RU2311452C2 - METHOD FOR PREPARING L-THREONINE USING MICROORGANISM BELONGING TO GENUS ESCHERICHIA WHEREIN OPERON phoBR IS INACTIVATED - Google Patents

Abstract

FIELD: biotechnology, microbiology, amino acids.

SUBSTANCE: invention relates to a method for preparing L-threonine using microorganism belonging to genus Escherichia that is modified and wherein operon phoBR is inactivated. Invention provides preparing L-threonine with high degree of effectiveness.

EFFECT: improved preparing method.

3 cl, 2 tbl, 2 dwg, 11 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 phoBR operon in said bacterium is weakened.

Description of the Related Art

The PhoB protein is a positive response regulator for the pho regulon and the asr gene.

The PhoB protein, a signal transmitter in the transcriptional regulation of the phosphate regulon, is part of the two-component regulatory system PhoR / PhoB. Proteins PhoR and CreC are sensors for this regulatory system. The PhoR / PhoB system is involved in the regulation of gene expression of the phosphate regulon. Under conditions of a limited amount of phosphate, the PhoB protein is phosphorylated using the phosphorylated form of the PhoR protein. In this phosphorylated state, PhoB acts as a transcriptional activator of the Pho regulon. There are also two phosphate-independent signaling pathways that control the Pho regulon. These paths are regulated by carbon and energy sources. In one way, the CreC catabolite kinase-phosphotransferase sensor can phosphorylate PhoB. In a different way, acetyl phosphate and another sensory kinase are needed for this (Makino, K. et al., J. Mol. Biol., 210 (3), 551-9 (1989); Wanner, BL, Chapter 78, p. 1357- 1381, in Escherichia coli and Salmonella, Second Edition, Editor in Chief: FC Neidhardt, ASM Press, Washington DC (1996)).

But there are currently no reports describing the use of phoBR operon 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 provide a method for producing L-amino acids using these strains.

The above goals were achieved by establishing the fact that inactivation of the phoBR operon can lead to increased 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.

An object of the present invention is to provide an L-amino acid producing bacterium of the Enterobacteriaceae family modified so that expression of the phoBR operon in said bacterium is attenuated.

It is also an object of the present invention to provide a bacterium as described above, wherein expression of said phoBR operon is attenuated by inactivation of the phoB gene.

It is also an object of the present invention to provide a bacterium as described above, wherein expression of said phoBR operon is attenuated by inactivating the phoBR operon.

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 the aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine and L-tryptophan.

Another objective of the present invention is the provision of the bacteria described above, while the 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 above bacteria in a nutrient medium for the purpose of production and accumulation of L-amino acids 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, 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 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 the Best Mode 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, modified so that expression of the phoBR operon in said bacterium is attenuated.

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 L-amino acid and causes the accumulation of L-amino acid in fermentation medium in large quantities, compared with the natural or parent E. coli strain, such as strain E .coli K-12, and preferably means that the microorganism is able to accumulate in the medium the target L-amino acid in an amount of not less than 0.5 g / l, more preferably not less than 1.0 g / l.

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

The 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. Most preferred 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) can be used.

(http://www.ncbi.nlm.nih.gov/htbinpost/Taxonomy/wgetorg?mode=Tree&id=1236&lvl=3&keep=1&srchmode=1&unlock). A bacterium belonging to the genera Escherichia or Pantoea is preferred.

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

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

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

The term “bacterium is modified in such a way that the expression of the phoBR operon is weakened” means that the bacterium has been modified so that, as a result of the modification, the bacterium contains a reduced amount of PhoB / PhoR proteins compared to an unmodified bacterium, or the specified bacterium is unable to synthesize PhoB proteins / PhoR.

Since the PhoB protein is a key protein of the pho regulon regulating system, according to the present invention, it is possible to inactivate only the phoB gene in said bacterium.

The terms "inactivation of the phoBR operon" and "inactivation of the phoB gene" means that the specified operon or gene is modified in such a way that such a modified operon or gene encodes a completely inactive protein (s). It is also possible that natural expression of the modified DNA region is not possible due to deletion of the target gene (s) or its part, shift of the reading frame of the given gene (s), introduction of missense / nonsense mutations (mutations) or modification of regions adjacent to the gene (s), which include sequences that control the expression of gene (s), such as promoter (s), enhancer (s), attenuator (s), ribosome binding site (s), etc.

Gene expression can be estimated by measuring the amount of mRNA transcribed from the target gene using various known techniques, including Northern blotting, quantitative RT-PCR and the like. The amount of protein encoded by this gene can be measured using known methods, including the SDS-PAGE method followed by Western blotting and the like.

The phoBR operon encodes the proteins PhoB (synonyms - B0399, PhoT) and PhoR (synonyms - B0400, NmpB), whereby PhoB is the regulator of the positive response for the pho regulon and the asr gene, that is, the protein regulator of transcription of the phosphate regulon, and PhoR is the sensory kinase phosphotransferase phosphate regulon. The phoB gene (nucleotide numbers 416366 to 417055 in the nucleotide sequence with inventory number NC_000913.2 in the GenBank database; gi: 49175990; SEQ ID NO: 1) and the phoR gene (nucleotide numbers 417113 through 418408 in the nucleotide sequence with inventory number NC_000913 .2 in the GenBank database; gi: 49175990; SEQ ID NO: 3) are located on the chromosome of strain E. coli K-12 between the sbcD and brnQ genes. The nucleotide sequences of the phoB nphoR genes and the corresponding amino acid sequences of the PhoB and PhoR proteins encoded by these genes are shown in the Sequence Listing Nos. 1 (SEQ ID NO: 1) and 3 (SEQ ID NO: 3), 2 (SEQ ID NO: 2 ) and 4 (SEQ ID NO: 4), respectively.

Since representatives of various genera and strains of the Enterobacteriaceae family may experience some variations in the nucleotide sequences, the concept of an inactivable phoBR operon is not limited to genes whose nucleotide sequences are listed in sequence numbers 1 or 3 (SEQ ID NO: 1 or SEQ ID NO: 3), but may also include genes homologous to them. Thus, protein variants encoded by the phoB gene or phoR gene can be represented by proteins with a homology of at least 80%, preferably at least 90% and, most preferably, at least 95%, with respect to the complete amino acid sequence shown in the Sequence Listing numbered 2 or 4 (SEQ ID N0: 2 or SEQ ID NO: 4), provided that the natural properties of the PhoB protein or PhoR protein, respectively, are retained.

In addition, the phoBR operon can be represented by a variant that hybridizes under stringent conditions with the nucleotide sequence shown in the List of sequences numbered 1 or 3 (SEQ ID NO: 1 or SEQ ID NO: 3), or with a probe that can be synthesized based on the specified nucleotide sequence, provided that the specified variant encodes a functional protein PhoB or PhoR. "Stringent conditions" include those conditions under which specific hybrids are formed, and non-specific hybrids are not formed. A practical example of harsh conditions is a one-time washing, preferably two or three times, at a salt concentration corresponding to standard washing conditions for Southern hybridization, for example, 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, at 60 ° C . The length of the probe can be selected depending on the hybridization conditions, usually it is from 100 bp up to 1 kb

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.”

The presence of PhoB protein activity can be determined by measuring the level of transcription from the pstS promoter containing pho boxes in the presence of a phosphorylated form of the PhoB protein, for example, using the method described by Makino, K. et al., (J. Mol. Biol. 210 (3), 551-9 (1989)). Thus, a decrease or absence of PhoB protein activity in bacteria according to the present invention can be determined by comparing said bacterium with a parent unmodified bacterium.

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).

L-amino acid producing bacterium

As a bacterium according to the present invention, modified so that the expression of the phoBR operon is impaired, a bacterium capable of producing an aromatic or non-aromatic L-amino acid can be used.

The bacterium of the present invention can be obtained by inactivating the phoBR operon in a bacterium already possessing the ability to produce L-amino acids. On the other hand, the bacterium of the present invention can be obtained by imparting a bacterium in which the phoBR operon is already inactivated to the ability to produce L-amino acids.

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 5,705,737) , E. coli strain NRRL-21593 (US patent 5939307), E. coli strain FERM BP-3756 (US patent 5474918), E. coli strains FERM BP-3519 and FERM BP-3520 (US patent 5376538), strain E. coli MG442 (Gusyatiner et al., Genetics, 14, 947-956 (1978)), E. coli strains VL643 and VL2055 (European patent application EP 1149911 A) and the like.

The TDH-6 strain is defective in the thrC gene, is capable of assimilating sucrose, and contains the ilvA gene with a leaky mutation. The specified strain contains a mutation in the rhtA gene, which causes resistance to high concentrations of threonine and homoserine. Strain B-3996 contains the plasmid pVIC40, which was obtained by introducing into the vector derived from the RSF1010 vector the thrA * BC operon including the thrA mutant gene encoding aspartokinase-homoserine dehydrogenase I, which has a significantly reduced feedback sensitivity to threonine inhibition. Strain B-3996 was deposited on November 19, 1987 at the All-Union Scientific Center for Antibiotics (RF, 117105 Moscow, Nagatinskaya St., 3-A) with accession 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.

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

- a 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 yaaX reading frame. All three of these genes function as one threonine operon.

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

The rhtA gene is located at the 18th minute of the E. coli chromosome near the 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 pexB and ompX genes. 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 (ABSTRACTS of 17 ^th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP 1013765 A).

The nucleotide sequence of the asd gene from E. coli is known (nucleotide numbers 3572511 to 3571408 in sequence with accession number NC_000913.1 in the GenBank database, gi: 16131307) and can be obtained 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, currently called the National Institute of Progressive 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, Ibara ki-ken, 305-8566, Japan), December 6, 1994 and received 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 (see US Pat. No. 5,827,698).

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 proteins that promote secretion of compounds toxic to the cell (US patent 5972663); E. coli strains containing cysteine desulfohydrase with reduced activity (Japanese patent JP 11155571 A2); E. coli strain W3110 with increased activity of the positive transcriptional regulator of the cysteine regulon encoded by the cysB gene (international PCT application WO0127307A1) 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 Publications JP 62-34397 B and JP 08-70879A), E. coli strains obtained using the genetic engineering methods described in PCT Application WO96 / 06926; E. coli strain H-9068 (JP8-70879A2), 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) (RF patent application 2001117632).

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 strains NRRL B-12116 - B12121 (US Pat. No. 4,388,405); E. coli strains H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (US patent 6344347); E. coli strain H-9341 (FERM BP-6674; European patent 1085087); E. coli strain AI80 / pFM201 (US patent 6258554) and the like.

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). 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 reduced 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 W3110sucA :: Kmr

E. coli AJ12624 (FERM BP-3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJ12949 (FERM BP-4881)

The E. coli strain W3110sucA :: 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 lack the activity of α-ketoglutarate dehydrogenase or have a reduced activity of α-ketoglutarate dehydrogenase, and can be obtained as described above. Examples of such strains are Pantoea ananatis strain AJ13356 (US patent 6331419), Pantoea ananatis strain AJ13356 deposited at the National Institute of Biological Sciences and Human Technologies, Agency for Industrial Science and Technology, Department of International Trade and Industry (National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry), currently called the National Institute of Advanced Industrial Science and Technology, International Organizational Depository for Patenting Purposes, Central 6, 1-1, Higashi 1-H me, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan (National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan), February 19, 1998, and received FERM P-16645. Then, the international deposit of this strain was made according to the conditions of the Budapest Treaty of January 11, 1999, and the strain received accession number FERM BP-6615. In the Pantoea ananatis strain AJ13356, α-KGDH activity is absent due to the destruction of the αKGDH-E1 subunit 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 in 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 the L-phenylalanine producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain AJ12739 (tyrA :: Tn10, tyrR; VKMP B-8197 ); E. coli strain HW1089 (ATCC-55371) containing the pheA34 gene (US patent 5354672); mutant E. coli strain MWEC101-b (KR8903681); E. coli strains NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (US 4407952) and the like. Also, bacteria belonging to the genus Escherichia, producers of L-phenylalanine, such as E. coli strain K-12 [W3110 (tyrA) / pPHAB] (FERM BP-3566), E. coli K strain, can be used as parent strains. -12 [W3110 (tyrA) / pPHAD] (FERM BP-12659), strain E. coli K-12 [W3110 (tyrA) / pPHATerm] (FERM BP-12662) and strain 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 with increased activity of the proteins encoded by the yedA gene or yddG gene can also be used (US patent applications 2003/0148473 A1 and 2003/0157667 A1).

L-tryptophan producing bacterium

Examples of parent strains used to produce L-tryptophan producing bacteria according to the present invention include, but are not limited to, L-tryptophan producing bacteria belonging to the genus Escherichia, such as E. coli strains JP4735 / pMU3028 (DSM10122) and JP6015 / pMU91 (DSM10123) lacking the activity of tryptophanyl tRNA synthetase encoded by the mutant trpS gene (US Pat. No. 5,756,345); E. coli strain SV164 (pGH5) containing an allele of the serA gene encoding an enzyme not inhibited by serine in a feedback manner (US Pat. No. 6,180,373); E. coli strains AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) lacking tryptophanase activity (US patent 4371614); E. coli strain AGX17 / pGX50, pACKG4-pps, which enhances the ability to synthesize phosphoenolpyruvate (international application 9708333, US patent 6319696), and the like.

It was previously shown that the natural allele of the yddG gene, which encodes a membrane protein that is not involved in the biosynthesis of any of the L-amino acids, amplified on a multi-copy vector in a microorganism, gives this microorganism resistance to L-phenylalanine and several analogues of this amino acid. In addition, the introduction of additional copies of the yddG gene into the cells of bacteria producing L-phenylalanine or L-tryptophan can positively affect the production of the corresponding amino acids (PCT international application WO03044192). Thus, it is desirable that the bacterium producing L-tryptophan be further modified so that expression of the open reading frame yddG is enhanced in this bacterium.

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) defective in the ilvA gene and capable of producing L-proline (European patent EP 1172433). The bacterium of the present invention can be improved by enhancing the expression of one or more genes involved in the biosynthesis of L-proline. Preferably, examples of such genes for bacterium-producing L-proline include the proB gene encoding glutamate kinase with desensitized feedback regulation of L-proline (German patent 3127361). In addition, the bacterium of the present invention can be improved by enhancing the expression of one or more genes encoding proteins that secrete the L-amino acid from a bacterial cell. Examples of such genes are the b2682 and b2683 genes (ygaZH genes) (European Patent Application EP 1239041 A2).

Examples of bacteria belonging to the genus Escherichia and having the ability to produce L-proline include the following E. coli strains: NRRL B-12403 and NRRL B-12404 (UK patent GB 2075056), VKPM B-8012 (RF patent application 2000124295), plasmid mutants described in German patent DE 3127361, plasmid mutants described 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 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 US2002058315) and its derivatives containing mutant N-acetylglutamate synthase (RF patent application 2001112869), E. coli 382 strain (VKPM B-7926; European patent application EP 1170358), arginine producing strain into which the argA gene encoding N-acetylglutamate synthetase is introduced ( Japan Patent Application 57-5693 A), 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 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. Carbon sources include various carbohydrates such as glucose and sucrose, as well as various organic acids. Depending on the nature of the assimilation of the microorganism used, alcohols such as ethanol and glycerin may be used. Various inorganic ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds, such as amines, natural nitrogen sources, such as peptone, soybean hydrolyzate, microorganism fermentolizate, can be used as a nitrogen source. As mineral additives, potassium phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium chloride and the like can be used. Thiamine and yeast extract can be used as vitamins.

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

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

Brief Description of Drawings

Figure 1 shows the relative positions of the phoBRL and phoBRR primers on the plasmid pACYC184 used to amplify the cat gene.

Figure 2 shows the construction of a chromosomal DNA fragment containing an inactivated phoBR operon.

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 an inactivated operon phoBR

1. Deletion of the phoBR operon

The strain containing the deletion of the phoBR operon was constructed using a technique developed by Datsenko, K.A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640-6645), known as "Red-dependent Integration". In accordance with this technique, phoBRL PCR primers (SEQ ID NO: 5) and phoBRR (SEQ ID NO: 6) were homologated both to the site adjacent to the phoBR operon and to the antibiotic resistance reporting gene on the plasmid used as a matrix for PCR. Plasmid pACYC184 (NBL Gene Sciences Ltd., UK; accession number X06403 in the GenBank / EMBL database) was used as a template 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 with a length of 1152 bp (Figure 1), purified on an agarose gel, can be used for electroporation into E. coli strain MG1655 (ATCC 700926) containing plasmid pKD46 with a heat-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 of 2154 nucleotides in length (positions 31088 through 33241 of the nucleotide sequence with accession number J02459 in the GenBank database), and also contains the λ genes of the Red 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 obtained as follows: an overnight culture of E. coli strain MG1655 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 ° C 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 and grown at 37 ° C for selection of Cm ^R recombinants. Then, to remove plasmid pKD46, 2 passages were performed on L-agar with Cm at 42 ° C, and the obtained colonies were tested for sensitivity to ampicillin.

2. Confirmation of the deletion of the phoBR operon by PCR.

The mutants with the deleted phoBR operon containing the Cm resistance gene were verified by PCR. Locus-specific primers phoBR1 (SEQ ID NO: 7) and phoBR2 (SEQ ID NO: 8) were used to verify deletion by PCR. The following temperature profile was used for PCR testing: denaturation at 94 ° C for 3 min; profile for 30 cycles: 30 sec at 94 ° C, 30 sec at 54 ° C, 1 min at 72 ° C; final step: 7 min at 72 ° C. The length of the PCR product obtained by the reaction using the parent strain phoBR ⁺ MG1655 as the cell matrix is 2270 bp The length of the PCR product obtained by the reaction using the mutant strain MG1655 ΔphoBR :: cat as a matrix of cells is 1451 bp (Figure 2).

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

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

Both strains of E. Coli, B-3996 and B-3996-ΔphoBR, were grown for 18-24 hours at 37 ° C on plates with L-agar containing chloramphenicol (30 μg / ml). To obtain a seed culture, these strains were grown at 32 ° C for 18 hours on a rotary shaker (250 rpm) in 20x200 mm test tubes containing 2 ml of L-broth with 4% sucrose. Then, 0.21 ml (10%) of the inoculum was added to the fermentation medium. Fermentation was carried out in 2 ml of minimal fermentation medium in test tubes measuring 20 × 200 mm. Cells were grown for 72 hours at 32 ° C with stirring (250 rpm).

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

The fermentation medium of the following composition was used (g / l): Glucose 80.0 (NH ₄ ) ₂ SO ₄ 22.0 NaCl 0.8 KN ₂ RO 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.

As can be seen from Table 1, strain B-3996-ΔphoBR accumulated a greater amount of L-threonine compared to strain B-3996.

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

To assess the effect of phoBR operon inactivation on lysine production, DNA fragments of the chromosome of the above E. coli strain MG1655 ΔphoBR :: cat can be transferred to E. coli WC196 L-lysine producer strain (pCABD2) using P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY). pCABD2 is a plasmid containing the dapA gene encoding a mutant dihydropicolinate synthase that is resistant to feedback inhibition by L-lysine, the lysC gene encoding mutant aspartokinase III, resistant to feedback by L-lysine inhibition, the dapB gene encoding digiducidase ddh gene encoding diaminopimelate dehydrogenase (US patent 6040160).

Both strains of E. coli, WC196 (pCABD2) and WC196 (pCABD2) ΔphoBR :: cat, can be grown in L medium containing 50 mg / l chloramphenicol and 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 can be calculated in terms of glucose consumed.

The composition of the fermentation medium (g / l): Glucose 40 (NH ₄ ) ₂ SO ₄ 24 K ₂ NRA ₄ 1.0 MgSO ₄ × 7H ₂ O 1.0 FeSO ₄ × 7H ₂ O 0.01 MnSO ₄ × 5H ₂ O 0.01 Yeast extract 2.0

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

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

To assess the effect of inactivation of the phoBR operon on L-cysteine production, DNA fragments of the chromosome of the E. coli MG1655 ΔphoBR :: cat strain described above 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), whereby the JM15 (ydeD) -ΔphoBR strain can be obtained.

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-ΔphoBR.

To assess the effect of inactivation of the phoBR operon on the production of L-leucine, DNA fragments of the chromosome of the E. coli strain MG1655 ΔphoBR :: cat described above can be transferred to the E. coli 51 L-leucine producer strain (VKPM B-7386, US patent 6124121) by P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby 57-pMW-ΔphoBR strain can be obtained.

Both strains of E. coli, 57 and 57-ΔphoBR, can be grown for 18-24 hours at 37 ° C on plates with L-agar containing chloramphenicol (30 μg / ml). To obtain a seed culture, these strains can be grown on a rotary shaker (250 rpm) at 32 ° C for 18 hours in 20x200 mm test tubes containing 2 ml of L-broth with 4% sucrose. Then, 0.21 ml (10%) of the seed culture can be added to the fermentation medium. Fermentation can be carried out in 2 ml of minimal fermentation medium in test tubes measuring 20 × 200 mm. Cells can be grown for 48-72 hours at 32 ° C with stirring (250 rpm). The amount of L-leucine can be measured using paper chromatography (mobile phase composition: butanol: acetic acid: water = 4: 1: 1).

Can be used in a fermentation medium of the following composition (g / l) (pH 7.2):

Glucose 60.0 (NH ₄ ) ₂ SO ₄ 25.0 K ₂ NRA ₄ 2.0 MgSO ₄ × 7H ₂ O 1.0 Thiamine 0.01

CaCO ₃ 25.0

Glucose and chalk should be sterilized separately.

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

To assess the effect of inactivation of the phoBR operon on L-histidine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔphoBR :: cat described above 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). Strain 80 is described in RF patent 2119536 and deposited in the All-Russian collection of industrial microorganisms (Russia, 117545 Moscow, 1st Dorozhniy passage, 1) with accession number VKPM V-7270.

Both strains of E. coli, 80 and 80-ΔphoBR, can be grown in L-broth containing chloramphenicol (30 mg / ml) 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 tubes, and the cultures can be grown at 29 ° C 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 Memeno 0.2 total nitrogen L-proline 1.0 (NH ₄ ) ₂ SO ₄ 25.0 KN ₂ RO 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 ⁺ -ΔphoBR.

To assess the effect of inactivation of the phoBR operon on the production of L-glutamic acid, DNA fragments of the chromosome of the above E. coli strain MG1655 ΔphoBR :: cat can be transferred to the L-glutamic acid producing strain E. VL334thrC ⁺ (EP 1172433) using P1- transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby strain VL334thrC ⁺ -ΔphoBR can be obtained.

Both strains, VL334thrC ⁺ and VL334thrC ⁺ -ΔphoBR, can be grown on L-agar plates containing chloramphenicol (30 μg / ml) 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 should 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 should be 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. The production of L-phenylalanine strain E. coli AJ12739-ΔphoBR.

To evaluate the effect of phoBR operon inactivation on L-phenylalanine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔphoBR :: cat described above can be transferred to the E. coli L-phenylalanine producing strain AJ12739 using P1 transduction (Miller, JH ( 1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY). 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.

Both strains, AJ12739-ΔphoBR and AJ12739, can be grown at 37 ° C for 18 hours in a nutrient broth containing chloramphenicol (30 μg / ml); 0.3 ml of the obtained cultures can be introduced into 3 ml of fermentation medium in 20 × 200 mm test tubes, and the cultures can be grown at 37 ° C for 48 hours on a rotary shaker. 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 NRA 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. The production of L-tryptophan strain E. coli SV164 (pGH5) - ΔphoBR.

To assess the effect of inactivation of the phoBR operon on the production of L-tryptophan, DNA fragments of the chromosome of the E. coli strain MG1655 ΔphoBR :: cat described above 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). Strain SV164 (pGH5) is described in detail in US Pat. No. 6,180,373 or European Patent No. 0,662,143.

Both strains, SV164 (pGH5) -ΔphoBR and SV164 (pGH5), can be grown with stirring at 37 ° C for 18 hours in 3 ml of nutrient broth with tetracycline (plasmid marker pGH5, 20 mg / ml) and chloramphenicol (30 μg / ml). 0.3 ml of the obtained cultures can be introduced into 3 ml of a fermentation medium containing tetracycline (20 mg / ml) in 20 × 200 mm test tubes; and cultures can be grown at 37 ° C. for 48 hours on a rotary shaker at 250 rpm. After growing, the amount of tryptophan accumulated in the medium can be determined using TLC, as described in Example 8. The components of the fermentation medium are shown in Table 2, but the groups of components A, B, C, D, E, F and H should be sterilized separately, as shown in Table 2 to avoid unwanted interactions during sterilization.

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

To assess the effect of inactivation of the phoBR operon on L-proline production, DNA fragments from the chromosome of the E. coli strain MG1655 ΔphoBR :: 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). Strain 702ilvA was deposited in the All-Russian collection of industrial microorganisms (VKPM; Russia, 117545 Moscow, ^1st Dorozhniy proezd, 1) with accession number VKPM V-8012.

Both strains of E. Coli, 702ilvA and 702ilvA-ΔphoBR, can be grown for 18-24 hours at 37 ° C on plates with L-agar containing chloramphenicol (30 μg / ml). Then fermentation using these strains can be carried out under the same conditions as described in Example 7.

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

To assess the effect of inactivation of the phoBR operon on L-arginine production, DNA fragments of the chromosome of the E. coli strain MG1655 described above ΔphoBR :: cat can be transferred to the E. coli 382 L-arginine producing strain using P1 transduction (Miller, JH ( 1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY). Strain 382 was deposited in the All-Russian collection of industrial microorganisms (VKPM; Russia, 117545 Moscow, ^1st Dorozhniy proezd, 1) on April 10, 2000 with accession number VKPM V-7926.

Both strains, 382-ΔphoBR and 382, can be grown with stirring at 37 ° C for 18 hours in 3 ml of nutrient broth containing chloramphenicol (30 mg / ml); 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 32 ° C for 48 hours on a rotary shaker.

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

The composition of the fermentation medium (g / l): Glucose 48.0 (NH ₄ ) ₂ SO ₄ 35.0 KN ₂ RO 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 180 ° C for 2 hours. The pH was adjusted to 7.0.

Although the invention has been described in detail with reference to the Best Mode for Carrying Out the Invention, it will be apparent to those skilled in the art that various changes can be made and equivalent replacements made, and such changes and replacements are not 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 OD ₅₄₀ L-threonine, g / l B-3996 25.2 ± 2.6 28.4 ± 0.7 B-3996-ΔphoBR 23.3 ± 0.5 32.7 ± 1.0 table 2 Solutions Component Final concentration, g / l BUT KN ₂ RO 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 × 7H ₂ O 0.0003 F Thiamine HCl 0.005 G CaCO ₃ 30.0 N Pyridoxine 0.03 The pH of solution A was adjusted to 7.1 with NH ₄ OH.

Claims (3)

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

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