanks to the use of this plasmid, a high level of glk gene expression can be achieved due to the lack of its regulation.

Plasmid pMW-Pp-glk was introduced into a streptomycin-resistant threonine-producing strain of E. coli B-3996 (US patent 5,175,107). Thus, strain B-3996 (pMW-PR-glk) was obtained.

Both strains of E. coli B-3996 and B-3996 (pMW-PR-glk) were grown for 18-24 hours at 37 ° C on L-agar plates containing streptomycin (100 μg / ml) and ampicillin (100 μg / ml). To obtain a seed culture, each of the strains was grown in 20x200 mm fermentation tubes containing 2 ml of L-broth with 4% sucrose on a rotary shaker (250 rpm) at a temperature of 32 ° C for 18 hours. Then 0.1 ml (5%) of the seed culture was introduced into the fermentation medium. Fermentation was carried out in 20x200 mm test tubes with 2 ml of minimal fermentation medium. Cells were grown for 24 hours at a temperature of 32 ° C on a rotary shaker at 250 rpm.

After fermentation was completed, the amount of L-threonine accumulated in the medium was determined by thin layer chromatography (TLC). Sorbfil plates (Sorbopolimer Joint-Stock Company, Krasnodar, Russia) were exhibited in the liquid phase of the following composition: isopropanol: acetone: water: 25% ammonia = 25:25: 7: 6 (volume ratio). A solution of (2%) ninhydrin in acetone was used as a visualizing reagent. The results are presented in Table 1.

Used the following composition of the medium for fermentation (g / l):

Glucose 40.0 (NH ₄ ) ₂ SO ₄ 10.0 KN ₂ PO ₄ 1.0 MgSO ₄ × 7H ₂ O 0.4 FeSO ₄ × 7H ₂ O 0.02 MnSO ₄ × 5H ₂ O 0.02 Thiamine HCl 0.0002 Yeast extract 1.0 CaCO ₃ 20.0 L-isoleucine 0.05

Sucrose and magnesium sulfate were sterilized separately. CaCO _{3 was} dry heat sterilized at 180 ° C for 2 hours. The pH value corresponded to 7.0. The antibiotic was added to the medium after sterilization.

Table 1.

Strain OD ₅₆₀ Threonine, g / l B-3996 9.7 ± 0.1 14.3 ± 0.1 B-3996 (pMW-PR-glk) 9.6 ± 0.1 14.8 ± 0.1

As can be seen from Table 1, increased expression of the glk gene increases the production of L-25 threonine by strain B-3996.

Example 2: Cloning of the pfkA gene from E. coli and the effect of increasing pjkA gene expression on L-threonine production.

The pfkA gene was obtained by PCR, 30 chromosomal DNA from E. coli strain MG 1655 (VKPM B-6195) was used as a template, and primers P3 (SEQ ID NO: 19) and P4 (SEQ ID NO: twenty). Primer P3 contains a BamHI restriction enzyme recognition site at the 5 ′ end. Primer P4 contains a Sad restriction enzyme recognition site at the 5 ′ end. The resulting DNA fragment (987 bp) containing the pfkA gene was directly cloned into the pCR 2.1 vector (Invitrogen) by ligation of the mixture of the vector and the fragment overnight at + 4 ° C. Then, the BamHI-Sad DNA fragment containing the pfkA gene was cloned into the plasmid vector pMWl 19, previously modified by replacing the P _lac promoter with the pr promoter of the phage lambda, also treated with the indicated restriction enzymes. Thus, the plasmid pMW-PR-pfkA containing the pfkA gene under the control of the pr promoter was obtained. Thanks to the use of this plasmid, a high level of pfkA gene expression can be achieved due to the lack of its regulation.

Plasmid pMW-PR-pfkA was introduced into streptomycin, a resistant producer strain of threonine E. coli B-3996 (US patent 5,175,107). Thus, strain B-3996 (pMW-PR-pfkA) was obtained.

The procedure for determining the amount of L-threonine accumulated in the nutrient medium by E. coli B-3996 and B-3996 strains (pMW-PR-pfkA) is described above (see Example 1). The results are presented in Table 2.

Table 2.

Strain OD ₅₆₀ Threonine, g / l B-3996 9.7 ± 0.1 14.3 ± 0.1 B-3996 (pMW-PR-pfkA 9.3 ± 0.1 14.4 ± 0.1

Since the effect of enhancing pfkA gene expression on the increase in L-threonine production turned out to be insignificant in vitro fermentation, fermentation was carried out using glucose depletion (batch fermentation) in laboratory fermenters with a capacity of 1.0 liter.

For this purpose, E. coli strains VKPM-3996 and VKPM-3996 (pM-PR-pfkA) were grown for 18-24 hours at 37 ° C at 37 ° C on L-agar plates containing streptomycin (100 μg / ml ) Then one microbiological loop with the cell culture was introduced into 50 ml of L-broth of the following composition: tryptone - 10 g / l, yeast extract - 5 g / l, NaCl - 5 g / l. Cells (50 ml, OD540 - 2 pu), grown at 37 ° C for 5 hours on a shaker (240 rpm), were used as seed for inoculation of 450 ml of fermentation medium. Fermentation (batch fermentation) was carried out in laboratory fermenters with a capacity of 1.0 liter with aeration (1/1 wm) and a stirrer speed of 1200 rpm at 37 ° C. pH stating was carried out automatically - a pH of 6.6 was maintained using an 8% aqueous ammonia solution. The results are presented in Table 3.

Used the following composition of the medium for fermentation (g / l):

Glucose 100.0 NH ₄ Cl 1.75 KH ₂ RO ₄ 1.0 MgSO ₄ × 7H ₂ O 0.8 FeSO ₄ × 7H ₂ O 0.01 MnSO ₄ × 5H ₂ O 0.01 Mameno (TN) 0.15 Betaine 1.0 L-isoleucine 0.2

Glucose and magnesium sulfate were sterilized separately. The pH value was 6.6.

Table 3

Strain OD ₅₆₀ (final) Threonine, g / l B-3996 B-3996 (pMW-PR-pfkA) 39.9 ± 2.1 34.4 ± 1.5 33.80 ± 3.1 39.57 ± 1.0

As can be seen from Table 3, increased expression of the pfkA gene increases the production of L-threonine by strain B-3996.

Example 3: Cloning of the fbaA gene from E. coli and the effect of increasing expression of TQH.Q.fbaA on L-threonine production.

TesfbaA was obtained by PCR, chromosomal DNA from strain MG 1655 (VKPM B-6195) E. coli was used as a template, and primers P5 (SEQ ID NO: 21) and P6 (SEQ ID NO: 22) were used as primers . Primer P5 contains a BamHI restriction enzyme recognition site at the 5 ′ end. Primer P6 contains a Sad restriction enzyme recognition site at the 5 ′ end. The obtained DNA fragment (1155 bp), including the fbaA gene, was treated with restriction enzymes BamHI and Sad, after which it was cloned into the plasmid vector pMWl 19, previously modified by replacing the P _lac promoter with the pr promoter of the lambda phage, also treated with said restriction enzymes. Thus, the plasmid pMW-PR-fbaA containing the fbaA gene under the control of the pr promoter was obtained. By using this plasmid, a high level of fbaA gene expression can be achieved due to the lack of its regulation.

Plasmid pMW-PR-fbaA was introduced into streptomycin, a resistant producer strain of threonine E. coli B-3996 (US patent 5,175,107). Thus, strain B-3996 (pMW-PR-fbaA) was obtained.

The procedure for determining the amount of L-threonine accumulated in the culture medium by E. coli B-3996 and B-3996 strains (pMW-PR-fbaA) is described above (see Example 1). The results are presented in Table 4.

Table 4.

Strain OD ₅₆₀ Threonine, g / l B-3996 9.7 ± 0.1 14.3 ± 0.1 B-3996 (pMW-PR-fbaA) 9.3 ± 0.2 15.1 ± 0.5

As can be seen from Table 4, increased expression of the fbaA gene increases the production of L-threonine by strain B-3996.

Example 4: Cloning of the tpiA gene from E. coli and the effect of increasing the expression of the tpiA gene on the production of L-threonine.

The tpiA gene was obtained by PCR, chromosomal DNA from strain MG 1655 (VKPM B-6195) E. coli was used as a template, and primers P7 (SEQ ID NO: 23) and P8 (SEQ ID NO: 24) were used as primers ) Primer P7 contains a BamHI restriction enzyme recognition site at the 5 ′ end. Primer P8 contains a Sad restriction enzyme recognition site at the 5 ′ end. The obtained DNA fragment (774 bp), including the tpiA gene, was digested with restriction enzymes BamHI and Sad, and then cloned into the plasmid vector pMWl 19, which was previously modified by replacing the fda promoter with the pr promoter of the lambda phage, also treated with the indicated restriction enzymes. Thus, the rmw-pr-tpiA plasmid was obtained containing the tpiA gene under the control of the pr promoter. Using this plasmid, a high level of tpiA gene expression can be achieved due to the lack of its regulation.

Plasmid pMW-PR-tpiA was introduced into streptomycin, a resistant producer strain of threonine E. coli B-3996 (US patent 5,175,107). Thus, strain B-3996 (pMW-PR-tpiA) was obtained.

The procedure for determining the amount of L-threonine accumulated in the nutrient medium by strains of E. coli B-3996 and B-3996 (pMW-PR-tpiA) is described above (see Example 1). The results are presented in Table 5.

Table 5.

Strain OD ₅₆₀ Threonine, g / l B-3996 9.7 ± 0.1 14.3 ± 0.1 B-3996 (pMW-PR-tpiA) 9.6 ± 0.5 14.6 ± 0.1

Since the effect of enhancing the expression of the tpiA gene on the increase in L-threonine production in vitro fermentation was not significant, fermentation was carried out using glucose depletion (batch fermentation) in laboratory fermenters with a capacity of 1.0 liter, as described above (see Example 2).

The results are presented in Table 6.

Table 6.

Strain OD ₅₆₀ (final) Threonine, g / l B-3996 B-3996 (pMW-PR-tpiA) 39.9 ± 2.1 34.9 ± 2.6 33.80 ± 3.1 37.16 ± 1.4

As can be seen from Table 6, increased expression of the tpiA gene increases the production of L-threonine by strain B-3996.

Example 5: Cloning of the gapA gene from E. coli and the effect of increasing gapA gene expression on L-threonine production.

To study the effect of the amplification effect of gapA gene expression on L-threonine production, gapA gene was cloned into pMWl 19 plasmid under the control of a mutant synthetic promoter (A3m) obtained from the A3 promoter of E. coli T3 phage (Yamada, M. et al. Promoter sequence analysis in Bacillus and Escherichia coli: construction of strong promoter in E. coli. Gene, 99 (1), 109-114 (1991)). Mutations introduced into the nucleotide sequence of the A3 promoter - constitutive polymerase-holoenzyme of the Est ⁷⁰ - dependent promoter, led to a decrease in its promoter strength. The nucleotide sequence of the oligonucleotides constituting the mutant synthetic promoter A3m are shown under the numbers SEQ ID NOs: 25 and 26. The structure of the A3m promoter is shown in the drawing.

The gapA gene was obtained by PCR, chromosomal DNA from E. coli strain MG 1655 (VKPM B-6195) was used as a template, and gapA-5 'primers (SEQ ID NO: 27) and gapA-ter ( SEQ ID NO: 28). The gapA-5 ′ primer contains a BamHI restriction enzyme recognition site at the 5 ′ end. The gapA-ter primer contains an Xbal restriction enzyme recognition site at the 5'-end. The obtained DNA fragment (1200 bp), including the gapA gene with a 5 'untranslated region to the P1 transcription start site and without its own promoter sequence, was treated with BamHI and Xbal restriction enzymes, ligated with a synthetic A3m promoter, whose DNA fragment had sticky ends EcoRI and BamHI, after which they were cloned into the plasmid vector pMWl 19 treated with restriction enzymes EcoRI and Xbal. Thus, the plasmid pMWl 19-PA3m-gapA was obtained. The structure of the obtained gapA gene was confirmed by sequencing.

Cells of E. coli strain HB101 were transformed with plasmid pMWl 19 - PA3m-gapA (Sambrook J. and Russell D.W. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). GAPDH activities in the middle of the logarithmic growth curve of strains HB101 and HB101 (pMW-PA3m-gapA) were determined according to the procedure described in Peng. L., and Shimizu. K. (Appl. Microbiol. Biotechnol. 61,163-178 (2003)). The results are presented in Table 7.

Table 7.

Strain Activity (nmol / mg · min) HB101 HB 101 (pMW-PA3m-gapA) 39 86

As can be seen from Table 7, the level of GAPDH activity in HB101 cells containing the plasmid is 2 times higher.

Then the plasmid pMW-PA3m-gapA was introduced into streptomycin, a resistant producer strain of threonine E. coli B-3996 (US patent 5,175,107). Thus, strain B-3996 (pMW-PA3m-gapA) was obtained.

Both strains of E. coli B-3996 and B-3996 (pMWl 19 - pA3-gapA) were grown overnight at 37 ° C on plates with L-agar, or, in the case of strain B-3996 (pMWl 19 - pA3- gapA), on L-agar plates containing ampicillin (100 μg / ml). One microbiological loop (OD595 ~ 2 - 3 p.u.) of the night culture of each strain was introduced into fermentation tubes with 2 ml of the minimum fermentation medium of the following composition: yeast extract - 2.0 g / l, (NH ₄ ) ₂ SO ₄ - 16.0 g / l, K ₂ HPO ₄ - 0.7 g / l, MgSO ₄ x 7H ₂ O - 1.0 g / l, MnSO ₄ x 5H ₂ O - 0.01 g / l, FeSO ₄ x 7H ₂ O - 0.01 g / l, thiamine HCl (Bi) - 0.2 mg / l, glucose - 4%, CaCO ₃ (chalk) -30.0 g / l, L-isoleucine - 50 mg / l, ampicillin - 100 μg / ml (only for strain 3996 ( pMWl 19 - pA3-gapA)). Cells were grown for 48 hours at a temperature of 32 ° C on a rotary shaker at 250 rpm.

After fermentation, the amount of L-threonine accumulated in the medium was determined by paper chromatography. The liquid phase of the following composition was used: n-butanol: acetic acid: water = 4: 1: 1 (volume ratio). Amino acids were stained with an alcoholic solution of ninhydrin (1%) containing CdCl ₂ (0.5%). After 1 hour incubation at 37 ° C, samples were measured with ODsog. The results are presented in Table 8.

Table 8.

Strain OD ₅₅₅ Threonine, g / l B-3996 13.2 ± 0.1 13.6 ± 0.2 B-3996 (pMW-PA3m-gapA) 13.3 ± 0.1 15.6 ± 0.3

As can be seen from Table 8, increased expression of the gapA gene increases the production of L-threonine by strain B-3996.

Example 6: Cloning of the eno gene from E. coli and the effect of increasing the expression of the eno gene on L-threonine production.

The eno gene was obtained by PCR, chromosomal DNA from strain MG 1655 (VKPM B-6195) E. coli was used as a template, and primers P9 (SEQ ID NO: 29) and P10 (SEQ ID NO: 30) were used as primers ) Primer P9 contains a BamHI restriction enzyme recognition site at the 5'-end. Primer P10 contains a Sad restriction enzyme recognition site at the 5'-end. The obtained DNA fragment (1298 bp), including the eno gene, was treated with BamYQ and Sad restriction enzymes, and then cloned into the plasmid vector pMWl 19, previously modified by replacing the Pfd promoter with the pr promoter of the lambda phage, also treated with the indicated restriction enzymes. Thus, the plasmid pMW-PR-eno containing the eno gene under the control of the pr promoter was obtained. By using this plasmid, a high level of expression of the eno gene can be achieved due to the lack of its regulation.

Plasmid pMW-Pp-eno was introduced into streptomycin, a resistant producer strain of threonine E. coli B-3996 (US patent 5,175,107). Thus, strain B-3996 (pMW-PR-eno) was obtained.

The procedure for determining the amount of L-threonine accumulated in the nutrient medium by E. coli B-3996 and B-3996 strains (pMW-PR-eno) is described above (see Example 1). The results are presented in Table 9.

Table 9.

Strain OD ₅₆₀ Threonine, g / l B-3996 9.7 ± 0.1 14.3 ± 0.1 B-3996 (pMW-PR-eno) 9.3 ± 0.2 14.9 ± 0.4

As can be seen from Table 9, increased expression of the fbaA gene increases the production of L-threonine by strain B-3996.

Example 7: Cloning of the pgi gene from E. coli and the effect of increasing the expression of the pgi gene on the production of L-threonine.

The pgi gene was obtained by PCR, chromosomal DNA from strain MO 1655 (VKPM B-6195) E. coli was used as a template, and primers P11 (SEQ ID NO: 31) and P12 (SEQ ID NO: 32) were used as primers ) Primer P11 contains a BamHI restriction enzyme recognition site at the 5 ′ end. Primer P12 contains a Sad restriction enzyme recognition site at the 5 ′ end. The obtained DNA fragment (1657 bp), including the pgi gene, was treated with restriction enzymes BamHI and Sad, and then cloned into the plasmid vector pMWI 19, previously modified by replacing the P _lac promoter with the pr promoter of the lambda phage, also treated with these restriction enzymes. Thus, the plasmid pMW-Pp-pgi containing the pgi gene under the control of the pr promoter was obtained. By using this plasmid, a high level of pgi gene expression can be achieved due to the lack of its regulation.

Plasmid pMW-PR-pgi was introduced into streptomycin, a resistant producer strain of threonine E. coli B-3996 (US patent 5,175,107). Thus, strain B-3996 (pMW-PR-pgi) was obtained.

The procedure for determining the amount of L-threonine accumulated in the culture medium by E. coli B-3996 and B-3996 strains (pMW-PR-pgi) is described above (see Example 1). The results are presented in Table 10.

Strain OD ₅₆₀ Threonine, g / l B-3996 B-3996 (pMW-PR-pgi) 8.7 ± 0.4 8.4 ± 0.2 18.4 ± 0.7 19.7 ± 0.4

As can be seen from Table 10, increased expression of the pgi gene increases the production of L-threonine by strain B-3996.

Example 8: Cloning of the pgk gene from E. coli and the effect of increasing pgk gene expression on L-threonine production.

The pgk gene was obtained by PCR, chromosomal DNA from strain MG 1655 (VKPM B-6195) E. coli was used as a template, and primers P13 (SEQ ID NO: 33) and P14 (SEQ ID NO: 34) were used as primers ) Primer P13 contains a BamHI restriction enzyme recognition site at the 5 ′ end. Primer P14 contains a Sad restriction enzyme recognition site at the 5 ′ end. The obtained DNA fragment (1163 bp), including the pgk gene, was treated with restriction enzymes BamHI and Sad, after which it was cloned into the plasmid vector pMWl 19, previously modified by replacing the P _lac promoter with the pr promoter of the lambda phage, also treated with these restriction enzymes. Thus, the rmw-pr-pgk plasmid containing the pgk gene under the control of the pr promoter was obtained. Thanks to the use of this plasmid, a high level of pgk gene expression can be achieved due to the lack of its regulation.

Plasmid pMW-Pp-pgk was introduced into streptomycin, a resistant producer strain of threonine E. coli B-3996 (US patent 5,175,107). Thus, strain B-3996 (pMW-PR-pgk) was obtained.

The procedure for determining the amount of L-threonine accumulated in the culture medium by E. coli B-3996 and B-3996 strains (pMW-PR-pgk) is described above (see Example 1). The results are presented in Table 11.

Table 11.

Strain OD ₅₆₀ Threonine, g / l B-3996 B-3996 (pMW-PR-pgk) 10.3 ± 0.5 10.2 ± 0.5 19.2 ± 0.2 20.3 ± 0.4

As can be seen from Table 11, increased expression of the pgk gene increases the production of L-threonine by strain B-3996.

Although the invention has been described in detail with reference to specific Examples, it will be apparent to those skilled in the art that various changes may be made and equivalent replacements may be made, and such changes and replacements are not outside the scope of the present invention.

Claims (7)

1. A bacterium belonging to the genus Escherichia is a producer of L-threonine, characterized in that said bacterium is modified so that expression of a gene selected from the group consisting of glk, pgi, pfkA, tpiA, gapA, pgk genes is enhanced eno and pykA encoding glycolysis enzymes. 2. The bacterium according to claim 1, characterized in that the expression of the gene is enhanced by increasing the number of copies of the gene, or by modifying the sequence that controls the expression of the specified gene in such a way that the expression of this gene is enhanced. 3. The bacterium according to claim 2, characterized in that the number of copies of the gene is increased due to the transformation of the bacterium using a low copy vector containing the specified gene. 4. The bacterium according to claim 1, characterized in that said genes are derived from a bacterium belonging to the genus Escherichia. 5. The bacterium according to claim 4, characterized in that said bacterium is additionally modified so that the specified bacterium has enhanced expression of one or more genes selected from the group consisting of: a mutant thrA gene encoding aspartokinase-homoserine dehydrogenase I resistant to threonine inhibition by feedback; thrB gene encoding homoserine kinase; thrC gene encoding threonine synthase; rhtA gene encoding a putative transmembrane protein. 6. The bacterium according to claim 5, characterized in that said bacterium is modified in such a way that the expression of the mutant thrA gene, thrB gene, thrC gene and rhtA gene is enhanced. 7. A method for producing L-threonine, comprising the steps of growing a bacterium producing L-threonine in a culture medium and isolating L-threonine from the culture fluid, characterized in that the bacterium according to claim 1 is used as the producer.

Images