RU2315808C2 - METHOD FOR PREPARING L-AMINO ACIDS USING MICROORGANISM BELONGING TO Escherichia GENUS WHEREIN yafA GENE IS INACTIVATED - Google Patents

Abstract

FIELD: biotechnology, microbiology, amino acids.

SUBSTANCE: invention proposes a method for preparing L-amino acid, such as L-threonine or L-tryptophan, using a microorganism belonging to Escherichia genus and modified so that gene yafA in this microorganism is inactivated. Invention provides preparing L-amino acids with higher degree of effectiveness.

EFFECT: improved preparing method of amino acids.

3 cl, 3 dwg, 3 tbl, 11 ex

Description

Technical field

The present invention relates to the microbiological industry, in particular to a method for producing L-amino acids using bacteria of the Enterobacteriaceae family, in which the expression of the yafA gene is impaired.

State of the art

Bacterial Phosphoenolpyruvate (PEP) System: Sugar Phosphotransferase (PTS) plays an important role in the transport of various sugars. The Escherichia coli PTS system consists of two main cytoplasmic proteins: enzyme I (enzyme I (EI)) and the phosphorylating histidine transporter protein, HPr, used for all sugars, and several sugar-specific components known under the general name enzymes II, including soluble IIA ^Glc enzyme and membrane-bound IICB ^Glc enzyme (Escherichia coli and Salmonella, Second Edition, Editor in Chief: FCNeidhardt, ASM Press, Washington DC, 1996, pp. 1149-1174). Glucose utilization is accompanied by a sequential transfer of the phosphorus group in the PTS system, in the following order: phosphoenolpyruvate (PEP) → EI → HPr → IIA ^Glc → IICB ^Glc → glucose.

It has been shown that each protein component of the PTS system in E. coli involved in glucose utilization regulates the activity of the partner protein through direct interaction of proteins with each other. These regulatory functions of the PTS system depend on the degree of phosphorylation of the component involved in the reaction. The product of the crr gene (enzyme IIA ^Glc ) serves as a link in some known regulatory processes. It has also been shown that the product of the yafA gene (also known as the frsA gene), the FrsA regulatory protein (fermentation / respiration switch protein) forms a complex that specifically interacts with the non-phosphorylated form of the IIA ^Glc enzyme (Co., B.M. et al. J Biol Chem. 2004 Jul 23; 279 (30): 31613-21). These authors also showed that disruption of the yafA gene increases cellular respiration during growth on certain sugars, including glucose.

There are currently no reports describing the use of yafA gene inactivation to produce L-amino acids.

Description of the invention

The objectives of the present invention are to increase the productivity of strains producing L-amino acids and the provision of a method for producing L-amino acids using these strains.

These goals were achieved by detecting the fact that weakening the expression of the yafA gene can increase the production of L-amino acids, such as L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L- phenylalanine, L-tryptophan, L-proline and L-arginine.

The present invention provides a bacterium of the Enterobacteriaceae family that is capable of increased production of amino acids such as L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L-phenylalanine, L-tryptophan, L Proline and L-Arginine.

The aim of the present invention is the provision of bacteria producing L-amino acids of the Enterobacteriaceae family, wherein said bacterium is modified so that the expression of the yafA gene in this bacterium is weakened.

It is also an object of the present invention to provide a bacterium as described above in which expression of the yafA gene is attenuated by inactivation of the yafA gene.

It is also an object of the present invention to provide the bacteria described above, wherein said bacterium belongs to the genus Escherichia.

It is also an object of the present invention to provide the bacteria described above, wherein said bacterium belongs to the genus Pantoea.

It is also an object of the present invention to provide the bacteria described above, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.

It is also an object of the present invention to provide the bacteria described above, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine and L-tryptophan.

It is also an object of the present invention to provide the bacteria described above, wherein said non-aromatic L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine , L-histidine, L-glycine, L-serine, L-alanine, L-asparagine, L-aspartate, L-glutamine, L-glutamic acid, L-proline and L-arginine.

Another objective of the present invention is the provision of a method for producing L-amino acids, which includes:

- growing the bacteria described above in a nutrient medium for the purpose of production and isolation of L-amino acids in a nutrient medium, and

- collection of the specified 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 selected from the group consisting of aromatic L-amino acids and non-aromatic L-amino acids.

It is also an object of the present invention to provide the method described above, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine and L-tryptophan.

It is also an object of the present invention to provide the method described above, wherein said non-aromatic L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine , L-histidine, L-glycine, L-serine, L-alanine, L-asparagine, L-aspartate, L-glutamine, L-glutamic acid, L-proline and L-arginine.

In more detail, the present invention is described below.

DETAILED DESCRIPTION OF BEST MODES FOR CARRYING OUT THE INVENTION

1. The bacterium according to the present invention.

The bacterium of the present invention is an L-amino acid producing bacterium of the Enterobacteriaceae family, the bacterium being modified in such a way that the expression of the yafA gene in such a bacterium is weakened.

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.

As used herein, the term “L-amino acid producing bacterium” also means a bacterium that is capable of producing the L-amino acid and causes the accumulation of the L-amino acid in the culture medium in large quantities, compared to 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-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.

The term “aromatic L-amino acid” includes L-phenylalanine, L-tyrosine and L-tryptophan. The term “non-aromatic L-amino acid” includes L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, L-glycine, L-serine, L-alanine, L-asparagine, L-aspartate, L-glutamine, L-glutamic acid, L-proline and L-arginine. L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L-phenylalanine, L-tryptophan, L-proline and L-arginine are most preferred.

The Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, etc. More precisely, a database (http://www.ncbi.nlm.nih.gov/htbinpost/Taxonomy/wgetorg?mode can be used to systematize the birth within the Enterobacteriaceae family in accordance with the taxonomy adopted by the NCBI (National Center for Biotechnology Information) = Tree & id = 1236 & lvl = 3 & keep = l & srchmode = l & 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. Examples of bacteria belonging to the genus Escherichia used in the present invention include, but are not limited to, the bacterium Escherichia coli (E. coli).

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 term “bacterium is modified, so the expression of the yafA gene is weakened,” means that the bacterium is modified so that the modified bacterium contains a reduced amount of FrsA protein compared to an unmodified bacterium, or becomes unable to synthesize FrsA protein.

The term "inactivation of the yafA gene" means that the modified gene encodes a fully non-functional protein. It is also possible that the modified portion of DNA is unable to express the gene normally as a result of deletion of a portion of the gene, shift of the reading frame of the gene, introduction of the missense / nonsense mutation (s), or modification of the region adjacent to the gene, inclusion of sequences that control gene expression, such as promoters, enhancers, attenuators, ribosome binding sites, etc.

The role of the FrsA regulatory protein encoded by the yafA gene in the production of L-amino acids remains unclear. However, it was shown that the destruction of the y-fA gene leads to increased cellular respiration with growth on several sugars, including glucose (Co., B.M. et al. J Biol Chem. 2004 Jul 23; 279 (30): 31613-21).

The yafA gene encodes a regulatory protein FrsA, which forms a specific complex with the non-phosphorylated form of the IIA ^Glc protein in a 1: 1 ratio. The yafA gene (gi: 16128225; nucleotide numbers 256527 to 257771 in sequence with accession number NC_000913.1 in the GenBank database) is located between the gpt and crl genes on the chromosome of E. coli K-12 strain. The nucleotide sequence of the yafA gene and the amino acid sequence of the FrsA regulatory protein encoded by it are shown in the List of sequences under the numbers SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

A BLAST search leads to the conclusion that the gene is orthologous. yafA exists in only a few gram-negative bacteria, such as E. coli, Salmonella typhimurium, Shigella flexneri, Yersinia pestis, Vibrio cholerae, V. vulnificus, V. parahaemolyticus and Photorhabdus luminescens (www.ncbi.nlm.nih.gov). All these types of bacteria are facultative anaerobes belonging to the group of γ-proteobacteria and most of them are highly pathogenic.

Based on statistical analysis of the database of transmembrane proteins found in nature, TMbase program built a model of the transmembrane topology of the FrsA protein (Figure 3). TMpred predicts transmembrane protein segments and their orientation. The prediction is based on the use of a combination of several weight matrices when calculating (TMbase - a database of transmembrane protein sections by K. Hofmann & W. Stoffel (1993) Biol. Chem. Hoppe-Seyler 374.166, http: //www.ch.embnet. Org / software /TMPRED_form.html).

This model found 6 stable transmembrane spirals with a total score of 6482 and prediction parameters: the length of the TM helix is between 14 and 35.

No. from before length score orientation one 205 223 (19) 513 o-i (outside-inside) 2 328 346 (19) 1059 i-o 3 435 454 (twenty) 1016 o-i four 472 487 (16) 980 i-o 5 823 841 (19) 1604 o-i 6 871 890 (twenty) 1310 i-o

Since there may be some differences in the DNA sequences of different genera or strains of the Enterobacteriaceae family, the nucleotide sequence of the yafA gene to be inactivated is not limited to the gene sequence shown in SEQ ID No: 1, but may also include gene sequences homologous to the sequence shown in SEQ ID No: 1 encoding a variant of the FrsA protein. The term “protein variant” as used in this discovery means a protein that has changes in sequence, such as deletions, insertions, additions or substitutions of amino acids, but as long as the activity of the protein product, namely the regulatory protein FrsA, is maintained. The number of changes in a protein variant depends on the position or type of amino acid residues in the tertiary spatial structure of the protein. It can vary in the range from 2 to 30, more preferably in the range from 2 to 15, and most preferably in the range from 2 to 5 in the full amino acid sequence shown in the list of sequences under the number SEQ ID NO.2. These changes in protein variants can occur at protein sites that are not essential for protein function. Such a change does not lead to a violation of the tertiary spatial structure or activity of the protein, since some amino acids have a high homology between each other. These changes in the protein variant may occur in areas of the protein that are not important for protein function. Thus, a variant of the protein encoded by the yafA gene can be represented by a protein with a homology of at least 80%, more preferably at least 90%, and most preferably at least 95%, with respect to the complete amino acid sequence encoded by the yafA gene and shown in The sequence listing under number 2 (SEQ ID NO.2), as long as the activity of the protein product, namely the regulatory protein FrsA, is maintained.

The homology between two amino acid sequences can be determined using well-known methods, for example, using the computer program BLAST 2.0, which calculates three parameters: score, identity and similarity. In addition, the yafA gene can be represented by a variant that hybridizes with the nucleotide sequence shown in Sequence Listing at number 1 (SEQ ID NO: 1), or with a probe that can be synthesized based on the specified nucleotide sequence, under stringent conditions, under provided that this option encodes a regulatory protein FrsA. "Stringent conditions" include those conditions under which specific hybrids are formed and non-specific hybrids are not formed. For example, a demonstration of harsh conditions can be a single or multiple washing, preferably two or three washing, at a salt concentration of 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, at a temperature of 60 ° C. The duration of washing depends on the type of membrane used for blotting, and, as a rule, on the recommendations of the manufacturer.

For example, the recommended duration of washing a Hybond N + nylon membrane (Amersham) under stringent conditions is 15 minutes. Preferably, the washing can be carried out from 2 to 3 times. The length of the probe can be selected accordingly, depending on the hybridization conditions, and usually varies in the range of 100 bp. up to 1 thousand bp

Inactivation of the indicated gene can be performed by traditional methods, such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, gene inactivation by homologous recombination, and / or insertion-deletion mutagenesis (Yu, D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 5978-83) and (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA , 2000.97: 12: 6640-45), also called "Red-Dependent Integration".

Inactivation of the yafA gene can be ascertained using Phenotype Microarrays matrices (PMs) (Biolog, Inc.) using cell respiration rate as a reporter system. The chemical reaction of this method is based on staining with a tetrazolium dye and subsequent colorimetric measurement of the cell respiration rate. Detailed information on RM experiments is available at www.biolog.com. An analysis of Phenotype Microarrays showed that inactivation of the yafA gene increases the rate of cellular respiration on several sugars, including arabinose, fructose, lactose, and glucose (Co., B.M. et al. J Biol Chem. 2004 Jul 23; 279 (30): 31613- 21).

The decrease or absence in the bacterium of the activity of the FrsA protein, according to the present discovery, can be established by comparison with the parent unmodified bacterium. In addition, the level of gene expression can be determined by measuring the amount of mRNA transcribed by the gene using various well-known methods, including Northern blotting, quantitative RT-PCR and similar methods. The amount or molecular weight of the protein encoded by the gene can be measured by well-known methods, including SDS-PAGE followed by Western blot analysis by Western blotting and similar methods.

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).

Bacteria - L-amino acid producer

As a bacterium according to the present invention, which is modified in such a way that expression of the yafA gene is impaired, a bacterium capable of producing L-aromatic or L-non-aromatic amino acids can be used.

The bacterium of the present invention can be obtained by attenuating the expression of the yafA gene in a bacterium already possessing the ability to produce L-amino acids. Alternatively, the bacterium of the present invention can be obtained by imparting a bacterium in which the expression of the yafA gene is already impaired to the ability to produce L-amino acids.

Bacteria - producer of L-threonine

Examples of parent strains for producing 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 Pat. No. 5,175,107 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 PERM BP-3520 (US patent 5,376,538), strain E. coli MG442 (Gusyatiner et al., Genetics (in Russia), 14, 947-956 (1978)), E. coli strains VL643 and VL2055 (European patent application EP 1149911 A) and the like.

Strain TDH-6 is deficient in the thrC gene, is capable of assimilating glucose, and contains the 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) with inventory number B-3996.

It is desirable that the bacterium according to the present invention, be further modified so as to have increased expression of one or more genes listed below:

- 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;

- asd gene encoding aspartate β-semialdehyde dehydrogenase;

- rhtA gene, presumably encoding a transmembrane protein; and

- aspC gene encoding aspartate aminotransferase (aspartate transaminase);

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

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

The rhtA gene is located for 18 min on the E. coli chromosome next to the glnHPQ operon, which encodes the components of the glutamine transport system. The rhtA gene is identical to ORF1 (ybiF gene, nucleotide numbers from 764 to 1651 in sequence with accession number AAA218541, gi: 440181 in the GenBank database) and is located between the pXB and opX genes. The protein expressing element encoded by ORF1 was designated as the rhtA gene (rht: resistance to homoserin and threonine). The rhtA23 mutation has also been shown to replace nucleotide residue A with nucleotide residue 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 E. coli asd gene has already been described (nucleotide numbers 3572511 to 3571408 in sequence with accession number 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. Asd genes from other microorganisms can be obtained in a similar way.

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

Bacteria - Producer of L-Lysine

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 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 conventional mutagens. Specific examples of bacterial strains used to produce L-lysine include Escherichia coli strain AJ 11442 (FERM BP-1543, NRRL B-12185; see US patent 4346170) 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 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, Ministry of International Trade and Industry) (currently called the National Institute of Advanced Industrial Science and Technology, International Depository of Organizations for Patenting Purposes, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan ( National in 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 received PERM accession number P-14690. Then, the strain was internationally deposited in accordance with the conditions of the Budapest Treaty of September 29, 1995, and the strain received accession number FERM BP-5252 (see U.S. Patent 5,827,698).

Bacteria - L-cysteine producer

Examples of parental strains used to produce the L-cysteine producing bacterium of 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 gene alleles encoding inhibition resistant feedback type serine acetyltransferase (US patent 6218168, patent application of the Russian Federation 2003121601); E. coli strain W3110 containing overexpressed genes encoding proteins involved in the secretion of compounds toxic to the cell (US patent 5972663); E. coli strains containing cysteine desulfohydrase with reduced activity (Japanese patent JP11155571 A2); E. coli strain W3110 with increased activity of the positive transcriptional regulator of the cysteine regulon encoded by the cysB gene (PCT application WO 0127307 A1) and the like.

Bacteria - Producer of L-Leucine

Examples of parental 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 resistant to leucine analogs, 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 the PCT application 96/06926; E. coli strain H-9068 (JP8-70879 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. 6,333,342). In addition, the bacterium of the present invention can be improved by enhancing the expression of one or more genes encoding proteins that export an L-amino acid from a bacterial cell. Examples of such genes include the b2682 and b2683 genes (ygaZH genes) (RF patent application 2001117632, European patent application EP 1239041 A2).

L-histidine producing bacterium

Examples of parental strains used to produce the L-histidine producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli 24 strains (VKPM B-5945, RF 2003677); E. coli 80 (VKPM B-7270, RF 2119536); E. coli NRRL B-12116-B12121 (U.S. Patent 4,388,405); E. coli H-9342 (FERM BP-6675) and E. coli H-9343 (FERM BP-6676) (US patent 6344347); E. coli H-9341 (FERM BP-6674) (European patent EP1085087); E. coli AI 80 / pFM201 (US patent 6258554), and the like.

L-glutamic acid producing bacterium

Examples of parent 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 VL334thrC ⁺ strains (European patent EP 1172433). Strain E. coli VL334 (VKPM B-1641) is auxotrophic for L-isoleucine and L-threonine with mutations in the thrC and ilvA genes (US patent 4278765). 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 K 12 strain (VKPM B-7). The result was a strain of VL334thrC ⁺ , auxotrophic for L-isoleucine. This strain has the ability to produce L-glutamic acid.

Examples of parental strains used to produce the L-glutamic acid producing bacterium of the present invention include mutant strains lacking α-ketoglutarate dehydrogenase activity or having reduced α-ketoglutarate dehydrogenase activity. Bacteria belonging to the genus Escherichia, lacking the activity of α-ketoglutarate dehydrogenase or having a decreased activity of α-ketoglutarate dehydrogenase and methods for their preparation are described in US patents 5378616 and 5573945. Specifically, examples of such strains include the following strains:

E.coli W3110 sucA :: Kmr

E. coli AJ12624 (PERM BP-3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJ12949 (PERM BP-4881)

The E. coli strain W3110 sucA :: Kmr was obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter referred to as the sucA gene) in the 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 Pantoea, which are devoid of α-ketoglutarate dehydrogenase activity or have reduced α-ketoglutarate dehydrogenase activity, and can be obtained as described above. An example of such strains is Pantoea ananatis strain AJ13356 (US Pat. No. 6,331,419). The strain Pantoea ananatis AJ13356 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, the Ministry of International Trade and Industry), National Institute of Biological Sciences and Human Technology (currently called the National Institute of Advanced Industrial Science and Technology, International Depository of Organizations for Patenting Goals, 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) on 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 FERM BP-6615. Strain Pantoea ananatis AJ 3356 does not have α-KGDH activity as a result of destruction of the subunit of the αKGDH-E1 gene (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 at the above depository, for the purposes of this description they will be referred to as Pantoea ananatis.

L-phenylalanine producing bacterium

Examples of parental strains used to produce L-phenylalanine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli AJ12739 strains (tyrA :: Tn10, tyrR) (VKPM B -8197); E. coli HW1089 (ATCC 55371) containing the pheA34 gene (US Pat. No. 5,354,672); E. coli MWEC101-b (KR 8903681); E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146, and NRRL B-12147 (U.S. Patent 4,407,952). E. coli K-12 [W3110 (tyrA) / pPHAB (FERM BP-3566), E. coli K-12 [W3110 (tyrA) / pPHAD] (FERM BP-12659) strains can also be used as the parent strain. E. coli K-12 [W3110 (tyrA) / pPHATerm] (FERM BP-12662), and E. coli K-12 [W3110 (tyrA) / pBR-aroG4, pACMAB] named AJ12604 (FERM BP-3579) (European Patent EP 488424 B1). In addition, L-phenylalanine producing bacteria belonging to the genus Escherichia and having increased activity of the protein encoded by the yedA or yddG genes (US patent applications 2003/0148473 A1 and 2003/0157667 A1) can be used.

L-tryptophan producing bacterium

Examples of parental strains used to produce L-tryptophan-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strains JP4735 / pMU3028 (DSM10122) and JP6015 / pMU91 (DSM10123 ) deficient in tryptophanyl tRNA synthetase encoded by the mutant trpS gene (US patent 5756345); E. coli strain SV164 (pGH5) having a serA allele encoding phosphoglycerate dehydrogenase resistant to feedback inhibition by serine and a trpE allele encoding anthranilate synthase resistant to feedback tryptophan inhibition (US Pat. No. 6,180,373); E. coli strains AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) deficient in the tryptophanase enzyme (US patent 4371614); E. coli strain AGX17 / pGX50, pACKG4-pps, in which phosphoenolpyruvate-producing ability is enhanced (PCT application WO 9708333, US patent 6319696), and strains like the like.

It was previously found that the yddG gene encodes a membrane protein that is not involved in the biosynthesis pathway of any L-amino acid and gives the microorganism resistance to L-phenylalanine and several amino acid analogs when the wild-type allele of this gene is amplified in the microorganism on a high copy vector. In addition, the yddG gene can enhance the production of L-phenylalanine or L-tryptophan when additional copies are introduced into the cells of the corresponding producer strain (PCT application WO 03044192). Therefore, it is desirable that the bacterium producing L-tryptophan be further modified so as to have enhanced expression of the open reading frame of yddG.

L-proline producing bacterium

Examples of parental strains used to produce L-proline producing bacteria according to the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as strains of E. coli 702ilvA (VKPM B-8012), which is deficient in the ilvA gene and is capable of producing L-proline (European application EP 1172433). According to the present invention, the bacterium can be improved by enhancing the expression of one or more genes involved in the biosynthesis of L-proline. Examples of such genes for L-proline producing bacteria include the proB gene encoding glutamate kinase, which has a reduced feedback sensitivity to L-proline inhibition (German patent 3127361). Additionally, the bacterium according to the present invention can be improved by enhancing the expression of one or more genes encoding proteins involved in the excretion of L-amino acids from a bacterial cell. Such genes are represented by the b2682 and b2683 genes (ygaZH genes) (European patent application EP 1239041 A2).

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

L-arginine producing bacterium

Examples of parent strains used to produce L-arginine producing bacteria 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) (US Patent Application 2002058315) ) and strains derived from this strain and incorporating mutant N-acetylglutamate synthase (RF patent application 2001112869), E. coli 382 strain (VKPM B-7926) (European patent application EP 1170358 A1), arginine producing strain, which has the argA gene encoding N-acetylglutamate synthase introduced cited therein (Japanese Patent Application Laid-Open JP57-5693A) and the like.

2. The method according to the present invention

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 in order to produce and isolate an L-amino acid in a culture medium and collect the L-amino acid from the culture medium.

According to the present invention, the cultivation, collection and purification of the L-amino acid from a culture or similar medium can be carried out in a manner similar to traditional fermentation methods in which the amino acid is produced using bacteria.

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 method of nutrient absorption by 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 may be used as vitamins.

The cultivation is preferably carried out under aerobic conditions, which are achieved by mixing the culture and shaking the culture with aeration in the temperature range from 20 to 40 ° C, preferably in the range from 30 to 38 ° C. The pH of the medium usually ranges between 5 and 9, preferably between 6.5 and 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases and buffers. Typically, growth of 1 to 5 days will result in the accumulation of the target L-amino acid in a liquid medium.

After growth, solid residues such as cells can be removed from the liquid medium by centrifugation or filtration through a membrane, and then the L-amino acid can be collected and purified using ion exchange chromatography, concentration and / or crystallization methods.

Brief Description of the Drawings

Figure 1 shows the relative positions of the yafAL and yafAR primers on the plasmid pACYC184, which is used to amplify the cat gene.

Figure 2 shows the construction of a DNA fragment of a chromosome containing an inactivated yafA gene.

Figure 3 shows a model of the transmembrane topology of the FrsA protein, built by the TMpred program based on statistical analysis of the database of transmembrane proteins.

Examples

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

Example 1. Construction of a strain with the inactivated yafA gene. one.

YafA gene deletion.

The strain with the yafA delegated gene was obtained using a method originally developed by Datsenko, K. A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640-6645) and entitled "Red-Dependent Integration". In accordance with this procedure, PCR primers yafAL (SEQ ID NO: 3) and yafAR (SEQ ID NO: 4) were synthesized, homologous to both regions of the chromosome adjacent to the yafA gene and the gene reporting antibiotic resistance on the plasmid used in as a matrix. Plasmid pACYC184 (NBL Gene Sciences Ltd., UK) (accession number X06403 in the GenBank / EMBL database) was used as such a matrix for PCR. The following temperature profile for PCR was used: denaturation at 95 ° C for 3 min; the first two cycles: 1 min at 95 ° C, 30 sec at 50 ° C, 40 sec at 72 ° C; and the following 25 cycles: 30 sec at 95 ° C, 30 sec at 54 ° C, 40 sec at 72 ° C; and final polymerization: 5 min at 72 ° C.

The resulting PCR product is a fragment of 965 bp in length. (Fig. 1, SEQ ID NO: 5) was purified on an agarose gel and was used for electroporation into E. coli strain MG1655 containing plasmid pKD46 containing a temperature-sensitive replicon. Plasmid pKD46 (Datsenko, KA and Wanner, BL, Proc. Natl. Acad. Sci. USA, 2000, 97: 12: 6640-45) contains a phage λ DNA fragment (sequence number J 02459 in the GenBank database) of 2.154 length nucleotide (31088-33241), and also contains A, Red genes of a homologous recombination system (γ, β, exo genes) under the control of the _{araB inducer} P induced by arabinose. 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 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 the ice with deionized H ₂ O three times. Electroporation was performed using 70 μl of cells and ≈100 ng of 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 fission of the yafA gene by PCR.

A mutant with a delegated yafA gene containing a Cm resistance gene was tested by PCR. Locus-specific primers yafA1 (SEQ ID NO: 6) and yafA2 (SEQ ID NO: 7) were used to verify deletion by PCR. The following temperature profile was used for PCR testing: denaturation at 94 ° C for 3 minutes; 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 parent strain yafA ⁺ MG1655 as a matrix of cells was 1403 bp The length of the PCR product obtained by the reaction using the mutant strain MG1655 ΔyafA :: cat as a matrix of cells was 1069 bp (Figure 2). The mutant strain was named MG1655 ΔyafA :: cat.

Example 2. Production of L-threonine by E. coli strain B-3996-ΔyafA

To test the effect of inactivation of the yafA gene on the production of threonine, DNA fragments from the chromosome of the E. coli MG1655 ΔyafA :: cat strain described above were transferred to the L-threonine E, coli VKPM B-3996 producer strain using P1 transduction (Miller, JH Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain B-3996-ΔyafA.

Both strains of E. coli, B-3996 and B-3996-ΔyafA, were grown for 18-24 hours at 37 ° C on plates with L-agar.

To obtain a seed culture, the strains were 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 supplemented with glucose to a final concentration of 4% . After that, inoculation material was introduced into the fermentation medium in a volume of 0.21 ml (10%). Fermentation was carried out in 2 ml of minimal medium for fermentation in test tubes with a volume of 20 × 200 mm. Cells were grown for 65 hours at 32 ° C with stirring on a rotary shaker at 250 rpm.

After cultivation, the amount of L-threonine that accumulated in the medium was determined by paper chromatography using a mobile phase of the following composition: butanol: acetic acid: water = 4: 1: 1 (v / v)). For visualization, a solution of ninhydrin (2%) in acetone was used. The spot containing L-threonine was excised, L-threonine was eluted in a 0.5% aqueous solution of CdCl ₂ , after which the amount of L-threonine was determined on a spectrophotometer at a wavelength of 540 nm. The results of 10 independent experiments are presented in Table 1. As follows from Table 1, strain B-3996-ΔyafA causes the accumulation of more L-threonine compared to strain B-3996.

For fermentation, a medium of the following composition was used (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

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

Table 1. Strain OD ₅₄₀ The amount of L-threonine, g / l B-3996 19.6 ± 0.8 26.0 ± 1.2 B-3996-ΔyafA 20.1 ± 0.5 27.8 ± 1.0

Example 3. Production of L-lysine by E. coli strain AJ11442-ΔuafA

To test the effect of inactivation of the yafA gene on lysine production, DNA fragments from the chromosome of the E. coli MG1655 ΔyafA :: cat strain described above can be transferred to the lysine E producing strain, coli WC196 (pCABD2) using P1 transduction (Miller, JH Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain WC196 (pCABD2) -ΔyafA. The plasmid pCABD2 contains the dapA gene encoding a dihydrodipicolinate synthase and carrying a mutation that reduces the sensitivity to feedback inhibition of L-lysine, the lysC gene encoding aspartokinase III that carries a mutation that reduces the sensitivity to feedback of L-lysine inhibition, the dapB gene encoding a dihydrodipicolinate reductase and a ddh gene encoding diaminopimelate dehydrogenase and a streptomycin resistance gene (US Pat. No. 6,040,160).

Both strains of E. coli, WC 196 (pCABD2) and WC196 (pCABD2) -ΔyafA can be grown in L-medium containing streptomycin (20 mg / L), at a temperature of 37 ° C and 0.3 ml of the resulting culture can be added to 20 ml of fermentation medium containing the necessary components into 500 ml vials. Cultivation can be carried out at 37 ° C for 16 hours using a return 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 by a known method (Biotech-analyzer AS210 manufactured by Sakura Seiki Co.). After that, the yield of L-lysine can be calculated in relation to the glucose consumed for each of the strains.

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

Glucose 40

(NH ₄ ) ₂ SO ₄ 24 K ₂ HPO ₄ 1.0 MgS0 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 value is set equal to 7.0 using KOH, and the medium is autoclaved at a temperature of 115 ° C for 10 minutes. Glucose and MgSO ₄ · 7H ₂ O are sterilized separately. CaCO _{3 is} sterilized by dry heat at a temperature of 180 ° C for 2 hours and added to the medium to a final concentration of 30 g / l.

Example 4. Production of L-cysteine by E. coli strain JM15 (ydeD) -ΔyafA

To test the effect of inactivation of the yafA gene on L-cysteine production, DNA fragments from the chromosome of the above E. coli strain MG1655 ΔyafA :: cat can be transferred to the L-cysteine producer strain JM15 (ydeD) using P1 transduction (Miller, JH Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain JM15 (ydeD) -ΔyafA strain.

The E. coli strain JM15 (ydeD) is a derivative of the E. coli strain JM15 (US patent 6218168), which can be transformed with DNA that carries the ydeD gene, which encodes a membrane protein and is not involved in the biosynthesis of any L-amino acid (US patent 5972663).

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

Example 5. The production of L-leucine strain E. coli 57-ΔyafA

To test the effect of inactivation of the yafA gene on the production of L-leucine, DNA fragments from the chromosome of the above E. coli strain MG1655 ΔyafA :: cat can be transferred to the producer strain L-leucine 57 (VKPM B-7386, US patent 6124121) using transduction P1 (Miller, JH Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain 57-pMW-ΔyafA.

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

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

Glucose 60.0 (NH ₄ ) ₂ SO ₄ 25.0 K ₂ HPO ₄ 2.0 MgSO ₄ * 7H ₂ O 1.0 Thiamine 0.01 CaCO ₃ 25.0

Glucose and CaCO _{3 are} sterilized separately. The pH of the medium is adjusted to 7.2.

Example 6. Production of L-histidine by E. coli strain 80-ΔyafA

To test the effect of inactivation of the yafA gene on L-histidine production, DNA fragments from the chromosome of the above E. coli strain MG1655 ΔyafA :: cat can be transferred to E. coli 80 histidine-producing strain using P1 transduction (Miller, JH Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the 80 ΔyafA strain. Strain 80 was described in RF patent 2119536 and deposited in the All-Russian collection of industrial microorganisms (VKPM) (RF, 117545 Moscow, 1st Dorozhniy proezd, 1) with accession number VKPM B-7270.

Both strains of E. coli, 80 and 80-ΔyafA, can be grown in L-broth at a temperature of 29 ° C for 6 hours. Further, 0.1 ml of the obtained culture can be introduced into 20 × 200-mm tubes containing 2 ml of fermentation medium and cultured at 29 ° C for 65 hours with shaking on a rotary shaker (at 350 rpm ) After cultivation, the amount of histidine that has accumulated in the medium can be determined using paper chromatography (composition of the mobile phase: butanol-acetic acid-water = 4: 1: 1). For visualization of amino acids in spots, a 0.5% solution of ninhydrin in acetone can be used.

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

Glucose 100.0 Mameno (soy bean hydrolyzate) 0.2 total nitrogen L-proline 1.0 (NH ₄ ) ₂ SO ₄ 25.0 KN 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. Before sterilization, the pH is adjusted to 6.0.

Example 7. The production of L-glutamate strain E. coli VL334thrC ⁺ -ΔvafA

To test the effect of inactivation of the yаfA gene on L-glutamate production, DNA fragments from the chromosome of the above E. coli strain MG1655 ΔyafA :: cat can be transferred to the E. coli L-glutamate producer strain VL334thrC ⁺ (European patent EP 1172433) using transduction P1 (Miller, JH Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain VL334thrC ⁺ -ΔyafA.

Both strains, VL334thrC ⁺ and VL334thrC ⁺ -ΔyafA, can be grown for 18-24 hours at 37 ° C on plates with L-agar. Further, one loop of cells can be transferred to tubes containing 2 ml of fermentation medium. The fermentation medium contains 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 chalk (25 g / l). The pH was adjusted to 7.2. Glucose and chalk are sterilized separately. Cultivation can be carried out at a temperature of 30 ° C for 3 days with shaking. After cultivation, the amount of L-glutamate produced can be determined using paper chromatography (mobile phase composition: butanol-acetic acid-water = 4: 1: 1), followed by staining with ninhydrin (1% solution in acetone) and further eluting the resulting compounds in 50% ethanol with 0.5% CdCl ₂ .

Example 8. The production of L-phenylalanine strain E. coli AJ12739-ΔyafA

To test the effect of inactivation of the yafA gene on L-phenylalanine production, DNA fragments from the chromosome of the above E. coli strain MG1655 ΔyafA :: cat can be transferred to the phenylalanine producing strain AJ 12739 using P1 transduction (Miller, JH Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain AJ12739-ΔyafA strain. Strain AJ12739 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF, 117545 Moscow, 1st Dorozhniy proezd, 1) November 6, 2001 with accession number VKPM B-8197.

Both strains, AJ12739-ΔyafA and AJ12739, can be cultured at 37 ° C for 18 hours in nutrient broth, and 0.3 ml of the resulting culture can be introduced into 20 × 200-mm tubes containing 3 ml of fermentation medium, and cultured at a temperature of 37 ° C for 48 hours with shaking on a rotary shaker. After cultivation, the amount of phenylalanine that has accumulated in the medium can be determined by TLC.

For fermentation can be used in the following medium, (g / l):

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 · 7H ₂ O 0.01 MnSO ₄ · 5H ₂ O 0.01 Thiamine HCl 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 is adjusted to 7.0.

Example 9. Production of L-tryptophan by E. coli strain SV164 (pGH5) -ΔyafA

To test the effect of inactivation of the yafA gene on L-tryptophan production, DNA fragments from the chromosome of the above E. coli strain MG1655 ΔyafA :: cat were transferred to the E. coli tryptophan producing strain SV164 (pGH5) using P1 transduction (Miller, JH Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain SV164 (pGH5) -ΔyafA. Strain SV164 has a trpE allele encoding anthranylate synthase, which is not inhibited by tryptophan in a feedback manner. Plasmid pGH5 carries the mutant serA gene encoding phosphoglycerate dehydrogenase, which is not inhibited by serine by the feedback principle. Strain SV164 (pGH5) was described in detail in US Pat. 6180373 or in European patent EP 0662143.

Both strains, SV164 (pGH5) -ΔyafA and SV164 (pGH5), were cultured with shaking at 37 ° C for 18 hours in 3 ml nutrient broth supplemented with tetracycline (5 μg / ml, marker of plasmid pGH5). The resulting cultures (in a volume of 0.3 ml each) were introduced into 20 × 200 mm tubes containing 3 ml of fermentation medium with tetracycline (5 μg / ml) and cultivated at 37 ° C for 72 hours on a rotary shaker at 250 rpm After cultivation, the amount of tryptophan that accumulated in the medium was determined using TLC as described in Example 8. The components of the fermentation medium are listed in Table 2 and are sterilized in separate groups (A, B, C, D, E, F, and H) in order to avoid adverse interactions during the sterilization process. The results of eight independent in vitro fermentation processes are shown in Table 3. As follows from Table 3, strain SV164 (pGH5) -ΔyafA caused the accumulation of more L-tryptophan compared to strain SV164 (pGH5).

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 N) 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 · H ₂ O 0.0003 F Thiamine HCl 0.005 G CaCO ₃ 30.0 H Pyridoxine 0.03 The pH of solution A was adjusted to 7.1 with NH ₄ OH. Table 3 Strain OD ₅₄₀ The amount of L-tryptophan, g / l SV164 (pGH5) 8.4 ± 0.2 4.4 ± 0.1 SV164 (pGH5) -ΔyafA 7.7 ± 0.4 4.8 ± 0.5

Example 10. The production of L-proline strain E. coli 702ilvA-ΔyafA

To test the effect of inactivation of the yafA gene on L-proline production, DNA fragments from the chromosome of the above E. coli MG1655 ΔyafA :: cat strain can be transferred to E. coli 702ilvA L-proline producing strain using P1 transduction (Miller, JH Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain 702ilvA-ΔyafA. Strain 702ilvA was deposited in the All-Russian collection of industrial microorganisms (VKPM) (RF, 117545 Moscow, 1st Dorozhniy proezd, 1) with accession number VKPM V-8012.

Both strains of E. coli, 702ilvA and 702ilvA-ΔyafA, can be grown for 18-24 hours at 37 ° C on plates with L-agar. Further, these strains can be cultured under the conditions described in Example 8.

Example 11. Production of L-arginine by E. coli 382-ΔyafA strain To test the effect of inactivation of the yafA gene on L-arginine production, DNA fragments from the chromosome of the above E. coli strain MG1655 ΔyafA :: cat can be transferred to arginine E-producing strain .coli 382 using P1 transduction (Miller, JH Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain 382-ΔyafA. Strain 382 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF, 117545 Moscow, 1st Dorozhniy proezd, 1) on April 10, 2000 with accession number VKPM V-7926.

Both strains, 382-ΔuafA and 382, can be cultured at 37 ° C for 48 hours with shaking on a rotary shaker.

After cultivation, the amount of L-arginine that has accumulated in the medium can be determined by paper chromatography using the following mobile phase: butanol: acetic acid: water = 4: 1: 1 (v / v). For visualization, a 2% solution of ninhydrin in acetone can be used. A stain containing L-arginine can be excised, L-arginine can be eluted in a 0.5% aqueous solution of CdCl _2, and the amount of L-arginine can be estimated on a spectrophotometer at a wavelength of 540 nm.

For fermentation can be used in the following medium, (g / l):

Glucose 48.0 (NH ₄ ) ₂ SO ₄ 35.0 KN 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 are sterilized separately. CaCO _{3 is} sterilized by dry heat at a temperature of 180 ° C for 2 hours. The pH is adjusted to 7.0.

Although the invention has been described in detail with reference to the best mode of 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 outside 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.

Industrial applicability

According to the present invention, the production of L-amino acids such as L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamine, L-phenylalanine, L-tryptophan, L-proline and bacterial L-arginine Enterobacteriaceae family can be enhanced.

Claims (3)

1. A bacterium belonging to the genus Escherichia is a producer of L-threonine or L-tryptophan, modified in such a way that the yafA gene is inactivated in this bacterium. 2. The bacterium according to claim 1, characterized in that said yafA gene is inactivated by deletion of the yafA gene in the bacterial chromosome. 3. A method of producing L-threonine or L-tryptophan, comprising growing the bacterium according to any one of claims 1 or 2 in a nutrient medium, causing production and accumulation of the L-amino acid in the culture fluid, and isolation of the L-amino acid from the culture fluid.

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