RU2311453C2 - METHOD FOR PREPARING NONAROMATIC L-AMINO ACIDS USING MICROORGANISMS BELONGING TO GENUS ESCHERICHIA WHEREIN GENE csrA IS INACTIVATED - Google Patents

Abstract

FIELD: biotechnology, microbiology, amino acids.

SUBSTANCE: invention relates to a method for preparing nonaromatic amino acid using a microorganism belonging to genus Escherichia that is modified and wherein gene csrA is inactivated. The claimed invention provides preparing nonaromatic amino acids with high degree of effectiveness.

EFFECT: improved preparing method.

3 cl, 2 dwg, 2 tbl, 9 ex

Description

Technical field

The present invention relates to the microbiological industry, in particular to a method for producing a non-aromatic L-amino acid using a bacterium of the Enterobacteriaceae family, modified in such a way that the expression of the csrA gene in this bacterium is weakened.

Description of the Related Art

CsrA protein, encoded by the pleiotropic csrA gene of Escherichia coli, is a regulator of carbohydrate metabolism, affecting glycogen biosynthesis, gluconeogenesis, cell sizes and cell surface properties (Romeo, T et al, J. Bacteriol., 175 (15): 4744-47-4755 (1993)), glycogen degradation (Yang, H. et al, J. BacterioL, 178 (4): 1012-7 (1996)), gluconeogenesis and glycolysis (Sabnis, NA et al, J. Biol. Chem., 270 49, 29096-29104 (1995)). The CsrA protein also plays a regulatory role in biofilm formation (Jackson, D.W., J. Bacteriol., 184 (1): 290-301 (2002)).

The csrA gene encoding the CsrA polypeptide of the bacterium Escherichia coli has been described, and it has been suggested that a change in the expression of the csrA gene can be used to regulate the synthesis of products of dependent metabolic pathways and, in itself or in combination with other mutations, can be used to obtain bacterial products (US patent 5684144).

A microorganism with increased synthesis of aromatic compounds has also been described, which is especially useful for the production of phenylalanine, which has a reduced activity of a carbon storage regulator protein (CsrA) and an increased content of erythrose-4-phosphate (W00073484A1).

A method for producing non-aromatic L-amino acids, in particular L-threonine, by increasing the expression of the csrA gene or similar nucleotide sequences has also been described (WO 03046184 A1).

But there are currently no reports describing the use of csrA gene inactivation to produce non-aromatic L-amino acids.

Description of the invention

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

The above goals were achieved by establishing the fact that inactivation of the csrA gene can lead to increased production of non-aromatic L-amino acids, such as L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L-Proline and L-Arginine.

The present invention provides a bacterium of the Enterobacteriaceae family that is capable of increased production of non-aromatic amino acids such as L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L-proline and L-arginine.

An object of the present invention is to provide a producer bacterium of a non-aromatic L-amino acid of the Enterobacteriaceae family, modified in such a way that expression of the csrA gene in said bacterium is attenuated.

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

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

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

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

It is also an object of the present invention to provide a method for producing a non-aromatic L-amino acid, which includes:

- growing the above bacteria in a nutrient medium for the purpose of production and accumulation of non-aromatic L-amino acids in a nutrient medium, and

- and the allocation of the specified non-aromatic L-amino acids from the culture fluid.

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.

1. The bacterium according to the present invention

The bacterium of the present invention is a bacterium producing a non-aromatic L-amino acid of the Enterobacteriaceae family, modified in such a way that the expression of the csrA gene 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 “non-aromatic L-amino acid producing bacterium” also means a bacterium that is capable of producing a non-aromatic L-amino acid and causes the non-aromatic L-amino acid to accumulate in the fermentation medium in large amounts compared to the natural or parent E. coli strain, such as E. coli strain K-12, and preferably means that the microorganism is capable of accumulating 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 “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-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) (http://www.ncbi.nlm.mh.gov/htbinpost/Taxonomy/ can be used. wgetorg? mode = Tree & id = 1236 & lvl = 3 & keep = l & srchmode = l & unlock). A bacterium belonging to the genera Escherichia or Pantoea is preferred.

The term "bacterium belonging to the genus Escherichia" means that the bacterium belongs to the genus Escherichia in accordance with the classification known to the person skilled in the field of microbiology. 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 so that csrA gene expression is weakened” means that the bacterium has been modified so that, as a result of the modification, the bacterium contains a reduced amount of CsrA protein compared to an unmodified bacterium, or the specified bacterium is not able to synthesize CsrA protein.

The term "inactivation of the csrA gene" means that the specified gene is modified in such a way that such a modified gene or operon encodes a completely inactive protein. It is also possible that natural expression of the modified DNA region is not possible due to deletion of the target gene or part thereof, shift of the reading frame of the gene, introduction of the missense / nonsense mutation, or modification of regions adjacent to the gene that include sequences that control gene expression, such as a 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 csrA gene encodes a CsrA protein, a carbon storage regulator. The csrA gene (nucleotide numbers 2816983 through 2817168 in sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990) is located on the chromosome of E. coli K-12 strain between the open reading frame of yqaB and the alaS gene. The nucleotide sequence of the csrA gene and the corresponding amino acid sequence of the CsrA protein are shown in the List of sequences under the numbers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2), respectively.

Since representatives of various genera and strains of the Enterobacteriaceae family may experience some variations in the nucleotide sequences, the concept of the inactivable csrA gene is not limited to the gene shown in the Sequence Listing Number 1 (SEQ ID NO: 1), but may also include genes homologous to it. Thus, a variant of the protein encoded by the csrA gene can be represented by a protein with a homology of at least 80%, preferably at least 90% and most preferably at least 95%, relative to the complete amino acid sequence shown in Sequence Listing No. 2 (SEQ ID NO: 2), provided that the activity of the CsrA protein is maintained.

In addition, the csrA gene can be represented by a variant that hybridizes under stringent conditions with the nucleotide sequence shown in Sequence Listing number 1 (SEQ ID NO: 1), or with a probe that can be synthesized based on the specified nucleotide sequence, provided that this option encodes a functional protein CsrA. "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 x SSC, 0.1% SDS, preferably 0.1 x 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 conventional methods, such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, inactivation of the gene 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 CsrA protein activity can be detected by complementing the csrA ^- mutation. Mutation of the csrA gene leads to a much higher level of glycogen accumulation in bacteria, which can be determined using the method described, for example, Romeo, T et al (J. Bacteriol., 175 (15): 4744-4755 (1993)). Thus, a decrease or absence of CsrA protein activity in bacteria according to the present invention can be determined by comparing said bacterium with a parent unmodified bacterium.

Methods for producing plasmid DNA, cutting and DNA leader, 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 csrA gene expression is weakened, a bacterium capable of producing a non-aromatic L-amino acid can be used.

The bacterium of the present invention can be obtained by inactivating the csrA gene in a bacterium already capable of producing L-amino acids. On the other hand, a bacterium according to the present invention can be obtained by giving a bacterium in which the csrA gene is already inactivated, 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 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)), strains of E. coli VL643 and VL2055 (European patent application EP 1149911 A) and the like.

The TDH-6 strain is thrC deficient, is able to assimilate sucrose, and contains the leaky UvA gene. 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 Escherichia coli aspartokinase homoserine dehydrogenase I 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 threonine synthase from Escherichia coli is known (nucleotide numbers 3734 to 5020 in sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990). The thrC gene is located on the chromosome of the E. coli K-12 strain between the thrB gene and the open uaaX reading frame. All three of these genes function as one threonine operon.

The mutant thrA gene encoding aspartokinase homoserine dehydrogenase I, 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 E. coli producer strain VKPM B-3996. Plasmid pVIC40 is described in detail in US Pat. No. 5,705,371.

The rhtA gene is located at the 18th minute of the E. coli chromosome near the ginHPQ operon, which encodes the components of the glutamine transport system, the rhtA gene is identical to the open reading frame ORF1 (reuybiF, nucleotide numbers 764 to 1651 in sequence with accession number AAA218541 in the GenBank database, gi: 440181), located between the genes PXB and OTP. A region of DNA expressed to form a protein encoded by an ORF1 open reading frame was called the rhtA gene (rht: resistance to homoserine and threonine). It has also been shown that the rhtA23 mutation is 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 inventory 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.

The nucleotide sequence of the E. coli aspC gene is known (nucleotide numbers 983742 through 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 hydroxamate, S- (2-aminoethyl) -L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam, and so on. Mutants that are resistant to these lysine analogues can be obtained by treating bacteria belonging to the genus Escherichia with traditional mutagens. Specific examples of bacterial strains used to produce L-lysine include Escherichia coli strain AL 1442 (PERM 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 AL 3069 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, Ibar aki-ken, 305-8566, Japan), December 6, 1994 with inventory number PERM P-14690. Then, the international deposit of this strain was made according to the conditions of the Budapest Treaty of September 29, 1995, and the strain received accession number FERM BP-5252 (US patent 5827698).

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 of serine acetyltransferase (US patent 6218168; RF patent application 2003121601); E. coli strain W3110 containing genes with increased expression encoding proteins capable of secretion of compounds toxic to the cell (US patent 5972663); E. coli strains with reduced cysteine desulfogadrase activity (Japanese Patent JP 11155571 A);

E. coli strain W3110 with increased activity of the positive transcriptional regulator of the cysteine regulon encoded by the cysB gene (international PCT application W00127307A1), and the like.

L-Leucine Producer Bacteria

Examples of parental strains used to produce the L-leucine producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strains resistant to leucine analogues, including, for example, P-2 -thienylalanine, 3-hydroxyleucine, 4-azaleucine and 5,5,5-trifluoroleucine (Japanese Patent Application Laid-open JP 62-34397 B and JP 8-70879 A), E. coli strains obtained using genetic engineering methods described PCT Application WO 96/06926; E. coli strain H-9068 (JP 08-70879A), 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) (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 EP 1085087); E. coli strain AI80 / pFM201 (US Pat. No. 6,258,554) 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 VL334thrC ⁺ (VKPM B-8961), auxotroph by L-isoleucine. This strain has the ability to produce L-glutamic acid.

Examples of parental strains used to produce the L-glutamic acid producing bacterium of the present invention include mutant strains lacking α-ketoglutarate dehydrogenase activity or having reduced α-ketoglutarate dehydrogenase activity. Bacteria belonging to the genus Escherichia, lacking the activity of α-ketoglutarate dehydrogenase or having 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 W 3110 sucA :: Kmr

E. coli AJ 12624 (FERM BP-3853)

E. coli AJ 12628 (FERM BP-3854)

E. coli AJ 12949 (FERM BP-4881)

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

Other examples of bacteria producing L-glutamic acid include bacteria belonging to the genus Pantoea, which are devoid of α-ketoglutarate dehydrogenase activity or have reduced α-ketoglutarate dehydrogenase activity, and can be obtained as described above. An example of such strains is Pantoea ananatis AJ 13356 strain (US Pat. No. 6,331,419). The strain Pantoea ananatis AJ 13356 was deposited at the National Institute of Bioscience and Human-Technology, the Agency of Industrial Science and Technology, the Ministry of International Trade and Industry, the National Institute of Biological Sciences and Human Technology ), currently called the National Institute of Progressive Industrial Science and Technology, International Depository of Organizations for Patenting Purposes, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan (National 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. In the Pantoea ananatis strain AJ13356, α-KGDH activity is absent due to the destruction of the αKGDH-El (sucA) subunit gene. The above strain, when isolated, was identified as Enterobacter agglomerans and deposited as Enterobacter agglomerans A J 13355. However, it was later classified as Pantoea ananatis based on the 16S rRNA nucleotide sequence and other evidence (see Examples section). Although strain AJ13356 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-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 702 ilvA strain (VKPM B-8012) deficient in the HvA 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 L-proline producing bacteria 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 secreting the L-amino acid from the 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 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 US 2002058315 ) and its derivatives containing mutant N-acetylglutamate synthase (RF patent application 2001112869), E. coli strain 382 (VKPM B-7926) (European patent application EP 1170358), an arginine producing strain into which the argA gene encoding N- acetylglutamate synthetase (Japanese Patent Laid-open 5 7-5693A), and the like.

2. The method according to the present invention.

The method according to the present invention is a method for producing a non-aromatic L-amino acid, comprising the steps of growing a bacterium according to the present invention in a nutrient medium in order to produce and accumulate an L-amino acid in a nutrient medium, and to isolate the L-amino acid from the culture fluid.

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

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

The cultivation is preferably carried out under aerobic conditions, 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 adding ammonia, calcium carbonate, various acids, bases and buffers. 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 the Drawings

Figure 1 shows the location of the csrAL and csrAR primers on the plasmid pACYC184 used to amplify the cat gene.

Figure 2 shows the construction of a chromosomal DNA fragment containing the inactivated csrA gene.

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 csrA gene.

1. Division of the csrA gene.

A strain containing a deletion in the csrA gene 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, csrAL (SEQ ID NO: 3) and csrAR (SEQ ID NO: 4) PCR primers homologous to the regions of the chromosome adjacent to the csrA gene and the antibiotic resistance gene of the plasmid used as a template for PCR were designed . The 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; subsequent 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 1093 bp (Figure 1) was purified on an agarose gel and can be used for electroporation into E. coli strain MG1655 (ATCC 700926) containing the plasmid pKD46 with a temperature-sensitive replicon. Plasmid pKD46 (Datsenko, K.A. and Wanner, BL, Proc. Natl. Acad. Sci. USA, 2000, 97: 12: 6640-45) contains a 2154 nucleotide phage A DNA fragment (positions 31088 to 33241 nucleotide sequence accession number J02459 database GenBank), and contains genes of the λ Red-homologous recombination system (genes γ, β, exo) under control of promoter P _agaves arabinose-inducible. 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 ° 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 L plates agar and grown at 37 ° C to select Cm ^R recombinants. Then, to remove plasmid pKD46, 2 passages were performed on L-agar with Cm at 42 ° C and the obtained colonies were tested for sensitivity to ampicillin.

2. Confirmation of deletion of the csrA gene by PCR.

Mutants with a delegated csrA gene containing a Cm resistance gene were verified by PCR. Locus-specific primers csrA1 (SEQ ID NO: 5) and csrA2 (SEQ ID NO: 6) 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 using the parental strain MG1655 csrA ⁺ as a matrix of cells is 547 bp The length of the PCR product obtained using the mutant strain MG1655 AcsrA :: cat as a matrix of cells is 1453 bp (Figure 2).

Example 2. Production of L-threonine by E. coli strain B-3996-AcsrA. To assess the effect of csrA gene inactivation on the production of threonine, DNA fragments of the chromosome of the E. coli strain MG1655 AcsrA :: cat described above were transferred to the E. coli L-threonine producer 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-AcsrA 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 20 × 200 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 65 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. The spot 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 10 independent test fermentation tubes are shown in Table 1. The following composition of the fermentation medium was used, g / l:

Glucose 80.0 (NH ₄ ) ₂ SO ₄ 22.0 NaCl 0.8 KN 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 MgSO ₄ · 7H ₂ O were sterilized separately. CaCO _{3 was} dry heat sterilized at 180 ° C for 2 hours. The pH was adjusted to 7.0. The antibiotic was added to the medium after sterilization.

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

Example 3. Production of L-lysine by E. coli strain AL 1442-AcsrA. To assess the effect of csrA gene inactivation on lysine production, DNA fragments of the chromosome of the E. coli strain MG1655 AcsrA :: cat described above 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 tysC gene encoding a mutant aspartokinase III that is resistant to feedback by L-lysine inhibition, the dapB gene encoding digiducidase and dihydredicol ddh gene encoding diaminopimelate dehydrogenase (US patent 6040160).

Both strains of E. coli, the parent WC196 (pCABD2) and the obtained WC196 (pCABD2) AcsrA :: 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 fermentation medium containing the necessary antibiotics in a 500 ml flask. 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 culture, the amounts 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 (NH ₄ ) ₂ SO ₄ 24 K ₂ HPO ₄ 1.0 MgSO ₄ · 7H ₂ O 1.0 FeSO ₄ · 7H ₂ O 0.01 MnSO ₄ · 5H ₂ O 0.01 Yeast extract 2.0

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

Example 4. Production of L-cysteine by E. coli strain JM15 (ydeD) -AcsrA. To assess the effect of csrA gene inactivation on L-cysteine production, DNA fragments of the chromosome of the E. coli strain MG1655 AcsrA :: cat 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 strain JM15 (ydeD) -ΔcsrA 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 Tw.yd.eD encoding a membrane protein not involved in the biosynthesis of any of the L-amino acids ( U.S. Patent 5,972,663).

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-ΔcsrA.

To assess the effect of csrA gene inactivation on L-leucine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔcsrA :: cat described above can be transferred to the E. coli 57 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-AcsrA strain can be obtained.

Both strains of E. coli, 57 and 57-AcsrA, 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 20 × 200 mm test tubes containing 2 ml of L-broth with 4% sucrose. Then, 0.21 ml (10%) of the seed culture can be added to the fermentation medium. Fermentation can be carried out in 2 ml of minimal fermentation medium in test tubes measuring 20 × 200 mm. Cells can be grown for 48-72 hours at 32 ° C with stirring (250 rpm).

The amount of L-leucine can be measured using paper chromatography (composition of the mobile phase: butanol: acetic acid: water = 4: 1: 1)

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

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

Glucose and chalk should be sterilized separately,

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

To assess the effect of csrA gene inactivation on L-histidine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔcsrA :: 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 National Collection of Industrial Microorganisms (Russia, 117545 Moscow, 1st Dorozhny passage, 1) with accession number VKPM B-7270.

For fermentation by glucose depletion (batch-fermentation) in mini-fermenters, one microbiological loop of each strain, 80 and 80-ΔcsrA grown on L-agar, was transferred to L-broth, and cultures were grown at 30 ° C on a rotary shaker (rotation speed 140 rpm) until an optical density of OD ₅₄₀ ≈2.0 is reached. After that, 25 ml of inoculum was introduced into 250 ml of fermentation medium and grown at 29 ° C on a rotary shaker (1500 rpm). The duration of the fermentation was approximately 35-40 hours. After growing, the amount of histidine accumulated in the medium was determined by paper chromatography. The paper was exposed with a mobile phase of the following composition: n-butanol: acetic acid: water = 4: 1: 1 (v / v). A 0.5% solution of ninhydrin in acetone was used as a reagent for visualization. The data obtained are presented in Table 2.

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

Glucose 100.0 Memeno 0.2 total nitrogen (NH ₄ ) ₂ SO ₄ 8.0 KN ₂ PO ₄ 1.0 MgSO ₄ · 7H ₂ O 0.4 FeSO ₄ · 7H ₂ O 0.02 MnSO ₄ 0.02 Thiamine 0.001 Betaine 2.0 L-proline 0.8 L-glutamate 3.0 L-aspartate 1.0 Adenosine 0.1

As can be seen from Table 2, strain 80-ΔcsrA accumulated a larger amount of L-histidine compared to strain 80.

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

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

Both strains, the parent VL334thrC ⁺ and the resulting VL334thrC ⁺ -ΔcsrA, 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. Production of L-Proline by E. coli strain 702ilvA-ΔcsrA. To assess the effect of csrA gene inactivation on L-proline production, DNA fragments of the chromosome of the E. coli MG1655 AcsrA :: cat strain described above can be transferred to the E. coli 702 ilvA 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 Russian National 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-ΔcsrA 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 9. The production of L-arginine strain E. coli 382-ΔcsrA.

To assess the effect of csrA gene inactivation on L-arginine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔcsrA :: cat described above 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 Russian National 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-ΔcsrA and 382, can be grown at 32 ° C for 18 hours in 2 ml of LB broth containing chloramphenicol (30 mg / ml), and 0.3 ml of the resulting culture can be transferred to 2 ml of fermentation medium in test tubes measuring 20 × 200 mm and grown at 32 ° C for 48 hours with stirring on a rotary shaker.

After growing, the amount of L-arginine accumulated in the medium can be determined using paper chromatography, using the following composition of the mobile phase: butanol: acetic acid: water = 4: 1: 1 (v / v). A solution (2%) of ninhydrin in acetone can be used for visualization. Stains containing L-arginine can be cut out, L-arginine can be eluted with a 0.5% aqueous solution of CdCl ₂ , after which the amount of L-arginine can be estimated 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 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 manganese 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 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 OD ₅₄₀ L-threonine, g / l B-3996 26.1 ± 0.5 23.4 ± 0.4 B-3996-AcsrA 27.2 ± 1.8 24.8 ± 0.9

table 2 Strain L-histidine, g / l The average yield of glucose,% 80 16.8 ± 0.5 20.3 80-ΔcsrA 18.0 ± 0.6 22.3

Claims (3)

1. A bacterium producing a non-aromatic L-amino acid, in particular L-threonine or L-histidine, belonging to the genus Escherichia, modified so that the csrA gene is inactivated in this bacterium. 2. The bacterium according to claim 1, characterized in that said csrA gene is inactivated due to deletion of the csrA gene in the bacterial chromosome. 3. A method for producing a non-aromatic L-amino acid, in particular L-threonine or L-histidine, comprising growing the bacterium according to any one of claim 1 or 2 in a nutrient medium, causing production and accumulation of the L-amino acid in the culture fluid, and isolation of the L-amino acid from the culture fluid.

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