RU2304615C2 - Method for production of l-amino acids belonging to genus eschericha - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to method for production of L-amino acids such as L-threonine, L-lysine, L-hystidine, L-phenylalanyne, L-arginine, or L-glutamic acid using modified bacteria belonging to genus Eschericha with increased activity of D-xylose permease.

EFFECT: high effective method for production of L-amino acids.

25 cl, 2 dwg, 4 tbl, 6 ex

Description

The field of technology.

The present invention relates to a method for producing L-amino acids by fermentation, and more particularly to genes that contribute to this fermentation. These genes positively affect the production of L-amino acids, for example, the production of L-threonine, L-lysine, L-histidine, L-phenylalanine, L-arginine and L-glutamic acid.

The prior art.

Traditionally, on an industrial scale, L-amino acids are obtained by fermentation of strains of microorganisms obtained from natural sources, or mutants of these microorganisms. Typically, these microorganisms are modified so that the yield of L-amino acids in these strains increases.

Many methods have been described to enhance the production of L-amino acids, including transformation of recombinant DNA microorganisms (see, for example, US Pat. No. 4,278,765). Other ways to increase the productivity of microorganisms include increasing the activity of enzymes involved in the biosynthesis of amino acids, and / or reducing the sensitivity of the target enzyme to reverse inhibition of the accumulated L-amino acid by feedback (see, for example, WO 95/16042 or US patent 4346170, 5661012 and 6040160).

Various strains are known to be used for the production of L-threonine by fermentation. These are strains with increased activity of enzymes involved in the biosynthesis of L-threonine (US patents 5175107; 5661012; 5705371 and 5939307; European patent 219027), strains resistant to certain chemicals, such as L-threonine and its analogues (PCT application 01 / 14525 A1, European application 301572A2, US patent 5,661,012), strains in which the sensitivity of the target enzyme to inhibition by the produced amino acid or its by-products is eliminated by feedback type (US patents 5175107 and 5661012), strains with inactivated enzymes of the threonine degradation system (US patents 5939307 and 6297031).

Known strain-producer of threonine VKPM B-3996 (US patents 5175107 and 5705371) is currently one of the best producers of threonine. To create a strain VKPM B-3996, several mutations and the plasmid described below were introduced into the parent strain E. coli K-12 (VKPM B-7). The mutant thrA gene (thrA442 mutation) encodes an aspartokinase homoserine dehydrogenase I protein, which is resistant to feedback threonine inhibition. The mutant ilvA gene (ilvA442 mutation) encodes a threonine deaminase protein that has reduced activity, which is expressed in a reduced level of isoleucine biosynthesis and in the leaky-type isoleucine deficient phenotype. In bacteria with the ilvA442 mutation, transcription of the thrABC operon is not repressed by isoleucine, which is very effective in the production of threonine. Inactivation of the tdh gene encoding threonine dehydrogenase leads to the prevention of threonine degradation. The genetic determinant of sucrose assimilation (scrKYABR genes) was introduced into this strain. To increase the expression of genes that control threonine biosynthesis, plasmid pVIC40 containing the thrA442BC mutant threonine operon was introduced into the intermediate strain TDH6. The amount of threonine accumulated during the fermentation of this strain reached 85 g / l.

To optimize the main pathways of biosynthesis of the target compound, further improvement of the strains producing L-amino acids can be made by increasing the supply of bacteria with increasing amounts of sugars serving as a carbon source, for example, glucose. Despite the effective PTS transport of glucose, the availability of carbon sources for cells of a highly productive strain may remain insufficient.

It is known that the active transport of sugars and other metabolites into the bacterial cell is carried out by several transport systems.

In particular, the XylE protein from E. coli is a permease of D-xylose, one of the two systems in E. coli responsible for the entry of D-xylose into the cell; another system is the ATP-dependent ABC transporter XylFGH. The cloned xylE gene has been shown to complement the in vivo xylE mutation (Davis EO and Henderson PJ, J. Biol. Chem., 262 (29); 13928-32 (1987)). Transport via XylE in an intact cell is inhibited by protonophores and causes a pH shift towards alkalization (Lam, VM et al., J. Bacteriol. 143 (1); 396-402 (1980)). Experiments with xylE and xylF mutants showed that in the XylE K _M protein, D-xylose is 63-169 μM (Sumiya. M. et al. Receptors Channels, 3 (2); 117-28 (1995)). The XylE protein is a member of the Major Facilitator Superfamily (MFS) transporter superfamily (Griffith, JK et al., Curr. Opin. Cell Biol. 4 (4); 684-95 (1992)) and functions as a D-xylose / proton symposer. Perhaps the xylE gene is part of the monocistronic operon, the expression of which is induced by D-xylose. Xylose imported into the cell is catabolized to xylulose-5-phosphate with the participation of the enzymes XylA (xylose isomerase) and XylB (xylulokinase). Under certain conditions, xylose isomerase encoded by the xylA gene is also capable of efficiently catalyzing the conversion of D-glucose to D-fructose (Wovcha, MG et al., Appl Environ Microbiol. 45 (4): 1402-4 (1983)).

However, to date, there are no reports of the use of bacteria of the Enterobacteriaceae family, which has increased D-xylose permease activity to increase the production of L-amino acids.

Brief Description of the Drawings

Figure 1 shows the structure of the nucleotide sequence in front of the xylE gene on the E. coli chromosome and the structure of an integrated DNA fragment containing the cat gene and the P _L-tac hybrid promoter.

Figure 2 shows the growth curves of E. coli strains MG1655, MG1655 ΔptsHI-crr and MG1655P _L-tac xylE grown on a medium with glucose. Legend: MG = E. coli MG1655; MG Δpts = E. coli MG1655 ΔptsHI-crr; MG Δpts P xylE = E. coli MG1655 ΔptsHI-crr P _L-tac xylE.

Description of the invention

The aim of the present invention is to increase the productivity of strains producing L-amino acids and the provision of a method for producing non-aromatic or aromatic L-amino acids using these strains.

This goal was achieved by detecting the fact that increased expression of the xylE gene encoding D-xylose permease increases L-amino acid production, for example L-threonine, L-lysine, L-histidine, L-phenylalanine, L-arginine or L- glutamic acid. Thus, the present invention has been completed.

The aim of the present invention is the provision of bacteria producing L-amino acids of the Enterobacteriaceae family, where the specified bacterium is modified so that the activity of D-xylose permease in the cells of the specified bacteria is increased. It is also an object of the present invention to provide a bacterium as described above, wherein the activity of said D-xylose permease is increased by enhancing the expression of a gene encoding D-xylose permease.

It is also an object of the present invention to provide a bacterium as described above, wherein the activity of said D-xylose permease is enhanced by modifying a sequence that controls the expression of a gene encoding D-xylose permease so that the expression of said gene is enhanced or the number of copies of the gene encoding D- permease is increased xyloses.

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

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

It is also an object of the present invention to provide a bacterium as described above, wherein said bacterium is modified in such a way that xylABFGHR locus expression is enhanced in the cells of this bacterium.

It is also an object of the present invention to provide a bacterium as described above, wherein said bacterium is selected from the group consisting of the genera 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 gene encodes a D-xylose permease selected from the group consisting of:

(A) a protein comprising the amino acid sequence shown in Sequence Listing No. 2 (SEQ ID NO: 2);

(B) a variant of a protein with the amino acid sequence shown in Sequence Listing No. 2 (SEQ ID NO: 2), which has D-xylose permease activity.

It is also an object of the present invention to provide a bacterium as described above, wherein said gene encoding a D-xylose permease includes DNA selected from the group consisting of:

(a) a DNA comprising a nucleotide sequence with nucleotide 1 to nucleotide 1476 shown in Sequence Listing No. 1 (SEQ ID NO: 1);

(b) DNA that hybridizes to a nucleotide sequence with nucleotide 1 to nucleotide 1476 shown in Sequence Listing No. 1 (SEQ ID NO: 1), or with a probe that can be synthesized based on the indicated nucleotide sequence under stringent conditions and encodes a protein having D-xylose permease activity.

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

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 so that expression of a gene selected from the group consisting of:

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

- thrB gene encoding homoserine kinase;

- thrC gene encoding threonine synthase;

- rhtA gene encoding the putative transmembrane protein;

- any combination of the above genes.

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

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-glutamic acid.

It is also an object of the present invention to provide a method for producing an L-amino acid, which comprises growing the above-described bacteria in a fermentation medium, which allows the accumulation of said L-amino acid in the culture fluid, as well as the isolation of said L-amino acid from the culture fluid.

It is also an object of the present invention to provide a process as described above, wherein the culture medium contains xylose.

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

Another objective of the present invention is the provision of the above method, where the L-amino acid is L-lysine.

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

Another objective of the present invention is the provision of the above method, where the L-amino acid is L-phenylalanine.

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

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

The best way of carrying out the invention.

According to the present invention, “bacterium producing L-amino acids” means a bacterium having the ability to accumulate L-amino acids in a nutrient medium, under the conditions when the bacterium of the present invention is grown in the specified nutrient medium. The ability to produce L-amino acids can be given or improved by selection. 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 refers to a microorganism capable of accumulating in the medium the target L-amino acid in amounts of not less than 0.5 g / l, more preferably not less than 1.0 g / l of the target L-amino acid. “L-amino acids” include L-alanine, L-arginine, L-asparagine, L-aspartate, 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. L-threonine, L-lysine, L-histidine, L-phenylalanine, L-arginine and L-glutamic acid are more preferred.

The Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, etc. In particular, bacterias classified as belonging to the Enterobacteriaceae family according to the taxonomic classification provided in the NCBI database (National Center for Biotechnological 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) may be included in their number 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. Based on the analysis of the nucleotide sequence of 16S rRNA, etc. some Enterobacter agglomerans species have recently been classified as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or similar (Int. J. Syst. Bacteriol. 43, 162-173 (1993)).

The bacterium according to the present invention includes a strain of the Enterobacteriaceae family having the ability to produce L-amino acids and which has been modified so that the activity of D-xylose permease is increased. In addition, the bacterium of the present invention includes a bacterium strain of the Enterobacteriaceae family, which is capable of producing L-amino acids and does not have the natural activity of D-xylose permease and which is transformed with a DNA fragment encoding D-xylose permease.

The term “D-xylose permease activity” refers to the activity of transporting sugars, such as xylose and glucose, into the cell. D-xylose permease activity can be determined by complementing growth retardation in bacteria with a broken PTS sugar transport system (see, for example, the ΔptsHI-crr mutant described in the Examples section) or by complementing in vivo xylE mutations (Davis, EO and Henderson, PJ, J. Biol. Chem., 262 (29); 13928-32 (1987)).

The term "bacterium is modified so that the activity of D-xylose permease is increased" means that the specified specific activity is higher than the same activity in an unmodified, for example, natural, strain. Examples of such a modification include an increase in the number of D-xylose permease molecules per cell, an increase in specific activity in terms of a D-xylose permease molecule, and so on. In addition, examples of a natural strain that can be used for comparison include, for example, Escherichia coli K-12 strain. In the present invention, the amount of accumulated L-amino acid, for example L-threonine or L-arginine, can be increased in the culture fluid by increasing the intracellular activity of D-xylose permease.

An increase in the activity of D-xylose permease in a bacterial cell can be achieved by enhancing the expression of the xylE gene encoding D-xylose permease. Any xylE gene obtained from bacteria belonging to the genus Escherichia, like any xylE gene obtained from other bacteria, such as coryneform bacteria, can be used as the D-xylose permease gene according to the present invention. XylE genes derived from bacteria belonging to the genus Escherichia are preferred.

The term "enhancing gene expression" means that the expressed amount of a given gene is higher than the expression of a given gene in an unmodified, for example, natural, strain. Examples of such modifications include increasing the number of copies of an expressed gene in terms of a cell, increasing the level of expression of this gene, and so on. The number of copies of an expressed gene is measured, for example, by cutting chromosomal DNA followed by Southern hybridization using a probe derived from the nucleotide sequence of the gene, in situ fluorescence hybridization (FISH) and the like. Gene expression can be measured using various methods, including Northern hybridization, quantitative reverse PCR (RT-PCR), and the like. In addition, examples of a natural strain that can be used as a control include, for example, the Escherichia coli K-12 strain or Pantoea ananatis PERM BP-6614 (US patent application US 2004180404 A1). The strain Pantoea ananatis FERM BP-6614 was deposited at the National Institute of Bioscience and Human-Technology of the Agency of Industrial Science and Technology of the Ministry of International Trade and Industry) (currently the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology) February 19, 1998 under accession number FERM P-16644. Then the strain was transferred to international deposit in accordance with the Budapest Treaty on January 11, 1999 under inventory number FERM BP-6614. Although this strain was described as Enterobacter agglomerans at the time of isolation, it was reclassified to Pantoea ananatis based on the analysis of the 16S rRNA nucleotide sequence as described above.

As a result of the increase in the intracellular activity of D-xylose permease, the accumulation of L-amino acids in the medium, for example L-threonine, L-lysine, L-histidine, L-phenylalanine or L-glutamic acid, increases.

The nucleotide sequence of the xylE gene encoding D-xylose permease, namely, D-xylose / proton symporter from Escherichia coli, is shown in the GenBank database in the sequence with accession number NC_000913.2, gi: 49175990 (nucleotide numbers 4240277 to 4238802). The xylE gene is located on the chromosome of E. coli K-12 strain between the open reading frame of yjbA ORF and the malG gene. The nucleotide sequences of other xylE genes encoding D-xylose permeases are also listed in the GenBank database under the numbers AAN45595. xylose-proton sym ... [gi: 24054686], AAM41050. MFS transporter ... [gi: 21112853]: Xanthomonas campestris: XCC1759. In the description of the present invention, the nucleotide sequence of the xylE gene from Escherichia coli is presented in Sequence Listing No. 1 (SEQ ID NO.1).

When transported to a cell, glucose is phosphorylated using the glukokinase encoded by the glk gene. Therefore, it is desirable to modify the bacterium so that the activity of glucokinase in the cells of the specified bacterium is increased. The nucleotide sequence of the glk gene, which encodes a glucokinase from Escherichia coli, is given in the GenBank database in sequence with accession number NC_000913.1, gi: 16127994 (nucleotide numbers 2506481 to 2507446). The glk gene is located on the chromosome of E. coli K-12 strain between the open reading frames of b2387 and b2389 ORF.

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., Appl Environ Microbiol. 45 (4): 1402-4 (1983)). Therefore, it is desirable to modify the bacterium so that xylose isomerase activity in the cells of said bacterium is increased. The nucleotide sequence of the xylA gene, which encodes xylose isomerase from Escherichia coli, is shown in the GenBank database in sequence with accession number NC_000913.2, gi: 49175990 (nucleotide numbers 3728788 through 3727466). The xylA gene is located on the chromosome of E. coli K-12 strain between the xylB and xylF genes.

In the case when the nutrient medium contains xylose as an additional source, it is necessary to increase the activity of xylose utilization enzymes. The term “xylose utilization enzymes” includes xylose transport proteins, xylose isomerization and phosphorylation enzymes, as well as regulatory proteins. Such enzymes include xylose isomerase, xylulokinase, xylose transporters, and transcriptional activators responsive to the presence of xylose. Xylose isomerase catalyzes the reaction of isomerization of D-xylose to D-xylulose. Xylulokinase catalyzes the phosphorylation of D-xylulose with ATP to form D-xylulose-5-phosphate and ADP. The presence of xylose utilization enzymes, such as xylose isomerase and xylulokinase, is determined by complementation of the corresponding minus mutations in xylose isomerase and xylulokinase in mutant E. coli strains, respectively.

Genes encoding the above xylose utilization enzymes are located at the xylABFGHR locus on the Escherichia coli chromosome. The gene encoding xylulokinase (EC numbers 2.7.1.17) is known and designated as xylB (nucleotide numbers from 3725546 to 3727000 in the sequence number NC_000913.1, gi: 16131435 in the GenBank database). The gene encoding the xylose binding protein of the transport system is known and designated as xylF (nucleotide numbers 3728760 through 3729752 in sequence numbered NC_000913.1, gi: 16131437 in the GenBank database). The gene encoding the supposedly ATP-binding protein of the xylose transport system is known and designated as xylG (nucleotide numbers 3729830 through 3731371 in sequence number NC_000913.1, gi: 16131438 in the GenBank database). The gene encoding the permease component in the ABC type xylose transport system is known and designated as xylH (nucleotide numbers 3731349 to 3732530 in the sequence number NC_000913.1, gi: 16131439 in the GenBank database). The gene encoding the transcriptional regulator of the xyl operon is known and designated as xylR (nucleotide numbers 3732608 through 3733786 in the sequence number NC_000913.1, gi: 16131440 in the GenBank database).

Thus, the xylE, glk, and xylABFGHR loci can be obtained by PCR (polymerase chain reaction; link to White, TJ et al., Trends Genet., 5, 185 (1989)) using primers synthesized based on known nucleotide sequences of genes. Genes encoding D-xylose permease from other microorganisms can be obtained in a similar manner.

The xylE gene obtained from Escherichia coli is represented by a DNA that encodes the following protein (A) or (B):

(A) a protein comprising the amino acid sequence shown in Sequence Listing No. 2 (SEQ ID NO: 2);

(B) a variant of a protein with the amino acid sequence shown in Sequence Listing No. 2 (SEQ ID NO: 2), and which has D-xylose permease activity.

As used in the present invention, the term “protein variant” means a protein with changes in its sequence, which may be deletions, insertions, additions of nucleotides or substitutions of amino acids, but at the same time maintains the desired activity at the necessary level, for example, necessary to increase the production of L-amino acids. The number of changes in a protein variant depends on the type of amino acid residue or on the position in the three-dimensional structure of this protein. This amount may be from 2 to 30, preferably from 2 to 15, and even more preferably from 2 to 5 for protein (A). These substitutions in protein variants can occur in regions of a given protein that are not critical to the functioning of the protein. This is due to the fact that some amino acids have high homology to each other, and the three-dimensional structure of the protein and its activity do not change as a result of such a replacement. Such substitutions in a protein variant can occur in regions of a given protein that are not critical to the functioning of the protein. Thus, the variant of protein (B) may be a protein having a homology of at least 70%, preferably 80%, more preferably 90% and even more preferably 95% with respect to the complete amino acid sequence of D-xylose permease, shown in the List of sequences under the number 2 (SEQ ID NO.2) until the activity of D-xylose permease is measured. The degree of homology between two amino acid sequences can be determined using known methods, for example, using the computer program BLAST 2.0, which calculates the following three parameters: score (score), identity (identity) and similarity (similarity).

DNA that encodes almost the same protein as the D-xylose permease described above can be obtained, for example, by modifying the nucleotide sequence of the DNA encoding D-xylose permease (SEQ ID NO: 1), for example, using site-directed mutagenesis , in such a way that at a specific site there is a substitution, deletion, insertion or addition of one or more amino acid residues. DNA modified by the method described above can be obtained using conventional mutagenesis methods. Such methods include treating the DNA encoding the protein of the present invention with hydroxylamine or treating the bacterium containing said DNA with UV radiation or a reagent such as N-methyl-N'-nitro-N-nitrosoguanidic or nitrous acid.

Substitutions, deletions, insertions and additions of one or more amino acid residues must be conservative mutations (mutations) in order to maintain protein activity. An example of a conservative mutation is a conservative substitution. Examples of conservative substitutions include replacing Ser or Thr with Ala, replacing Gln, His or Lys with Arg, replacing Glu, Gln, Lys, His or Asp with Asn, replacing Asn, Glu or Gln with Asp, replacing Ser or Ala with Cys, replacing Asn, Glu, Lys, His, Asp or Arg to Gln, replace Asn, Gln, Lys or Asp to Glu, replace Pro to Gly, replace Asn, Lys, Gln, Arg or Tyr to His, replace Leu, Met, Val or Phe with Ile, replace Ile, Met, Val or Phe with Leu, replace Asn, Glu, Gln, His or Arg with Lys, replace Ile, Leu, Val or Phe with Met, replace Trp, Tyr, Met, Ile or Leu with Phe, replacing Thr or Ala with Ser, replacing Ser or Ala with Thr, replacing Phe or Tyr with Trp, replacing His, Phe or Trp with Tyr, and replacing Met, Ile or Leu with Val.

DNA encoding almost the same protein as D-xylose permease can be obtained by expression of DNA containing the indicated mutation in the corresponding host cell, followed by determination of the activity of the expressed product. DNA encoding almost the same protein as D-xylose permease can also be obtained by isolating DNA that hybridizes with a probe whose nucleotide sequence contains, for example, the nucleotide sequence shown in Sequence Listing No. 1 (SEQ ID NO: 1 ), under severe conditions, and which encodes a protein having D-xylose permease activity. The term "stringent conditions", mentioned here, means the conditions under which the so-called specific hybrids are formed, and non-specific ones are not formed. For example, stringent conditions include conditions under which DNA with a high degree of homology hybridizes, for example DNA, preferably having a homology of at least 50% relative to each other, preferably 80%, more preferably 90% and most preferably 95%, but DNA, having a homology of less than 50% relative to each other, do not hybridize. On the other hand, an example of stringent conditions is the conditions under which DNA hybridizes with each other, at a salt concentration that meets standard washing conditions for Southern hybridization, for example 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS at 60 ° C. The duration of washing depends on the type of filter used for blotting and, as a rule, on the recommendations of the manufacturer. For example, the recommended washing time for a Hybond ^™ N + nylon filter (Amersham) under harsh conditions is 15 minutes. Preferably, washing is carried out 2-3 times.

Also, as a probe, a part of the nucleotide sequence shown in the sequence list under number 1 (SEQ ID NO: 1) can be used. Probes can be obtained by PCR using oligonucleotides as primers obtained based on the nucleotide sequence at number 1 and a DNA fragment containing the nucleotide sequence at number 1 as a matrix. When a DNA fragment with a length of about 300 base pairs is used as a probe, the washing conditions during hybridization correspond, for example, to 50 ° С, 2 × SSC, and 0.1% SDS.

The nucleotide substitutions, deletions, insertions, or additions described above also include naturally occurring mutations (mutant or variant), for example, due to the natural diversity within the species or genus of the bacterium containing D-xylose permease.

The term "transformation of a bacterium with DNA encoding a protein" means the introduction of the specified DNA into the bacterium, for example, by traditional methods. Transformation of this DNA leads to increased expression of the gene encoding the protein and thus increases the activity of the specified protein in the bacterial cell. Transformation methods include all methods described or known to date. For example, a method of treating recipient cells with calcium chloride can be used to increase DNA permeability across the cell membrane described for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)).

Methods for enhancing gene expression include increasing the number of copies of the gene. The introduction of a gene into a vector capable of functioning in bacteria of the Enterobacteriaceae family increases the number of copies of the gene. The use of low copy genes is preferred. Examples of low copy vectors include, but are not limited to, pSC 101, pMW 118, pMW 119, and the like. The term "low copy vector" is used for vectors whose number of copies in the cell does not exceed 5.

Enhanced gene expression can also be achieved by introducing multiple copies of the gene into the bacterial chromosome, for example, using methods of homologous recombination, Mu integration and the like. For example, one round of Mu integration allows you to integrate up to 3 copies of the target gene into the bacterial chromosome.

An increase in the number of copies of the D-xylose permease gene can be achieved by integrating several copies of the D-xylose permease gene in the bacterial chromosomal DNA. In order to integrate several copies of this gene into the bacterial chromosome, homologous recombination is performed using a sequence whose multiple copies are present in the chromosomal DNA and are used as targets. Sequences present as multiple copies in chromosomal DNA include, but are not limited to, DNA repeats or inverted repeats present at the ends of transposition elements. Also, as described in US Pat. No. 5,595,889, it is possible to incorporate the D-xylose permease gene into the transposon, allowing the integration of several copies of this gene into the chromosomal DNA.

Enhanced gene expression is also achieved by placing the DNA of the present invention under the control of a strong promoter. For example, the lac promoter, trp promoter, trc promoter, P _R or P _L lambda phage promoters are known as strong promoters. Enhanced gene expression can also be achieved by placing a strong terminator after the DNA according to the present invention. The use of a strong promoter and / or terminator can be combined with an increase in the number of copies of the gene.

On the other hand, the influence of the promoter can be enhanced, for example, by introducing a mutation into the promoter to increase the level of transcription of the structural gene (coding region of the gene) located behind the promoter. Similarly, the effect of the terminator can be enhanced, for example, by introducing a mutation into the terminator to increase the number of transcription initiation of the gene located in front of the terminator.

Moreover, it is known that the replacement of several nucleotides in the gap between the ribosome binding site (RBS) and the start codon, especially the sequences immediately before the start codon, significantly affect mRNA translatability. For example, a 20-fold variation in expression levels was found, depending on which three nucleotides preceded the start codon (Gold et al., Annu. Rev. Microbiol., 35, 365-403, 1981; Hui et al., EMBO J., 3, 623-629, 1984). The rhtA23 mutation was previously shown to be an A-to-G substitution at position -1 relative to the ATG start codon (ABSTRACTS of 17 ^th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology , San Francisco, California August 24-29, 1997, abstract No. 457). Thus, we can assume that the rhtA23 mutation enhances the expression of the rhtA gene and, therefore, increases resistance to threonine, homoserine, and other substances released from the cell.

Moreover, it is also quite possible to introduce a nucleotide substitution into the promoter or terminator region of the D-xylose permease gene in the bacterial chromosome, which will lead to an increase in the promoter. A change can be made to the sequence that regulates gene expression, for example, in the same way as replacing a gene using a temperature-sensitive plasmid, as described in published PCT application WO 00/18935 and Japanese Patent Application Laid-open No. 1-215280.

Methods for obtaining plasmid DNA include, but are not limited to cutting and ligation of DNA, transformation, selection of oligonucleotides as primers and similar methods, or other methods well known to a person skilled in the art. These methods are described, for example, in Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).

The bacterium according to the present invention can be obtained by introducing the aforementioned DNA into a bacterium that already has a hereditary trait - the ability to produce L-amino acids. On the other hand, the bactria according to the present invention can be obtained by imparting the ability to produce L-amino acids of bacteria that already contain these DNA.

Bacteria producing L-threonine.

Examples of the parent strain are strains producing threonine belonging to the genus Escherichia, such as E. coli strain TDH-6 / pVIC40 (VKPM B-3996) (US patents 5175107 and 5,705.371), 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), E. coli strain MG442 (Gusyatiner et al., Genetics, 14, 947-956 (1978)), strains of E. coli VL643 and VL2055 (European patent application EP 1149911 A) and the like, but are not limited to these strains.

Strain TDH-6 is deficient in the thrC gene, is able to assimilate 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, 113105 Moscow, Nagatinskaya St., 3-A) with inventory number RIA 1867. This strain was also deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF , 113545 Moscow, 1st Road passage, 1) with inventory number B-3996.

Preferably, the bacterium of the present invention is further modified in order 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 encoding a presumably transmembrane protein;

- asd gene encoding aspartate β-semi-aldehyde dehydrogenase;

- aspC gene encoding aspartate aminotransferase (aspartate transaminase).

The nucleotide sequence of the thrA gene encoding aspartokinase homoserine dehydrogenase I from Escherichia coli is presented in the GenBank database in the sequence with accession number NC_000913.2, gi: 49175990 (nucleotide numbers 337 to 2799). The thrA gene is located on the E. coli K-12 chromosome between the thrL and thrB genes. The nucleotide sequence of the thrB gene encoding homoserine kinase from Escherichia coli is presented in the GenBank database in the sequence with accession number NC_000913.2, gi: 49175990 (nucleotide numbers from 2801 to 3733). The thrB gene is located on the E. coli K-12 chromosome between the thrA and thrC genes. The nucleotide sequence of the thrC gene encoding threonine synthase from Escherichia coli is presented in the GenBank database in the sequence with accession number NC_000913.2, gi: 49175990 (nucleotide numbers from 3734 to 5020). The thrC gene is located on the E. coli K-12 chromosome between the thrB gene and the open uaaX reading frame. All three of these genes function as one threonine operon.

Both the mutant thrA gene encoding aspartokinase homoserine dehydrogenase I, which is resistant to threonine inhibition by feedback type, and the thrB and thrC genes can be obtained as part of an operon from the known plasmid pVIC40 present in the deposited E. coli VKPM B-threonine producer strain 3996. Plasmid pVIC40 is described in detail in US Pat. No. 5,705,371.

The rhtA gene is located at the 18th minute of the E. coli chromosome near the gInHPQ operon, which encodes the components of the glutamine transport system. The rhtA gene is identical to ORF1 (ybiF gene, positions 764 to 1651 in the sequence numbered AAA218541, gi: 440181 in the GenBank database) and is located between the pXB and ompX genes. The module expressing the protein encoded by ORF1 was designated as the rhtA gene (rht: resistance to homoserine and threonine - resistance to homoserin and threonine). It was also found that the rhtA23 mutation is a substitution of A for G at position -1 relative to the ATG start codon (ABSTRACTS of the 17 ^th International Congress of Biochemistry and Molecular Biology in conjugation with the 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 asd gene from E. coli is known (nucleotide numbers 3572511 to 3571408 in sequence numbered NC_000913.1, gi: 16131307 in the GenBank database) and can be obtained by PCR (polymerase chain reaction; link to White, TJ et al. , Trends Genet., 1989, 5: 185) using primers designed based on the nucleotide sequence of a gene. The asd gene from other microorganisms can be obtained in a similar way.

The aspC gene from E. coli is also known (nucleotide numbers 983742 to 984932 in sequence numbered NC_000913.1, gi: 16128895 in the GenBank database) H can be obtained by PCR. The aspC gene from other microorganisms can be obtained in a similar way.

Bacteria producing L-lysine.

Examples of parental strains used to produce L-lysine producing bacteria belonging to the genus Escherichia include mutant strains that are resistant to L-lysine analogues. L-lysine analogs inhibit the growth of bacteria belonging to the genus Escherichia, but this inhibition is completely or partially removed when L-lysine is present in the medium. Examples of L-lysine analogues include, but are not limited to, oxalysine, lysine hydroxamate, S- (2-aminoethyl) -L-cysteine (AEC), γ-methyllysine, 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 mutagenesis methods. Particular examples of bacterial strains used to produce L-lysine include Escherichia coli AJ11442 strains (FERM BP-1543, NRRL B-12185; see US Pat. No. 4,346,170) and Escherichia coli VL611. In these microorganisms, inhibition of aspartokinase by L-lysine in the type of feedback was removed.

Strain WC196 can be used as an example of the bacterium producing L-lysine of Escherichia coli. This bacterial strain was obtained by conferring AEC resistance to strain W3110, derived from Escherichia coli K-12. The resulting strain was called Escherichia coli AJ13069 and was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International, and the National Institute of Biological Sciences and Human Technologies. Trade and Industry) (currently called the National Institute of Advanced Industrial Science and Technology, 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, Ibaraki-ken, 305-8566, Japan) December 6, 1994 and was assigned an inventory FERM number P-14690. Then, on September 29, 1995, the strain was internationally deposited in accordance with the terms of the Budapest Treaty, and the strain received accession number FERM BP-5252 (see US Pat. No. 5,827,698).

Bacteria producing L-histidine.

Examples of parental strains used to produce L-histidine producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli 24 strain (VKPM B-5945, RU 2003677); E. coli strain 80 (VKPM B-7270, RU 2119536); E. coli NRRL B-12116-B12121 strains (US Pat. No. 4,388,405); E. coli strains H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (US patent 6,344,347); E. coli strain H-9341 (FERM BP-6674) (European Patent 1085087); E. coli strain AI80 / pFM201 (US patent 6258554) and the like.

Bacteria producing L-phenylalanine.

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 AJ 12739 (tyrA :: Tn10, tyrR) (VKPM B-8197); strain HW1089 (ATCC accession number 55371) containing the pheA34 gene (US patent 5,354,672); mutant strain MWEC101-b (South Korean patent 8903681); strains NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (US patent 4407952) and the like. Also, the parent strain for the subsequent increase in the activity of the protein according to the present invention can be used bacteria-producers of L-phenylalanine belonging to the genus Escherichia, strains of E. coli K-12 [W3110 (tyrA) / pPHAB (FERM BP-3566) E. coli K-12 [W3110 (tyrA) / pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA) / pPHATerm] (FERM BP-12662) and E. coli K-12 [ W3110 (tyrA) / pBR-aroG4, pACMAB], also called AJ 12604 (FERM BP-3579) (European patent 488424 B1). In addition, L-phenylalanine producing bacteria belonging to the genus Escherichia with increased activity of the protein encoded by the yedA gene or the yddG gene can be used (US patent applications 2003/0148473 A1 and 2003/0157667 A1).

Bacteria producing L-arginine.

Examples of parent 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 mutants resistant to α-methylmethionine, p-fluorophenylalanine, D- arginine, arginine hydroxamate, S- (2-aminoethyl) -cysteine, α-methylserine, β-2-thienylalanine or sulfaguanidine (Japanese Patent Application Laid-Open No. 56-106598); a producer strain of L-arginine into which the argA gene encoding N-acetylglutamate synthetase has been introduced (European Patent 1170361 A1); strains 237 (VKPM B-7925) and 382 (VKPM B-7926) described in European patent EP 1170358 A1 and the like.

Bacteria producing L-glutamic acid.

Examples of parent strains used to produce the L-glutamic acid producing bacterium of the present invention include mutant strains lacking α-ketoglutarate dehydrogenase activity or having a reduced level of α-ketoglutarate dehydrogenase activity. Bacteria belonging to the genus Escherichia, lacking the activity of α-ketoglutarate dehydrogenase or having a reduced level of activity of α-ketoglutarate dehydrogenase, as well as methods for their preparation are described in US patents 5378616 and 5573945. In particular, such strains can be represented by 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)

The E. coli strain W3110sucA :: Km ^r is a strain resulting from the destruction of a gene encoding α-ketoglutarate dehydrogenase (hereinafter referred to as the “sucA gene”) in the E. coli strain W3110. The activity of α-ketoglutarate dehydrogenase in the obtained strain is completely absent.

Also, examples of bacteria producing L-glutamic acid include bacteria belonging to the genus Escherichia and resistant to antimetabolite - aspartic acid. Also, such strains may not have alpha-ketoglutaric acid dehydrogenase activity and include, for example, strain AF13199 (FERM BP-5807) (US Pat. No. 5,908,768) or strain FFRM P-12379, further having a reduced ability to degrade L-glutamine acids (US Pat. No. 5,393,671); E. coli strain AJ13138 (FERM BP-5565) (US Pat. No. 6,110,714) and the like.

Examples of a bacterium producing L-glutamic acid belonging to the genus Pantoea include mutant strains lacking α-ketoglutarate dehydrogenase activity or having reduced α-ketoglutarate dehydrogenase activity, and can be obtained as described above. Examples of such strains are Pantoea ananatis strain AJ13356 (US patent 6331419), Pantoea ananatis strain AJ13356 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 me, 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 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 FERM 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 AJ13355 strain. However, it was later classified as Pantoea ananatis based on the nucleotide sequence of 16S rRNA and other evidence (see the Examples section). Although both strains — AJ13355 and the strain AJ13356 obtained from it — were deposited as Enterobacter agglomerans in the above depository, for the purposes of this description they will be referred to as Pantoea ananatis.

Obtaining L-amino acids.

Oxaloacetate (OAA) serves as a substrate for reactions that are part of the Thr and Lys biosynthesis pathways. OAA is formed from phosphoenolpyruvate (PEP) with phosphoenolpyruvate carboxylase (PEPC) as a catalyst. Therefore, increasing the concentration of PEPC in the cell can be very significant for the enzymatic production of these amino acids. When glucose is used as a carbon source for fermentation, glucose enters the cell through the glucose phosphotransferase (Glc-PTS) system. This system consumes PEP, proteins of the PTS system are encoded by the ptsG and ptsHIcrr genes. In the process of glucose uptake, one PEP molecule and one pyruvate molecule (Pyr) are formed from one glucose molecule.

The L-threonine producing strain and the L-lysine producing strain, which were modified to be able to utilize sucrose (Scr-PTS), had increased productivity of these amino acids when cultured on sucrose compared to glucose (European Patent 1149911 A2). Using Scr-PTS, it is believed that three PEP molecules and one Pyr molecule are formed from one sucrose molecule, thereby increasing the PEP / Pyr ratio and thereby facilitating the synthesis of Thr and Lys from sucrose. In addition, gene expression of the Glc-PTS system has been reported to be regulated (Postma PW et al., Microbiol Rev., 57 (3), 543-94 (1993); Clark 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 thus, it is possible that glucose uptake alone may be a rate-limiting factor in the enzymatic production of amino acids.

An even greater increase in the PEP / Pyr ratio due to increased expression of the xylE gene in the threonine producer strain, in the lysine producer strain, in the histidine producer strain, in the phenylalanine producer strain and / or in the glutamic acid producer strain should increase the amino acid production. Since four PEP molecules are formed from two glucose molecules, the PEP / Pyr ratio is expected to increase significantly. Due to increased xylE gene expression, the expression control of glc-PTS is expected to be removed.

A method for producing an L-amino acid according to the present invention includes the steps of growing a bacterium according to the present invention in a fermentation medium, which allows the L-amino acid to accumulate in the culture fluid, and the isolation of the L-amino acid from the culture fluid. Further, the method according to the present invention includes methods for producing L-threonine, L-lysine, L-histidine, L-phenylalanine, L-arginine or L-glutamic acid, including the steps of growing the bacteria of the present invention in a fermentation medium, which allows L-threonine , L-lysine, L-histidine, L-phenylalanine, L-arginine or L-glutamic acid accumulate in the culture fluid, and secretions of L-threonine, L-lysine, L-histidine, L-phenylalanine, L-arginine or L- glutamic acid from the culture fluid.

According to the present invention, the cultivation, isolation and purification of L-amino acids from the culture fluid can be carried out by conventional fermentation methods in which the L-amino acid is obtained by fermentation of a microorganism.

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

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

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

Examples

The present invention will be described in more detail below with reference to the following examples, not limiting the scope of its application.

Example 1: Replacement of the natural promoter region of the xylE gene in E. coli with the hybrid promoter P _L-tac .

To replace the natural promoter region of the xylE gene, a DNA fragment containing the P _L-tac hybrid promoter and the chloramphenicol resistance marker (Cm ^R ) encoded by the cat gene was integrated into this promoter region of the chromosome of E. coli strain MG1655 (ATCC 700926) using the method described by Datsenko KA and Wanner BL (Proc. Natl. Acad. Sci. USA, 2000, 97, 6640-6645), also known as "Red-dependent integration" and / or "Red-directional integration". The recombinant plasmid pKD46 (Datsenko, KA, Wanner, BL, Proc. Natl. Acad. Sci. USA, 2000, 97, 6640-6645) with a thermosensitive replicon was used as a donor of genes derived from phage λ, responsible for the Red-dependent recombination system . The Escherichia coli strain BW25113 containing the recombinant plasmid pKD46 can be obtained from E. coli Genetic Stock Center, Yale University, New Haven, USA, accession number CGSC7630.

The hybrid promoter P _L-tac was synthesized chemically. The nucleotide sequence of the replaced promoter is presented in the List of sequences under the number 3 (SEQ ID NO: 3). The synthesized DNA fragment contains the hybrid promoter P _L-tac , which in turn contains the BglII restriction enzyme recognition site at the 5'-end, necessary for further attachment of the cat gene and 36 nucleotides homologous to the 5'-end of the xylE gene introduced for further integration of the fragment into the bacterial chromosome.

The DNA fragment containing the Cm ^R marker encoded by the cat gene was obtained by PCR using the commercially available plasmid pACYC184 (GenBank / EMBL accession number X06403, Fermentas, Lithuania) and PI primers (SEQ ID NO: 4) and P2 (SEQ ID NO: 5). Primer P1 contains the BglII restriction enzyme recognition site at the 5'-end, necessary for further attachment of the P _L-tac hybrid promoter, and P2 primer contains 36 nucleotides at the 5'-end homologous to the 217 bp region of the chromosome. before the start of the codon of the xylE gene introduced into the primer for further integration of the resulting fragment into the bacterial chromosome.

PCR was performed using a TermoHybaid PCR Express Amplifier. The reaction mixture (total volume - 50 μl) consisted of 5 μl of 10 × PCR buffer with 15 mM MgCl ₂ (Fermentas, Lithuania), 200 μM of each of dNTP, 25 pM of each of the primers used and 1 unit. Taq polymerases (Fermentas, Lithuania). Approximately 5 ng of plasmid DNA was added to the reaction mixture as a template for PCR. The following temperature profile was used: initial DNA denaturation at 95 ° C for 5 min, then 25 denaturation cycles at 95 ° C for 30 s, annealing at 55 ° C for 30 s, chain extension at 72 ° C for 30 s and final completion of the chain at + 72 ° C for 7 min. Next, the amplified DNA fragment was concentrated by agarose gel electrophoresis and purified using GenElute Spin Columns (Sigma, USA) and reprecipitated with ethanol.

Both of the above DNA fragments were treated with BglII and ligated. The ligation product was amplified by PCR; for this, primers P2 (SEQ ID NO: 5) and P3 (SEQ ID NO: 6) were used. Primer P3 contains at the 5'end of 36 nucleotides homologous to the 5'end of the xylE gene introduced into the primer for further integration of the resulting fragment into the bacterial chromosome.

The amplified DNA fragment was concentrated by agarose gel electrophoresis, purified using GenElute Spin Columns (Sigma, USA) and reprecipitated with ethanol. The obtained DNA fragment was used for electroporation and Red-dependent integration into the bacterial chromosome of E. coli strain MG1655 / pKD46.

Cells of strain MG1655 / pKD46 were grown overnight at 30 ° C in LB liquid medium with the addition of ampicillin (100 μg / ml), then diluted with SOB medium at a ratio of 1: 100 (Yeast extract, 5 g / l; NaCl, 0.5 g / l; tryptone, 20 g / L; KCl, 2.5 mm; MgCl ₂ , 10 mm) with the addition of ampicillin (100 μg / ml) and L-arabinose (10 mm) (arabinose is used to induce a plasmid containing the Red system genes) and were grown at 30 ° C until the optical density of the bacterial culture is achieved OD ₆₀₀ = 0.4-0.7. Cells from 10 ml of the grown bacterial culture were washed 3 times with ice-cold deionized water, followed by cell resuspension in 100 μl of water. 10 μl of the DNA fragment (100 ng) was dissolved in deionized water and added to the cell suspension. Electroporation was carried out on a Bio-Rad electroporator (USA) (No. 165-2098, version 2-89) in accordance with the manufacturer's instructions. The parried cells were placed in 1 ml SOC medium (Sambrook et al., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989)), incubated for 2 hours at 37 ° C and then plated on L-agar plates containing 25 μg / ml chloramphenicol. Colonies grown in 24 hours were tested for the presence of a Cm ^R marker instead of the natural promoter region of the xylE gene using NDP using primers P4 (SEQ ID NO: 7) and P5 (SEQ ID NO: 8). For this purpose, freshly grown colonies were resuspended in 20 μl of water, after which 1 μl of the resulting suspension was used for PCR. The following temperature profile was used: initial DNA denaturation at 95 ° C for 10 min, then 30 denaturation cycles at 95 ° C for 30 s, annealing at 55 ° C for 30 s, chain extension at 72 ° C for 1 min and final completion of the chain at + 72 ° C for 7 min. Several tested Cm ^R colonies containing the desired ~ 2000 bp DNA fragment instead of the 192 bp native xylE gene promoter region. (see figure 1). One of the obtained strains was cured of the temperature-sensitive plasmid pKD46 by growing at 37 ° C; the obtained strain was named E. coli MG1655P _L-tac xylE.

Example 2. The effect of enhancing xylE gene expression on the growth of E. coli strain with impaired PTS transport system.

In order to demonstrate the effect of increased xylE gene expression on the growth of E. coli strain, an E. coli strain with a disrupted PTS transport system was constructed.

For this purpose, a DNA fragment containing a kanamycin resistance marker (Km ^R ) was integrated into the chromosome of E. coli strain MG1655 / pKD46 instead of the ptsHI-crr operon by the method described by K. Datsenko and Wanner BL (Proc. Natl. Acad. Sci. USA, 2000, 97, 6640-6645), also known as “Red-dependent integration” and / or “Red-directional integration”, also described in Example 1.

The ptsHI-crr operon is known (nucleotide numbers 2531786 to 2532043, 2532088 to 2533815 and 2533856 to 2534365 for the ptsH, ptsI and err genes, respectively, in the GenBank database, sequence with accession number NC_000913.2, gi: 49175990). The ptsHI-crr operon is located between the cysK and pdxK genes on the chromosome of E. coli K-12 strain.

The DNA fragment containing the Km ^R gene was obtained by PCR using the commercially available plasmid pUC4KAN (GenBank / EMBL accession number X06404, Fermentas, Lithuania) and primers P6 (SEQ ID NO: 9) and P7 (SEQ ID NO: 10). Primer P6 contains 36 nucleotides homologous to the 5 ′ end of the ptsH gene, and primer P7 contains 36 nucleotides homologous to the 3 ′ end of the err gene. These sequences were introduced into primers P6 and P7 for further integration into the bacterial chromosome.

The methodology for PCR is described in Example 1.

Next, the amplified DNA fragment was concentrated by agarose gel electrophoresis, purified using GenElute Spin Columns (Sigma, USA) and reprecipitated with ethanol. The obtained DNA fragment was used for electroporation and Red-dependent integration into the bacterial chromosome of E. coli strain MG1655 / pKD46, similarly to that described in Example 1, except that after electroporation the cells were plated on plates with L-agar containing 50 μg / ml kanamycin.

Colonies grown for 24 hours were tested for the presence of the Km ^R marker instead of the ptsHI-crr operon using PCR using primers P8 (SEQ ID NO: 11) and P9 (SEQ ID NO: 12). For this purpose, freshly grown colonies were resuspended in 20 μl of water, after which 1 μl of the resulting suspension was used for PCR. The PCR conditions were similar to those described in Example 1. Several tested Km ^R colonies contained the desired DNA fragment ~ 1300 bp in size, which confirmed the replacement of the ptsHI-crr operon with the Km ^R gene. One of the obtained strains was cured of the temperature-sensitive plasmid pKD46 by growing at 37 ° C; the obtained strain was named E. coli MG1655 ΔptsHI-crr.

Further, a DNA fragment from the chromosome of the aforementioned E. coli strain MG1655P _L-tac xylE was transferred to E. coli strain MG1655 ΔptsHI-crr by P1 by transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview , NY), resulting in the obtained strain MG1655 ΔptsHI-crr P _L-tac xylE.

Four E. coli strains MG1655, MG1655 ΔptsHI-crr and MG1655 ΔptsHI-crr P _L-tac xylE were tested for their growth ability on Adams minimal medium with glucose (4%) as a carbon source. As can be seen from figure 2, the strain E. coli MG1655 ΔptsHI-crr poorly grows on minimal Adams medium containing glucose (μ ~ 0.06). Enhanced xylE gene expression significantly improves the growth characteristics of recipient strains on Adams minimal glucose medium.

Example 3. The effect of enhancing xylE gene expression on threonine production.

To identify the effect of the amplification of xylE gene expression under the control of the P _L-tac promoter on the production of threonine, DNA fragments from the chromosome of the above E. coli strain MG1655P _L-tac xylE were transferred to the E. coli threonine producing strain VKPM B-3996 P1 by transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY)). The VKPM strain B-3996 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF, 117545 Moscow, 1st Dorozhniy proezd, 1) on April 7, 1987 under the number B-3996.

Both strains of E. coli B-3996 and B-3996 P _L-tac xylE were grown for 18-24 hours at 37 ° C on plates with L-agar. To obtain a seed culture, the strain was grown in 20 × 200 mm test tubes containing 2 ml of L-broth with 4% sucrose on a rotary shaker (250 rpm) at 32 ° C for 18 hours. Next, inoculum 0.21 ml (10%) was introduced into the fermentation medium. Fermentation was carried out in 20 × 200 mm tubes with 2 ml of minimal fermentation medium. Cells were grown for 48 hours at 32 ° C using a shaker with a rotation speed of 250 rpm.

After cultivation, the amount of L-threonine accumulated in the medium was determined by paper chromatography. The following composition of the liquid phase was used: butanol: acetic acid: water = 4: 1: 1 (v / v). For visualization, a solution of ninhydrin in acetone (2%) was used. The spots with L-threonine were excised, L-threonine was eluted in a 0.5% aqueous solution of CdCl ₂ , and the amount of L-threonine was measured using a spectrophotometer at 540 nm. The results are presented in Table 1. The production of threonine increased due to the introduction of the promoter P _L-tac xylE.

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

Glucose 80.0 (NH ₄ ) ₂ SO ₄ 22.0 NaCl 0.8 KH ₂ PO ₄ 2.0 MgSO ₄ · 7H ₂ O 0.8 FeSO ₄ · 7H ₂ O 0.02 MnSO ₄ · 5H ₂ O 0.02 Thiamine HCl 0,0002 Yeast extract 1,0 CaCO ₃ 30.0

Sucrose and magnesium sulfate were sterilized separately. CaCO _{3 was} sterilized by dry heat at 180 ° C for 2 h, the pH was adjusted to 7.0. Antibiotics were added to the medium after sterilization.

Example 4. The effect of enhancing xylE gene expression on L-lysine production.

The complete nucleotide sequence of the chromosomal DNA of E. coli strain W3110 is known (Science, 277, 1453-1474 (1997)). Based on the published nucleotide sequence, primers were synthesized and the xylE gene was amplified by PCR, as described below.

Chromosomal DNA was obtained in the traditional way (Sambrook, J., Fritsch E.F. and Maniatis T. (1989): Molecular Cloning: A laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Primer 10 (SEQ ID NO: 13) was created as the nucleotide sequence 7479-7508 in sequence with accession number AE000476, the EcoRI restriction enzyme recognition site was introduced at the 5'-end thereof, and primer 11 (SEQ ID NO: 14) was created as a sequence complementary to nucleotide sequence 8963-8992, in sequence with accession number AE000476, at the 5'end of which a Sail restriction enzyme recognition site was introduced. These primers were used to amplify the xylE gene under standard conditions as described in "PCR protocols. Current methods and applications" (White, B.A., ed., Humana Press, Totowa, New Jersey, 1993).

The PCR product was purified in a conventional manner. The resulting fragment was digested with SalI and EcoRI restriction enzymes, and using the ligation kit, cloned into the pSTV29 vector pretreated with the same restriction enzymes.

Competent cells of E. coli strain JM109 were transformed with a ligation product (Sambrook, J., Fritsch EF and Maniatis T. (1989) Molecular Cloning: A laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), after whereby the cells were plated on plates with L-agar (Bacto-tryptone: 10 g / l, yeast extract: 5 g / l, NaCl: 5 g / l, agar: 15 g / l, pH 7.0) containing 10 μg / ml IPTG (isopropyl-β-D-thiogalactopyranoside), 40 μg / ml X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 50 μg / ml chloramphenicol, and grown overnight. The resulting white colonies were sterilely resected, thus transformants were obtained. Plasmids from transformants were isolated by alkaline extraction, thereby obtaining the pSTV29-xylE plasmid, in which the xylE gene was placed under the lac promoter.

The strain E. coli WC196 was used as an example of a producer strain of L-lysine belonging to the genus Escherichia.

WC196 was transformed with either pSTV29-xylE plasmid or pSTV29 vector, resulting in strains of WC196 / pSTV29-xylE and WC196 / pSTV29. Each of these strains was grown in L medium containing 50 mg / L chloramphenicol at 37 ° C until an optical density OD of about 0.6 was reached at a wavelength of 600 nm. Then equal volumes of a 40% glycerol solution were added to the cell culture, after which this mixture was placed in a certain volume and stored at -80 ° C. This mixture was called the "glycerin museum."

To assess the effect of increased xylose permease activity on L-lysine production, strain WC196 was transformed with plasmids pSTV29-xylE and pCABD2 in accordance with the method described above. pCABD2 is a plasmid containing the dapA gene encoding a mutant dihydropicolinate synthase that is not inhibited by feedback L-lysine, the lysC gene encoding mutant aspartokinase III, which is not inhibited by feedback L-lysine and a dapB gene encoding gene digidropic encoding diaminopimelate dehydrogenase (US patent 6040160). For control, strain WC196 was transformed with plasmids pSTV29 and pCABD2. Each of the obtained transformants was cultured in L medium containing 50 mg / L of chloramphenicol and 20 mg / L of streptomycin at 37 ° С until the OD value of about 0.6 was reached at a wavelength of 600 nm. Then equal volumes of a 40% glycerol solution were added to the cell culture, after which this mixture was placed in a specific volume and stored at -80 ° C.

The glycerin museum of strains WC196 / pSTV29-xylE and WC196 / pSTV29 was melted and 100 μl from each sample were uniformly scattered on L-agar plates containing 50 mg / l chloramphenicol and grown at 37 ° C for 24 hours. In addition, each of the strains WC196 / (pCABD2, pSTV29-xylE) and WC196 / (pCABD2, pSTV29) were uniformly seeded on L-agar plates containing 50 mg / l chloramphenicol and 20 mg / l streptomycin and grown at 37 ° With in 24 hours. About 1/8 of the number of cells per plate was added to 20 ml of fermentation medium containing the necessary additives in a 500 ml flask. Cultivation was carried out at 37 ° C for 16 hours using a reciprocating shaker with a rotation speed of 115 rpm. After growing, the amount of L-lysine and residual glucose accumulated in the medium was measured by known methods (Biotech-analyzer AS210, manufactured by Sakura Seiki Co.). After that, the yield of L-lysine was calculated with respect to the glucose used for each of the strains.

Used the following composition of the fermentation medium (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 were sterilized separately. 30 g / L CaCO _{3 was} also added, which was sterilized by dry heat at 180 ° C for 2 hours.

The results are shown in Table 2. The strain WC196 / pSTV29-xylE accumulates a greater amount of L-lysine compared to strain WC196 / pSTV29, in which the expression amount of the xylose permease gene is not increased. In addition, it was noted that an increase in xylose permease activity positively affects the accumulation and yield of L-lysine in the strain WC196 / pCABD2, in which the yield of L-lysine increased.

Example 5. The effect of enhancing xylE gene expression on L-arginine production.

To identify the effect of enhancing xylE gene expression under the control of the P _L-tac promoter on arginine production, DNA fragments from the chromosome of the E. coli MG1655P _L-tac xylE strain described above were transferred to E. coli 382 P1 arginine producing strain by transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY). Strain 382 was deposited with the All-Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) on April 10, 2000 with accession number VKPM B-7926.

The obtained strain 382 P _L-tac xylE and the parent strain 382 were cultured at 32 ° C for 18 hours in 2 ml of LB nutrient broth and 0.3 ml of the obtained culture were introduced into 20 × 200 mm tubes with 2 ml of fermentation medium and cultured at 32 ° C for 48 hours on a rotary rocking chair.

After cultivation, the amount of L-arginine accumulated in the medium was determined by paper chromatography. The following composition of the liquid phase was used: butanol: acetic acid: water = 4: 1: 1 (v / v). For visualization, a solution of ninhydrin in acetone (2%) was used. L-arginine stains were excised, L-arginine was eluted in a 0.5% aqueous solution of CdCl _2, and the amount of L-arginine was measured using a spectrophotometer at a wavelength of 540 nm.

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

Glucose 48.0 (NH ₄ ) ₂ SO ₄ 35.0 KH ₂ PO ₄ 2.0 MgSO ₄ · 7H ₂ O 1,0 Thiamine HCl 0,0002 Yeast extract 1,0 L-isoleucine 0.1 CaCO ₃ 5,0

Glucose and magnesium sulfate were sterilized separately. CaCO _{3 was} sterilized by dry heat at 180 ° C for 2 h, the pH was adjusted to 7.0.

The results of 10 independent experiments are presented in Table 3.

As can be seen from Table 3, strain 382 P _L-tac xylE accumulates a greater amount of L-arginine compared to strain 382, in which the expression amount of D-xylose permease is not increased.

Example 6: Production of L-histidine by a bacterium-producer of L-histidine by fermentation of a mixture of glucose and xylose.

Strain E. coli 80 - producer of L-histidine was used for the production of L-histidine by fermentation of a mixture of glucose and xylose. Strain E. coli 80 (VKPM B-7270) is described in detail in the patent of the Russian Federation No. 2119536.

To test the effect of enhancing the expression of the xylE gene under the control of the P _L-tac promoter on histidine production, the DNA fragment of their chromosome of the above E. coli strain MG1655P _L-tac xylE was transferred to E. coli 80 strain, a histidine producer by P1 transduction method (Miller , JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY). Transformation of strain 80 and the resulting strain of 80 P _L-tac xylE with plasmid pMW119mod-xylA-R was carried out by the standard method to obtain strains 80 / pMW119mod-xylA-R and 80 P _L-tac xylE / pMW119mod-xylA-R. Cloning of the xylABFGHR locus from the chromosome of E. coli strain MG1655 is described in Russian Patent Application No. 2005106720.

To obtain a seed culture, both strains 80 / pMW119mod-xylA-R and 80 P _L-tac xylE / pMW119mod-xylA-R were grown on a rotary stirrer (250 rpm) at 27 ° С for 6 hours in 40 ml tubes (⌀ 18 mm) containing 2 ml of L-broth with 1 g / l of streptomycin and 100 mg / l of ampicillin. Then 2 ml (5%) of seed was transferred to a nutrient medium for fermentation. Fermentation was carried out on a rotary shaker (250 rpm) at 27 ° C for 50 hours in 40 ml tubes containing 2 ml of culture medium for fermentation.

After growing, the amount of L-histidine accumulated in the culture fluid was determined by paper chromatography. The composition of the mobile phase was as follows: butanol: acetate: water = 4: 1: 1 (v / v). A solution of ninhydrin in acetone (0.5%) as imaging. The results are presented in Table 4.

The composition of the nutrient medium for fermentation (g / l):

Carbohydrates (total) 100.0 Mameno (Soya Bean Hydrolyzate) 0.2 total nitrogen L-proline 0.8 (NH ₄ ) ₂ SO ₄ 25.0 K ₂ HPO ₄ 2.0 MgSO ₄ · 7H ₂ O 1,0 FeSO ₄ · 7H ₂ O 0.01 MnSO ₄ · 5H ₂ O 0.01 Thiamine HCl 0.001 Betaine 2.0 CaCO ₃ 6.0 Streptomycin 1,0

Carbohydrates (glucose, xylose), L-proline, betaine and magnesium sulfate were sterilized separately. CaCO _{3 was} sterilized by heating to 110 ° C for 30 minutes. The pH of the medium was maintained at 6.0 by the addition of KOH after sterilization.

As can be seen from Table 4, strain 80 P _L-tac xylE / pMW119mod-xylA-R accumulated a greater amount of L-histidine in a medium containing a mixture of glucose and xylose, compared with strain 80 / pMW119mod-xylA-R, in which permease expression D-xylose has not been enhanced.

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

Each of the documents mentioned above is part of the description in its entirety.

Claims (25)

1. The bacterium producing L-amino acids belonging to the genus Escherichia, characterized in that the bacterium is modified so that the activity of D-xylose permease in this bacterium is increased. 2. The bacterium according to claim 1, characterized in that the indicated D-xylose permease activity is increased by enhancing the expression of a gene encoding a D-xylose permease. 3. The bacterium according to claim 2, characterized in that the indicated activity of D-xylose permease is increased by modifying the sequence that controls the expression of the gene encoding D-xylose permease, or by increasing the number of copies of the gene encoding D-xylose permease. 4. The bacterium according to claim 1, characterized in that the bacterium is further modified so that the activity of glucokinase in this bacterium is increased. 5. The bacterium according to claim 1, characterized in that the bacterium is further modified so that the activity of xylose isomerase in this bacterium is increased. 6. The bacterium according to claim 1, characterized in that the bacterium is further modified so that expression of the xylABFGHR locus is enhanced in it. 7. The bacterium according to claim 2, characterized in that said gene encodes a D-xylose permease selected from the group consisting of (A) a protein comprising the amino acid sequence under the number SEQ ID NO: 2; and (B) a variant protein with the amino acid sequence number SEQ ID NO: 2, 8. The bacterium according to claim 2, characterized in that said gene encoding D-xylose permease includes DNA selected from the group consisting of (a) DNA comprising the nucleotide sequence from nucleotide 1 to nucleotide 1476 in the sequence of SEQ ID NO: 1; and (b) DNA that hybridizes with the nucleotide sequence from nucleotide 1 to nucleotide 1476 in the sequence of SEQ ID NO: 1, or with a probe that can be prepared based on the specified nucleotide sequence under stringent conditions, and encodes a protein having permease activity D- xyloses. 9. The bacterium of claim 8, characterized in that the stringent conditions are conditions under which washing is carried out at 60 ° C, at a salt concentration of 1 × SSC and 0.1% SDS, and the washing time is 15 minutes 10. The bacterium according to claim 1, characterized in that said bacterium is a producer of L-threonine. 11. The bacterium of claim 10, characterized in that the bacterium is further modified in such a way that the expression of a gene selected from the group consisting of a mutant thrA gene encoding aspartokinase homoserine dehydrogenase I and resistant to threonine inhibition by the feedback principle; thrB gene encoding homoserine kinase; thrC gene encoding threonine synthase; rhtA gene encoding a hypothetical transmembrane protein; and any combination of these genes. 12. The bacterium according to claim 11, wherein said bacterium is modified so that the expression of said mutant thrA gene, said thrB gene, said thrC gene, and said rhtA gene is enhanced. 13. The bacterium according to claim 1, characterized in that said bacterium is a producer of L-lysine. 14. The bacterium according to claim 1, characterized in that said bacterium is a producer of L-histidine. 15. The bacterium according to claim 1, characterized in that said bacterium is a producer of L-phenylalanine. 16. The bacterium according to claim 1, characterized in that said bacterium is a producer of L-arginine. 17. The bacterium according to claim 1, characterized in that said bacterium is a producer of L-glutamic acid. 18. A method for producing an L-amino acid, comprising the steps of growing the bacterium according to claim 1 in a fermentation medium, leading to the accumulation of the L-amino acid in the culture fluid, and the isolation of the L-amino acid from the culture fluid. 19. The method according to p, characterized in that the nutrient medium contains xylose. 20. The method according to p, characterized in that the specified L-amino acid is L-threonine. 21. The method according to p, characterized in that the specified L-amino acid is L-lysine. 22. The method according to p. 18, characterized in that the specified L-amino acid is L-histidine. 23. The method of claim 18, wherein said L-amino acid is L-phenylalanine. 24. The method of claim 18, wherein said L-amino acid is L-arginine. 25. The method according to p, characterized in that the specified L-amino acid is L-glutamic acid.

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