MXPA06006334A - L-threonine producing bacterium belonging to the genus escherichia and method for producing l-threonine - Google Patents

Abstract

There is disclosed a method for producing L-threonine using bacterium belonging to the genus Escherichia wherein the bacterium has been modified to enhance an activity of aspartate-&bgr;-semialdehyde dehydrogenase.

Description

BACTERIA THAT PRODUCES THREONIN WHICH BELONGS TO THE GENRE ESCHERICHIA AND METHOD TO PRODUCE L-TREONINE Technical Field The present invention relates to a method for producing an L-amino acid by fermentation, and more specifically to a gene derived from Escherichia coli that helps in this fermentation. The gene is useful for the improvement of L-amino acid production, and specifically, for example, for the production of L-threonine. BACKGROUND ART Conventionally, L-amino acids are produced industrially by fermentation methods using strains of microorganisms obtained from natural or mutant sources thereof, which are modified to increase the production yields of L-amino acids. Many techniques to increase production yields of L-amino acids have been reported, including the transformation of microorganisms with recombinant DNA (see, for example, U.S. Patent No. 4,278,765). Other techniques for increasing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and / or desensitizing target enzymes of feedback inhibition by the resulting L-amino acid (see, for example, WO 95/16042 or North American patents 4,346,170, 5,661,012 and 6,040,160). Strains useful in the production of L-threonine by fermentation are known, including strains with increased activities of enzymes involved in the biosynthesis of L-threonine (U.S. Patent Nos. 5,175,107; 5,661,012 / 5,705,371; 5,939,307; EPO219027), drug-resistant strains Chemicals such as L-threonine and its analogs (WO 0114525A1, EP301572A2, US 5,376,538), strains with target enzymes desensitized to the retroaligned inhibition by the L-amino acid produced or its by-products (US patents 5,175,107; 5,661,012) and strains with enzymes of inactivated threonine degradation (U.S. Patents 5,939,307; 6,297,031). The known threonine strain VKPM B-3996 (US patents 5,175,107 and 5,705,371) is the best threonine producer known today. For the construction of strain VKPM B-3996, several mutations and a plasmid, described below, were introduced into the strain of E. coli K-12 origin (VKPM B-7). The imitative thrA gene (thrA442 mutation) encodes aspartokinase homoserine dehydrogenase I, which is resistant to threonine-fed inhibition. The imitating ilvA gene (ilvA442 mutation) encodes threonine desasease that has decreased activity which results in a decreased proportion of isoleucine biosynthesis and a poorly isolated underfeed phenotype. isoleucine In bacteria containing the ilvA442 mutation, the transcription of the thrABC operon is not repressed by isoleucine, and therefore is very efficient for the production of threonine. Inactivation of the tdh gene results in the prevention of threonine degradation. The genetic determinant of sucrose uptake (genes scrKYABR) was transferred to the strain. To increase the expression of the genes controlling threonine biosynthesis, plasmid pVIC40 containing the threonine mutant operon thrA442BC was introduced into the intermediate strain TDH6. The amount of L-threonine accumulated during the fermentation of the strain can be up to 85 g / 1. The present inventors obtained, with respect to E. coli K-12, a mutant, thrR (referred to herein as rhtA23) having resistance to high concentrations of threonine or homoserin in the minimal medium (Astaurova, OB et al., Appl. Bioch. And Microbiol., 21, 611-616 (1985)). The mutation resulted in the improvement in the production of L-threonine (SU patent No. 974817), homoserin and gluta ato (Astaurova, OB et al., Appl. Bioch.And Microbiol., 27, 556-561, 1991, EP 1013765 A) by the strain that produces the respective E. coli, such as strain VKPM B-3996. In addition, the present inventors have revealed that rhtA exists in 18 min in the E. coli chromosome close to the glnHPQ operon encoding components of the transport system of glutamine, and that the rhtA gene is identical to 0RF1 (gene ybiF numbers 764 to 1651 in accession number of GenBank AAA218541, gi: 440181), located between the genes pexB and ompX. The unit expressing a protein encoded by ORF1 has been designated as rhtA gene (rht: homoserin and threonine resistance). Also, the present inventors have found that the rhtA23 mutation is a substitution of A-for-G in the -1 position with respect to the ATG start codon (ABSTRACTS of 17th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of American Society for Biochemistry and Molecular Biology, San Francisco, California, August 24-29, 1997, extract No. 457, EP 1013765A). Under conditions of optimization of the mainstream threonine biosynthetic pathway, further improvement of threonine producing strains can be performed by supplementing the bacteria with increased amounts of threonine distant precursors, such as aspartate. It is known that aspartate is a carbon donor for the synthesis of the amino acids of the aspartate family (threonine, ethionine, usina) and diaminopimelate, a constituent compound of the bacterial cell wall. This synthesis is carried out through a complex route that has several branch points and an extremely sensitive regulatory scheme. At branching points (aspartate, aspartate senialdehyde, homoserine), there are as many isoenzymes as there are amino acids that are derived from this biosynthetic stage. The aspartokinase homoserine dehydrogenase I encoded by the part of the thripe OPABC causes the first and the third reaction of threonine biosynthesis. Threonine and isoleucine regulate the expression of aspartokinase homoserine dehydrogenase I, and threonine which inhibits both activities to catalyze the reactions described in the above. { Escherichia coli and Salmonella ,. Second Edition, Editor in Chief: F.C.Neidhardt, ASM Press, Washington D.C., 1996). The asd gene encodes aspartate-β-semialdehyde dehydrogenase (Asd; EC 1.2.1.11), which is a key enzyme in the biosynthetic pathways for usina, methionine, threonine and diaminopimelate. Aspartate-β-semialdehyde dehydrogenase reversibly converts L-aspartyl-4-P to L-aspartate semialdehyde together with the reduction of NADP. The effect of the asd gel amplification on the production of L-lysine, an amino acid of the aspartate family, by the E. coli strain is disclosed (North American patent 6,040,160). It has also been reported that aspartate-β-semialdehyde dehydrogenase could be useful for the production of L-lysine, L-threonine and L-isoleucine by the corineforme bacterium (EP 0219027 A). However, there has been no report to date of the use of a bacterium belonging to the genus Escherichia with increased activity of aspartate-β-semialdehyde dehydrogenase for the production of L-threonine. BRIEF DESCRIPTION OF THE INVENTION An objective of the present invention is to increase the productivity of strains producing L-threonine and to provide a method for producing L-threonine using these strains. This objective was achieved by discovering that the asd gene encoding aspartate-β-semialdehyde dehydrogenase cloned in a low copy vector increases the production of L-threonine. Thus, the present invention has been completed. An object of the present invention is to provide a bacterium that produces L-threonine belonging to the genus Escherichia, wherein the bacterium has been modified to increase an activity of aspartate-β-semialdehyde dehydrogenase. A further objective of the present invention is to provide the bacterium described in the foregoing, wherein the activity of aspartate-β-semialdehyde dehydrogenase is increased by increasing the expression of an aspartate-β-semialdehyde dehydrogenase gene. A further objective of the present invention is to provide the bacterium described above, wherein the activity of aspartate-β-semialdehyde dehydrogenase is increased by increasing a copy number of the aspartate-β-semialdehyde dehydrogenase gene or by modifying a sequence controlling the expression of the gene so that the expression of the gene is increased. A further objective of the present invention is to provide the bacterium as described above, wherein the number of copies is increased by transforming the bacterium with a vector containing the gene. A further objective of the present invention is to provide the bacterium as described above, wherein the aspartate-β-semialdehyde dehydrogenase gene is derived from a bacterium belonging to the genus Escherichia. A further objective of the present invention is to provide the bacterium as described above, wherein the aspartate-β-semialdehyde dehydrogenase gene encodes a protein selected from the group consisting of: (A) a protein comprising the amino acid sequence of SEQ ID NO: 2; and (B) a protein, comprising an amino acid sequence that incl the deletion, substitution, insertion or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 2 and having an aspartate-β- activity semialdehyde dehydrogenase. A further objective of the present invention is to provide the bacterium as described above, wherein the aspartate-β-semialdehyde dehydrogenase gene it comprises a DNA selected from the group consisting of: (a) a DNA comprising a nucleotide sequence of nucleotides 1 to 1196 in SEQ ID NO: 1; Y (b) a DNA that is hybridizable with a nucleotide sequence of nucleotides 1-1196 in SEQ ID NO: 1, or a probe that can be prepared from the nucleotide sequence under severe conditions, and encodes a protein that It has an activity of aspartate-β-semialdehyde dehydrogenase. A further objective of the present invention is to provide the bacteria as described above, wherein the severe conditions comprise those in which the washing is performed at 60 ° C at a salt concentration of lxSSC and 0.1% SDS, and during 15 minutes. A further objective of the present invention is to provide the bacterium as described above, wherein the bacterium has been further modified to increase the expression of one or more of the genes selected from the group consisting of - the mutant thrA gene encoding for aspartokinase homoserine dehydrogenase I and is resistant to threonine-fed inhibition; the thrB gene that codes for homoserin kinase. - the thrC gene coding for threonine synthase; Y the rhtA gene encoding a putative transmembrane protein. A further objective of the present invention is to provide the bacterium as described above, wherein the bacterium has been modified to increase the expression of the mutant thrA gene, the thrB gene, the thrC gene and the rh tA gene. A further objective of the present invention is to provide a method for producing L-threonine which comprises culturing the bacterium as described above in a culture medium to cause accumulation of L-threonine in the culture medium, and collecting the L-threonine. threonine from the culture medium. DESCRIPTION OF THE PREFERRED MODALITIES In the present invention, "bacteria producing L-threonine" means a bacterium having an ability to cause accumulation of L-threonine in a medium when the bacterium is grown in the medium. The ability to produce L-threonine can be imparted or enhanced by reproduction. The phrase "L-threonine-producing bacterium" as used herein also means a bacterium that is capable of producing and causing accumulation of L-threonine in a culture medium in a larger amount than a wild type strain or source. of E. coli, such as strain K12 of E. coJi.

The phrase "a bacterium belonging to the genus Escherichia" means that the bacterium is classified in the genus Escherichia according to the known classification > for a person skilled in the microbiology technique. Examples of a microorganism belonging to the genus Escherichia as used in the present invention inclbut are not limited to Escherichia coli [E. coli). The bacterium belonging to the genus Escherichia that can be used in the present invention is not particularly limited, however for example, the bacteria described by Neidhardt, F.C. and collaborators . { Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1) are encompassed by the present invention. The phrase "aspartate-β-semialdehyde dehydrogenase activity" means an activity that catalyzes the reversible substrate-dependent reduction of NADP in the presence of phosphate or arsenate. The activity of aspartate-β-semialdehyde dehydrogenase can be measured by the method described by, for example, Preiss, J. et al (Curr Microbiol., 7: 263-268 (1982)). The phrase "modified to increase an aspartate-β-semialdehyde dehydrogenase activity" means that the activity per cell is higher than that of an unmodified strain, for example, a wild-type strain. Examples such modifications include increasing the number of aspartate-β-semialdehyde dehydrogenase molecules per cell, increasing the specific activity per molecule of aspartate-β-semialdehyde dehydrogenase and so on. In addition, a wild-type strain that can be used for comparison purposes includes, for example, Escherichia coli K-12. As a result of the increased intracellular activity of aspartate-β-semialdehyde dehydrogenase, the amount of accumulation of L-threonine in a medium is increased. The increased activity of aspartate-β-semialdehyde dehydrogenase in a bacterial cell can be achieved by increasing the expression of a gene encoding aspartate-β-semialdehyde dehydrogenase. Any gene derived from bacteria belonging to the genus Escherichia, as well as any gene derived from another bacterium, such as corinefor e bacteria, can be used as the aspartate-β-semialdehyde dehydrogenase gene. Among these, genes aerivated from bacteria belonging to the genus Escherichia are preferred. As the gene coding for aspartate-β-semialdehyde dehydrogenase from Escherichia coli, the asd gene has already been made clear (nucleotide numbers 3572511 to 3571408 in the access sequence of GenBank NC_000913.1, gi: 16131307). Therefore, the asd gene can be obtained by PCR (polymerase chain reaction, refer to White, T.J. et al, Trends Genet., 5, 185 (1989)) using primers prepared based on the nucleotide sequence of the gene. Genes encoding aspartate-β-semialdehyde dehydrogenase from other organisms can be obtained in a similar manner. The asd gene derived from Escherichia coli is exemplified by a DNA encoding the following protein (A) or (B): (A) a protein having the amino acid sequence shown in SEQ ID NO: 2; or (B) a protein, having an amino acid sequence that includes the deletion, substitution, insertion or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 and having an activity of aspartate-β -semi-aldehyde dehydrogenase. The number of "several" amino acids differs depending on the position or type of amino acid residues in the three-dimensional structure of the protein. This may be from 2 to 30, preferably 2 to 15, and more preferably 2 to 5 for the protein (A). The deletion, substitution, insertion or addition of amino acids can occur in regions of the protein that are not critical for the function of the protein. This is because some amino acids have high homology to each other so that the Three-dimensional structure or activity is not affected by such a change. Therefore, the protein variant (B) may be one having homology of not less than 70%, preferably not less than 80%, more preferably not less than 90%, and much more preferably not less than 95 % with respect to the complete amino acid sequence of aspartate-β-semialdehyde dehydrogenase shown in SEQ ID NO: 2, as long as the activity of aspartate-β-semialdehyde dehydrogenase is maintained. The homology between two amino acid sequences can be determined using well-known methods, for example, the BLAST 2.0 computer program, which calculates three parameters: registration, identity and similarity. Substitution, deletion, insertion or addition of one or more amino acid residues must be a conservative mutation (s) so that activity is maintained. The representative conservative mutation is a conservative substitution. Examples of conservative substitutions include the substitution of Ser or Thr for Ala, substitution of Gln, His or Lys for Arg, substitution of Glu, Gln, Lys, His or Asp for Asn, substitution of Asn, Glu or Gln for Asp, substitution of Ser or Ala by Cys, substitution of Asn, Glu, Lys, His, Asp or Arg by Gln, substitution of Asn, Gln, Lys or Asp by Glu, substitution of Pro by Gly, substitution of Asn, Lys, Gln, Arg or Try for His, substitution for Leu, Met, Val or Phe for lie, substitution for lie, Met, Val or Phe for Leu, substitution of Asn, Glu, Gln, His or Arg by Lys, substitution of Lie, Leu, Val or Phe by Met, substitution of Trp, Tyr, Met, lie or Leu by Phe, substitution of Thr or Ala by Ser, substitution of Ser or Ala by Thr, substitution of Phe or Tyr by Trp, substitution of His, Phe or Trp by Tyr and substitution of Met, lie or Leu by Val. The DNA, which encodes substantially the same protein as the aspartate-β-semialdehyde dehydrogenase described in the above, can be obtained, for example, by modifying the nucleotide sequence of DNA encoding aspartate-β-semialdehyde dehydrogenase (SEQ ID NO- .1), for example, by means of the site-directed mutagenesis method so that one or more amino acid residues at a specific site involve deletion, substitution, insertion or addition. The modified DNA as described above can be obtained by conventionally known mutation treatment. Such treatments include treatment with hydroxylamine of the proteins encoding DNA of the present invention, or treatment of the bacteria containing the DNA with UV irradiation or a reagent such as N-methyl-N '-nitro-N-nitrosoguanidine or acid nitrous. A DNA encoding substantially the same protein as aspartate-β-semialdehyde dehydrogenase can be obtained by expressing DNA having the mutation as described above in an appropriate cell, and Investigate the activity of any expressed product. A DNA that encodes substantially the same aspartate-β-semialdehyde dehydrogenase protein can also be obtained by isolating a DNA from the mutant DNA encoding aspartate-β-semialdehyde dehydrogenase or from a mutant-containing cell, which is hybridizable with a probe that has a nucleotide sequence containing, for example, the nucleotide sequence shown as SEQ ID NO: 1, under severe conditions, and encoding a protein having the activity of aspartate-β-semialdehyde dehydrogenase. The "severe conditions" referred to herein are conditions under which so-called specific hybrids are formed, and non-specific hybrids are not formed. It is difficult to express this condition clearly when using some numerical value. However, for example, severe conditions can be exemplified by conditions under which DNAs having high homology, for example, DNAs having the homology of not less than 50% are capable of hybridizing to each other, but DNAs having a lower homology than the previous one are not able to hybridize with each other. Alternatively, severe conditions can be exemplified by conditions under which the DNA is capable of hybridizing one salt concentration equivalent to the ordinary washing conditions in Southern hybridization, i.e., 1 x SSC, 0.1% SDS, 0.1 preference x SSC, 0.1% SDS, at 60 ° C. The duration of the wash depends on the type of membrane used for staining, as a rule, it is recommended by the manufacturer. For example, the recommended duration of washing with the Hybond ™ N + nylon membrane (Amersham) under severe conditions is 15 minutes. A partial sequence of the nucleotide sequence of SEQ ID NO: 1 can also be used as a probe. The probe can be prepared by PCR using primers based on the nucleotide sequence of SEQ ID NO: 1 and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 1 as a template. When a DNA fragment having a length of about 300 bp is used as the probe, the hybridization conditions for washing include, for example, 50 ° C, 2 x SSC and 0.1% SDS. Substitution, deletion, insertion or addition of nucleotides as described above also includes the mutation, which naturally occurs (mutant or variant), for example, due to a variety in the species or genus of bacteria, which contains aspartate- β-semialdehyde dehydrogenase. "Transformation of a bacterium with a DNA encoding a protein" means the introduction of the DNA into a bacterium, for example, by conventional methods. The Transformation of this DNA will result in an increase in the expression of the gene encoding the protein of the present invention, and will increase the activity of the protein in the bacterial cell. Methods of increasing gene expression include increasing the number of copies of the gene. The introduction of a gene into a vector that is capable of functioning in a bacterium belonging to the genus Escherichia increases the number of copies of the gene. Preferably, low copy vectors are used. Examples of low copy vectors include but are not limited to pSClOl, pMWlld, PMW119, and the like. The term "low copy vector" is used for vectors, the number of copies of which is up to 5 copies per cell. The transformation methods included in any of the known methods that have been reported so far. For example, a method for treating recipient cells with calcium chloride to increase the permeability of cells to DNA has been reported for K-12 from Escherichia coli (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)) and can be used. Increasing gene expression can also be achieved by introducing multiple copies of the gene into a bacterial chromosome by, for example, a homologous recombination method. Integration of Mu or similar. For example, an act of integration of Mu allows to introduce in the bacterial chromosome up to 3 copies of the gene. The increase in gene expression can also be achieved by placing the DNA of the present invention under the control of a potent promoter. For example, the lac promoter, the po trp promoter, the trc promoter and the PR and PL promoters of lambda phage are known as potent promoters. The use of a potent promoter can be combined with the multiplication of copies of the gene. Alternatively, the effect of a promoter can be increased, for example, by introducing a mutation in the promoter to increase a level of transcription of a gene located downstream of the promoter. Furthermore, it is known that the replacement of several nucleotides in the spacer between the ribosome binding site (RBS) and the start codon, especially the sequences immediately upstream of the start codon, profoundly affect the translatability of the RNA. For example, a 20-fold range in expression levels was found, depending on the nature of the three nucleotides preceding the start codon (Gold et al, Annu, Rev. Microbiol., 35, 365-403, 1981; et al., EMBO J., 3, 623-629, 1984). Previously, it was shown that the rhtA23 mutation is an A-to-G substitution at position 1 relative to the ATG start codon (ABSTRACTS of 17th International Congress of Biochemistry and Molecular Biology in Conjunction with 1997).

Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California Augusi "24-29, 1997, abstract No. 457. Therefore, it can be suggested that the rhtA23 mutation increases the expression of the rhtA gene and, as a consequence, it increases the resistance to threonine, homoserine and some other substances transported out of the cells.In addition, it is also possible to introduce a nucleotide substitution in a promoter region of the aspartate-β-semialdehyde dehydrogenase gene on the bacterial chromosome that gives as a result a stronger promoter function.Altering the expression control sequence can be performed, for example, in the same way as the replacement of the gene using a temperature-sensitive plasmid, as disclosed in the international publication WO 00/18935 and Japanese Patent Publication No. 1-215280. Increase in the copy number of the aspartate-β-semialdehyde dehydrog gene Enasa can also be achieved by introducing multiple copies of the aspartate-β-semialdehyde dehydrogenase gene into the chromosomal DNA of the bacterium. In order to introduce multiple copies of the aspartate-β-semialdehyde dehydrogenase gene into the bacterial chromosome, homologous recombination is carried out using a sequence whose multiple copies exist as targets in chromosomal DNA. Sequences that have multiple copies in the chromosomal DNA include, but are not limited to, repetitive DNA, or inverted repeats that exist at the end of a transposable element. Also, as disclosed in U.S. Patent No. 5,595,889, it is possible to incorporate the aspartate-β-semialdehyde dehydrogenase gene into a transposon, and allow it to be transferred to introduce multiple copies of the gene into the chromosomal DNA. Methods for the preparation of plasmid DNA include, but are not limited to, DNA digestion and ligation, transformation, selection and an oligonucleotide such as a primer and the like, or other methods well known to those skilled in the art. These methods are described, for example, in Sambrook, J., Fritsh, E.F., and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition," Cold Spring Harbor Laboratory Press (1989). The bacterium of the present invention can be obtained by introducing the DNAs mentioned in the above into the bacterium which evidently has the ability to produce L-threonine. Alternatively, the bacterium of the present invention can be obtained by imparting an ability to produce L-threonine to the bacterium that already contains the DNAs. Examples of origin strains included by the present invention include, but are not limited to, the threonine producing bacteria belonging to the genus Escherichia such as the E. coli strain TDH-6 / pVIC40 (VKPM B-3996) (U.S. Patent 5,175,107, U.S. Patent 5,705,371, E strain). Coli NRRL-21593 (U.S. Patent 5,939,307), E. coli strain FERM BP-3756 (U.S. Patent 5,474,918), E. coli strains FERM BP-3519 and FERM BP-3520 (U.S. Patent 5,376,538), E strain. coli MG442 (Gusyatiner et al., Genetics (in Russia), 14, 947-956 (1978)), strains of E. coli VL643 and VL2055 (EP 1149911 A) and the like.The strain TDH-6 is deficient in the gene This strain is also assimilated to sucrose, and the ilvA gene has a poorly isolated mutation.This strain has a mutation in the rhtAr gene that imparts resistance to high concentrations of threonine or homoserin.The strain B-3996 contains the plasmid pVIV40 that has been obtained by inserting the thrA * BC operon including the thrA mut gene ante-encoding aspartokinase homoserin dehydrogenase I that has been substantially desensitized from threonine-fed inhibition in the vector derived from RSF1010. Strain B3996 was deposited on November 19, 1987 at the All-Union Scientific Center of Antibiotics (Nagatinskaya Street 3.A, 113105 Moscow, Russian Federation) under accession number RIA 1867. The strain was also deposited on April 7, 1987 in Russian National Collection of Industrial Microorganisms (VKPM) (Doroshny proezd.I, Moscow 113545, Russian Federation) under accession number B-3996. Preferably, the bacterium of the present invention is further modified to effect the expression of one or more of the following genes as well as the asd gene: the mutant thrA gene encoding aspartokinase homoserin dehydrogenase I resistant to threonine-fed inhibition; the thrB gene that codes for homoserin kinase; the thrC gene encoding threonine synthase; Another preferred embodiment of the present invention is the bacterium modified to augment the rhtA gene encoding a putative transmembrane protein in addition to the increase in the asd gene. The most preferred embodiment of the present invention is a bacterium modified to increase the expression of the asd gene, the mutant thrA gene, the thrB gene, the thrC gene and the rhtA gene. The method for producing L-threonine of the present invention includes the steps of culturing the bacterium of the present invention in a culture medium, which allows the L-Threonine accumulates in the culture medium, and collect the L-threonine from the culture medium. In the present invention, the cultivation, harvesting and Purification of L-threonine from the medium and the like can be performed in a manner similar to conventional fermentation methods wherein L-threonine is produced using a microorganism. A medium used for cultivation can be either a synthetic or natural medium, while the medium includes a heat source and a source of nitrogen and minerals, and if necessary, appropriate amounts of nutrients that the microorganism requires for growth. The carbon source may include various carbohydrates such as glucose and sucrose and various organic acids. Depending on the mode of assimilation of the selected microorganism, alcohol that includes ethanol and glycerol can be used. As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a source of natural nitrogen such as peptone, soybean-hydrolyzate and the digested fermentative microorganism are used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like are used. As vitamins, thiamine, yeast extract and the like are used. Additional nutrients can be added to the medium, if necessary. For example, if the mechanism requires isoleucine for growth (isoleucine auxotrophy), it can be add a sufficient amount of isoleucine to the culture medium. The cultivation is preferably carried out under aerobic conditions such as the agitated culture, and the agitation culture with aeration, at a temperature of 20 a 40 ° C, preferably 30 to 38 ° C. The pH of the culture is usually between 5 and 9, preferably between 6. 5 and 7.2. The pH of the crop can be adjusted as ammonia, calcium carbonate, various acids, various bases and buffer solutions. Usually, a culture of 1 to 5 days leads to the accumulation of L-threonine in the liquid medium. After cultivation, solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration, and then L-threonine can be harvested and purified by ion exchange, concentration and crystallization methods. E ploses The present invention will be more specifically explained below with reference to the following non-limiting examples. Example 1: Cloning of the asd gene of E. coli in the vector pM The asd gene was cloned from the chromosomal DNA of the E strain. coli (K12 Mu cts62 Mud 5005) (VKPM B-6804) obtained from Russian National Collection of Industrial Microorganism (VKPM) (Dorozhny proezd.1, Moscow 113545, Russian Federation).

First, the mini-Mu phage in the E. coli strain (K12 Mu cts62 Mud5005) (VKPM B-6804) was induced. Then, the set of derivatives obtained from the plasmids pMud5005 containing parts of the chromosome was used for the transformation of the strain asd SH 309. The strain SH 309 (VKPM B-3899) obtained from Russian National Collection of Industrial Microorganisms (VKPM) (Dorozhny proezd.1, Moscow 113545, Russian Federation) has the following phenotype: F ~ araD139 rpsL150 deoCl ptsF25 relAl feb5301 rbsR ugpA704:: nlO Del (argF-lac) U169 Del (bad-asd) TetR StrR. The SH309 strain of asd "can not grow in the L medium and requires diaminopimellenic acid (DAPA) for growth." The asd + SH309 clones harboring the pMud5005-asd plasmid were selected in the L medium. Plasmid pMud5005-asd was isolated and the DNA fragment of Bamñl-Pstl (1646 bp) containing the asd gene was recloned in the plasmid pMW119 previously modified to a Piac substitute promoter by the PR promoter. Thus, the pMW-asd plasmid containing the asd gene under the control of the PR promoter was constructed. Plasmid pMW-asd is compatible with plasmid pVIC40 (pRSFlOlO replicon), therefore the two plasmids pVIC40 and pMW-asd could be maintained in the bacteria simultaneously. Plasmid pMW-asd was introduced into strain B-3996 of streptomycin-resistant threonine producing E. coli. Thus, strain B-3996 (pMW-asd) was obtained. Example 2. Example of the amplification of the asd gene in the production of threonine. Both strains B-3996 and B-3996 (pMW-asd) of E. coli were cultured for 18-24 hours at 37 ° C on plates of L agar containing streptomycin (100 μg / ml) and ampicillin (100 μg / ml). ). To obtain the seed culture, the strain was cultivated on a rotary shaker (250 rpm) at 32 ° C for 18 hours in 20x200 mm test tubes containing 2 ml of the L broth with 4% sucrose. Then, the fermentation medium was inoculated with 0.1 ml (5%) of the seed material. Fermentation was carried out in 2 ml of minimum medium for fermentation in 20x200 mm test tubes. The cells were cultured for 24 hours at 32 ° C with shaking at 250 rpm. After cultivation, an accumulated amount of L-threonine in the medium was determined by TLC. Sorbfil plates (Stock Company Sorbopolymer, Kransnodar, Russia) were developed with a mobile phase: propan-2-ol: acetone: water: 25% aqueous ammonia = 25: 25: 7: 6 (v / v). A solution of (2%) ninhydrin in acetone was used as a visualization reagent. The results are presented in Table 1. The composition of the fermentation medium (g / 1) is as follows: Sucrose 40.0 (NH4) 2S04 10.0 KH2P04. 1.0 MgS0 • 7H20 0.4 FeS04-7H20 0.02 MnS04 • 5H20 0.02 Tia ina HC1 0.0002 Yeast Extract 1.0 CaC03 20.0 L-Isoleucine 0.05 Sucrose and magnesium sulfate are sterilized separately. CaCO3 is sterilized with dry heat at 180 ° C for 2 h. The pH is adjusted to 7.0. The antibiotic is introduced into the medium after sterilization. While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. Each of the documents mentioned in the foregoing are incorporated by reference herein in their entirety. Table 1 Field of Industrial Application L-threonine can be produced efficiently.

Claims (11)

1. CLAIMS 1. A bacterium that produces L-threonine belonging to the genus Escherichia, characterized in that the bacterium has been modified to increase an activity of aspartate-β-semialdehyde dehydrogenase.
2. 2. The bacterium according to claim 1, characterized in that the activity of aspartate-β-semialdehyde dehydrogenase is increased by increasing the expression of an aspartate-β-semialdehyde dehydrogenase gene.
3. 3. The bacterium in accordance with the claim 1, characterized in that the activity of aspartate-β-semialdehyde dehydrogenase is increased by increasing the number of copies of the aspartate-β-semi-aldehyde dehydrogenase gene or by modifying a gene expression control sequence so that the expression of the gen.
4. 4. The bacterium according to claim 3, characterized in that the number of copies is increased by transforming the bacterium with a vector containing the gene.
5. 5. The bacterium in accordance with the claim 2, characterized in that the aspartate-β-semialdehyde dehydrogenase gene is derived from a bacterium belonging to the genus Escherichia.
6. 6. The bacterium according to claim 5, characterized in that the aspartate-β-semialdehyde dehydrogenase gene encodes a protein selected from the group consisting of: (A) a protein comprising the amino acid sequence of the SEO ID NO: 2; and (B) a protein, comprising an amino acid sequence that includes the deletion, substitution, insertion or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 2, and which has an aspartate activity. β-semialdehyde dehydrogenase. The bacterium according to claim 5, characterized in that the aspartate-β-semialdehyde dehydrogenase gene comprises a DNA selected from the group consisting of: (a) a DNA comprising a nucleotide sequence of nucleotides 1 to 1196 in SEQ ID NO: l; Y (b) a DNA that is hybridizable with a nucleotide sequence of nucleotides 1-1196 in SEQ ID NO: 1; or a probe that can be prepared from the nucleotide sequence under severe conditions, and encodes a protein having an aspartate-β-semialdehyde dehydrogenase activity. 8. The bacterium in accordance with the claim 7, characterized in that the severe conditions comprise those in which the washing is carried out at 60 ° C at a salt concentration of IxSSC and 0.1% SDS, and for 15 minutes. 9. The bacterium in accordance with the claim 1, characterized in that the bacterium has been further modified to increase the expression of one or more of the genes selected from the group consisting of the mutant thrA gene encoding aspartokinase homoserine dehydrogenase I and is resistant to threonine-fed inhibition; the thrB gene that codes for homoserin kinase. the thrC gene encoding threonine synthase; and - the rhtA gene encoding a putative transmembrane protein. 10. The bacterium according to claim 9, characterized in that the bacterium has been modified to increase the expression of the mutant thrA gene, the thrBr gene, the thrC gene and the rhtA gene. 11. A method for producing L-threonine, characterized in that it comprises culturing the bacterium according to claim 1 in a culture medium to cause accumulation of L-threonine in the culture medium, and collecting the L-threonine from the medium of culture.