RU2351646C2 - METHOD OF PRODUCING L-threonine WITH USING BACTERIUM OF Escherichia TYPE - Google Patents

Abstract

FIELD: chemistry, biochemistry.

SUBSTANCE: invention refers to biotechnology and represents method of producing L-threonine by glucose fermentation with using bacterium of Escherichia type modified so that the specified bacterium is characterised with intensified activity of high-affinity D-allose carrier coded with operon alsABC.

EFFECT: high-efficient production of L-threonine.

10 cl, 6 dwg, 2 tbl, 12 ex

Description

Technical field

The present invention relates to a method for producing L-amino acids by fermentation and, in particular, to genes that contribute to the fermentation process. These genes are useful for improving the productivity of L-amino acids, such as, for example, L-threonine, L-lysine, L-leucine, L-histidine, L-cysteine, L-phenylalanine, L-arginine, L-tryptophan, L- glutamic acid, L-valine, and L-isoleucine.

Description of the Related Art

Typically, L-amino acids are produced on an industrial scale using fermentation methods using strains of microorganisms isolated from natural sources, or their mutants. In most cases, microorganisms are modified in such a way as to increase the production yield of L-amino acids.

To date, many methods have been described to increase the production yield of L-amino acids, including the transformation of microorganisms using recombinant DNA (see, for example, US patent 4,278,765). Other methods for increasing production yield include enhancing the activity of enzymes involved in amino acid biosynthesis and / or reducing the sensitivity of the target enzyme to feedback inhibition by the final L-amino acid (see, for example, PCT application WO 95/16042 or US patents 4,346,170; 5,661,012 and 6,040,160).

The strains used to produce L-threonine are known, including 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 and European patent EP 0219027), strains resistant to chemical products such as L-threonine and its analogues (PCT application WO 01 / 14525A1, European application EP 301572 A2, US patent 5,376,538), strains in which key enzymes are not sensitive to feedback inhibition produced by L-amino acid or its derivatives (US patents 5,175,107 ; 5,661,012), and strains in which the farm nts involved in the destruction of threonine are inactivated (US patents 5,939,307; 6,297,031).

Currently, one of the most famous producers of threonine is the producer strain of threonine Escherichia coli VKPM B-3996 (US patents 5,175,107 and 5,705,371). When constructing the VKPM B-3996 strain, several mutations and the plasmid described below were inserted into the parent E. coli K-12 strain (VKPM B-7). The mutant thrA gene (thrA442 mutation) encodes aspartokinase homoserine dehydrogenase I, which is resistant to feedback threonine inhibition. The mutant ilvA gene (ilvA442 mutation) encodes a threonine deaminase, which has increased activity, which leads to a decrease in the rate of isoleucine biosynthesis and the manifestation of the leaky isoleucine starvation phenotype. In bacteria containing the ilvA442 mutation, transcription of the thrABC operon is not inhibited by isoleucine; therefore, this mutation leads to a very effective production of threonine. Inactivation of the tdh gene encoding threonine dehydrogenase prevents the destruction of threonine. The genetic determinant of sucrose assimilation (scrKYABR genes) has been transferred to this strain. To enhance the expression of genes that control the threonine biosynthesis, plasmid pVIC40 containing the thrA442BC mutant threonine operon was introduced into the intermediate strain TDH6. The amount of L-threonine accumulated during the fermentation of the strain can reach 85 g / l.

By optimizing the main biosynthesis pathway of the desired compound, further improvement of the L-amino acid producing strains can be achieved by supplying the bacteria with an increased amount of sugars as a carbon source, for example glucose or arabinose. Despite the efficient glucose transport by the PTS system, the reduction of a highly productive strain by a carbon source, however, may not be sufficient.

It is known that the active transport of sugars and other metabolites in bacterial cells is carried out by several transport systems. One of them is the AlsABC carrier.

ABC transport systems designed to administer or remove nutrients and other substances through the cell membrane are widespread in nature. For most bacterial systems, the main factor determining the specificity of the transport complex as a whole is the periplasmic component (Chaudhuri BN, Co. J, Park C, Jones TA, Mowbray SL .; J Mol Biol. 1999 Mar 12; 286 (5): 1519-31 )

Periplasmic protein-binding transport systems consist of a periplasmic substrate-binding protein, two (sometimes one) very hydrophobic integral membrane proteins, and one (sometimes two) hydrophilic surface membrane protein that binds and hydrolyzes ATP. These systems form the superfamily of ABC carriers (Saurin W, Dassa E .; Protein Sci. 1994 Feb; 3 (2): 325-44).

The AlsABC carrier belongs to the superfamily of ATP-binding cassette transport proteins (ABC-ATP-binding Cassette). He is responsible for the uptake of D-allose, hexose with hydroxyl groups in the cis position, which can be used by the E.coli bacterium as the sole carbon source. Deletion of als genes led to the inability of the bacterium to grow on D-allose. Using fluorescence spectroscopy, it was found that the AlsB protein is a periplasatic protein that binds D-allose with a dissociation constant K _d = 0.33 μM. According to sequence similarity analysis, the AlsA protein is an ATP-binding component, and the AlsC protein is a membrane component of an ABC carrier. The AlsABC system also tolerates low affinity ribose. Complementary analysis of als mutants showed that cloned als genes can restore bacterial growth in minimal ribose medium. A study of transcriptionally fusion proteins with β-galactosidase led to the assumption that the AlsR protein is a negative regulator of the alsABC genes, and the transcription of the alsR gene is regulated by allose (Kim C, Song S, Park C .; J Bacteriol. 1997 Dec; 179 (24) : 7631-7).

The operon responsible for the metabolism of D-allose was localized at 92.8 minutes on the linkage map of the genetic traits of E. coli. It consists of six genes, alsRBACEK, which are induced by D-allose and are controlled by the alsR repressor regulatory gene encoding the repressor protein. This operon is also subject to catabolite repression. Three genes, alsB, alsA, and alsC, are required for D-allose transport. The D-allose binding protein, which is encoded by the alsB gene, is a periplasmic protein with Mr = 32.779, which has affinity for D-allose with K _d = 0.33 μM. By analogy with another intermediate ABC transporter binding protein, it was found that the allose transport system includes an ATP binding component (AlsA) and a transmembrane protein (AlsC). The AlsA protein has a molecular weight of Mr = 56.614 and is encoded by the alsA gene, which is located after the alsB gene. A protein with a molecular weight of Mr = 34.185 is encoded by the alsC gene, which is located after the alsA gene. It was found that the AlsE protein (presumably D-allose-6-phosphate-3-epimerase), but not the AlsK protein (presumably D-allose kinase), is required for allose metabolism. The D-allose transporter is partially involved in the low-affinity transport of D-ribose (Kirn C, Song S, Park C .; J Bacteriol. 1997 Dec; 179 (24): 7631-7).

However, there are currently no reports describing the use of Enterobacteriaceae family bacteria with enhanced expression of the alsABC operon to increase L-amino acid production by glucose fermentation.

Brief Description of the Drawings

Figure 1 shows the relative position of the primers P1 and P2 on the plasmid pMW 118-attL-Cm-attR.

Figure 2 shows the construction of a chromosomal DNA fragment containing the inactivated ptsHI-crr operon.

Figure 3 shows the replacement of the natural promoter region of the alsABC operon in E. coli with the hybrid promoter P _L-tac .

Figure 4 shows the influence of P _L-tac alsABC on growth PTS ^- strain. MG1655 means E. coli strain MG1655; MGΔpts means E. coli strain MG1655ΔptsHI-crr; and MGΔpts-P-alsABC means E. coli strain MG1655 ΔptsHI-crr P _L-tac alsABC.

Figure 5 shows the result of alignment of the primary AlsB sequences from Yersinia pseudotuberculosis (YERPS), Yersinia pestis (YERPE), Bacillus subtilis (BACSU), Bacillus cereus (BACC1), Escherichia coli (ECOLI), Symbiobacterium thermophilum (SYMTH). Alignment was performed using the PIR Multiple Alignment program (http://pir.georgetown.edu). Identical amino acids are marked with an asterisk (*), similar amino acids are marked with a colon (:).

Figure 6 shows the result of alignment of the primary AlsA sequences from Yersinia pestis (YERPE), Bacillus cereus (BACC1), Bacillus subtilis (BACSU), Yersinia pseudotuberculosis (YERPS), Escherichia coli (ECOLI), Symbiobacterium thermophilum (SYMTH). Alignment was performed using the PIR Multiple Alignment program (http://pir.georgetown.edu). Identical amino acids are marked with an asterisk (*), similar amino acids are marked with a colon (:).

Figure 7 shows the alignment result of the primary Als C sequences from Escherichia coli (ECOLI), Symbiobacterium thermophilum (SYMTH), Bacillus cereus (BACC1), Bacillus subtilis (BACSU), Yersinia pestis (YERPE), Yersinia pseudotuberculosis (YERPS). Alignment was performed using the PIR Multiple Alignment program (http://pir.georgetown.edu). Identical amino acids are marked with an asterisk (*), similar amino acids are marked with a colon (:).

Description of the invention

The objectives of the present invention are to increase the productivity of strains producing L-amino acids and providing a method for producing non-aromatic and aromatic L-amino acids using these strains of bacterial cells.

The above objectives were achieved by establishing the fact that increased expression of the alsABC operon encoding the D-allose transporter can lead to increased productivity of L-amino acids such as L-threonine, L-lysine, L-leucine, L-histidine, L- cysteine, L-glutamic acid, L-phenylalanine, L-tryptophan, L-valine, L-isoleucine and L-arginine by fermentation using glucose as a carbon source. The carbon source deficiency was modeled by removing the PTS transport system (ptsHI-crr) in the L-amino acid producing strain.

The aim of the present invention is the provision of bacteria producing L-amino acids of the Enterobacteriaceae family, modified in such a way that the expression of the alsABC operon is enhanced in the bacterium.

It is also an object of the present invention to provide the bacterium described above, wherein said alsABC operon expression is enhanced by modifying the sequence controlling the alsABC operon expression so that gene expression is enhanced, or by increasing the number of copies of the alsABC operon.

It is also an object of the present invention to provide the bacteria described above, wherein said bacterium is selected from the group consisting of bacteria of the genus Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Providencia, Salmonella, Serratia, Shigella and Morganella.

It is also an object of the present invention to provide a bacterium as described above, wherein said operon encodes:

(A) a protein comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof;

(B) a protein comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof; and

(C) a protein comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof;

however, in the case when these options are present in the specified operon, such options have the activity of high-affinity carriers of D-allose, when these options are combined together.

It is also an object of the present invention to provide a bacterium as described above, wherein said operon includes:

(A) a DNA fragment containing a nucleotide sequence with nucleotide numbers 1 to 936 in SEQ ID NO: 1, or a DNA fragment that hybridizes with the specified sequence or with a probe synthesized based on the specified sequence under stringent conditions;

(B) a DNA fragment comprising a nucleotide sequence with nucleotide numbers 1 to 1533 in SEQ ID NO: 3, or a DNA fragment that hybridizes with the specified sequence or with a probe synthesized based on the specified sequence under stringent conditions; and

(C) a DNA fragment comprising a nucleotide sequence with nucleotide numbers 1 to 981 in SEQ ID NO: 5, or a DNA fragment that hybridizes with said sequence or with a probe synthesized based on said sequence under stringent conditions; and

in this case, when said DNA fragments are present in said operon that hybridize with said sequences or with said probes, such DNA fragments encode for proteins having high affinity D-allose transporter activity when said proteins are combined together.

It is also an object of the present invention to provide the bacteria described above, wherein the stringent conditions are those under which washing is carried out at a temperature of 60 ° C. at a salt concentration of 1 × SSC and 0.1% SDS, for approximately 15 minutes .

It is also an object of the present invention to provide a bacterium as described above, wherein said bacterium is further modified so as to increase glucokinase activity.

It is also an object of the present invention to provide a bacterium as described above, wherein said bacterium is further modified so as to increase xylose isomerase activity.

It is also an object of the present invention to provide a bacterium as described above, wherein said bacterium is a bacterium producing L-threonine.

It is also an object of the present invention to provide a bacterium as described above, wherein said bacterium is further modified in such a way that expression of a gene selected from the group including:

- a mutant thrA gene that encodes aspartokinase-homoserine dehydrogenase I and is resistant to feedback threonine inhibition;

- thrB gene, which encodes homoserine kinase;

- thrC gene, which encodes threonine synthase;

- the rhtA gene, which encodes a presumably transmembrane protein;

- the asd gene, which encodes aspartate β-semi-aldehyde dehydrogenase;

- aspC gene, which encodes aspartate aminotransferase (aspartate transaminase); and

a combination of these genes.

It is also an object of the present invention to provide a bacterium as described above, wherein said bacterium is a bacterium producing L-lysine.

It is also an object of the present invention to provide a bacterium as described above, wherein said bacterium is a bacterium producing L-histidine.

It is also an object of the present invention to provide a bacterium as described above, wherein said bacterium is a bacterium producing L-phenylalanine.

It is also an object of the present invention to provide a bacterium as described above, wherein said bacterium is a bacterium producing L-arginine.

It is also an object of the present invention to provide a bacterium as described above, wherein said bacterium is a bacterium producing L-tryptophan.

It is also an object of the present invention to provide a bacterium as described above, wherein said bacterium is a bacterium producing L-glutamic acid.

Another objective of the present invention is the provision of a method for producing L-amino acids, which includes the cultivation of the bacteria described above in a nutrient medium containing glucose as a carbon source, and the allocation of L-amino acids from the culture fluid.

It is also an object of the present invention to provide the method described above, wherein said L-amino acid is an L-threoin.

It is also an object of the present invention to provide the method described above, wherein said L-amino acid is L-lysine.

It is also an object of the present invention to provide the method described above, wherein said L-amino acid is L-histidine.

It is also an object of the present invention to provide a process as described above, wherein said L-amino acid is L-phenylalanine.

It is also an object of the present invention to provide the method described above, wherein said L-amino acid is L-arginine.

It is also an object of the present invention to provide the method described above, wherein said L-amino acid is L-tryptophan.

It is also an object of the present invention to provide a process as described above, wherein said L-amino acid is L-glutamic acid.

Detailed Description of the Best Mode for Carrying Out the Invention

According to the present invention, “L-amino acid producing bacterium” means a bacterium capable of producing and secreting an L-amino acid into a culture medium when the bacterium of the present invention is grown in said culture medium. The ability of a bacterium to produce an L-amino acid can be imparted or increased by excretion. As used herein, the term “L-amino acid producing bacterium” also means a bacterium that is capable of producing L-amino acid and causes the accumulation of L-amino acid in fermentation medium in large quantities, compared with the natural or parent E. coli strain, such as strain E .coli K-12, and preferably means that the microorganism is able to accumulate in the medium the target L-amino acid in an amount of not less than 0.5 g / l, more preferably not less than 1.0 g / l. The term “L-amino acid” includes L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine , L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine. The amino acids L-threonine, L-lysine, L-histidine, L-phenylalanine, L-arginine, L-tryptophan, and L-glutamic acid are preferred.

The Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Enterobacter, Envinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, etc. More specifically, bacteria classified as belonging to the Enterobacteriaceae family according to the taxonomy used in the NCBI database (National Center for Biotechnology Information) (http://www.ncbi.nlm.nih.gov/htbinpost/Taxonomy/ can be used. wgetorg? mode = Tree & id = 1236 & lvl = 3 & keep = 1 & srchmode = 1 & unlock). A bacterium belonging to the genera Escherichia or Pantoea is preferred.

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, the bacterium Escherichia coli (E. coli) may 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), can be included in the number of bacteria according to the present invention.

The term "bacterium belonging to the genus Pantoea" means that the bacterium belongs to the genus Pantoea in accordance with the classification known to the specialist in the field of microbiology. Recently, several Enterobacter agglomerans species have been classified as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or the like based on analysis of the 16S rRNA nucleotide sequence, etc. (Int. J. Syst. Bacteriol., 43, 162-173 (1993)).

The bacterium of the present invention includes a strain of the Enterobacteriaceae family that is capable of producing an L-amino acid and is modified so that the expression of the alsABC operon in this bacterium is enhanced. In addition, the bacterium of the present invention includes a strain of the Enterobacteriaceae family that is capable of producing an L-amino acid and is transformed with a DNA fragment encoding the alsABC operon so that the D-allose transporter components encoded by this DNA fragment are expressed.

The phrase “high affinity D-allose transporter activity” means the activity of transferring sugars, such as D-allose and glucose, into a cell. The activity of the high affinity D-allose transporter can be detected and measured using membrane vesicles, such as those described by Darwalla et al (Biochem J., 200 (3), 611-27 (1981)) or by complementing a strain with delegated operon genes alsABC of the high affinity D-allose transporter (Horazdovsky, BF and Hogg, RW, J. Bacteriol; 171 (6): 3053-9 (1989)).

The term “enhancing operon expression” means that the expression level of the genes that make up the operon is higher than that of an unmodified strain, for example, a natural strain. Examples of such a modification include increasing the number of copies of the operon gene (s) per cell and / or increasing the expression level of the operon gene (s) and the like. The number of copies of the operon gene is measured, for example, using a number of known methods, such as Southern blotting using probes synthesized based on the nucleotide sequence of the operon genes, in situ fluorescence hybridization (FISH), and similar methods. The level of expression of the operon genes can be measured using a number of known methods, including Northern blotting, quantitative RT-PCR and the like. In addition, natural strains that can act as a control include, for example, Escherichia coli K-12 or Pantoea ananatis FERM BP-6614 strains (PCT application WO 2004099426, AU 2004236516 A1). The strain Pantoea ananatis FERM BP-6614 was deposited at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, currently called the National Institute of Progressive Industrial Science and Technology, International Depository of Organisms for Patenting Goals, February 19, 1998 and received accession number PERM P-16644. Then, the strain was internationally deposited in accordance with the conditions of the Budapest Treaty of January 11, 1999, and the strain received accession number PERM BP-6614. Although this strain was identified as Enterobacter upon isolation, however, it was later classified as Pantoea ananatis based on the 16S rRNA nucleotide sequence and other evidence previously provided. As a result of the increased intracellular activity of the D-allose transporter, L-amino acids, for example, L-threonine, L-lysine, L-histidine, L-phenylalanine, L-tryptophan or L-glutamic acid, are found in the culture medium.

The alsABC operon includes three genes in the following sequence. The alsB gene (synonyms - ESK4081, b4088, yjcX) encodes a protein that binds D-allose (synonyms - B4088, YjcX). The alsB gene (nucleotides complementary to nucleotides numbered 4,309,130 to 4,310,065 in the nucleotide sequence with accession number NC_000913 in the GenBank database; gi: 49175990) is located on the chromosome of E. coli K-12 strain between the rpiR and alsA genes. The alsA gene (synonyms - ECC4080, b4087, yjcW) encodes the fused subunits of the D-allose transporter of the ABC superfamily: ATP-binding components of the D-allose transporter (synonyms - B4087, YjcW). The alsA gene (nucleotides complementary to nucleotides numbered 4,307,471 to 4,309,003 in the nucleotide sequence with accession number NC_000913.2 in the GenBank database; gi: 16131913) is located on the chromosome of E. coli strain K-12 between the alsB and alsC genes. The alsC gene (synonyms - yjcV, ECK4079, b4086, JW4047) encodes a subunit of the D-allose transporter (synonyms - B4086, YjcV). The alsC gene (nucleotides complementary to nucleotides numbered 4,306,512 to 4,307,492 in the nucleotide sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located on the chromosome of E. coli strain K-12 between the alsA and alsE genes. Other alsABC operons are known as: the alsABC operon from Shigella dysenteriae serotype I, Shigella sonney, Erwinia carotovora subsp. atroseptica, Yersinia pestis, Yersiniapseudotuberculosis, Pseudomonas pseudomallei, Pseudomonas mallei, Pseudomonas solanacearum. The nucleotide sequences of the alsB, alsA, and alsC genes from Escherichia coli are shown in the Sequence Listing Numbers: SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively. Amino acid sequences encoded by the alsB, alsA, and alsC genes are listed in the Sequence Listing Numbers SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively

When transported into a cell, glucose is phosphorylated by glucokinase, which is encoded by the glk gene. Therefore, it is desirable to modify the bacterium in such a way as to enhance the activity of glucokinase. The glk gene that encodes glucokinase in Escherichia coli is known (nucleotide numbers 2506481 to 2507446 in the nucleotide sequence with accession number NC_000913.1 in the GenBank database; gi: 16127994) located on the chromosome of E. coli strain K-12 between reading frames b2387 and b2389.

Under certain conditions, xylose isomerase encoded by the xylA gene also effectively catalyzes the conversion of D-glucose to D-fructose (Wovcha, M.G. et al, AppI Environ Microbiol. 45 (4): 1402-4 (1983)). Therefore, it is desirable to modify the bacterium in such a way as to enhance the activity of xylose isomerase. The xy1A gene, which encodes xylose isomerase in Escherichia coli, is known (nucleotide numbers 3728788 to 3727466 in the nucleotide sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) located on the chromosome of E. coli K-12 strain between xylB genes and xylF.

The alsABC, glk, and xylA genes can be obtained by PCR (polymerase chain reaction; link to White, T.J. et al., Trends Genet., 5, 185 (1989)) using primers synthesized based on known nucleotide gene sequences. Genes encoding a D-allose transporter from other microorganisms can be obtained in a similar manner.

Examples of alsABC operon genes are DNA fragments isolated from Escherichia coli that encode the following proteins:

(A) a protein comprising the amino acid sequence shown in the List of sequences under the number SEQ ID NO: 2 and its variants;

(B) a protein comprising the amino acid sequence shown in the List of sequences under the number SEQ ID NO: 4 and its variants;

(B) a protein comprising the amino acid sequence shown in the List of sequences under the number SEQ ID NO: 6 and its variants.

The term "protein variant" in the sense in which it is used in the present invention means a protein that has changes in the amino acid sequence, namely deletion, insertion, addition or replacement of amino acids, provided that this maintains the necessary level of protein activity, for example , a level of activity that provides enhanced production of L-amino acids. A number of changes in the variant of the protein depends on the position or type of amino acid residue in the three-dimensional structure of the protein. 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 protein sequences numbered 2, 4, 6 (SEQ ID NO: 2 , 4, 6). These changes can occur in areas of the protein that are not critical to its function. This is possible due to the fact that amino acids have high homology with each other, and therefore the tertiary structure or activity of the protein is not violated by such a change. Therefore, as a variant of the protein can be a protein that has a homology of not less than 70%, preferably not less than 80%, more preferably not less than 90% and most preferably not less than 95% with respect to the complete amino acid sequence represented on SEQ ID NOs. 2, 4, 6, provided that the activity of the D-allose transporter is maintained when this protein is combined with the corresponding components of the high-affinity D-allose transporter. The following components can serve as an example of the components with which the high-affinity D-allose transporter can be combined: the protein variant shown in the Sequence Listing under the number SEQ ID NO: 2 is combined with proteins having the amino acid sequence shown in the Sequence Listing under the SEQ numbers ID NO: 4 and SEQ ID NO: 6, a variant of the protein shown in the List of sequences under the number SEQ ID NO: 4 is combined with proteins having the amino acid sequence shown in the List of sequences numbers under the numbers SEQ ID NO: 2 and SEQ ID NO: 6, and the protein variant shown in the List of sequences under the number SEQ ID NO: 6 is combined with proteins having the amino acid sequence shown in the List of sequences under the numbers SEQ ID NO: 2 and SEQ ID NO: 4. Homology between amino acid sequences can be established using well-known methods, for example, using sequence alignment in the BLAST 2.0 computer program, which calculates three parameters: count, identity, and similarity.

Substitution, deletion, insertion, addition or inversion of one or more amino acid residues will be a conservative mutation or conservative mutations, provided that the protein activity is maintained. An example of a conservative mutation (s) is a conservative substitution (s). Examples of conservative substitutions include replacing Ala with Ser or Thr, replacing Arg with Gin, His or Lys, replacing Asn with Glu, Gin, Lys, His or Asp, replacing Asp with Asn, Glu or Gin, replacing Cys with Ser or Ala, replacing Gin with Asn, Glu, Lys, His, Asp or Arg, replacing Glu with Asn, Gin, Lys or Asp, replacing Gly with Pro, replacing His with Asn, Lys, Gin, Arg or Tyr, replacing Not with Leu, Met, Val or Phe, replace Leu with He, Met, Val or Phe, replace Lys with Asn, Glu, Gin, His or Arg, replace Met with Ile, Leu, Val or Phe, replace Phe with Trp, Tyr, Met, He or Leu, replacing Ser with Thr or Ala, replacing Thr with Ser or Ala, replacing Trp with Phe or Tyr, replacing Tyr with His, Phe or Trp, and replacing Val with Met, He or Leu.

Comparison results of the primary amino acid sequences of alsABC proteins from Escherichia coli (ECOLI), Symbiobacterium thermophilum (SYMTH), Bacillus cereus (BACC1), Bacillus subtilis (BACSU), Yersinia pestis (YERPE), Yersinia pseudotuberculosis or high YERPS proteins (see Figure 5, Figure 6, Figure 7). From this point of view, substitutions or deletions of amino acid residues that are identical (marked with an asterisk (*)) in all of the above proteins may be critical for their function. It is possible that replacing similar amino acid residues (marked by a colon (:)) with similar amino acid residues will not lead to disruption of protein activity. But modifications of other non-conservative amino acid residues may not lead to disruption of the activity of the high-affinity D-allose transporter.

DNA fragments that encode essentially the same proteins that are components of the D-allose transporter can be obtained, for example, by modifying the nucleotide sequences of DNA fragments encoding the components of the D-allose transporter (SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, respectively), for example, by a site-directed mutagenesis method, such that one or more amino acid residues at a particular site are involved in deletion, substitution, insertion, or addition. DNA fragments modified as described above can be obtained using conventional mutagenic processing techniques. Such treatment includes hydroxylamine treatment of DNA molecules encoding the proteins of the present invention or treatment of bacteria containing said DNA with UV rays or a reagent such as N-methyl-N'-nitro-N-nitrosoguanidine or nitrous acid,

DNA fragments that encode essentially the same proteins that are components of the D-allose transporter can be obtained by expressing DNA fragments having the mutation described above in the corresponding cell and establishing the activity of the expressed product. DNA fragments that encode essentially the same proteins that are components of the D-allose transporter can also be obtained by isolating DNA fragments that hybridize with probes having a nucleotide sequence that contains, for example, any of the nucleotide sequences listed in the List sequences under the numbers SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 under stringent conditions, and encodes proteins having the activity of components of the D-allose transporter. "Stringent conditions", as used in this expression in the framework of the present invention, means those conditions under which the so-called specific hybrids are formed and non-specific hybrids are not formed. For example, a demonstration of stringent conditions may be conditions under which DNA fragments having high homology, for example, DNA fragments having a homology of not less than 50%, more preferably not less than 60%, more preferably not less than 70%, even more preferable not less than 80%, even more preferably not less than 90% and most preferably not less than 95% are able to hybridize with each other, and DNA fragments having a homology lower than that described above are unable to hybridize with each other. In addition, conditions that can be used to demonstrate stringent conditions are those under which the DNA fragments hybridize at a salt concentration equivalent to the single wash conditions for Southern hybridization, which are 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 is generally recommended by the kit manufacturer. For example, the recommended washing time for a Hybond ™ N + nylon membrane (Amersham, USA) under severe conditions is approximately 15 minutes. It is advisable to repeat the washing 2-3 times. Partial nucleotide sequences shown in the Sequence Listing under the numbers SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 can also be used as probes. Probes can be prepared by PCR using primers synthesized based on the nucleotide sequences shown in the sequence Listing numbers SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and based on DNA fragments containing nucleotide sequences, given in the List of sequences under the numbers SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 as a matrix. In cases where a DNA fragment having a length of about 300 bp is used as a probe, washing conditions during the hybridization process include, for example, 50 ° C, 2 × SSC and 0.1% SDS.

Substitution, deletion, insertion, or addition of nucleotides, as described above, also includes a mutation that occurs in nature (mutant or variant), for example, due to the variability of species or genera of bacteria that contain components of the D-allose transporter.

"Transformation of a bacterium using a DNA fragment encoding a protein" means the introduction of a DNA fragment into a bacterium, for example, by well-known methods. Transformation of this DNA will increase expression of the gene encoding the protein of the present invention and increase the activity of the protein in the bacterial cell. Transformation methods include any known methods that have been described to date. For example, a method for treating recipient cells with calcium chloride in order to increase cell permeability for DNA has been described for strain Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970 )) and can be used.

Methods for enhancing gene expression include increasing the number of copies of the gene. The insertion of a gene into a vector that is able to function in bacteria of the Enterobacteriaceae family increases the number of copies of this gene. It is preferable to use low-copy vectors. Examples of low copy vectors include, but are not limited to, plasmids pSC101, pMW118, pMW119, and the like. The term "low copy vector" is used to denote vectors whose number of copies does not exceed five copies per cell.

Enhanced gene expression can also be achieved by introducing multiple copies of the gene into the bacterial chromosome using, for example, homologous recombination method, Mu integration and the like. For example, one Mu integration cycle allows you to embed up to three copies of a gene in the bacterial chromosome.

Enhanced gene expression of the alsABC operon can also be achieved by introducing multiple copies of the alsABC operon genes into the bacterial chromosome. To introduce multiple copies of the indicated operon into the bacterial chromosome, homologous recombination is carried out using a sequence whose multiple copies are present as targets in the chromosomal DNA. Sequences having multiple copies in chromosomal DNA include, but are not limited to, repeating DNAs or inverted repeats present at the end of a transposable element. As described in US Pat. No. 5,595,889, it is possible to incorporate the alsABC operon into the transposon and thereby transfer and incorporate multiple copies of the gene into the chromosomal DNA.

Enhanced gene expression of the specified operon can also be produced by controlling gene expression using a strong promoter. For example, the lac, trp, and trc promoters, as well as the P _R or P _L lambda phage promoters, are known as strong promoters. Using a strong promoter can be combined with an increase in the number of copies of the gene.

On the other hand, the promoter can be enhanced, for example, by introducing a mutation into the specified promoter in order to increase the level of transcription of the gene located on the chromosome after the promoter. Further, it is known that the replacement of several nucleotides in the region between the binding site of the ribosome (RBS) and the start codon, and especially in the sequence immediately before the start codon, significantly affects the translatability of mRNA. For example, a 20-fold change in expression level was detected depending on the nature of the three nucleotides prior to the start of the codon (Gold et al., Annu. Rev. Microbiol., 35, 365-403, 1981; Hui et al., EMBO J., 3, 623-629, 1984), As described above, the authors of the present invention found that the rhtA23 mutation is a substitution of A for G at 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). 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 substrates exported from the cell.

Moreover, it is possible to introduce nucleotide substitutions into the promoter region of the BCAT gene in the bacterial chromosome in such a way that this 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 00/18935 or Japanese Patent Application Laid-open No. 1-215280 A.

Methods for obtaining plasmid DNA, cutting and ligation of DNA, transformation, selection of oligonucleotides as primers 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 methods and guidelines described above for increasing the activity of an allose transporter are also applicable for increasing the activity of xylose isomerase and glucokinase.

The bacterium of the present invention can be obtained by incorporating the above DNA fragments into a bacterium that already has the ability to produce L-amino acids. Moreover, the bacterium according to the present invention can be obtained by giving the bacterium containing these DNA fragments the ability to produce L-amino acids.

L-amino acid producing bacterium

As a bacterium according to the present invention, modified so that alsABC gene expression is enhanced, a bacterium capable of producing L-amino acids can be used.

The bacterium of the present invention can be obtained by enhancing the expression of alsABC genes in bacteria already capable of producing L-amino acids. On the other hand, the bacterium of the present invention can be obtained by imparting a bacterium in which the expression of alsABC genes has already been enhanced to produce L-amino acids.

L-threonine producing bacterium

Examples of the parent strain for the production of the L-threonine-producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain TDH-6 / pVIC40 (VKPM B-3996) (US patents 5175107 and 5705371 ), E. coli strain NRRL-21593 (US patent 5939307), E. coli strain FERM BP-3756 (US patent 5474918), E. coli strains FERM BP-3519 and FERM BP-3520 (US patent 5376538), 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.

The TDH-6 strain is defective in the thrC gene, is capable of assimilating sucrose, and contains the ilvA gene with a leaky mutation. 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, 117105 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 , 117545 Moscow, 1st Road passage, 1) April 7, 1987 with inventory number VKPM B-3996.

The E. coli strain VKPM B-5318 (EP 0593792 B) can also be used as the parent strain to obtain the L-threonine-producing bacteria according to the present invention. Strain B-5318 is prototrophic against isoleucine, and the temperature-sensitive repressor of phage lambda C1 and the PR promoter replace the regulatory region of the threonine operon in plasmid pVIC40. The strain VKPM B-5318 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF, 117545 Moscow, 1st Dorozhniy proezd, 1) on May 03, 1990 with accession number VKPM B-5318.

Preferably, the bacterium of the present invention is further modified so as to have increased expression of one or more of the following genes:

- 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;

- rhtA gene, presumably encoding a transmembrane protein;

the asd gene encoding aspartate β-semialdehyde dehydrogenase, and

- aspC gene encoding aspartate aminotransferase (aspartate transaminase).

The nucleotide sequence of the thrA gene encoding aspartokinase-homoserine dehydrogenase I from Escherichia coli is known (nucleotide numbers 337 to 2799 in sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990). The thrA gene is located on the chromosome of E. coli K-12 strain between the thrL and thrB genes. The nucleotide sequence of the thrB gene encoding homoserine kinase from Escherichia coli is known (nucleotide numbers 2801 to 3733 in sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990). The thrB gene is located on the chromosome of E. coli K-12 strain between the thrA and thrC genes. The nucleotide sequence of the thrC gene encoding a threonine synthase from Escherichia coli is known (nucleotide numbers 3734 to 5020 in the sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990). The thrC gene is located on the chromosome of the E. coli K-12 strain between the thrB gene and the open uaaX reading frame. All three of these genes function as one threonine operon. To enhance expression of the threonine operon, it is desirable to remove the region of the attenuator that disrupts transcription from the operon (PCT application WO 2005/049808, WO 2003/097839).

The mutant thrA gene encoding aspartokinase homoserine dehydrogenase I, which is resistant to threonine inhibition by feedback, as well as the thrB and thrC genes, can be obtained as a single operon from the well-known plasmid pVIC40, which is represented in the producer strain E. coli VKPM B-3996. Plasmid pVIC40 is described in detail in US patent 5,705,371.

The rhtA gene is located at the 18th minute of the E. coli chromosome near the glnHPQ operon, which encodes the components of the glutamine transport system, the rhtA gene is identical to ORF1 (ybiF gene, nucleotide numbers 764 to 1651 in sequence with accession number AAA218541 in the GenBank database, gi: 440181) , located between the genes pXB and ompX. A region of DNA expressed to form the protein encoded by the ORF1 reading frame was named the rhtA gene (rht: resistance to homoserine and threonine). The rhtA13 mutation has also been shown to represent an A-to-G substitution at position -1 with respect 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, EP 1013765 A).

The nucleotide sequence of the asd gene from E. coli is known (nucleotide numbers 3572511 to 3571408 in sequence with accession number NC_000913.1 in the GenBank database, gi: 16131307) and can be obtained using PCR (polymerase chain reaction; link to White, TJ et al., Trends Genet., 5, 185 (1989)) using primers synthesized based on the nucleotide sequence of the gene. Asd genes from other microorganisms can be obtained in a similar way.

Also, the nucleotide sequence of the aspC gene from E. coli is known (nucleotide numbers 983742 to 984932 in the sequence with accession number NC_000913.1 in the GenBank database, gi: 16128895) and can be obtained by PCR. AspC genes from other microorganisms can be obtained in a similar way.

L-lysine producing bacterium

Examples of bacteria producing L-lysine belonging to the genus Escherichia include mutants that are resistant to the L-lysine analogue. The L-lysine analogue inhibits the growth of bacteria belonging to the genus Escherichia, but this inhibition is completely or partially removed when L-lysine is also present in the medium. Examples of the L-lysine analogue include, but are not limited to, oxalysine, lysine hydroxyamate, S- (2-aminoethyl) -L-cysteine (AEC), γ-methyllysis, α-chlorocaprolactam, and so on. Mutants that are resistant to these lysine analogues can be obtained by treating bacteria belonging to the genus Escherichia with traditional mutagens. Specific examples of bacterial strains used to produce L-lysine include Escherichia coli strain AJ11442 (FERM BP-1543, NRRL B-12185; see US Pat. No. 4,346,170) and Escherichia coli strain VL611. In these microorganisms, aspartokinase is resistant to feedback inhibition by L-lysine.

Strain WC196 can be used as a bacterium producer of L-lysine Escherichia coli. This bacterial strain was obtained by selection of the phenotype of resistance to AEC in strain W3110, derived from the strain Escherichia coli K-12. The resulting strain was named Escherichia coli AJ13069 and was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, currently called the National Institute of Advanced Industrial Science and Technologies, International Organizational Depository for Patenting, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan (National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibarak i-ken, 305-8566, Japan), December 6, 1994, and received FERM P-14690 inventory number. Then, the strain was internationally deposited in accordance with the terms of the Budapest Treaty on September 29, 1995, and the strain received accession number FERM BP-5252 (see US Pat. No. 5,827,698).

Examples of parental strains for producing L-lysine producing bacteria of the present invention also include strains in which the expression of one or more genes encoding L-lysine biosynthesis enzymes is enhanced. Examples of enzymes involved in the biosynthesis of L-lysine include, but are not limited to, dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate carboxylase (lysA), diaminopromedolate ppc), aspartate semialdehyde dehydrogenase (asd), nicotinamide adenine dinucleotide transhydrogenase (pntAB) and aspartase (aspA) (European application EP 1 253 195 A). In addition, parental strains may have an increased expression level of the gene involved in the respiration process (cyo) (European application EP 1170376 A), the gene encoding nicotinamide nucleotranshydrogenase (pntAB) (US patent 5,830,716), the ybjE gene (PCT application WO 2005/073390) , or combinations of these genes.

Examples of parent strains for the production of L-lysine producing bacteria according to the present invention also include strains in which enzyme activity is reduced or absent which catalyze the formation of compounds other than L-lysine branching off from the main L-lysine biosynthesis pathway. Examples of enzymes that catalyze the formation of non-L-lysine compounds branching off from the main L-lysine biosynthesis pathway include homoserine dehydrogenase, lysine decarboxylase (US Pat. No. 5,827,698) and malate dehydrogenase (PCT application WO 2005/010175).

L-cysteine producing bacterium

Examples of parental strains used to produce L-cysteine-producing bacteria according to the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain JM15 transformed with various cysE alleles encoding inhibition resistant cysE feedback type serine acetyltransferase (US patent 6,218,168, patent application of the Russian Federation 2003121601); E. coli strain W3110 containing overexpressed genes encoding a protein capable of secretion of compounds toxic to the cell (US Pat. No. 5,972,663); E. coli strains containing cysteine desulfohydrase with reduced activity (Japanese patent JP 11155571 A2); E. coli strain W3110 with increased activity of the positive transcriptional regulator of the cysteine regulon encoded by the cysB gene (international application PCT WO 0127307 A1) and the like.

L-Leucine Producer Bacteria

Examples of parent strains used to produce the L-leucine producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strains that are resistant to leucine analogues, including, for example, β-2 -thienylalanine, 3-hydroxyleucine, 4-azaleucine and 5,5,5-trifluoroleucine (Japanese Patent Laid-open 62-34397 and 8-70879), E. coli strains obtained using the genetic engineering methods described in PCT 96 / 06926; E. coli strain H-9068 (JP 8-70879 A2), and the like.

The bacterium of the present invention can be improved by enhancing the expression of one or more genes involved in the biosynthesis of L-leucine. Examples of such genes include the leuABCD operon genes, and are preferably represented by the mutant leuA gene encoding feedback feedback depleted L-leucine isopropyl malate synthase (US Pat. No. 6,403,342). In addition, the bacterium of the present invention can be improved by enhancing the expression of one or more genes encoding proteins that export the L-amino acid from a bacterial cell. Examples of such genes include the b2682 and b2683 genes (ygaZH genes) (European application EP 1239041 A2).

L-histidine producing bacterium

Examples of parent strains used to produce L-histidine producing bacteria of the present invention include, but are not limited to, L-histidine producing bacteria belonging to the genus Escherichia, such as E. coli 24 strain (VKPM B-5945, patent RF 2003677); E. coli strain 80 (VKPM B-7270, RF patent 2119536); E. coli strains NRRL B-12116 - B12121 (US Pat. No. 4,388,405); E. coli strains H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (US Pat. No. 6,344,347); E. coli strain H-9341 (FERM BP-6674) (European Patent 1085087); E. coli strain AI80 / pFM201 (US patent 6,258,554) and the like. Examples of parent strains for producing bacteria producing L-histidine according to the present invention also include strains in which the expression of one or more genes encoding L-histidine biosynthesis enzymes is enhanced. Examples of enzymes involved in the biosynthesis of L-histidine include ATP-phosphoribosyltransferase (hisG), phosphoribosyl-AMP-cyclohydrolase (hisi), phosphoribosyl-ATP-phosphohydrolase (hisIE), phosphoribosylformimino-5-aminamide amide amide amide amide amide amide amide azide) histidinolphosphate aminotransferase (hisC), histidinolphosphatase (hisB), histidinol dehydrogenase (hisD), etc.

It is known that genes encoding L-histidine biosynthesis enzymes (hisG, hisBHAFI) are inhibited by L-histidine, therefore, the ability to produce L-histidine can also be significantly enhanced by introducing a feedback inhibition mutation into the ATP-gene phosphoriboside transferase (hisG) (RF patents. 2003677 and 2119536).

Specific examples of strains with the ability to produce L-histidine include E. coli FERM-P 5038 and 5048, into which a vector containing DNA encoding the L-histidine biosynthesis enzyme (Japanese application 56-005099 A), strains E. coli into which the rht gene has been introduced for the export of amino acids (European application EP 1016710 A), strain E. coli 80, which is given resistance to sulfaguanidine, DL-1,2,4-triazole-3-alanine and streptomycin (VKPM B- 7270, RF patent 2119536), etc.

L-glutamic acid producing bacterium

Examples of parental strains used to produce the L-glutamic acid producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain VL334 thrC ⁺ (European patent EP 1172433). Strain E. coli VL334 (VKPM B-1641) is an auxotroph of L-isoleucine and L-threonine with mutations in the thrC and ilvA genes (US patent 4,278,765). The natural allele of the thrC gene was transferred to this strain by the general transduction method using bacteriophage P1 grown on cells of the natural E. coli K12 strain (VKPM B-7). The result was a strain, auxotroph for L-isoleucine, VL334 thrC ⁺ (VKPM B-8961). This strain has the ability to produce L-glutamic acid.

Examples of parent strains used to produce the L-glutamic acid producing bacterium of the present invention include, but are not limited to, strains in which the expression of one or more genes encoding L-glutamic acid biosynthesis enzymes is enhanced. Examples of the enzymes involved in the biosynthesis of L-glutamic acid include glutamate dehydrogenase, glutamine synthetase, glutamate synthetase, isocitrate dehydrogenase, aconitate hydratase, citrate synthase, phosphoenolpyruvate carboxylase, pyruvate carboxylase, pyruvate dehydrogenase, pyruvate kinase, phosphoenolpyruvate synthase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, fructose, phosphofructoninase and glucose phosphatisomerase.

Examples of strains modified in such a way that the expression of the citrate synthetase gene, phosphoenolpyruvate carboxylase gene and / or glutamate dehydrogenase gene is enhanced include EP 1078989 A, EP 955368 A and EP 952221 A described in European applications.

Examples of parental strains for producing L-glutamic acid producing bacteria according to the present study also include strains in which enzyme activity is reduced or absent that catalyzes the synthesis of compounds other than L-glutamic acid branching from the main pathway of L-glutamic acid biosynthesis. Examples of such enzymes include isocitrate lyase, α-ketoglutarate dehydrogenase, phosphotransacetylase, acetate kinase, acetohydroxy acid synthase, acetolactate synthase, format acetyl transferase, lactate dehydrogenase and glutamate decarboxylase. Bacteria belonging to the genus Escherichia, lacking the activity of α-ketoglutarate dehydrogenase or having reduced activity of α-ketoglutarate dehydrogenase and methods for their preparation are described in US patents 5,378,616 and 5,573,945. Specifically, examples of such strains include the following strains:

E.coli W3110sucA :: Km ^R

E. coli AJ12624 (FERM BP-3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJ12949 (FERM BP-4881)

E. coli W3110sucA :: Km ^R is a strain obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter referred to as the “sucA gene”) in E. coli strain W3110. In this strain, the activity of α-ketoglutarate dehydrogenase is completely absent.

Other examples of bacteria producing L-glutamic acid include bacteria belonging to the genus Escherichia and resistant to aspartic acid antimetabolites and deficient in the activity of α-ketoglutarate dehydrogenase, for example, strain AJ13199 (FERM BP-5807) or US patent 5,908,768) strain FERM P-12379, additionally having low activity for the cleavage of L-glutamic acid (US patent 5,393,671); E. coli strain AJ13138 (FERM BP-5565) (US patent 6,110,714) and the like.

Examples of the L-glutamic acid producing bacterium include mutant strains belonging to the genus Pantoea that are devoid of α-ketoglutarate dehydrogenase activity or have reduced α-ketoglutarate dehydrogenase activity, and can be obtained as described above. Examples of such strains are Pantoea ananatis AJ13356 strain (US patent 6,331,419), Pantoea ananatis AJ13356 strain deposited at the National Institute of Biological Sciences and Human Technologies, Agency for Industrial Science and Technology, Department of International Trade and Industry, National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry) (currently called the National Institute of Advanced Industrial Science and Technology, International Organizational Depository for Patenting Purposes, Central 6, 1-1, Higashi 1-Ch Ome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan - National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) February 19, 1998 and received accession number FERM P-16645. Then, the international deposit of this strain was made according to the conditions of the Budapest Treaty of January 11, 1999, and the strain received accession number PERM BP-6615. The Pantoea ananatis strain AJ13356 does not have α-KGDH activity as a result of the destruction of the αKGDH-E1 gene subunit (sucA). The above strain, when isolated, was identified as Enterobacter agglomerans and deposited as Enterobacter agglomerans AJ13356 strain. However, it was later classified as Pantoea ananatis based on the 16S rRNA nucleotide sequence and other evidence. Although the strain AJ13356 was deposited in the above depository as Enterobacter agglomerans, for the purposes of this description it will be referred to as Pantoea ananatis.

L-phenylalanine producing bacterium

Examples of parental strains used to produce the L-phenylalanine-producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as strain AJ12739 (tyrA :: Tn10, tyrR) (VKMP B-8197); strain HW1089 (ATCC-55371) containing the pheA34 gene (US Pat. No. 5,354,672); mutant strain MWEC101-b (KR8903681); strains NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (US patent 4,407,952) and the like. Also, bacteria belonging to the genus Escherichia, producers of L-phenylalanine, such as E. coli K-12 strain [W3110 (tyrA) / pPHAB] (PERM BP-3566), E. coli K strain, can be used as parent strains. -12 [W3110 (tyrA) / pPHAD] (FERM BP-12659), E. coli K-12 strain [W3110 (tyrA) / pPHA Term] (FERM BP-12662) and E. coli K-12 strain [W3110 ( tyrA) / pBR-aroG4, pACMAB], named as AJ12604 (FERM BP-3579) (European patent EP 488424 B1). In addition, L-phenylalanine producing bacteria belonging to the genus Escherichia with increased activity of the proteins encoded by the yedA gene or yddG gene can also be used (US patent applications 2003/0148473 A1 and 2003/0157667 A1).

L-tryptophan producing bacterium

Examples of parent strains used to produce L-tryptophan producing bacteria according to the present invention include, but are not limited to, L-tryptophan producing bacteria belonging to the genus Escherichia, such as E. coli strains JP4735 / pMU3028 (DSM10122) and JP6015 / pMU91 (DSM10123) lacking the activity of tryptophanyl tRNA synthetase encoded by the mutant trpS gene (US Pat. No. 5,756,345); E. coli strain SV164 (pGH5) containing an allele of the serA gene encoding an enzyme not inhibited by serine by feedback type (US patent 6,180,373); E. coli strains AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) lacking tryptophanase activity (US Pat. No. 4,371,614); E. coli strain AGX17 / pGX50, pACKG4-pps, which enhances the ability to synthesize phosphoenolpyruvate (PCT application WO 9708333, US patent 6,319,696), and the like.

It was previously shown that the natural allele of the yddG gene, which encodes a membrane protein that is not involved in the biosynthesis of any of the L-amino acids, amplified on a multi-copy vector in a microorganism, gives this microorganism resistance to L-phenylalanine and several analogues of this amino acid. In addition, the introduction of additional copies of the yddG gene into the cells of bacteria producing L-phenylalanine or L-tryptophan can positively affect the production of the corresponding amino acids (PCT international application WO 03044192). Thus, it is desirable that the bacterium producing L-tryptophan be further modified so that expression of the open reading frame yddG is enhanced in this bacterium.

Examples of parent strains used to produce the L-tryptophan producing bacterium of the present invention also include strains in which the activity of one or more enzymes selected from the group consisting of anthranilate synthase, phosphoglycerate dehydrogenase, and tryptophan synthase is increased. Both anthranilate synthase and phosphoglycerate dehydrogenase are feedback inhibited by L-tryptophan and L-serine, so mutations can be introduced into these enzymes that reduce the sensitivity to feedback inhibition. Specific examples of strains with such a mutation include E. coli SV164, whose anthranilate synthase is not sensitive to feedback inhibition, and a transformant strain obtained by introducing the plasmid pGH5 into E. coli SV164 (PCT application WO 94/08031), which contains the mutant gene serA encoding phosphoglycerate dehydrogenase, which is not sensitive to feedback inhibition.

Examples of parental strains used to produce the L-tryptophan producing bacterium of the present invention also include strains that have introduced a tryptophan operon containing a gene encoding anthranilate synthase that is not sensitive to feedback inhibition (Japanese application 57- 71397 A, Japanese Patent Application 62-244382 A, U.S. Patent 4,371,614). In addition, the ability to produce L-tryptophan can be imparted by enhancing the expression of a gene (from the tryptophan operon) encoding tryptophan synthase (trpBA). Tryptophan synthase consists of two subunits α and β, which are encoded by trpA and trpB, respectively. In addition, the ability to produce L-tryptophan can be increased by enhancing the expression of the isocytratliase-malatesynthase operon (PCT application WO 2005/103275).

L-proline producing bacterium

Examples of L-proline producing bacteria used as the parent strain of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 702ilvA (VKPM B-8012) deficient in the ilvA gene and capable of producing L-proline (European patent EP 1172433). The bacterium of the present invention can be improved by enhancing the expression of one or more genes involved in the biosynthesis of L-proline. Preferably, examples of such genes for bacterium-producing L-proline include the proB gene encoding glutamate kinase with desensitized feedback regulation of L-proline (German patent 3127361). In addition, the bacterium of the present invention can be improved by enhancing the expression of one or more genes encoding proteins that secrete the L-amino acid from a bacterial cell. Examples of such genes are the b2682 and b2683 genes (ygaZH genes) (European Patent Application EP 1239041 A2).

Examples of bacteria belonging to the genus Escherichia and having the ability to produce L-proline include the following E. coli strains: NRRL B-12403 and NRRL B-12404 (UK patent GB 2075056), VKPM B-8012 (RF patent application 2000124295), plasmid mutants described in German patent DE 3127361, plasmid mutants described in Bloom FR et al (The 15 ^th Miami winter symposium, 1983, p. 34), and the like.

L-arginine producing bacterium

Examples of parental strains used to produce the L-arginine producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 237 (VKPM B-7925) and its derivatives containing mutant N -acetylglutamate synthase (RF patent application 2001112869), an arginine producing strain into which the argA gene encoding N-acetylglutamate synthetase (Japanese Patent Application Laid-Open No. 57-5693) and the like are introduced.

Examples of parental strains used to produce the L-arginine producing bacterium of the present invention also include strains in which the expression of one or more genes encoding L-arginine biosynthesis enzymes is enhanced. Examples of L-arginine biosynthesis enzymes include N-acetylglutamylphosphate reductase (argC), ornithine acetyl transferase (argJ), N-acetyl glutamate kinase (argB), acetylornithine transaminase (argD), ornithinecarbamoyl transferase (argN), argin acid, argN) and carbamoyl phosphate synthetase (carAB).

L-Valine producing bacteria

Examples of parental strains used to excrete L-valine producing bacteria of the present invention include, but are not limited to strains that are modified such that expression of the ilvGMEDA operon is enhanced (US Pat. No. 5,998,178). It is desirable to remove the region of the ilvGMEDA operon necessary for attenuation so that the expression of the opreron is not reduced by the produced L-valine. In addition, it is desirable to destroy the ilvA gene in the operon so that threonine deaminase activity is reduced.

Examples of parent strains used to excrete L-valine producing bacteria of the present invention also include mutant strains having a mutation of the aminoacyl-t-RNA synthetase enzyme (US Pat. No. 5,658,766). For example, you can use the E. coli strain VL1970, carrying a mutation in the ileS gene, which encodes the isoleucine t-RNA synthetase enzyme. The strain E. coli VL1970 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF, 117545 Moscow, 1st Dorozhniy proezd, 1) June 24, 1988 with accession number VKPM B-4411.

In addition, mutant strains that require lipoic acid and / or the absence of H ⁺ -ATPase for their growth can also be used as parent strains (PCT application WO 96/06926).

L-isoleucine-producing bacteria

Examples of parental strains used for the excretion of bacteria-producers of L-valine according to the present invention include, but are not limited to strains having mutations of resistance to 6-dimethylaminopurine (Japanese patent application JP 5-304969 A) having mutations of resistance to isoleucine analogues, such as thioisoleucine and isoleucine hydroxamate and mutant strains having additional resistance to DL-ethionine and / or arginine hydroxate (Japanese Patent Application JP 5-130882 A). Additionally, recombinant strains transformed with genes encoding proteins involved in the biosynthesis of L-isoleucine, such as threonine deaminase and acetohydroxate synthase (Japanese patent application JP 2-458 A, French patent FR 0356739, and US patent 5,998,178) can be used as parent strains. .

The method according to the present invention

Oxaloacetate (OAA) serves as a substrate for the reaction, which leads to the synthesis of threonine (Thr) and lysine (Lys). OAA is formed from phosphoenolpyruvate (PEP) in the presence of phosphoenolpyruvate carboxylase (PEPC), which acts as a catalyst. Therefore, increasing the concentration of PEPC in the cell can be very important for the enzymatic production of these amino acids. When glucose is used as a carbon source in the fermentation process, glucose is absorbed by the cell through the glucose phosphotransferase system (Glc-PTS). This system consumes PEP, and the proteins in the PTS are encoded by the ptsG and ptsHIcrr genes. During internalization, one PEP molecule and one pyruvate (Pyr) molecule are formed from one glucose molecule.

The strains producing L-threonine and L-lysine, which are modified in such a way that they acquire the ability to absorb sucrose (Scr-PTS), have higher productivity with respect to these amino acids when grown on sucrose than when grown on glucose (European patent application EP 1149911 A2). It is believed that three PEP molecules and one Pyr molecule are formed from one sucrose molecule with the participation of Scr-PTS, thereby increasing the PEP / Pyr ratio and therefore facilitating the synthesis of threonine (Thr) and lysine (Lys) from sucrose. In addition, Glc-PTS has been reported to be regulated by several expression control systems (Postma PW et al., Microbiol Rev., 57 (3), 543-94 (1993); dark B. et al. J. Gen. Microbiol 96 (2), 191-201 (1976); Plumbridge J., Curr. Opin. Microbiol., 5 (2), 187-93 (2002); Ryu S. et al ,, J. Biol. Chem., 270 (6): 2489-96 (1995)), and therefore it is possible that the incorporation of glucose as such may be a rate-limiting step in the process of producing an amino acid during fermentation.

Increasing the PEP / Pyr ratio to an even greater extent by enhancing the expression of the alsABC operon in the threonine producer strain, in the lysine producer strain, in the histidine producer strain, in the phenylalanine producer strain, in the arginine producer strain, in the tryptophan producer strain, and / or in a glutamic acid producer strain, should further increase amino acid production. Since four PEP molecules are formed from two glucose molecules, it is expected that the PEP / Pyr ratio will increase significantly. Due to increased expression of the alsABC operon, glc-PTS expression control is expected to weaken.

The method according to the present invention is a method for producing an L-amino acid, comprising the steps of growing a bacterium according to the present invention in a culture medium for the purpose of producing and accumulating an L-amino acid in a culture fluid, and isolating the L-amino acid from the culture fluid.

According to the present invention, the cultivation, isolation and purification of an L-amino acid from a culture or similar liquid may be carried out in a manner similar to traditional fermentation methods in which the amino acid is produced using a bacterium.

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. As vitamins, thiamine, yeast extract and the like can be used.

The cultivation is preferably carried out under aerobic conditions, for example, by stirring 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 ° C 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 with ammonia, calcium carbonate, various acids, bases and buffers. Usually, cultivation 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 the L-amino acid can be isolated and purified by ion exchange chromatography, concentration and / or crystallization.

Examples

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

Example 1. Construction of E. coli strain with impaired PTS transport system.

1. Removal of the ptsHI-crr operon

A strain containing a deletion of the ptsHI-crr operon was constructed using a method first developed by Datsenko, K.A. and Wanner, BL (Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640-6645) and named " Red-driven integration. " A DNA fragment containing the Cm ^R marker encoded by the cat gene was obtained by PCR using primers P1 (SEQ ID NO: 7) and P2 (SEQ ID NO: 8) and plasmid pMW118-attL-Cm-attR as a template ( PCT Application WO 05/010175). Primer P1 contains both a region complementary to the 36 nucleotide region localized at the 5 'end of the ptsHI-crr operon and a region complementary to the 24 nucleotides of the attL region. Primer P2 contains both a region complementary to the 36 nucleotide region localized at the 3 'end of the ptsHI-crr operon and a region complementary to the 24 nucleotides of the attR region. The following temperature profile was used for PCR testing: denaturation at 95 ° C for 3 minutes; profile for the first 2 cycles: 1 min at 95 ° C, 30 sec at 50 ° C, 40 sec at 72 ° C; profile for the last 25 cycles: 30 sec at 95 ° C, 30 sec at 54 ° C, 40 sec at 72 ° C; final step: 5 min at 72 ° C.

The resulting PCR product is a 1699 bp fragment. (Figure 2) was purified on an agarose gel and used for electroporation into E. coli strain MG1655 (ATCC 700926) containing the plasmid pKD46, the replication of which is sensitive to high temperature. Plasmid pKD46 (Datsenko, KA and Wanner, BL, Proc. Natl. Acad. Sci. USA, 2000, 97: 12: 6640-45) contains a phage λ DNA fragment (accession sequence number J02459 in the GenBank database) of 2.154 nucleotides (31088-33241), and also contains the λ Red genes of a homologous recombination system (γ, β, exo genes) under the control of the _{araB inducer} P _araB . Plasmid pKD46 is required for integration of the PCR product into the chromosome of strain MG1655,

Electrocompetent cells were prepared as follows: an overnight culture of E. coli strain MG1655 / pKD46 was grown at 30 ° C in LB medium containing ampicillin (100 mg / L), diluted 100 times with 5 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 (1 mM). The resulting culture was grown with aeration at 30 ° C until it reached an optical density of OD ₆₀₀ ≈0.6, after which the cells were made electrocompetent by concentrating them 100 times and washing three times with ice-cold deionized H ₂ O. Electroporation was performed using 70 μl of cells and ≈100 ng of the 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 chloramphenicol at a concentration of 30 μg / ml and grown at 37 ° C to select Cm ^R recombinants. Then, to remove plasmid pKD46, 2 passages were performed on L-agar with Cm at 42 ° C and the obtained colonies were tested for sensitivity to ampicillin.

2. Confirmation of the division of the ptsHI-crr operon by PCR

A mutant with the ptsHI-crr delegated operon containing the Cm resistance gene was checked by PCR. Locus-specific primers P3 (SEQ ID NO: 9) and P4 (SEQ ID NO: 10) were used to verify fission by PCR. The following temperature profile was used to verify fission by PCR: denaturation at 94 ° C for 3 min ; profile for 30 cycles: 30 sec at 94 ° C, 30 sec at 54 ° C, 1 min at 72 ° C; final step: 7 min at 72 ° C. The length of the PCR product obtained by the reaction using the parental strain ptsHI-crr ⁺ MG1655 as a matrix of cells was ~ 3.0 kb. (Figure 3). The mutant strain was named MG1655 Δ ptsHI-crr :: cat.

3. Removal of the Cm chloramphenicolol resistance gene (cat gene) from the chromosome of E. coli strains MG1655-Δ ptsHI-crr :: cat

The Cm resistance gene (cat gene) was removed from the chromosome of strain MG1655 Δ ptsHI-crr :: cat using the int-xis system. For this purpose, E. coli strain MG1655 ΔptsHI-crr :: cat was transformed with the plasmid pMWts-Int / Xis (PCT application WO 05/010175). Transformed clones were selected on LB medium containing ampicillin at a concentration of 100 μg / ml. Cells were incubated at 30 ° C. overnight. Transformed clones were freed from the cat gene by growing individual colonies at 37 ° C (at this temperature, the Cits repressor is inactivated to some extent, while the int / xis genes are activated), followed by selection of Cm ^S Ap ^R variants. Removal of the cat gene from the chromosome of the strains was confirmed by PCR. Locus-specific primers P3 (SEQ ID NO: 9) and P4 (SEQ ID NO: 10) were used for this confirmation by PCR. The PCR conditions were the same as described above. The PCR product obtained in the case of using cells with the deleted cat gene as a matrix was ~ 0.4 kb in length. Thus, a L-amino acid producing strain with the inactivated ptsHI-crr operon was obtained, from which the cat gene was deleted. This strain was named MG1655 Δ ptsHI-crr.

Example 2. Replacing the natural promoter of the alsABC operon with the hybrid P _L-tac promoter in E. coli.

To replace the natural promoter of the alsABC operon, a DNA fragment containing the P _L-tac hybrid promoter and the chloramphenicol resistance marker (Cm ^R ) encoded by the cat gene was inserted into the chromosome of E. coli strain MG1655 ΔptsHI-crr instead of the natural promoter using the method, described by Datsenko K.A. and Wanner BL (Proc. Natl. Acad. Sci. USA, 2000, 97: 6640-6645) and dubbed "Red-mediated integration" and / or "Red-dependent integration", which is also described in Example 1.

The hybrid promoter was obtained by PCR using the chromosomal DNA of E. coli strain B-3996P _L-tac xylE (PCT application WO 2006043730) as a matrix and primers P5 (SEQ ID NO 11 and P6 (SEQ ID NO: 12). described in Example 1.

The amplified DNA fragment was purified by agarose gel electrophoresis, extracted using GenElute Spin Columns (Sigma, USA), and reprecipitated using ethanol. The obtained DNA fragment was used for electroporation and Red-mediated integration into the chromosome of E. coli strain MG1655 ΔptsHI-crr / pKD46, as described in Example 1.

Colonies were grown for 24 hours and then tested for the presence of the Cm ^R marker instead of the natural promoter of the alsABC operon using PCR using primers P7 (SEQ ID NO: 13) and P8 (SEQ ID NO: 14). For this, the colony was suspended in water (20 μl) and the resulting suspension (1 μl) was used for PCR. The PCR conditions are described in Example 1. Several tested Cm ^R colonies contained the desired DNA fragment ~ 2.1 kb in size, which confirmed the presence of the P _L-tac hybrid promoter and the Cm ^R marker instead of the natural alsABC operon promoter of ~ 0.3 thousand bp (Figure 3). One of the obtained strains was freed from the heat-sensitive plasmid pKD46 by culturing at 37 ° C, whereby E. coli strain MG1655 ΔptsHI-crr P _L-tac alsABC was obtained.

The ability to grow on a minimal medium containing glucose (4%) as a carbon source was tested for three strains of E. coli: MG1655, MG1655ΔptsHI-crr, MG1655ΔptsHI-crr P _L-tac alsABC. As follows from Figure 4, the E. coli strain MG1655ΔptsHI-crr had a low growth rate (μ ~ 0.06) on minimal Adam medium containing glucose. The enhanced expression of the alsABC operon significantly improved the growth characteristics of the recipient strains on Adams minimal medium containing glucose.

Example 3. The effect of enhanced expression of the alsABC operon in E. coli strain with impaired PTS transport system on the production of L-threonine

In order to disrupt the PTS transport system in the E. coli VKPM B-3996 threonine-producing strain strain, the operon ptsH1-crr was inactivated on the chromosome of this strain. For this purpose, DNA fragments from the chromosome of the above E. coli strain MG1655 ΔptsHI-crr :: cat were transferred to E. coli strain VKPM B-3996 using P1 transduction (Miller, JH Experiments in Molecular Genetics, Cold Spring Harbor Lab, Press, 1972, Plainview, NY) to obtain strain B-3996-Δ ptsHI-crr :: cat.

Mutants with the deleted ptsHI-crr operon marked by the Cm resistance gene were checked by PCR. Locus-specific primers P3 (SEQ ID NO: 9) and P4 (SEQ ID NO: 10) were used in PCR for this check. The conditions for PCR testing are described above. The PCR product obtained in the reaction, where the parent strain ptsHI-crr ⁺ B-3996 was used as a matrix, was ~ 3.0 kb in length. The PCR product obtained in the reaction using the mutant strain B-3996 Δ ptsHI-crr :: cat as a matrix was ~ 2.0 kb in length. (Figure 2).

The Cm resistance gene (cat gene) was removed from the chromosome of E. coli strain B-3996 ΔptsHI-crr :: cat using the int-xis system. For this purpose, E. coli strain B-3996 ΔptsHI-crr :: cat was transformed with the plasmid pMWts-Int / Xis (PCT application WO 05/010175).

Transformed clones were selected on LB medium containing ampicillin at a concentration of 100 μg / ml. Cells were incubated at 30 ° C. overnight. Transformed clones were released from the cat gene by growing individual colonies at 37 ° C (at this temperature, the CIts repressor is inactivated to some extent, while the int / xis genes are activated), followed by selection of Cm ^S Ap ^R variants. Removal of the cat gene from the chromosome of the strains was confirmed by PCR. Locus-specific primers P3 (SEQ ID NO: 9) and P4 (SEQ ID NO: 10) were used for this confirmation by PCR. The PCR conditions were the same as described above. The PCR product obtained when cells with the cat gene were used as a matrix was ~ 0.3 kb in length. Thus, an Lts-threonine producer strain with the inactivated ptsHI-crr operon was obtained from which the cat gene was removed. This strain was named B-3996 ΔptsHI-crr.

To enhance the expression of the alsABC operon in the E. coli strain B-3996 ΔptsHI-crr, the natural promoter of the alsABC operon was replaced by the P _L-tac hybrid promoter. For this purpose, DNA fragments from the chromosome of the above E. coli strain MG1655 ΔptsHI-crr P _L-tac alsABC were transferred to E. coli strain B-3996 ΔptsHI-crr using P1 transduction (Miller, JH Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain E. coli strain B-3996-Δ ptsHI-crr P _L-tac alsABC.

Removal of the ptsHI-crr operon in E. coli strain B-3996-ΔptsHI-crr P _L-tac alsAB was verified by PCR. Locus-specific primers P3 (SEQ ID NO: 9) and P4 (SEQ ID NO: 10) were used in PCR for verification. The PCR conditions for verification are described above. The PCR product obtained in the reaction, where the strain B-3996-ΔptsHI-crr P _L-tac alsAB was used as a matrix, was ~ 0.3 kb in length.

The replacement of the natural promoter of the alsABC operon with the P _L-tac hybrid promoter and the Cm ^R marker in the E. coli strain B-3996-ΔptsHI-crr P _L-tac alsABC was verified by PCR. Locus-specific primers P7 (SEQ ID NO: 13) and P8 (SEQ ID NO: 14) were used in PCR for verification. The conditions of this PCR are given above. The PCR product obtained in the reaction where the strain B-3996-ΔptsHI-crr P _L-tac alsABC was used as a matrix was ~ 2.1 thousand bp in length.

Then, E. coli strains B-3996, B-3996-ΔptsHI-crr and B-3996-ΔptsHI-crr P _L-tac alsABC were each grown at 37 ° C for 18 hours in nutrient broth and then 0.3 ml each from the obtained cultures were inoculated in 3 ml of fermentation medium of the following composition into 20 × 200 mm test tubes and cultured at a temperature of 37 ° C for 72 hours on a rotary shaker.

After growing, the amount of L-threonine accumulated in the medium was determined by paper chromatography using the mobile phase of the following composition: butanol: acetic acid: water = 4: 1: 1 (v / v). A solution (2%) of ninhydrin in acetone was used for visualization. A spot containing L-threonine was excised; L-threonine was suirable 0.5% aqueous solution of CdCl ₂ , after which the amount of L-threonine was estimated spectrophotometrically at a wavelength of 540 nm. The results of five independent in vitro fermentations are shown in Table 1.

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

Glucose 80.0 (NH ₄ ) ₂ SO ₄ 22.0 K ₂ HPO ₄ 2.0 MgSO ₄ · 7H ₂ O 0.8 MnSO ₄ · 5H ₂ O 0.02 FeSO ₄ · 7H ₂ O 0.02 Thiamine 0.002 Yeast extract 1.0 CaCO ₃ 30.0

MgSO ₄ · 7H ₂ O and CaCO _{3 were} sterilized separately.

As follows from Table 1, strain B-3996-ΔptsHI-crr P _L-tac alsABC accumulated a greater amount of L-threonine compared to strain B-3996.

Example 4. Production of L-lysine by E. coli strain AJ11442-P _L-tac alsABC

To assess the effect of increased expression of the alsABC operon on lysine production, DNA fragments of the chromosome of the E. coli strain MG1655-ΔptsHI-crr P _L-tac alsAB described above can be transferred to the E. coli AJ11442 L-lysine producer strain using P1 transduction ( Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab, Press, Plainview, NY), whereby AJ11442-P _L-tac alsABC strain can be obtained.

Both strains of E. coli, AJ11442 and AJ11442-P _L-tac alsABC, can be grown in L-medium at 37 ° C; and 0.3 ml of the obtained cultures can be introduced into 20 ml of a fermentation medium containing the necessary antibiotics into 500 ml flasks. Cultivation can be carried out at 37 ° C for 16 hours using a reciprocating rocking chair with a stirring speed of 115 rpm. After growing, the amount of L-lysine and residual glucose in the medium can be measured in a known manner (Biotech-analyzer AS210, manufacturer - Sakura Seiki Co.). Then, for each of the strains, the yield of L-lysine in terms of glucose consumed can be calculated.

Can be used in a fermentation medium of the following composition (g / l):

Glucose 40 (NH ₄ ) ₂ SO ₄ 24 K ₂ HPO ₄ 1.0 MgSO ₄ · 7H ₂ O 1.0 FeSO ₄ · 7H ₂ O 0.01 MnSo ₄ · 5H ₂ O 0.01 Yeast extract 2.0

The pH was adjusted to 7.0 with KOH and the medium was autoclaved at 115 ° C for 10 minutes. Glucose and MgSO ₄ 7H ₂ O are sterilized separately. CaCO _{3 is} also added to a concentration of 30 g / l, previously sterilized by dry heat at 180 ° C for 2 hours.

Example 5. Production of L-cysteine by E. coli strain JM15 (ydeD) P _L-tac alsABC

To assess the effect of increased expression of the alsABC operon on L-cysteine production, DNA fragments of the chromosome of the E. coli strain MG1655-ΔptsHI-crr P described above _L-tac alsABC can be transferred to the E. coli JM15 L-cysteine producer strain (ydeD) c using P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby the JM15 (ydeD) -P _L-tac alsABC strain can be obtained.

The E. coli strain JM15 (ydeD) is a derivative of the E. coli strain JM15 (US patent 6,218,168), which can be transformed with DNA containing the ydeD gene encoding a membrane protein not involved in the biosynthesis of any of the L-amino acids (US patent 5,972,663 ) Strain JM15 (CGSC # 5042) can be obtained from the genetic collection of the University of New Haven in the USA (The E. coli Genetic Stock Center, Yale University, New Haven, USA), (http://cgsc.biology.yale.edu/ )

Fermentation conditions for evaluating L-cysteine production are described in detail in Example 6 of US Pat. No. 6,218,168.

Example 6. Production of L-leucine by E. coli strain 57-P _L-tac alsABC

To assess the effect of increased expression of the alsABC operon on the production of L-leucine, DNA fragments of the chromosome of the above E. coli strain MG1655-ΔptsHI-crr P _L-tac alsABC can be transferred to the E. coli 57 L-leucine producer strain (VKPM B- 7386, U.S. Patent 6,124,121) by P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby 57-P _L-tac alsABC strain can be obtained. Strain 57 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF, 117545 Moscow, 1st Dorozhniy proezd, 1) on May 19, 1997 with accession number VKPM B-7386.

Both strains of E. coli, 57 and 57-P _L-tac alsABC can be grown for 18-24 hours at 37 ° C on plates with L-agar. To obtain a seed culture, these strains can be grown on a rotary shaker (250 rpm) at 32 ° C for 18 hours in 20 × 200 mm test tubes containing 2 ml of L-broth with 4% sucrose. Then, 0.21 ml (10%) of the seed culture can be added to the fermentation medium. Fermentation can be carried out in 2 ml of minimal fermentation medium in test tubes measuring 20 × 200 mm. Cells can be grown for 48-72 hours at 32 ° C with stirring (250 rpm). The amount of L-leucine can be measured using paper chromatography (composition of the mobile phase: butanol - acetic acid - water = 4: 1: 1).

A fermentation medium (pH 7.2) of the following composition (g / l) can be used:

Glucose 60.0 (NH ₄ ) ₂ SO ₄ 25.0 K ₂ HPO ₄ 2.0 MgSO ₄ · H ₂ O 1.0 Thiamine 0.01 CaCO ₃ 25.0

Glucose and chalk are sterilized separately.

Example 7. The production of L-histidine strain E. coli 80-P _L-tac alsABC

To assess the effect of increased expression of the alsABC operon on L-histidine production, DNA fragments of the chromosome of the E. coli strain described above MG1655-ΔptsHI-crr P _L-tac alsABC can be transferred to the E. coli 80 L-histidine producer strain using P1- transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby the 80-P _L-tac alsABC strain can be obtained. Strain 80 is described in RF patent 2119536 and deposited in the All-Russian collection of industrial microorganisms (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) October 15, 1999 with accession number VKPM B-7270. Then, the international deposit of this strain was made according to the conditions of the Budapest Treaty on July 12, 2004.

Both strains of E. coli, 80 and 80-P _L-tac alsABC can be grown in L-broth at 29 ° C for 6 hours. Then, 0.1 ml of the obtained cultures can be introduced into 2 ml of fermentation medium in 20 × 200 mm test tubes, and the cultures can be grown at 29 ° С for 65 hours on a rotary shaker (350 rpm). After growing, the amount of histidine accumulated in the medium can be determined by paper chromatography. The mobile phase of the following composition can be used: p-butanol - acetic acid - water = 4: 1: 1 (v / v). A solution of ninhydrin (0.5%) in acetone can be used for visualization.

A fermentation medium (pH 6.0) of the following composition (g / l) can be used:

Glucose 100.0 Mameno (soybean hydrolyzate) 0.2 total nitrogen L-proline 1.0 (NH ₄ ) ₂ SO ₄ 25.0 KH ₂ PO ₄ 2.0 MgSO ₄ · 7H ₂ O 1.0 FeSO ₄ · 7H ₂ O 0.01 MnSO ₄ 0.01 Thiamine 0.001 Betaine 2.0 CaCO ₃ 60.0

Glucose, proline, betaine and CaCO _{3 are} sterilized separately. The pH is adjusted to 6.0 before sterilization.

Example 8. The production of L-glutamic acid strain E. coli VL334thrC ⁺ -P _L-tac alsABC

To assess the effect of increased expression of the alsABC operon on the production of L-glutamic acid, DNA fragments of the chromosome of the above E. coli strain MG1655-ΔptsHI-crr P _L-tac alsABC can be transferred to the L-glutamic acid producing strain E. VL334thrC ⁺ (EP 1172433) by P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby strain VL334thrC ⁺ -P _L-tac alsABC can be obtained. The strain VL334thrC ⁺ was deposited in the All-Russian collection of industrial microorganisms (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) on December 6, 2004 with accession number VKPM B-8961. Then, the international deposit of this strain was made according to the conditions of the Budapest Treaty on December 8, 2004.

Both strains, VL334thrC ⁺ and VL334thrC ⁺ -P _L-tac alsABC, can be grown on L-agar plates at 37 ° C for 18-24 hours. Further, one loop of cells can be transferred to tubes containing 2 ml of fermentation medium. The fermentation medium may contain glucose - 60 g / l, ammonium sulfate - 25 g / l, KH ₂ PO ₄ - 2 g / l, MgSO ₄ - 1 g / l, thiamine - 0.1 mg / ml, L-isoleucine - 70 μg / ml and CaCO ₃ - 25 g / l (pH 7.2). Glucose and chalk are sterilized separately. Cultivation can be carried out at 30 ° C for 3 days with stirring. After growing, the amount of L-glutamic acid obtained can be determined using paper chromatography (composition of the mobile phase: butanol - acetic acid - water = 4: 1: 1), followed by staining with ninhydrin (1% solution in acetone) and further eluting the obtained compounds in 50% ethanol with 0.5% CdCl ₂ .

Example 9. Production of L-phenylalanine by E. coli strain AJ12739-P _L-tac alsABC

To assess the effect of increased expression of the alsABC operon on L-phenylalanine production, DNA fragments of the chromosome of the E. coli strain described above MG1655-ΔptsHI-crr P _L-tac alsABC can be transferred to the E. coli A-J12739 L-phenylalanine producer strain using P1- transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby AJ12739-P _L-tac alsABC strain can be obtained. Strain AJ12739 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, ^1st Road Road, 1) November 6, 2001 with accession number VKPM B-8197. Then, the international deposit of this strain was made according to the conditions of the Budapest Treaty on August 23, 2002.

Both strains, AJ12739-P _L-tac alsABC and AJ12739, can be grown at 37 ° C for 18 hours in nutrient broth; 0.3 ml of the obtained cultures can be introduced into 3 ml of fermentation medium in 20 × 200 mm test tubes, and the cultures can be grown at 37 ° C for 48 hours on a rotary shaker. After fermentation, the amount of phenylalanine accumulated in the medium can be determined using thin layer chromatography (TLC). For this purpose, TLC plates 10 × 15 cm in size coated with a 0.11 mm layer of Sorbfil silica gel without a fluorescent indicator can be used (Sorbpolymer Joint-Stock Company, Krasnodar , Russia). Sorbfil plates can be exposed in the mobile phase of the following composition: propan-2-ol: ethyl acetate: 25% aqueous ammonia: water = 40: 40: 7: 16 (v / v). A solution (2%) of ninhydrin in acetone can be used for visualization.

A fermentation medium of the following composition (g / l) can be used:

Glucose 40.0 (NH ₄ ) ₂ SO ₄ 16.0 K ₂ HPO ₄ 0.1 MgSO ₄ · 7H ₂ O 1.0 FeSO ₄ · 5H ₂ O 0.01 MnSO ₄ · 5H ₂ O 0.01 Thiamine 0.0002 Yeast extract 2.0 Tyrosine 0.125 CaCO ₃ 20.0

Glucose and magnesium sulfate are sterilized separately. CaCO _{3 is} sterilized by dry heat at 180 ° C for 2 hours. The pH was adjusted to 7.0.

Example 10. Production of L-tryptophan by E. coli strain SV164 (pGH5) -P _L-tac alsABC

To assess the effect of increased expression of the alsABC operon on L-tryptophan production, DNA fragments of the chromosome of the above E. coli strain MG1655-ΔptsHI-crr P _L-tac alsABC can be transferred to the E. coli L-tryptophan producer strain coli SV164 (pGH5) with using P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby strain SV164 (pGH5) -P _L-tac alsABC can be obtained. Strain SV164 (pGH5) is described in detail in US patent 6,180,373 or European patent EP 0662143.

Both strains, SV164 (pGH5) -P _L-tac alsABC and SV164 (pGH5), can be grown with stirring at 37 ° C for 18 hours in 3 ml of nutrient broth with tetracycline (plasmid marker pGH5, 20 mg / ml) . 0.3 ml of the obtained cultures can be introduced into 3 ml of a fermentation medium containing tetracycline (20 mg / ml) in 20 × 200 mm test tubes; and cultures can be grown at 37 ° C. for 48 hours on a rotary shaker at 250 rpm. After growing, the amount of tryptophan accumulated in the medium can be determined by TLC, as described in Example 10.

The components of the fermentation medium that can be used are presented in Table 2; the groups of components A, B, C, D, E, F and H are sterilized separately, as shown in Table 2, in order to avoid unwanted interactions during sterilization.

Example 11. Production of L-Proline by E. coli strain 702ilvA-P _L-tac alsABC

To assess the effect of increased expression of the alsABC operon on L-proline production, DNA fragments from the chromosome of the E. coli strain described above MG1655-ΔptsHI-crr P _L-tac alsABC can be transferred to the E. coli 702ilvA L-proline producer strain using P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby strain 702ilvA-P _L-tac alsABC can be obtained. Strain 702ilvA was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, ^1st Road passage, 1) July 18, 2000 with accession number VKPM B-8012. Then, the international deposit of this strain was made according to the conditions of the Budapest Treaty on May 18, 2001.

Both strains of E. coli, 702ilvA and 702ilvAP _L-tac alsABC, can be grown for 18-24 hours at 37 ° C on plates with L-agar. Then fermentation using these strains can be carried out under the same conditions as described in Example 8.

Example 12. The production of L-arginine strain E. coli 382-P _L-tac alsABC

To assess the effect of enhanced expression of the alsABC operon on L-arginine production, DNA fragments of the chromosome of the E. coli strain described above MG1655-ΔptsHI-crr P _L-tac alsABC can be transferred to the E. coli 382 L-arginine producing strain using P1- transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby strain 382 -P _L-tac alsABC can be obtained. Strain 382 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, ^1st Road Road, 1) on April 10, 2000 with accession number VKPM B-7926. Then, the international deposit of this strain was made according to the conditions of the Budapest Treaty on May 18, 2001.

Both strains, 382-P _L-tac alsABC and 382, can be grown with stirring at 37 ° C for 18 hours in 3 ml of nutrient broth; 0.3 ml of the resulting cultures can be introduced into 2 ml of fermentation medium in 20 × 200 mm tubes, and cultures can be grown at 32 ° C for 48 hours on a rotary shaker.

After growing, the amount of L-arginine accumulated in the medium can be determined using paper chromatography, and the following composition of the mobile phase can be used: butanol: acetic acid: water = 4: 1: 1 (v / v). A solution of ninhydrin (2%) in acetone can be used for visualization. A stain containing L-arginine can be excised; L-arginine can be eluted with a 0.5% aqueous solution of CdCl ₂ , after which the amount of L-arginine can be determined spectrophotometrically at a wavelength of 540 nm.

A fermentation medium of the following composition (g / l) can be used:

Glucose 48.0 (NH ₄ ) ₂ SO ₄ 35.0 KN ₂ PO ₄ 2.0 MgSO ₄ · 7H ₂ O 1.0 Thiamine 0.0002 Yeast extract 1.0 L-isoleucine 0.1 CaCO ₃ 5.0

Glucose and magnesium sulfate are sterilized separately. CaCO _{3 is} sterilized by dry heat at 180 ° C for 2 hours. The pH was adjusted to 7.0.

Although the invention has been described in detail with reference to the Best Mode for Carrying Out the Invention, it will be apparent to those skilled in the art that various changes can be made and equivalent replacements made, and such changes and replacements are not beyond the scope of the present invention. Each of the above documents has a link, and all cited documents are part of the description of the present invention.

Table 1 The effect of P _L-tac alsABC on the production of threonine strain B3996ΔptsHI-crr in test tubes Strain 48 hours 72 hours OD ₅₄₀ Threonine, g / l OD ₅₄₀ Threonine, g / l B3996ΔptsHI-crr 0.53 ± 0.01 traces 0.72 ± 0.01 traces B3996ΔptsHI-crr P _L-tac alsABC 4.4 ± 0.2 0.5 ± 0.1 6.2 ± 0.1 1.4 ± 0.1

table 2 Groups Component Final concentration, g / l BUT KN ₂ PO ₄ 1.5 NaCl 0.5 (NH ₄ ) ₂ SO ₄ 1.5 L-Methionine 0.05 L-Phenylalanine 0.1 L-tyrosine 0.1 Mameno (total nitrogen) 0,07 AT Glucose 40.0 MgSO ₄ · 7H ₂ O 0.3 FROM CaCl ₂ 0.011 D FeSO ₄ · 7H ₂ O 0.075 Sodium citrate 1.0 E Na ₂ MoO ₄ · 2H ₂ O 0.00015 H ₃ IN ₃ 0.0025 CoCl ₂ · 6H ₂ O 0.00007 CuSO ₄ · 5H ₂ O 0.00025 MnCl ₂ · 4H ₂ O 0.0016 ZnSO ₄ · 7H ₂ O 0.0003 F Thiamine 0.005 G CaCO ₃ 30.0 N Pyridoxine 0.03 Group A has a pH of 7.1, which is adjusted with ammonia. Each group is sterilized separately.

Claims (10)

1. The bacterium-producer of L-threonine, belonging to the genus Escherichia, modified in such a way that the expression of the alsABC operon is enhanced in said bacterium. 2. The bacterium according to claim 1, characterized in that in said bacterium the expression of the alsABC operon is enhanced by modifying the sequence controlling the expression of the alsABC operon so that gene expression is enhanced, or by increasing the number of copies of the alsABC operon. 3. The bacterium according to claim 1, characterized in that said operon encodes:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof;
(B) a protein comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof;
(B) a protein comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof;
characterized in that in the case when these variants are present in the specified operon, such variants have the activity of a high affinity D-allose transporter, when these variants are combined together. 4. The bacterium according to claim 1, characterized in that said operon includes:
(A) a DNA fragment comprising a nucleotide sequence with nucleotide numbers 1 to 936 in SEQ ID NO: 1, or a DNA fragment capable of hybridizing under stringent conditions with the specified sequence, or a probe synthesized based on the specified sequence;
(B) a DNA fragment comprising a nucleotide sequence with nucleotide numbers 1 to 1533 in SEQ ID NO: 3, or a DNA fragment capable of hybridizing under stringent conditions with the specified sequence, or a probe synthesized based on the specified sequence;
(B) a DNA fragment comprising a nucleotide sequence with nucleotide numbers 1 to 981 in SEQ ID NO: 5, or a DNA fragment capable of hybridizing under stringent conditions with the specified sequence, or a probe synthesized based on the specified sequence, and characterized in that when said DNA fragments are present in said operon that hybridize with said sequences or with said probes, such DNA fragments encode proteins having high affinity D-allose transporter activity When these proteins are combined together. 5. The bacterium according to claim 4, characterized in that the harsh conditions include those conditions under which washing is carried out at a temperature of 60 ° C at a salt concentration of 1 × SSC and 0.1% SDS for approximately 15 minutes 6. The bacterium according to claim 1, characterized in that said bacterium is further modified in such a way that glucokinase activity is enhanced in it. 7. The bacterium according to claim 1, characterized in that said bacterium is further modified in such a way that xylose isomerase activity is enhanced in it. 8. The bacterium according to claim 1, characterized in that said bacterium is further modified so that expression of a gene selected from the group consisting of a mutant thrA gene encoding aspartokinase-homoserine dehydrogenase I, resistant to threonine inhibition by feedback type, is enhanced; thrB gene encoding homoserine kinase; thrC gene encoding threonine synthase; rhtA gene, presumably encoding a transmembrane protein; asd gene encoding aspartate p-semialdehyde dehydrogenase; the aspC gene encoding aspartate aminotransferase (aspartate transaminase); and combinations of these genes. 9. The bacterium of claim 8, wherein said bacterium is modified in such a way that expression of said mutant thrA gene, said thrB gene, said thrC gene and said rhtA gene is enhanced. 10. A method of producing L-threonine, comprising growing the bacterium according to claim 1 in a nutrient medium containing glucose as a carbon source, and isolating L-threonine from the culture fluid.

Images