RU2482188C2 - METHOD FOR PREPARING L-ARGININE WITH USE OF BACTERIA OF GENUS Escherichia WHEREIN astCADBE OPERON IS INACTIVATED - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention relates to biotechnology, and represents a method for preparing L-arginine with the use of the bacteria of to the genus Escherichia, which is modified in such a way that the astCADBE operon in the mentioned bacterium is inactivated. The bacterium is grown in a culture medium, after that L-arginine is recovered from a culture fluid.

EFFECT: method enables providing higher productivity of the bacteria producing L-arginine and producing high-yield L-arginine.

4 cl, 2 tbl, 14 ex

Description

Technical field

The present invention relates to the microbiological industry, in particular to a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family, modified in such a way that expression of the astCADBE operon in said bacterium is weakened.

Description of the Related Art

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

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

Other methods for increasing the production of L-amino acids are to weaken the expression of one or more genes involved in the degradation of the target L-amino acid; genes whose expression leads to the distraction of the precursors of the target amino acid from the pathway of biosynthesis of L-amino acids; genes involved in the redistribution of carbon, nitrogen and phosphorus streams; genes encoding toxins, etc.

The search for the pathway of arginine catabolism with the formation of ammonium in Escherichia coli led to the discovery of the arginine succinyl transferase pathway (AST - arginine succinyltransferase) with the formation of ammonium in E. coli and the astCADBE operon. The genes of this operon encode five enzymes of the AST pathway. The succinylornithine transaminase gene was detected based on the identity of the protein sequence and the derived gene product. This gene was designated astC, and it is obviously the first gene of the operon. A homology search of the second, third, and fifth open reading frames (OPCs) of this operon led to the assumption that they code for AST, succinyl glutamine semi-aldehyde dehydrogenase and succinyl glutamate de succinylase, the first, fourth, and fifth enzymes of the AST pathway, respectively. A homology search did not lead to an assumption of the function of the fourth ORS, but the destruction of this ORS led to a specific disappearance of the activity of the second pathway enzyme, succinyl arginine dihydrolase. The fact that each gene overlaps with the subsequent one confirms their organization into an operon and suggests that the translation of polycistronic mRNA is probably carried out by one ribosome. The destruction of the first gene leads to a weakening of the synthesis of all enzymes of the AST pathway, which is also consistent with the operon structure. Finally, these observations, together with the fact that in the cells containing the plasmid with this putative operon, the levels of all five enzymes increase, prove the existence of the astCADBE operon. Mutants deficient in enzymes of the AST pathway cannot utilize arginine, which indicates that the AST pathway is required for catabolism of arginine as the only source of nitrogen in the exponential growth phase during aerobic cultivation. The AST pathway also obviously contributes to the degradation of ornithine and aspartate. The nitrogen restriction led to the induction of this pathway in both E. coli and Klebsiella aerogenes, but the activation mechanisms clearly differed in the two organisms. Regulation of the AST pathway under conditions other than the nitrogen limit suggests additional functions. The AST pathway is induced by growth on broth. The AST pathway also appears to be induced upon entering the stationary growth phase. The function of the AST path under such conditions is not clear. Unlike other organisms that have an AST pathway, E. coli does not utilize arginine as a carbon source. It is possible that E. coli cannot utilize arginine as a carbon source due to the lack of an appropriately controlled promoter of the ast genes or due to inadequate transport during growth under carbon restrictions (Schneider BL et al., J Bacteriol .; 180 (16 ): 4278-86 (1998)).

There are currently no reports describing the use of inactivation of the astCADBE operon 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.

The aforementioned goals were achieved thanks to the discovery that the weakening of the expression of the astCADBE operon can lead to an increase in the production of L-amino acids such as L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L -valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-arginine, L-citrulline, L-ornithine , L-phenylalanine, L-tyrosine and L-tryptophan.

The present invention provides a bacterium belonging to the Enterobacteriaceae family with enhanced ability to produce amino acids such as L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine , glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-arginine, L-citrulline, L-ornithine, L-phenylalanine, L tyrosine and L-tryptophan.

An object of the present invention is to provide an L-amino acid producing bacterium belonging to the Enterobacteriaceae family, modified in such a way that expression of the astCADBE operon in said bacterium is attenuated.

It is also an object of the present invention to provide the bacterium described above, characterized in that the astCADBE operon is inactivated.

It is also an object of the present invention to provide a bacterium as described above, characterized in that said bacterium belongs to the genus Escherichia.

It is also an object of the present invention to provide a bacterium as described above, characterized in that said bacterium belongs to the genus Pantoea.

It is also an object of the present invention to provide a bacterium as described above, characterized in that 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 a bacterium as described above, characterized in that 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 a bacterium as described above, characterized in that 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-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-arginine, L-citrulline and L -ornithine.

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

- growing the bacteria described above in a nutrient medium; and

- the allocation 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, characterized in that 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 method described above, characterized in that 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, characterized in that 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-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-arginine, L-citrulline and L -ornithine.

In more detail, the present invention is described below.

Detailed Description of the Best Mode for Carrying Out the Invention

1. Bacteria

A bacterium according to the present invention is an L-amino acid producing bacterium belonging to the Enterobacteriaceae family, characterized in that the astCADBE operon in said bacterium is inactivated.

The phrase “L-amino acid producing bacterium” may mean a bacterium capable of producing and secreting an L-amino acid into a culture medium when the bacterium is grown in said culture medium.

The term “bacterium-producer of L-amino acids” can also mean a bacterium that is capable of producing and accumulating L-amino acids in a fermentation medium in large quantities in comparison with the natural or parent E. coli strain, such as E. coli K-12 strain , and may also mean that the bacterium is able to accumulate in the medium the target L-amino acid in an amount of not less than 0.5 g / l or not less than 1.0 g / l in another example. The term “L-amino acid” includes, for example, L-alanine, L-arginine, L-citrulline, L-ornithine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, L- glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.

The term “aromatic L-amino acid” includes, for example, L-phenylalanine, L-tyrosine and L-tryptophan. The term “non-aromatic L-amino acid” includes, for example, 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-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-arginine, L-citrulline and L-ornithine. Specific examples are L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L-phenylalanine, L-tryptophan, L-proline and L-arginine.

The Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, etc. More specifically, bacteria classified as belonging to the Enterobacteriaceae family according to the taxonomy used in the NCBI database (National Center for Biotechnology Information) (https://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 genus Escherichia or Pantoea is preferred.

The term "bacterium belonging to the genus Escherichia" means that the bacterium belongs to the genus Escherichia in accordance with the classification known to the person skilled in the field of microbiology. As an example of a microorganism belonging to the genus Escherichia used in the present invention, the bacterium Escherichia coli (E. coli) may be mentioned.

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

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

The term “bacterium is modified in such a way that the expression of the astCADBE operon is weakened” means that the bacterium is modified in such a way that, as a result of the modification, such a bacterium contains a reduced amount of proteins AstC, AstA, AstD, AstB and AstE compared to an unmodified bacterium, or it may mean that the modified bacterium is not able to synthesize proteins AstC, AstA, AstD, AstB and AstE. The term “bacterium is modified in such a way that expression of the astCADBE operon is weakened” can also mean that the bacterium is modified in such a way that the modified genes encode mutant proteins AstC, AstA, AstD, AstB and AstE with reduced activity.

The presence or absence of the astCADBE operon on the chromosome can be determined by well-known methods, including PCR, Southern blotting, etc. In addition, the level of gene expression can be estimated by determining the amount of RNA transcribed from the gene using various known methods, including Northern blotting, quantitative RT-PCR, etc. The amount and molecular weight of the proteins encoded by the genes can be determined by known methods, including electrophoresis in SDS-PAGE with subsequent immunoblotting (Western blotting), etc.

The phrase "inactivation of the astCADBE operon" may mean that the modified genes encode completely inactive proteins. It is also possible that natural expression of the modified DNA region is not possible due to deletion of a portion of the gene, shift of the reading frame, insertion of the missense / nonsense mutation (s), or modification of regions adjacent to the gene that include sequences that control gene expression, such as promoters, enhancers , attenuators, etc.

The astC gene encodes the AstC protein, acetylornithine transaminase / succinylornithine transaminase (synonym - In 1748). The astC gene from E. coli (nucleotides complementary to nucleotides from 1,828,786 to 1,830,006 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the astA gene and xthA gene, oriented in the opposite direction to the astC gene, on the chromosome E. coli K-12 strain. The nucleotide sequence of the astC gene and the amino acid sequence of the AstC protein encoded by the astC gene are shown in the Sequence Listing Numbers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2), respectively.

The astA gene encodes the AstA protein, arginine succinyl transferase (synonym - In 1747). The astA gene from E. coli (nucleotides complementary to nucleotides from 1,827,755 to 1,828,789 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the astD gene and the astC gene on the chromosome of E. coli K-12 strain. The nucleotide sequence of the astA gene and the amino acid sequence of the AstA protein encoded by the astA gene are shown in the Sequence Listing Numbers 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4), respectively.

The astD gene encodes the AstD protein, aldehyde dehydrogenase (synonym - B1746). The astD gene from E. coli (nucleotides complementary to nucleotides from 1,826,280 to 1,827,758 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the astB gene and the astA gene on the chromosome of E. coli K-12 strain. The nucleotide sequence of the astD gene and the amino acid sequence of the AstD protein encoded by the astD gene are shown in the Sequence Listing Nos. 5 (SEQ ID NO: 5) and 6 (SEQ ID NO: 6), respectively.

The astB gene encodes the AstB protein, succinyl arginine dihydrolase (synonym - B1745). The astB gene from E. coli (nucleotides complementary to nucleotides from 1,824,940 to 1,826,283 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the astE gene and astD gene on the chromosome of E. coli K-12 strain. The nucleotide sequence of the astB gene and the amino acid sequence of the AstB protein encoded by the astB gene are shown in the Sequence Listing Numbers 7 (SEQ ID NO: 7) and 8 (SEQ ID NO: 8), respectively.

The astE gene encodes the AstE protein, succinylglutamate desuccinylase (synonym - B1744). The astE gene from E. coli (nucleotides complementary to nucleotides from 1,823,979 to 1,824,947 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located between the spy gene and astB gene on the chromosome of E. coli strain K-12. The nucleotide sequence of the astE gene and the amino acid sequence of the AstE protein encoded by the astE gene are shown in the Sequence Listing Nos. 9 (SEQ ID NO: 9) and 10 (SEQ ID NO: 10), respectively.

Since representatives of various genera and strains of the Enterobacteriaceae family may experience some variations in the nucleotide sequences, the concept of the inactivated operon astCADBE is not limited to the genes whose sequences are listed in the Sequence Listing under the numbers SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, but may also include genes homologous to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, coding variants of proteins AstC, AstA, AstD, AstB and AstE. The term "protein variant" can mean a protein with changes in the sequence, whether deletion, insertion, addition or substitution of amino acids, in which the product retains the functional activity of the proteins AstC, AstA, AstD, AstB and AstE. The number of changes in a protein variant depends on the position or type of amino acid residue in the tertiary structure of the protein. It can be from 1 to 30, or in another example from 1 to 15, or in another example from 1 to 5 in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10. These variations in variants may occur in areas not critical to protein function. These changes are possible because some amino acids have a high homology to each other, therefore, such changes do not affect the tertiary structure or activity. Therefore, a variant of the protein encoded by the astCADBE operon gene can be represented by proteins with a homology of at least 80%, or in another example, at least 90%, or in another example, at least 95%, relative to the complete amino acid sequence given in the Sequence Listing under the number SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, provided that the proteins AstC, AstA, AstD, AstD, AstB, and AstE are preserved until inactivation.

Homology between two amino acid sequences can be determined using known methods, for example, the BLAST 2.0 computer program, which counts three parameters: amino acid number, identity, and similarity.

In addition, the astCADBE operon genes can be variants that hybridize under stringent conditions with the nucleotide sequence shown in the Sequence Listing Nos. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 7 ID NO: 9, or with a probe that can be synthesized based on the indicated nucleotide sequence, provided that it encodes the functional protein AstC, AstA, AstD, AstB, or AstE prior to inactivation. "Stringent conditions" include those conditions under which specific hybrids, for example, hybrids with a homology of at least 60%, or in another example, at least 70%, or in another example, at least 80%, or in another example, at least 90%, or in another example, at least 95% are formed, and non-specific hybrids, for example, hybrids with less homology than indicated above, are not formed. A practical example of harsh conditions is a single wash, preferably two or three times, at a salt concentration of 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, at 60 ° C. The duration of washing depends on the type of membrane used for blotting and, as a rule, is as recommended by the manufacturer. For example, the recommended washing time for Hybond ™ N + nylon membrane (Amersham) under stringent conditions is 15 minutes. Two to three times washing is preferred. The length of the probe can be selected depending on hybridization conditions and is usually about 100-1000 bp.

The expression of the astCADBE operon can be attenuated by introducing such a mutation into the gene that the intracellular activity of the protein encoded by the gene is reduced compared to the unmodified strain. Mutations that result in diminished gene expression include the substitution of one or more bases for amino acid substitution in the encoded protein (missense mutation), the introduction of a stop codon (nonsense mutation), deletion of one or more bases for frame shift reading, inserting the antibiotic resistance gene, or deleting the gene or part thereof (Qiu, Z. and Goodman, MF, J. Biol. Chem., 272, 8611-8617 (1997); Kwon, DH et al, J. Antimicrob. Chemother., 46, 793-796 (2000)). The expression of the astCADBE operon can also be attenuated by modifying the expression of regulatory sequences such as a promoter, a Shine-Dalgarno (SD) sequence, etc. (PCT application WO95 / 34672; Carrier, T.A. and Keasling, J.D., Biotechnol Prog 15, 58-64 (1999)).

For example, the following methods can be used to introduce mutations by gene recombination. A mutant gene is constructed that encodes a mutant protein with reduced activity, and the bacterium for its modification is transformed with a DNA fragment containing the mutant gene. Then the native gene on the chromosome is replaced by homologous recombination with the mutant gene, and the resulting strain is selected. Substitution of a gene using homologous recombination can be carried out using a linear DNA method known as "Red-dependent integration" or "Red-system integration" (Datsenko KA, Wanner BL, Proc. Natl. Acad. Sci. USA, 97, 12, 6640-6645 (2000)) or by a method using a plasmid whose replication is temperature sensitive (US Pat. No. 6,303,383 or Japanese Patent Application JP 05-007491A). In addition, the introduction of a site-specific mutation by replacing a gene using the aforementioned homologous recombination can also be carried out using a plasmid with reduced replication ability in the host cell.

Gene expression can also be attenuated by inserting a transposon or IS factor into the coding region of the gene (US Pat. No. 5,175,107), or by conventional methods such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine).

Gene inactivation can also be carried out by such traditional methods as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-specific mutagenesis, gene destruction using homologous recombination and / or mutagenesis for deletion insertion account (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".

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

L-amino acid producing bacterium

As a bacterium in accordance with the present description of the invention, a bacterium modified in such a way that the expression of the astCADBE operon in said bacterium is weakened and capable of producing an aromatic or non-aromatic L-amino acid can be used.

The specified bacterium in accordance with the present description of the invention can be obtained by attenuating the expression of the astCADBE operon in bacteria already possessing the ability to produce L-amino acids. On the other hand, said bacterium can be obtained by imparting the ability to produce a L-amino acid to a bacterium in which expression of the astCADBE operon is already attenuated.

L-threonine producing bacterium

Examples of the parent strain 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. Nos. 5,175,107 and 5,705,3371 ), E. coli strain NRRL-21593 (US patent 5939307), E. coli strain PERM BP-3756 (US patent 5474918), E. coli strains FERM BP-3519 and FERM BP-3520 (US patent 5376538), strain E .coli MG442 (Gusyatiner et al., Genetics, 14, 947-956 (1978)), E. coli strains VL643 and VL2055 (European patent application EP 1149911 A) and the like.

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, 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 Dorozhniy proezd, 1) April 7, 1987 with inventory number B-3996.

The E. coli strain VKPM B-5318 (European application 0593792 B) can also be used as the parent strain for producing the L-threonine producing bacterium according to the present invention. Strain B-5318 is prototrophic with respect to isoleucine, and the temperature-sensitive C1 repressor of the phage λ and P _R promoter replaces the regulatory region in the threonine operon on the plasmid pVIC40. Strain VKPM B-5318 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF, 117545 Moscow, 1st Dorozhniy proezd, 1) May 3, 1990 with accession number B-5318.

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

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

- thrB gene encoding homoserine kinase;

- thrC gene encoding threonine synthase;

- rhtA gene, presumably encoding a transmembrane protein;

the asd gene encoding aspartate β-semialdehyde dehydrogenase, and

- aspC gene encoding aspartate aminotransferase (aspartate transaminase).

The nucleotide sequence of the thrA gene encoding aspartokinase homoserine dehydrogenase I from Escherichia coli is known (nucleotide numbers 337 to 2799 in sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990). The thrA gene is located on the chromosome of E. coli K-12 strain between the thrL and thrB genes. The nucleotide sequence of the thrB gene encoding homoserine kinase from Escherichia coli is known (nucleotide numbers 2801 to 3733 in sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990). The thrB gene is located on the chromosome of E. coli K-12 strain between the thrA and thrC genes. The nucleotide sequence of the thrC gene encoding a threonine synthase from Escherichia coli is known (nucleotide numbers 3734 to 5020 in the sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990). The thrC gene is located on the chromosome of E. coli K-12 strain between the thrB gene and the open uaaX reading frame. All three of these genes function as one threonine operon. To enhance the expression of a threonine operon, it is desirable to remove an attenuator region from the operon that affects transcription (PCT application WO2005 / 049808, PCT application WO2003 / 097839).

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

The rhtA gene is located 18 minutes from the E. coli chromosome near the glnHPQ operon, which encodes the components of the glutamine transport system, the rhtA gene is identical to ORF1 (ybiF gene, nucleotide numbers 764 to 1651 in sequence with accession number AAA218541 in the GenBank database, gi: 440181) , located between the genes pXB and ompX. A region of DNA expressed to form the protein encoded by the ORF1 reading frame was named the rhtA gene (rht: resistance to homoserine and threonine). The rhtA23 mutation has also been shown to represent an A-to-G substitution at position -1 with respect to the ATG start codon (abstract 17 ^th International Congress of Biochemistry and Molecular Biology, abstract 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457; European application EP 1013765 A).

The nucleotide sequence of the asd gene from E. coli is known (nucleotide numbers 3572511 to 3571408 in sequence with accession number NC_000913.1 in the GenBank database, gi: 161313 07) and can be obtained by PCR (polymerase chain reaction; link to White TJ et al., Trends Genet., 5, 185 (1989)) using primers synthesized based on the nucleotide sequence of the gene. Asd genes from other microorganisms can be obtained in a similar way.

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

L-lysine producing bacterium

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

Strain WC196 can be used as a bacterium producer of L-lysine Escherichia coli. This bacterial strain was obtained by selection of the phenotype of resistance to AEC in strain W3110, derived from 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, the Ministry of International 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 received accession number FERM P -14690. Then, the strain was internationally deposited in accordance with the terms of the Budapest Treaty on September 29, 1995, and the strain received accession number FERM BP-5252 (US patent 5827698).

Examples of parental strains for producing L-lysine producing bacteria of the present invention also include strains in which the expression of one or more genes encoding L-lysine biosynthesis enzymes is enhanced. Examples of enzymes involved in the biosynthesis of L-lysine include, but are not limited to, dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate carboxylase (lysA), diaminopromenolephenolate ), aspartate semialdehyde dehydrogenase (asd), nicotinamide adenine dinucleotide transhydrogenase (pntAB) and aspartase (aspA) (European application EP 1253195 A). In addition, parental strains may have an increased level of expression of the gene involved in the respiration process (suo) (European application EP 1170376 A), the gene encoding nicotinamide nucleotranshydrogenase (pntAB) (US patent 5,830,716), the ybjE gene (PCT application WO 2005/073390) , or combinations of these genes.

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

L-cysteine producing bacterium

Examples of parental strains used to produce L-cysteine-producing bacteria according to the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain JM15 transformed with various cysE alleles encoding inhibition resistant cysE feedback type serine acetyltransferase (US patent 6218168, patent application of the Russian Federation 2003121601); E. coli strain W3110 containing overexpressed genes encoding a protein capable of secretion of compounds toxic to the cell (US patent 5972663); E. coli strains containing cysteine desulfohydrase with reduced activity (Japanese patent JP11155571A2); E. coli strain W3110 with increased activity of the positive transcriptional regulator of the cysteine regulon encoded by the cysB gene (international PCT application WO 0127307A1) and the like.

L-Leucine Producer Bacteria

Examples of parent strains used to produce the L-leucine producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strains that are resistant to leucine analogues, including, for example, β-2 -thienylalanine, 3-hydroxyleucine, 4-azaleucine and 5,5,5-trifluoroleucine (Japanese Patent Laid-open 62-34397 and 8-70879), E. coli strains obtained using the genetic engineering methods described in PCT 96 / 06926; E. coli strain H-9068 (JP8-70879A), 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 the L-amino acid from a bacterial cell. Examples of such genes include the b2682 and b2683 genes (ygaZH genes) (European application EP 1239041 A2).

L-histidine producing bacterium

Examples of parent strains used to produce L-histidine producing bacteria of the present invention include, but are not limited to, L-histidine producing bacteria belonging to the genus Escherichia, such as E. coli strain 24 (VKPM B-5945, patent RF 2003677); E. coli strain 80 (VKPM B-7270, RF patent 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 6344347); E. coli strain H-9341 (FERM BP-6674) (European Patent 1085087); E. coli strain AI80 / pFM201 (US Pat. No. 6,258,554) and the like.

Examples of parent strains for producing bacteria producing L-histidine according to the present invention also include strains in which the expression of one or more genes encoding L-histidine biosynthesis enzymes is enhanced. Examples of such genes include genes encoding ATP-phosphoribosyltransferase (hisG), phosphoribosyl-AMP-cyclohydrolase (hisI), phosphoribosyl-ATP-phosphohydrolase (hisIE), phosphoribosylformimino-hisimonosulfonamide (isomerizide aminoside) isomerase histidinolphosphatase (hisB), histidinol dehydrogenase (hisD), etc.

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

Specific examples of strains with the ability to produce L-histidine include E. coli FERM-P 5038 and 5048, into which a vector containing DNA encoding the L-histidine biosynthesis enzyme (Japanese application 56-005099 A), strains E. coli, into which the rht gene is introduced, for the export of amino acids (European application EP1016710A), strain E. coli 80, which is given resistance to sulfaguanidine, DL-1,2,4-triazole-3-alanine and streptomycin (VKPM B-7270, RF patent. 2119536), etc.

L-glutamic acid producing bacterium

Examples of parental strains used to produce the L-glutamic acid producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain VL334 thrC ⁺ (European patent EP 1172433). Strain E. coli VL334 (VKPM B-1641) is an auxotroph of L-isoleucine and L-threonine with mutations in the thrC and ilvA genes (US patent 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 K12 strain (VKPM B-7). The result was a strain, auxotroph for L-isoleucine, VL334thrC ⁺ (VKPM B-8961), which is capable of producing L-glutamic acid.

Examples of parent strains used to produce the L-glutamic acid producing bacterium of the present invention include, but are not limited to, strains defective in α-ketoglutarate dehydrogenase activity, or strains in which the expression of one or more genes encoding L-glutamic acid biosynthesis enzymes. Examples of such enzymes include glutamate dehydrogenase (gdh), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydrate, acnA, acnu zarzudenosefruzidazufarzudenosephase (glutamine), phosphate synthase pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglycerometase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogen phosphate dehydrogenase (gapA) isofluosophosphate pfkB) and glucose phosphatisomerase (pgi).

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

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

E.coli W3110sucA :: Km ^R

E. coli AJ12624 (FERM BP-3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJ12949 (FERM BP-4881).

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

Other examples of bacteria producing L-glutamic acid include bacteria belonging to the genus Escherichia and resistant to aspartic acid antimetabolites and deficient in the activity of α-ketoglutarate dehydrogenase, for example, strain AJ 13199 (FERM BP-5807), US patent 5,908,768), or strain FERM P-12379, additionally having low activity for the cleavage of L-glutamic acid (US patent 5,393,671); E. coli strain AJ 13138 (FERM BP-5565) (US patent 6,110,714) and the like.

Examples of the L-glutamic acid producing bacterium include mutant strains belonging to the genus Pantoea that are devoid of α-ketoglutarate dehydrogenase activity or have reduced α-ketoglutarate dehydrogenase activity, and can be obtained as described above. Examples of such strains are Pantoea ananatis AJ 13356 strain (US patent 6,331,419), Pantoea ananatis AJ 13356 strain deposited at the National Institute of Biological Sciences and Human Technologies, Agency for Industrial Science and Technology, Department of International Trade and Industry (National Institute of Bioscience and Human- Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry) (currently called the National Institute for 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) 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. The strain Pantoea ananatis AJ 13356 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 AJ 13356. However, it was later classified as Pantoea ananatis based on the 16S rRNA nucleotide sequence and other evidence. Although strain AJ 13356 has been deposited as Enterobacter agglomerans at the above depository, for the purposes of this description it will be referred to as Pantoea ananatis.

L-phenylalanine producing bacterium

Examples of parental strains used to produce the L-phenylalanine producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as strain AJ 12739 (tyrA :: Tn10, tyrR) (VKMP B-8197) ; strain HW1089 (ATCC-55371) containing the pheA34 gene (US patent 5354672); mutant strain MWEC101-b (KR8903681); strains NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (US patent 4407952) and the like. Also, bacteria belonging to the genus Escherichia, producers of L-phenylalanine, such as E. coli K-12 strain [W3110 (tyrA) / pPHAB] (FERM BP-3566), E. coli K strain, can be used as parent strains. -12 [W3110 (tyrA) / pPHAD] (FERM BP-12659), E. coli K-12 strain [W3110 (tyrA) / pPHATerm] (FERM BP-12662) and E. coli K-12 strain [W3110 (tyrA ) / pBR-aroG4, pACMAB], named as AJ12604 (FERM BP-3579) (European patent EP488424 B1). In addition, L-phenylalanine producing bacteria belonging to the genus Escherichia with increased activity of the proteins encoded by the yedA gene or yddG gene can also be used (US patent applications 2003/0148473 A1 and 2003/0157667 A1).

L-tryptophan producing bacterium

Examples of parent strains used to produce L-tryptophan producing bacteria of the present invention include, but are not limited to, L-tryptophan producing bacteria belonging to the genus Escherichia, such as E. coli strains JP4735 / pMU3028 (DSM10122) and JP6015 / pMU91 (DSM10123) lacking the activity of tryptophanyl tRNA synthetase encoded by the mutant trpS gene (US patent 5756345); E. coli strain SV164 (pGH5) containing a serA allele encoding a phosphoglycerate dehydrogenase non-feedback inhibitable serine and a trpE allele encoding anthranilate synthase non-feedback inhibitory tryptophan (US Pat. No. 6,180,373); E. coli strains AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) in which tryptophanase activity is absent (US patent 4371614); E. coli strain AGX17 / pGX50, pACKG4-pps, which enhances the ability to synthesize phosphoenolpyruvate (PCT application WO 9708333, US patent 6319696), and the like. L-tryptophan-producing bacteria belonging to the genus Escherichia can also be used in which the activity of the protein encoded by the yedA gene or yddG gene is increased (US patent applications 2003/0148473 A1 and 2003/0157667 A1).

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

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

L-proline producing bacterium

Examples of L-proline producing bacteria used as the parent strain of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 702ilvA (VKPM B-8012), deficient in the ilvA gene and capable of producing L-proline (European patent EP 1172433). The bacterium of the present invention can be improved by enhancing the expression of one or more genes involved in the biosynthesis of L-proline. Preferably, examples of such genes for bacterium-producing L-proline include the proB gene encoding glutamate kinase with desensitized feedback regulation of L-proline (German patent 3127361). In addition, the bacterium of the present invention can be improved by enhancing the expression of one or more genes encoding proteins that secrete the L-amino acid from a bacterial cell. Examples of such genes are the b2682 and b2683 genes (ygaZH genes) (European Patent Application EP1239041A2).

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

L-arginine producing bacterium

Examples of parental strains used to produce the L-arginine producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 237 (VKPM B-7925) (US patent application 2002 / 058315 A1) and its derivatives containing mutant N-acetylglutamate synthase (RF patent application 2001112869), E. coli 382 strain (VKPM B-7926) (European patent application EP 1170358A1), arginine producing strain into which the argA gene encoding the gene encoding N-acetylglutamatesynthetase (European Patent Application EP 117036 1A1), and the like.

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

Citrulline Producing Bacteria

Examples of parental strains used to produce the citrulline producer bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as N-acetylglutamate synthase mutant strains E. coli 237 / pMADS11, 237 / pMADS12 and 237 / pMADS13 (RU 2215783, EP 1170361 B1, US 6790647B2).

Also, the citrulline producing bacterium can be easily obtained from any arginine producing bacterium, for example, from E. coli 382 strain (VKPM B-7926), by inactivation of arginine succinate synthase encoded by the argG gene.

The phrase "inactivation of arginine succinate synthase" means that the bacterium is modified so that the modified bacterium contains inactive arginine succinate synthase, or it may also mean that the bacterium is not capable of synthesizing arginine succinate synthase. Inactivation of arginine succinate synthase can be accomplished by inactivation of the argG gene.

The phrase "inactivation of the argG gene" means that the modified gene encodes a fully non-functional protein. It is also possible that the region of the modified DNA is not capable of naturally expressing the gene due to deletion of part of the gene or the entire gene, shift of the reading frame of the gene, insertion of the missense / nonsense mutation (s), or modification of regions adjacent to the gene, including sequences that control gene expression such as promoter, enhancer, attenuator, ribosome binding site, etc.

The presence or absence of the argG gene on the bacterial chromosome can be determined by well-known methods, including PCR, Southern blotting, and the like. In addition, the level of gene expression can be estimated by determining the amount of RNA transcribed from the gene using various known methods, including Northern blotting, quantitative RT-PCR, etc. The amount of protein encoded by the argG gene can be determined by known methods, including SDS-PAGE electrophoresis followed by immunoblotting (Western blotting), etc.

The expression of the argG gene can be attenuated by introducing a mutation into the gene on the chromosome so that the intracellular activity of the protein encoded by the gene is reduced compared to that in the unmodified strain. Such a gene mutation may be the replacement of one or more bases for amino acid substitution in the encoded protein (the Missense mutation), the introduction of a stop codon (the "nonsense" mutation), the deletion of one or more bases for reading frame shift, insertion of the resistance gene an antibiotic or deletion of a gene or part thereof (Qiu, Z. and Goodman, MF, J. Biol. Chem., 272, 8611-8617 (1997); Kwon, DH et al, J. Antimicrob. Chemother., 46, 793 -796 (2000)). The expression of the argG gene can also be attenuated by modifying the expression of regulatory sequences such as a promoter, Shine-Dalgarno (SD) sequence, etc. (PCT application WO 95/34672; Carrier TA and Keasling JD, Biotechnol Prog 15, 58-64 ( 1999)).

For example, the following methods can be used to introduce mutations by gene recombination. A mutant gene is constructed that encodes a mutant protein with reduced activity, and the bacterium for its modification is transformed with a DNA fragment containing the mutant gene. Then the native gene on the chromosome is replaced by homologous recombination with the mutant gene, and the resulting strain is selected. Such gene substitution using homologous recombination can be carried out using a linear DNA method known as “Red-dependent integration” or “Red-system integration” (Datsenko KA, Wanner BL, Proc. Natl. Acad. Sci. USA, 97 , 12, 6640-6645 (2000), PCT application WO 2005/010175) or by a method using a plasmid whose replication is temperature sensitive (US patent 6,303,383 or Japanese patent application JP 05-007491A). Further, the introduction of a site-specific mutation by gene replacement using the aforementioned homologous recombination can also be carried out using a plasmid with reduced replication ability in the host cell.

Gene expression can also be attenuated by inserting a transposon or IS factor into the coding region of the gene (US Pat. No. 5,175,107), or by conventional methods such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine).

Ornithine-producing bacteria

The ornithine producing bacterium can be easily obtained from any arginine producing bacterium, for example, E. coli 382 strain (VKPM B-7926), by inactivating the ornithine carbamoyltransferase encoded by the argF and argI genes. Methods for inactivating ornithine carbamoyltransferase are described above.

L-valine producing bacterium

Examples of parental strains used to produce the L-valine producing bacterium of the present invention include, but are not limited to, strains modified to overexpress the ilvGMEDA operon (US Pat. No. 5,998,178). It is desirable to remove the region of the ilvGMEDA operon that is necessary to attenuate expression so that the expression of the operon is not attenuated by the resulting L-valine. Further, it is desirable to destroy the ilvA gene in the operon in order to reduce the activity of threonine deaminase.

Examples of parental strains used to produce the L-valine producing bacterium of the present invention also include mutant strains having an aminoacyl tRNA synthetase mutation (US Pat. No. 5,658,766). For example, E. coli strain VL1970, which has a mutation in the ileS gene encoding isoleucine-tRNA synthetase, can be used. The strain E.coli VL1970 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) on June 24, 1988 with accession number VKPM B-4411.

Further, mutant strains that require lipoic acid and / or with an insufficient amount of H ⁺ -ATPase (PCT application WO 96/06926) can also be used as parent strains.

L-isoleucine producing bacterium

Examples of parent strains used to produce the L-isoleucine producing bacterium of the present invention include, but are not limited to, mutant strains resistant to 6-dimethylaminopurine (Japanese application 5-304969A), mutant strains resistant to isoleucine analogue such as thiaisoleucine and isoleucine hydroxamate, and mutant strains additionally resistant to DL-ethionine and / or arginine hydroxamate (Japanese application 5-130882A). In addition, recombinant strains transformed with genes encoding proteins involved in the biosynthesis of L-isoleucine, such as threonine deaminase and acetohydroxate synthase (Japanese application 2-458A, French patent 0356739 and US patent 5998178) can also be used as parent strains.

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 nutrient medium for the purpose of producing and accumulating an L-amino acid in a nutrient medium and isolating the L-amino acid from the culture fluid.

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

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

The cultivation is preferably carried out under aerobic conditions, 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 medium.

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

Examples

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

Example 1. Construction of a strain with inactivated operon astCADBE.

1. The divisions of the astCADBE

The strain containing the deletion of the astCADBE operon was constructed using the method of "Red-dependent integration." A DNA fragment containing the Cm ^R marker encoded by the cat gene was obtained by PCR using primers P1 (SEQ ID NO: 11) and P2 (SEQ ID NO: 12) and plasmid pMW118-attL-Cm-attR as a template. The following temperature profile for PCR was used: denaturation at 94 ° C for 30 sec; subsequent 25 cycles: 30 sec at 94 ° C, 30 sec at 55 ° C, 90 sec at 72 ° C; and final polymerization: 2 min at 72 ° C.

The obtained PCR product was purified on an agarose gel and used for electroporation into E. coli strain MG1655 containing the plasmid pKD46 with a heat-sensitive replicon.

Electrocompetent cells were obtained as follows: the overnight culture of E. coli strain MG1655 / pKD46 was grown at 30 ° C in LB medium supplemented with ampicillin (100 mg / L), diluted 100 times by adding 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 stirring at 30 ° С until OD ₆₀₀ ≈0.6 was reached, after which the cells were made electrocompetent by 100-fold concentration and three times washing with ice-cold deionized H ₂ O. 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 plates with L-agar containing 30 μg / ml chloramphenicol and grown at 37 ° C to select Cm ^R recombinants. Then, to remove plasmid pKD46, two passages were performed on L-agar with Cm at 42 ° C, and the obtained colonies were tested for sensitivity to ampicillin.

2. Confirmation of the deletion of the astCADBE operon using PCR.

Mutants with the astCADBE delegated operon containing the Cm resistance gene were verified by PCR. Locus-specific primers P3 (SEQ ID NO: 13) and P4 (SEQ ID NO: 14) were used to verify deletion by PCR. The following temperature profile was used for PCR testing: denaturation at 94 ° C for 30 sec; profile for 30 cycles: 30 sec at 94 ° C, 30 sec at 54 ° C, 1 min at 72 ° C; final step: 7 min at 72 ° C. The length of the PCR product obtained by the reaction using the parental strain MG1655 as a matrix of cells is ~ 6.1 kb. The length of the PCR product obtained by the reaction using a mutant strain as a matrix of cells is ~ 1.7 kb. The mutant strain was named MG1655 Δ astCADBE :: cat.

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

To assess the effect of inactivation of the astCADBE operon on the production of threonine, DNA fragments of the chromosome of the E. coli strain MG1655 ΔastCADBE :: cat described above can be transferred to the E. coli B-3996 L-threonine producer strain using P1 transduction (Miller, JH ( 1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain B-3996-ΔchaC / B-3996-ΔastCADBE. 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 Dorozhniy proezd, 1) April 7, 1987 with inventory number B-3996.

Both strains of E. coli, B-3996 and B-3996-ΔastCADBE, can be grown for 18-24 hours at 37 ° C on plates with L-agar. To obtain a seed culture, these strains can be grown at 32 ° C for 18 hours on a rotary shaker (250 rpm) in 20 × 200 mm test tubes containing 2 ml of L-broth with 4% sucrose. Then, 0.21 ml (10%) of the seed culture can be added to the fermentation medium. Fermentation can be carried out in 2 ml of minimal fermentation medium in test tubes measuring 20 × 200 mm. Cells can be grown for 65 hours at 32 ° C with stirring (250 rpm).

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

The 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 hydrochloride 0.0002 Yeast extract 1.0 CaCO ₃ 30.0

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

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

To assess the effect of inactivation of the astCADBE operon on lysine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔastCADBE :: cat described above can be transferred to E. coli AJ11442 L-lysine producer strain using P1 transduction (Miller JH (1972). Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain AJ11442-ΔastCADBE. The plasmid pCABD2 contains the dapA gene that encodes a dihydrodipicolinate synthase with a mutation that removes feedback inhibition of L-lysine, the lysC gene that encodes aspartokinase III with a mutation that removes feedback by inhibition of L-lysine, the dapB gene that encodes dihydrodipzucine and dihydrodipzicoline ddh encoding diaminopimelate dehydrogenase (US Patent 6,040,160).

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

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

Glucose 40.0 (NH ₄ ) ₂ SO ₄ 24.0 K ₂ HPO ₄ 1.0 MgSO ₄ 7H ₂ O 1.0 FeSO ₄ 7H ₂ O 0.01 MnSO ₄ 5H ₂ O 0.01 Yeast extract 2.0

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

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

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

The E. coli strain JM15 (ydeD) is a derivative of the E. coli strain JM15 (US patent 6218168), which can be transformed with DNA containing the ydeD gene encoding a membrane protein not involved in the biosynthesis of any of the L-amino acids (US patent 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-ΔchaC / E. coli 57-ΔastCADBE.

To assess the effect of inactivation of the astCADBE operon on L-leucine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔastCADBE :: cat described above can be transferred to the E. coli 57 L-leucine producer strain (VKPM B-7386, US patent 6124121) using P1 transduction (Miller JH (1972). Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain 57-ΔastCADBE. Strain 57 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF, 117545 Moscow, 1st Dorozhniy proezd, 1) May 19, 1997 with accession number VKPM B-7386.

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

The composition of the fermentation medium (pH 7.2) (g / l):

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

Glucose and CaCO _{3 are} sterilized separately.

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

To assess the effect of inactivation of the astCADBE operon on L-histidine production, DNA fragments of the chromosome of the E. coli strain MG1655 described above ΔastCADBE :: cat can be transferred to the E. coli 80 L-histidine producing strain using P1 transduction (Miller JH (1972 ), Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain 80-ΔastCADBE. Strain 80 is described in RF patent 2119536 and deposited in the All-Russian collection of industrial microorganisms (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) October 15, 1999 with accession number VKPM B-7270, then July 12, 2004 the international deposit of this strain in accordance with the terms of the Budapest Treaty.

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

The composition of the fermentation medium (pH 6.0) (g / l):

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

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

Example 7. The production of L-glutamic acid strain E. coli VL334thrC ⁺ -ΔastCADBE.

To assess the effect of inactivation of the astCADBE operon on the production of L-glutamic acid, DNA fragments of the chromosome of the above E. coli strain MG1655 ΔastCADBE :: cat can be transferred to the E. coli L-glutamic acid producer strain VL334thrC ⁺ (EP 1172433) using P1 transduction (Miller JH (1972), Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain VL334thrC ⁺ -ΔastCADBE. The strain VL334thrC ^{+ was} deposited in the All-Russian collection of industrial microorganisms (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) on December 6, 2004 with accession number VKPM B-8961, then on December 8, 2004 this strain was internationally deposited in accordance with the conditions Budapest Treaty.

Strains of E. coli VL334thr C ⁺ and VL334thrC ⁺ - ΔastCADBE can be grown on plates with L-agar at 37 ° C for 18-24 hours. Further, one loop of cells can be transferred to tubes containing 2 ml of fermentation medium. The fermentation medium may contain glucose - 60 g / l, ammonium sulfate - 25 g / l, KH ₂ PO ₄ - 2 g / l, MgSO ₄ - 1 g / l, thiamine - 0.1 mg / ml, L-isoleucine - 70 μg / ml and chalk - 25 g / l (pH 7.2). Glucose and chalk are sterilized separately. Cultivation can be carried out at 30 ° C for 3 days with stirring. After growing, the amount of L-glutamic acid obtained can be determined by 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 obtained compounds in 50% ethanol with 0.5% CdCl ₂ .

Example 8. Production of L-phenylalanine by E. coli strain AJ12739-ΔastCADBE.

To assess the effect of inactivation of the astCADBE operon on the production of L-phenylalanine, DNA fragments of the chromosome of the E. coli strain MG1655 ΔastCADBE :: cat described above can be transferred to the E. coli L-phenylalanine producer strain AJ12739 using P1 transduction (Miller JH (1972 ), Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain AJ12739-AastCADBE. Strain AJ12739 was deposited in the All-Russian collection of industrial microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) November 6, 2001 with accession number VKPM B-8197, then on August 23, 2002, this strain was internationally deposited according to terms of the Budapest Treaty.

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

The composition of the fermentation 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 was adjusted to 7.0.

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

To assess the effect of inactivation of the astCADBE operon on L-tryptophan production, DNA fragments of the chromosome of the E. coli strain MG1655 described above ΔastCADBE :: cat can be transferred to the E. coli L-tryptophan producing strain SV164 (pGH5) using P1 transduction (Miller) , JH (1972), Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain SV164 (pGH5) -ΔastCADBE. Strain SV164 contains the trpE allele encoding anthranylate synthase that is not susceptible to tryptophan inhibition by feedback. Plasmid pGH5 contains the mutant gene serA, encoding phosphoglycerate dehydrogenase, not susceptible to serine inhibition by feedback. Strain SV164 (pGH5) is described in detail in US Pat. No. 6,180,373 or European Patent No. 0,662,143.

Strains of E. coli SV164 (pGH5) and SV164 (pGH5) - ΔastCADBE can be grown with stirring at 37 ° C for 18 hours in 3 ml of nutrient broth with tetracycline (plasmid marker pGH5, 10 μg / ml). 0.3 ml of the resulting cultures can be introduced into 3 ml of tetracycline-containing fermentation medium (10 μg / ml) in 20 × 200 mm test tubes, and the cultures can be grown at 37 ° C for 48 hours on a rotary shaker at 250 rpm. / min After growing, the amount of tryptophan accumulated in the medium can be determined using TLC, as described in Example 7.

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

Example 10. Production of L-Proline by E. coli strain 702ilvA-ΔastCADBE.

To assess the effect of inactivation of the astCADBE operon on L-proline production, DNA fragments from the chromosome of the E. coli strain MG1655 ΔastCADBE :: cat described above can be transferred to the E. coli 702ilvA L-proline producer strain using P1 transduction (Miller JH ( 1972), Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain 702ilvA-ΔastCADBE. Strain 702ilvA was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Road passage, 1) with accession number VKPM B-8012, then on May 18, 2001, this strain was internationally deposited in accordance with the conditions of the Budapest Treaty.

Strains of E. coli 702ilvA and 702ilvA-ΔastCADBE can be grown for 18-24 hours at 37 ° C on plates with L-agar. Then fermentation using these strains can be carried out under the same conditions as described in Example 6.

Example 11. The production of L-arginine strain E. coli 382-ΔastCADBE.

To evaluate the effect of inactivation of the astCADBE operon on L-arginine production, DNA fragments of the chromosome of the E. coli strain MG1655 described above ΔastCADBE :: cat were transferred to the E. coli 382 L-arginine producing strain using P1 transduction (Miller JH (1972) , Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain 382-ΔastCADBE. Strain 382 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhny proezd, 1) on April 10, 2000 with accession number VKPM B-7926, then on May 18, 2001, this strain was internationally deposited according to terms of the Budapest Treaty.

Both strains, 382 and 382 ΔastCADBE, were grown with stirring at 37 ° C for 18 hours in 3 ml of nutrient broth, 0.3 ml of the resulting cultures were introduced into 3 ml of fermentation medium in 20 × 200 mm tubes, and cultures were grown at 32 ° C for 48 hours on a rotary rocking chair.

After growing, the amount of L-arginine accumulated in the medium was determined by paper chromatography, and the following composition of the mobile phase was used: butanol: acetic acid: water = 4: 1: 1 (v / v). A solution of ninhydrin (2%) in acetone was used for visualization. A spot containing L-arginine was excised; L-arginine was eluted with a 0.5% aqueous solution of CdCl ₂ , after which the amount of L-arginine was determined spectrophotometrically at a wavelength of 540 nm. The results of ten independent in vitro fermentation tests are shown in Table 1. As follows from Table 1, strain 382-ΔastCADBE accumulated more L-arginine than strain 382.

The fermentation medium of the following composition was used (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} dry heat sterilized at 180 ° C for 2 hours. The pH was adjusted to 7.0.

Example 12. Production of L-citrulline by E. coli strain 382ΔargG ΔastCADBE.

To assess the effect of inactivation of the astCADBE operon on L-citrulline production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔastCADBE :: cat described above can be transferred to the E. coli 382ΔargG citrulline producing strain using P1 transduction (Miller JH (1972), Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the 382ΔargG ΔastCADBE strain. Strain 382ΔargG can be obtained by deletion of the argG gene on the chromosome of strain 382 (VKPM B-7926) using the method proposed by Datsenko K.A. and Wanner B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640-6645), called "Red-dependent Integration". In accordance with this procedure, PCR primers homologous to both the region adjacent to the argG gene and the gene responsible for antibiotic resistance can be designed. Plasmid pMW118-attL-Cm-attR (WO 05/010175) can be used as a template in PCR.

Both strains of E. coli, 382ΔargG and 382ΔargG ΔastCADBE, can be grown with stirring at 37 ° C for 18 hours in 3 ml of culture medium, and 0.3 ml of the resulting cultures can be transferred to 2 ml of fermentation medium in 20 × 200 mm tubes and can be cultured at 32 ° C for 48 hours on a rotary shaker.

After growing, the amount of citrulline obtained can be determined by paper chromatography using a mobile phase of the following composition: butanol-acetic acid-water = 4: 1: 1. Subsequent staining can be performed with ninhydrin (2% solution in acetone). A stain containing citrulline can be excised, citrulline can be eluted using a 0.5% aqueous solution of CdCl ₂ , and the amount of citrulline can be determined spectrophotometrically at a wavelength of 540 nm.

Possible 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 L-arginine 0.1 CaCO ₃ 5.0

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

Example 13. The production of ornithine strain E. coli 382 ΔargFΔargI ΔastCADBE.

To assess the effect of inactivation of the astCADBE operon on the production of ornithine, DNA fragments of the chromosome of the E. coli strain MG1655 ΔastCADBE :: cat described above can be transferred to the E. coli 382ΔargFΔargI ornithine producer strain using P1 transduction (Miller JH (1972). Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain 382 ΔargFΔargI ΔastCADBE. Strain 382ΔargFΔargI can be obtained by successive deletions of the argF and argI genes on the chromosome of strain 382 (VKPM B-7926) using the method proposed by Datsenko K.A. and Wanner B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640-6645), called "Red-dependent Integration". In accordance with this procedure, two pairs of PCR primers can be constructed that are homologous to both the regions adjacent to the argF and argI genes and the gene responsible for antibiotic resistance. Plasmid pMW118-attL-Cm-attR (WO 05/010175) can be used as a template in PCR.

Both strains of E. coli, 382ΔargFΔargI and 382ΔargFΔargI ΔastCADBE, can be grown with stirring at 37 ° C for 18 hours in 3 ml of culture medium, and 0.3 ml of the resulting cultures can be transferred to 2 ml of fermentation medium in 20 × 200 mm and can be cultured at 32 ° C for 48 hours on a rotary shaker.

After growing, the amount of ornithine obtained can be determined by paper chromatography using a mobile phase of the following composition: butanol-acetic acid-water = 4: 1: 1. Subsequent staining can be performed with ninhydrin (2% solution in acetone). A stain containing citrulline can be excised, citrulline can be eluted using a 0.5% aqueous solution of CdCl ₂ , and the amount of citrulline can be determined spectrophotometrically at a wavelength of 540 nm.

Possible 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 L-arginine 0.1 CaCO ₃ 5.0

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

Example 14. The production of L-valine strain E. coli H-81 ΔastCADBE.

To assess the effect of inactivation of the astCADBE operon on L-valine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔastCADBE :: cat described above can be transferred to the E. coli H-81 valine producing strain by transduction P1 (Miller JH Experiments in Molecular Genetics , Cold Spring Harbor Lab. Press, 1972, Plainview, NY) for the production of strain H-81 ΔastCADBE.

Both strains of E. coli, H-81 and H-81 ΔastCADBE, can be grown at 37 ° C for 18 h in nutrient broth and 0.1 ml of each of the obtained cultures can be inoculated in 2 ml of fermentation medium in 20 × tubes 200 mm and grown at 32 ° C for 72 hours on a rotary shaker. After culturing for 48 hours and for 72 hours, the amount of accumulated L-valine can be determined using TLC. 10 x 15 cm TLC plates coated with a 0.11 mm layer of Sorbfil silica gel without a fluorescent indicator can be used (Sorbpolymer Joint Stock Company, Krasnodar, Russia). Sorbfil plates can be exposed in the mobile phase of the following composition: propan-2-ol: ethyl acetate: 25% aqueous ammonia: water = 40: 40: 7: 16 (v / v). A solution (2%) of ninhydrin in acetone can be used for visualization.

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

Glucose 60.0 (NH ₄ ) ₂ SO ₄ 15.0 KH ₂ PO ₄ 1.5 MgSO ₄ · 7H ₂ O 1.0 Mameno (TN) 0.4 CaCO ₃ 25.0

CaCO _{3 is} dry-sterilized at 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.

Table 1 Strain OD540 The amount of L-arginine, g / l 382 15.7 ± 0.5 8.0 ± 0.2 382-ΔastCADBE 15.0 ± 0.5 10.5 ± 0.9

table 2 Solutions Component Final concentration, g / l BUT KH 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 0.03 CaCl ₂ 0.011 FROM FeSO ₄ 7H ₂ O 0.075 D Na ₂ MoO ₄ 2H ₂ O 0.00015 H ₃ IN ₃ 0.0025 CoCl ₂ 6H ₂ O 0.00007 CuSO ₄ 5H ₂ O 0.00025 MnCl ₂ 4H ₂ O 0.0016 ZnSO ₄ 7H ₂ O 0.0003 E Thiamine HCl 0.005 F CaCO ₃ 30.0 G Pyridoxine 0.03 The pH of solution A was adjusted to 7.1 using NH ₄ OH.

Claims (4)

1. L-arginine producing bacterium belonging to the genus Escherichia, modified in such a way that the astCADBE operon in the bacterium is inactivated. 2. The bacterium according to claim 1, characterized in that said bacterium belongs to the species Escherichia coli. 3. The bacterium according to any one of claims 1 and 2, characterized in that said astCADBE operon is inactivated due to deletion of the astCADBE operon in the bacterial chromosome. 4. A method of obtaining L-arginine, including:
growing bacteria according to any one of claims 1 to 3 in a nutrient medium; and
isolation of L-arginine from the culture fluid.

Images