KR20090106365A - Microorganism producing L-methionine precursor and the method of producing L-methionine precursor using the microorganism - Google Patents

Abstract

PURPOSE: An L-methionine which is converted from O-succinylhomoserine from L-methionine precursor is provided to use as a animal feed or animal feed additive and food or food additive for human. CONSTITUTION: An Escherichia sp. derived strain which produces O-succinylhomoserine is formed by inducing and enhancing homoserine O-succinyltransferase activity (EC2.3.1.46), having feed back resistance to the methinonine, and enhancing aspatokinase or homoserine dihydrogenase activity (EC2.7.2.4 or 1.1.1.3).

Description

L-methionine precursor producing strain and method for producing L-methionine precursor using the same {Microorganism producing L-methionine precursor and the method of producing L-methionine precursor using the microorganism}

The present invention relates to a strain for producing O-succinylhomoserine, which is a precursor of L-methionine precursor, and a method for producing L-methionine precursor using the strain.

Methionine is one of the essential amino acids for humans and is used as a synthetic raw material for medical and medical supplements as well as feed and food additives. Methionine acts as a precursor of compounds such as choline (lecithin) and creatine, and is also used as a synthetic raw material for cysteine and taurin. Methionine also serves to provide sulfur. S-adenosyl-methionine is derived from L-methionine, serves to provide methyl groups in vivo, and is involved in the synthesis of various neurotransmitters in the brain. Methionine and / or S-adenosyl-methionine (SAM) play a variety of roles in vivo, such as inhibiting fat accumulation in the liver and arteries and alleviating depression, inflammation, liver disease and muscle pain.

The in vivo functions of methionine and / or S-adenosyl-methionine known to date are as follows.

1) It inhibits fat accumulation in the liver and arteries that promote fat metabolism and improves blood circulation in the brain, heart and kidney (J Hepatol. Jeon BR et al., 2001 Mar; 34 (3): 395-401).

2) It promotes digestive action, detoxification or release of toxic substances, and the release of heavy metals such as lead (Pb).

3) may be administered as an antidepressant at a dose of 800-1600 mg per day (Am J Clin Nutr. Mischoulon D. et al., 2002 Nov; 76 (5): 1158S-61S).

4) Enhances liver function (FASEB J. Mato JM., 2002 Jan; 16 (1): 15-26) and is particularly effective for alcohol-induced liver disease (Cochrane Database Syst Rev., Rambal A. , 2001; (4): CD002235).

5) show anti-inflammatory effects on osteoarthritis and promote joint recovery (ACP J Club. Sander O., 2003 Jan-Feb; 138 (1): 21, J Fam Pract., Soeken KL et al., 2002 May ; 51 (5): 425-30)

6) Essential nutrients for hair. Prevent hair loss by nourishing hair (Audiol Neurootol., Lockwood DS et al., 2000 Sep-Oct; 5 (5): 263-266).

Methionine can be synthesized chemically or biologically for application in animal feed, food and pharmaceuticals.

Chemical synthesis produces methionine mainly through the hydrolytic reaction of 5- (β-methylmercaptoethyl) -hydantoin (5- (β-methylmercaptoethyl) -hydantoin). However, the chemically synthesized methionine has the disadvantage that the L- and D-forms are produced in a mixed form.

Biological synthesis is a method of using proteins involved in methionine synthesis. L-methionine is biosynthesized from homoserine by the action of enzymes expressed from genes such as metA, metB, metC, metE and metH in E. coli. In particular, metA is a gene encoding homoserine O-succinyltransferase, the first enzyme of methionine biosynthesis, and homoserine is O-succinyl-L-homoserine. Function to switch to. O-succinylhomoserine lyase or cystathionine gamma synthase encoded by the metB gene converts O-succinyl-L-homoserine to cystathionine. Cystathionine veta lyase, encoded by metC, functions to convert cystathionine to L-homocysteine. Cobalamine-independent methionine synthase encoded by metE and cobalamine-independent methionine synthase encoded by metH convert L-homocysteine to L-methionine. The 5,10-methylenetetrahydrofolate reductase encoded by metF and the serine hydroxymethytransferase encoded by glyA work together to form L-methionine. It functions to synthesize N (5) -methyltetrahydrofolate, which provides the methyl group essential for synthesis.

L-methionine is synthesized by a chain organic reaction by the enzyme, and when genetic manipulation is applied to the protein or a protein affecting the protein, L-methionine synthesis may be increased. For example, Japanese Patent Laid-Open No. 2000/139471 discloses Escherichia. A method for producing L-methionine is described by removing thrBC and metJ on the genome of sp.), overexpressing metBL, and producing metK in Ricky. U.S. Patent Publication No. US2003 / 0092026 also describes a method using Corynerbacterium sp. And strains knocked out of the metD (L-methionine synthesis inhibitor) gene. . US Patent Publication No. 2002/0049305 describes a method of increasing L-methionine production by increasing 5,10-methylenetetrahydrofolate reductase (metF) expression.

In addition, methionine produced by biological methods has the advantage of L-type, but the amount is very small. This is because the methionine biosynthetic pathway has a very well regulated feedback system. When methionine is synthesized to a certain level or more, the final product, methionine, suppresses the metA gene transcription by feedback, which is the initial protein that initiates methionine biosynthesis. The metA gene is inhibited by methionine in the transcriptional stage and is degraded by intracellular proteases in the translational stage to regulate the amount of methionine. Therefore, the metA gene expression alone can increase the amount of methionine above a certain level. None (Dvora Biran, Eyal Gur, Leora Gollan and Eliora Z. Ron: Control of methionine biosynthesis in Escherichia coli by proteolysis: Molecular Microbiology (2000) 37 (6), 1436-1443). Thus, many previous patents have focused on the study of releasing feedback of metA genes from feedback control systems (International Patent Publications 2005/108561 and 1403813).

U.S. Patent Publication No. 2005/0054060 discloses cystathionine neochases (O-succinate) modified to use methylmercaptan (CH ₃ SH) or hydrogen sulfide (H ₂ S) directly without the use of cysteine as a source of sulfur. A method for synthesizing homocysteine or methionine by nilhomoserine lyase) is described. However, cystathionine synthase is known to be able to bind various methionine precursors in cells and thereby produce high levels of byproducts. For example, cystathionine synthase has been reported to accumulate high levels of homolanthionin by side reaction of O-succinyl homoserine and homocysteine (J. Bacteriol (2006) vol 188: p609- 618). Thus, overexpression of cystathionine synthase can reduce the efficiency of intracellular responses due to increased side reactions. In addition, this method uses the methionine metabolism pathway in the cell, which is ineffective because the reaction process of the enzyme is inefficient and uses H ₂ S or CH ₃ SH which is severely toxic to the cell. There is a problem that a sufficient amount of methionine cannot be synthesized.

In order to solve these problems, the present inventors have carried out two steps, (1) producing L-methionine precursor by E. coli fermentation, and (2) converting L-methionine precursor to L-methionine by enzymatic reaction. A process has been developed that consists of conversion steps (PCT / KR2007 / 003650). According to the above two steps, sulfide-specific substrate toxicity problem, feedback control of strains by methionine and SAMe, intracellular enzymes (for example, cystathionine gamma synthase, O-succinyl homoserine sulfhydrila) The problem of degradation activity of intermediates by aze and O-acetylhomoserine sulfhydrylase) can be solved. In addition, the two-step process is very effective for selectively producing only L-methionine, compared to chemical synthesis methods that produce a mixed form of D-methionine and L-methionine.

In the second step, the production of methionine precursor is one of the key factors to increase the production of methionine. In order to increase the amount of synthesis of methionine precursor O-acetyl homoserine, the combination of potent aspartokinase, homoserine transferase and O-acetyl homoserine transferase is really important.

Under this background, the present inventors 1) introduced and enhanced homoserine O-succinyltransferase activity (EC2.3.1.46), and said homoserine O-succinyltransferase has feedback resistance to methionine. ; And 2) O-succinyl homoserine producing strains with enhanced aspartokinase or homoserine dehydrogenase activity (EC2.7.2.4 or 1.1.1.3). This strain has the ability to produce high concentrations of L-methionine precursors regardless of the concentration of methionine in the culture and can significantly increase the production of L-methionine precursors.

It is an object of the present invention to 1) introduce and enhance homoserine O-succinyltransferase activity (EC2.3.1.46), wherein the homoserine O-succinyltransferase has feedback resistance to methionine; And 2) Escherichia sp. Derived strains capable of producing O-succinyl homoserine, with enhanced aspartokinase or homoserine dehydrogenase activity (EC2.7.2.4 or 1.1.1.3). To provide.

Another object of the present invention is to provide a method for producing an L-methionine precursor using the strain.

In one embodiment, the present invention is directed to the introduction and enhancement of homoserine O-succinyltransferase activity (EC2.3.1.46), wherein the homoserine O-succinyltransferase has a resistance to feedback against methionine. Has; And 2) Escherichia sp. Derived strains capable of producing O-succinyl homoserine, with enhanced aspartokinase or homoserine dehydrogenase activity (EC2.7.2.4 or 1.1.1.3). It is about.

As used herein, the term “L-methionine precursor” refers to a metabolite that is part of a methionine-specific metabolic pathway or a substance derived from this metabolite. In particular, the L-methionine precursor herein means O-succinyl homoserine.

As used herein, the term “L-methionine precursor producing strain” refers to a prokaryotic or eukaryotic microorganism strain capable of producing an L-methionine precursor in an organism, and a strain capable of accumulating L-methionine precursor by an operation according to the present invention. Say. For example, the strain Escherichia genus (Escherichia sp.), Air Winiah in (Erwinia sp.), Serratia genus (Serratia sp.), Providencia genus (Providencia sp.), Genus Corynebacterium ( Corynebacteria sp.), Pseudomonas species (Pseudomonas sp.), leptospira in (leptospira), Salmonella genus (Salmonellar sp.), in breather ratio bacteria (Brevibacteria sp.), Scientific Pseudomonas genus (Hypomononas sp.), chromotherapy tumefaciens Microorganism strains belonging to the genus Chromobacterium sp . And the genus Norcardia or fungi or yeast. Preferably, microorganism strains of the genus Escherichia, Corynebacterium, Leptospira and yeast. More preferably, it is a microbial strain of the genus Escherichia, most preferably E. coli ( E. coli ).

In various embodiments, the present invention is directed to the synthesis of intact O-succinyl homoserine or O-succinyl homoserine, which is involved in the degradation of O-succinyl homoserine or O-acetyl homoserine, and is resistant to feedback. The strain into which the gene to be introduced or enhanced is provided as an L-methionine precursor producing strain. The present invention also provides strains that block or attenuate the threonine biosynthetic pathway in order to enhance O-succinylhomoserine production. The present invention also provides a strain overexpressed or enhanced activity of aspartokinase or homoserine dehydrogenase. In addition, the present invention provides a strain in which aspartokinase or homoserine dehydrogenase is overexpressed or enhanced upon the introduction, overexpression, or activation of homoserine O-succinyltransferase free from a feedback control system. do.

More specifically, the present invention provides an L-methionine precursor-producing strain that lacks the metB gene involved in L-methionine precursor degradation, the thrB gene involved in threonine biosynthetic pathways, and the metJ gene that regulates transcription of the L-methionine precursor producing gene. To provide. The present invention also provides an L-methionine precursor producing strain which knocks out the original metA or metX gene involved in the synthesis of methionine precursor and introduces an external metA gene whose feedback is released. The present invention also provides an L-methionine precursor producing strain that enhances the activity encoded by the thrA gene.

More preferably, the present invention provides L-methionine precursor producing strains that knock out native metA or metX genes, introduce feedback resistant metA genes, and enhance thrA genes.

As used herein, the term “inactive” refers to attenuation or deletion of a gene. Deletion of a gene is performed by modifying the protein's sequence by cleaving the gene or inserting a particular gene sequence on a chromosome. As used herein, the term "attenuation" or "weakening" refers to the removal or reduction of intracellular activity of one or more enzymes encoded by corresponding DNA in a microbial strain, e.g., a promoter of a gene. The activity of the protein can be attenuated by altering the nucleotide sequence of the site) and the 5′-UTR site, or by attenuating the expression of the protein or by introducing a mutation into the ORF site of the gene. According to the attenuation measurement, the concentration or activity of the corresponding protein is generally 0-75%, 0-50%, 0-25%, 0-10% relative to the concentration or activity of the wild-type or early microbial strain. Or 0 to 5%.

To achieve attenuation, for example, the expression of a gene or the catalytic activity of an enzyme protein can be reduced or eliminated, and the two methods can be optionally combined.

Gene expression can be reduced by appropriate culture, by altering the signal structure of gene expression, or by antisense-RNA techniques. For example, the signal structure of gene expression can be, for example, a suppressor gene, an activator gene, an operator, a promoter, an attenuator, a ribosomal binding site, a start codon and Terminators. In this regard, Jensen and Hammer (Biotechnology and Bioengineering 58: 191 195 (1998)), Carrier and Keasling (Biotechnology Progress 15: 58 64 (1999)), Franch and Gerdes (Current Opinion in Microbiology 3: 159 164 (2000) And Knippers ("Molecular Genetics", 6th edition, 1995) or Winnacker ("Genes and Clones", 1990), which are well-known textbooks of genetics and molecular biology.

Mutations that lead to changes or reductions in catalytic activity of enzyme proteins are well known in the art. For example, Qiu and Goodman (Journal of Biological Chemistry 272: 8611 8617 (1997)), Yano et al. (Proceedings of the National Academy of Sciences, USA 95: 5511 5515 (1998)), and Wente and Schachmann (Journal of Biological Chemistry 266: 20833 20839 (1991), or Hagemann ("General Genetics", 1986), etc. For reference.

Possible mutations are mutations due to transitions, transitions, insertions and deletions. Depending on whether the amino acid sequence changes in relation to enzyme activity can be divided into missense (nonsense) mutation or nonsense (nonsense) mutation. At least one base pair in the gene, the mutation introduces the wrong amino acid and leads to premature translational disruption. If stop codons form in the coding region as a result of the mutation, this also leads to premature termination of the translation. Typically, the deletion of several codons results in complete loss of enzymatic activity. Specific details on the occurrence of these mutations can be found in prominent genetic and molecular biology such as Knippers ("Molecular Genetics", 6th edition, 1995), Winnacker ("Genes and Clones",, 1990) or Hagemann ("General Genetics", 1986). You can refer to the textbook.

As used herein, the term "enhancement" refers to an increase in the intracellular activity of an enzyme encoded by the corresponding DNA. Enhancement of the enzyme's intracellular activity can be achieved by overexpression of the gene. Overexpression of the gene of interest can enhance protein expression by modifying the nucleotide sequence of the promoter region or 5'-UTR region of the gene, can enhance the expression by additional introduction of the gene on the chromosome, vector The expression level of the protein can be enhanced by introducing into a phase with a self promoter or enhanced separate promoter and transforming the strain. In addition, enhancement of the enzyme's intracellular activity can also be achieved by introducing mutations into the open reading frame (ORF) region of the gene of interest. Enhancement measures indicate that the activity or concentration of the corresponding protein is generally at least 10%, 25%, 50%, 75%, 100%, 150%, based on the activity or concentration in the wild type protein or the initial microbial strain. It can be seen that 200%, 300%, 400% or 500%, up to 1000% or 2000% increase.

As a specific embodiment of the present invention, a method for preparing a L-methionine precursor producing strain is as follows;

In step 1, to accumulate L-methionine precursors such as O-succinyl homoserine or O-acetylhomoserine, cystathionine gamma synthase, O-succinyl homoserine sulfhydrylase or O-acetylhomoserine Genes encoding proteins such as sulfhydrylases are deleted or attenuated in strains.

The gene encoding cystathionine gamma synthase is collectively referred to as metB, the gene encoding O-succinyl homoserine sulfhydrylase is collectively referred to as metZ, and the gene encoding O-acetylhomoserine sulfhydrylase The gene is collectively referred to as metY. Genes encoding proteins having the above-mentioned activity are, for example, Escherichia coli .), metB. The genomic base sequence of this gene can be obtained from E coli's genome base (Accession no. AAC75876) published by Blattner et. Al., Science 277: 1453-1462 (1997). The gene sequence can also be obtained from the National Center for Biotechnology Information (NCBI) and the DNA Data Bank Japan (DDBJ). In addition, as genes having the same activity, metB and metY of Corynebacterium (Coynebacterium.), MetZ derived from Pseudomonas ( Pseudomonas .), And the like can be exemplified.

Cystathionine gamma synthase or O-succinylhomoserine sulfhydrylase or O-acetylhomoserine sulfhydrylase can be used to cystathionine O-succinylhomoserine or O-acetylhomoserine as shown in the following scheme. Or has the ability to convert to homocysteine. Therefore, when the gene with this activity is attenuated or attenuated, O-succinyl homoserine or O-acetyl homoserine is accumulated in the culture medium.

L-cysteine + O-succinyl-L-homoserine <=> succinate + cystathionine

L-cysteine + O-acetyl-L-homoserine <=> Acetate + cystathionine

HS ^- + O- succinyl homoserine -L- <=> succinate + homocysteine

HS ^- + O- acetyl -L- homoserine <=> acetate + homocysteine

As a second step, the strain prepared in step 1 is removed or attenuated with the thrB gene encoding homoserine kinase. The ThrB gene involves the synthesis of O-phosphohomoserine from homoserine and is subsequently converted to threonine by the thrC gene. The thrB gene is deleted or attenuated so that the synthesized homoserine can be used for precursor synthesis of the whole methionine.

As a third step, the transcriptional regulator of methionine synthesis pathway is deleted or attenuated. The metA, metB, metC, metE and metF genes involved in methionine synthesis are inhibited by a feedback regulatory system. The metJ gene is a typical transcriptional regulator gene in E. coli . MetA or metX genes must be overexpressed to synthesize methionine precursors, which requires the removal of metJ. Therefore, the removal of the metJ gene from E. coli always promotes the expression of metA or metX genes, enabling the mass production of L-methionine precursors.

Steps 2 and 3 may vary depending on the precursor producing strain and are not essential for producing the precursor producing strain. More preferably it can be carried out to enhance the precursor production route in the microorganism of the genus Escherichia sp.

As a fourth step, feedback resistant homoserine O-succinyltransferase is introduced that mediates the first course of the methionine biosynthetic pathway. metA is a generic name for genes encoding proteins with the activity of homoserine O-succinyltransferase. Feedback resistant metA gene mutations result in feedback resistant homosine O-succinyl activity. PCT / US2007 / 009146 describes a method for producing a feedback resistant metA gene. General information contained in the above international patent is included in the scope of the invention in the claims. In the present invention, the amino acid sequence encoded by the feedback resistant metA 10 gene is shown in SEQ ID NO: 14, metA 11 gene in SEQ ID NO: 16, metA 32 gene in SEQ ID NO: 18, metA 37 gene in SEQ ID NO: 20, metA 41 The gene is shown in SEQ ID NO: 22 (PCT / US2007 / 009146). In addition, the present invention, the homoserine O-succinyltransferase is 24, 29, 79, 114, 140, 163, 222, 275 in amino acid sequence of SEQ ID NO: 30 (amino acid sequence according to GenBank Accession No: AP 004514) And have mutations in one or more of positions 290, 291, 295, 297, 304, or 305.

Introduction and enhancement of the metA gene can be accomplished by introduction of the gene, by modification of the nucleotide sequence of the 5'-UTR and the promoter region of the gene, or by introducing a mutation into the ORF region of the gene of interest. The introduction of a mutant metA gene released in a feedback control system results in increased production of L-methionine precursors regardless of the concentration of methionine in the medium.

In addition, O-succinylhomoserine production can be further increased by removing the original metA gene from the chromosome and introducing the mutant metA gene therein. Under the metA promoter where promoter activity is enhanced by a deficiency of the metJ gene, the feedback resistant metA gene can produce more O-succinylhomoserine.

As a five step, it enhances the activity of aspartokinase or homoserine dehydrogenase to increase the synthesis of homoserine, a precursor of O-succinyl homoserine. thrA is a collective name for genes encoding peptides having the activity of aspartokinase and homoserine dehydrogenase. Enhancement of thrA gene activity is performed by introducing mutations into the thrA gene, by introducing a large number of thrA genes on the chromosome, or by introducing plasmids containing thrA.

Preferably, aspartokinase and homoserine dehydrogenase are encoded by the gene of Unipro database No. AP_000666 (Unipro database No: AP_000666). More preferably, the amino acid sequence encoded by the thrA gene is shown in SEQ ID NO: 29 with a mutation at amino acid position 345. Enhancement of ThrA activity can be achieved by additionally introducing a processed plasmid by additionally introducing a target gene onto the chromosome by introducing a mutation into the thrA gene.

O-succinylhomoserine, an L-methionine precursor, can be accumulated in the strain by using the entire process or part of steps 1 to 5 above.

L-methionine precursor producing strains can be prepared using strains that produce L-lysine (Lysine), L-threonine, L-isoleucine (isoleusine). It is preferably prepared by using a strain that produces L-threonine. In this case, as a strain that has been successfully synthesized up to homoserine, it is possible to synthesize a higher amount of methionine precursor. Thus, methionine precursors can be accumulated by using a threonine producing strain to delete or attenuate genes involved in threonine biosynthetic pathways and metA, metY, metZ.

As used herein, the term “L-threonine-producing strain” refers to a prokaryotic or eukaryotic microbial strain capable of producing L-threonine in vivo. For example, the genus Escherichia (Escherichia sp.), Air Winiah in (Erwinia sp.), Serratia genus (Serratia sp.), Providencia genus (Providencia sp.), The genus Corynebacterium (Corynebacteria sp .), Or L-threonine producing microorganism strains belonging to the Brevibacteria sp . Among them, Escherichia sp .) is preferred, Escherichia coli ) is more preferred.

As a preferred embodiment of the invention, L-threonine production and independent strain CJM002 transformed from E. coli mutant strain TF4076 (KFCC 10718, Korean Patent Publication No. 92-8365) producing L-threonine was used. . TF4076 is required for methionine requirement, methionine analogs (eg, α-amino-β-hydroxy valeric acid, AHV), lysine analogs (eg, S- (2-aminoethyl) -L-cysteine, AEC) Resistance to isoleucine analogs (eg, α-aminobutyric acid) and the like. TF4076 is a methionine-required strain that does not synthesize methionine in cells. In order to release the methionine requirement and use it as a methionine producing strain, the present inventors prepared a threonine producing strain E. coli CJM002 whose methionine requirement was released using artificial mutagenesis using NTG. E. coli CJM002 is Escherichia It was named coli MF001 and deposited on KCCM (Korean Culture Center of Microorganism) on April 9, 2004 as KCCM-10568 in Yurim Building, 361-221 Hongje 1-dong, Seodaemun-gu, Seoul, Korea.

In the present invention, the metB, thrB, metJ and metA genes of the E. coli CJM002 strain were deleted followed by the introduction of feedback resistant metA, and the final L-methionine precursor producing strain thus prepared was named CJM-A11. The CJM-X strain was transformed with a thrA expression vector and named CJM-A11 (pthrA (M) -CL). O-succinyl homoserine producing strain Escherichia thus prepared coli CJM-A11 was deposited on Jan. 23, 2008 with accession number KCCM 10922P.

As a preferred embodiment of the present invention, FTR2533, an L-threonine producing strain described in PCT / KR2005 / 00344, can be used. FTR2533 is Escherichia derived from the Escherichia coli Accession No. KCCM10236 derived from coli TFR7624. Escherichia coli accession number KCCM10236 is Escherichia from coli TF4076. Esherichia coli Accession No. KCCM10236 is a ppc gene that catalyzes the formation of oxaloacetate from PEP and enzymes required for biosynthesis of threonine from aspartate, for example thrA: aspartokinaze 1-homoserine dehydro Genome, thrB: homoserine kinase, thrC: high expression of threonine neochaase may increase L-threonine production. In addition, Escherichia coli FTR7624 (KCCM10538) has an inactivated tyrR gene that inhibits the expression of the tyrB gene required for L-threonine biosynthesis. In addition, Escherichia coli FTR2533 (KCCM10541) is an L-threonine producing strain with an inactivated galB gene and an L-threonine producing E. coli mutant strain.

In a specific embodiment of the present invention, after the metB, thrB, metJ and metA genes of the E. coli FTR2533 strain were deleted, a feedback resistant metA gene was introduced, and the final L-methionine precursor thus prepared was named CJM2-A11. It was. The CJM2-A11 strain was transformed with thrA expression vector and named CJM2-A11 (pthrA (M) -CL). Escherichia prepared by the above method coli CJM2-A11 (pthrA (M) -CL) was deposited on February 12, 2008 under accession number KCCM 10924P.

In another aspect, the present invention relates to a method for producing an L-methionine precursor, specifically: a) fermenting the microbial strains; b) culturing O-succinylhomoserine in medium or strains; And c) isolating O-succinyl homoserine.

Cultivation of the L-methionine precursor producing strain prepared above may be made according to suitable media and culture conditions known in the art. This culture process can be easily adjusted and used by those skilled in the art according to the strain selected. Examples of the culture method include, but are not limited to, batch, continuous and fed-batch cultures. These various culture methods are described, for example, in "Biochemical Engineering" by James M. Lee, Prentice-Hall International Editions, pp 138-176.

The medium used for culturing must adequately meet the requirements of the particular strain. Various microbial culture media are described, for example, in "Manual of Methods for General Bacteriology" by the American Society for Bacteriology, Washington D.C., USA, 1981. These media include various carbon sources, nitrogen sources and trace element components. Carbon sources include carbohydrates such as glucose, lactose, sucrose, fructose, maltose, starch and cellulose; Fats such as soybean oil, sunflower oil, castor oil and coconut oil; Fatty acids such as palmitic acid, stearic acid and linoleic acid; Alcohols such as glycerol and ethanol and organic acids such as acetic acid. These carbon sources may be used alone or in combination. Nitrogen sources include organic nitrogen sources and urea, such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and soy flour (bean flour). inorganic nitrogen sources such as urea), ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. These nitrogen sources may be used alone or in combination. The medium may further comprise potassium dihydrogen phosphate, potassium dihydrogen phosphate and corresponding sodium-containing salts as phosphoric acid sources. The medium may also include metals such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins and suitable precursors may be added. These media or precursors may be added batchwise or continuously to the culture.

In addition, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid in an appropriate manner during the culture. In addition, by using an antifoaming agent such as a fatty acid polyglycol ester during the culture, it is possible to suppress the generation of bubbles during the culture. In addition, in order to maintain aerobic conditions of the culture solution, oxygen or a gas containing oxygen (eg, air) may be injected into the culture solution. The temperature of the culture is generally 20-45 ° C, preferably 25-40 ° C. The duration of the incubation can continue until the production of L-methionine precursor reaches the desired level and the preferred incubation time is 10-160 hours.

When using the methionine precursor producing strain of the present invention, methionine can be produced more environmentally friendly than typical chemical methionine synthesis methods. In addition, L-methionine converted from O-succinyl homoserine produced from the strain according to the present invention can be widely used in the production of food or food additives in humans as well as animal feed or animal feed additives.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are merely to illustrate the present invention is not limited to the contents of the present invention.

Example  1: Preparation of Methionine Precursor Strains

<1-1> Deletion of the metB Gene

In order to delete the metB gene encoding cystathionine synthase in E. coli strain, FRT-one-step PCR deletion method was performed (PNAS (2000) vol97: P6640-6645). Using the primers of SEQ ID NO: 1 and SEQ ID NO: 2, the pKD3 vector (PNAS (2000) vol97: P6640-6645) was used as a template to form a deletion cassette by PCR reaction. PCR was repeated 30 times of denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and extension for 1 minute at 72 ° C.

The resulting PCR product was electrophoresed on a 1.0% agarose gel, followed by purification of DNA from a 1.2 kbp sized band. The recovered DNA fragments were electroporated to the E. coli (K12) W3110 strain previously transformed with pKD46 vector (PNAS (2000) vol97: P6640-6645). W3110 strain transformed with pKD46 for electroporation was incubated at 30 ° C. until OD ₆₀₀ reached 0.6 using LB medium containing 100 ug / L ampicillin and 5 mM arabinose. It was. Sterile distilled water was used twice, and washed once with 10% glycerol (glycerol). Electroporation was applied at 2500V. The recovered strains were plated in LB plate medium containing 25 μg / L chloramphenichol and incubated overnight at 37 ° C., and strains showing resistance were selected.

Selected strains were PCR strains under the same conditions using the same primers as the strain as a template, and confirmed the deficiency of the metB gene by confirming that the gene size was observed to 1.2Kb on 1.0% agarose gel. The identified strain was transformed with pCP20 vector (PNAS (2000) vol97: P6640-6645) and cultured in LB medium, and the gene size was reduced to 150 bp on 1.0% agarose gel through PCR under the same conditions. Gene final metB gene deletion strains were constructed and confirmed that chloramphenicol markers were removed. The produced strain was named W3-B.

<1-2> Deletion of the thrB Gene

An attempt was made to increase the amount of O-succinyl homoserine synthesized from homoserine by deleting the thrB gene, a gene encoding homoserine kinase. In particular, the use of threonine-producing strains requires a deficiency of this gene because of the high activity of homoserine. In order to delete the thrB gene in the W3-B strain, the same FRT one step PCR deletion method was used as in the metB gene deletion.

Using the pKD4 vector (PNAS (2000) vol97: P6640-6645) as a template to prepare a thrB deletion cassette, the denaturation step using the primers of SEQ ID NOs: 3 and 4 was performed at 94 ° C. for 30 seconds, annealing. The step was carried out for 30 seconds at 55 ℃, the extension (extension) step for 1 minute at 72 ℃, and proceeded with a PCR reaction to perform this 30 times.

The resulting PCR product was electrophoresed on a 1.0% agarose gel, followed by purification of DNA from a 1.6 kbp band. The recovered DNA fragments were electroporated to a W3-B strain previously transformed with pKD46 vector. The recovered strain was plated in LB plate medium containing 50 μg / L kanamycin and cultured overnight at 37 ° C., and then strains showing resistance were selected.

The selected strains were PCR-treated under the same conditions using the primers of SEQ ID NOs: 3 and 4, using the strains as templates, and then selected for strains having a size of 1.6 Kb on a 1.0% agarose gel. Confirmed. The identified strains were transformed with pCP20 vector and cultured in LB medium, and again, the final thrB gene deletion strain was reduced to 150 bp on 1.0% agarose gel by PCR under the same conditions, and kanamycin ( kanamycin) marker was removed. The produced strain was named W3-BT strain.

<1-3> deletion of the metJ gene

In order to delete the metJ gene, which is a regulatory gene of the metA gene involved in the synthesis of methionine precursor, the same FRT one step PCR deletion method as in the metB gene deletion was used. To prepare the metJ gene deletion cassette, the denaturation step was 30 seconds at 94 ° C, the annealing step was 30 seconds at 55 ° C, and the extension step was 72 using primers of SEQ ID NOs: 5 and 6. It was carried out at 1 ° C. for 1 minute, which was carried out 30 times.

The resulting PCR product was electrophoresed on a 1.0% agarose gel, followed by purification of DNA from a 1.2 kbp sized band. The recovered DNA fragments were electroporated to a W3-BT strain previously transformed with the pKD46 vector. The recovered strains were plated in LB plate medium containing chloramphenicol and cultured overnight at 37 ° C, and strains showing resistance were selected.

Selected strains were PCR the same conditions using the primers of SEQ ID NO: 7, 8 using the strain as a direct template, and confirmed the deficiency of the metJ gene by confirming that the gene size was changed to 1.6 Kb on a 1.0% agarose gel. It was. The identified strain was transformed into pCP20 vector and cultured in LB medium, and again, the final metJ gene-deficient strain was reduced to 600 bp on 1.0% agarose gel by PCR under the same conditions, and the chloramphenicol marker was added. It was confirmed that it was removed. The produced strain was named W3-BTJ.

<1-4> Deletion of the metA Gene

To introduce the feedback resistant metA gene into the chromosome, the original metA gene was deleted on the chromosome of the W3-BTJ strain. In order to remove the metA gene, FRT one step PCR deletion method was performed.

To prepare the metA gene deletion cassette, the denaturation step was 30 seconds at 94 ° C, the annealing step was 30 seconds at 55 ° C, and the extension step was 72 using primers of SEQ ID NOs: 9 and 10. It was carried out at 1 ° C. for 1 minute, which was carried out 30 times.

The resulting PCR product was electrophoresed on a 1.0% agarose gel, followed by purification of DNA from a 1.2 kbp sized band. The recovered DNA fragments were electroporated to a W3-BT strain previously transformed with the pKD46 vector. The recovered strains were plated in LB plate medium containing chloramphenicol and cultured overnight at 37 ° C, and strains showing resistance were selected.

Selected strains were PCR as a template by using the primers of SEQ ID NOs: 9 and 10 under the same conditions, and then confirmed the deficiency of the metA gene by confirming that the gene size was changed to 1.1 Kbp on a 1.0% agarose gel. It was. The identified strains were transformed into pCP20 vector and cultured in LB medium, and again, the final metA gene-deficient strains were reduced to 100 bp on 1.0% agarose gel by PCR under the same conditions, and the chloramphenicol marker was added. It was confirmed that it was removed. The produced strain was named W3-BTJA.

<1-5> Introduction of feedback resistant metA gene

In order to increase the production of the L-methionine precursor O-succinyl homoserine, the feedback resistant metA gene encoding homoserine O-succinyltransferase was overexpressed.

The feedback resistant metA 10 gene is shown in SEQ ID NO: 13 and the amino acid sequence encoding it is shown in SEQ ID NO: 14. The metA 11 gene is shown in SEQ ID NO: 15 and the amino acid sequence encoding it is shown in SEQ ID NO: 16. The metA 32 gene is shown in SEQ ID NO: 17 and the amino acid sequence encoding it is shown in SEQ ID NO: 18. The metA 37 gene is shown in SEQ ID NO: 19 and the amino acid sequence encoding it is shown in SEQ ID NO: 20. The metA 41 gene is shown in SEQ ID NO: 21 and the amino acid sequence encoding it is shown in SEQ ID NO: 22 (PCT / US2007 / 009146).

PCR using the genes SEQ ID NO: 11, 12 as a template, the denaturation step is 30 seconds at 94 ℃, the annealing step is 55 seconds at 30 ℃, the extension step is 72 ℃ 2 minutes at 25 cycles.

The resulting PCR product was electrophoresed on 1.0% agarose gel, and then DNA was purified from the 1.2 kbp band. The recovered DNA fragment was linked to pCL1920 vector digested with restriction enzyme SmaIdm. Linked vectors were transformed into E. coli and cultured in LB medium containing 50 μg / L spectinomycin and selected. The configuration of the prepared vector is shown in FIG. 3.

The vectors prepared above were electroporated to the W3-BTJA strain, respectively, and the production capacity of the strain was assayed using flask culture as described in Example 2-1. O-succinyl homoserine production of the modified strain was measured based on the O-succinyl homoserine production capacity of the wild-type strain as 100%, respectively. As a result, all of the modified strains were markedly increased compared to wild type production. In particular, the most effective production at metA 11, metA 10 and metA 32.

Feedback resistance of mutant metA in the presence of 100 mM methionine % of specific activity retention Wild type # 10 # 11 # 32 # 37 # 41 Control 100 100 100 100 100 100 w / 100mM Met 7.6 97 94 79 83 74

O-succinylhomoserine Production of Feedback Resistant metA in W3-BTJA Strains metA O-succinyl homoserine production (%) Wild type (WT) 100   # 10 392   # 11 425   # 32 396   # 37 227   # 41 389

For the stable expression of pMetA10-CL, pMetA11-CL and pMetA32-CL showing the most effective production, these genes were introduced into E. coli chromosomes.

First, the pKD3 vector was used, and chloramphenicol marker gene was used as a template, and SEQ ID NOs: 23 and 24 were PCR amplified using primers. PCR fragments were purified by gel-elution. The metA gene was PCR amplified using primers SEQ ID NOs: 9 and 10. The 3'-UTR region of the metA gene was PCR amplified using primers SEQ ID NOs: 25 and 26 using a W3110 wt chromosome as a template. The resulting PCR products were separated by gel-extraction and the mixture of fragments was PCR amplified SEQ ID NOs: 9, 26 with primers to prepare metA gene segments containing chloramphenicol labels. The recovered DNA fragments were electroporated to the W3BTJA strain transformed with the pKD46 vector, chloramphenicol resistant strains were selected, and it was confirmed that metA was successfully cloned using the primers of SEQ ID NOs: 9 and 10. Strains found to have introduced the modified metA genes 10, 11, 32 were named W3BTJ-A10, 11 and 32, respectively. O-succinyl homoserine production of each strain was similar to that of W3-BTJA containing each metA plasmid.

<1-6> Overexpression of the thrA Gene

To more effectively increase the production of O-succinylhomoserine, the thrA gene was overexpressed.

To this end, PCR amplification was carried out using primers of SEQ ID NOs: 27 and 28 using the chromosome of L. threonine producing strain E. coli CJM002 (KCCM10568) as a template. The amplified DNA fragments were isolated via gel-extraction and linked to the CJ1 promoter of the pCL1920 vector using restriction enzyme EcoRV and selected in LB medium containing 50 μg / L spectinomycin. The selected vector was named pCJ-thrA (M) -CL. The amino acid sequence encoded by the thrA gene is shown in SEQ ID NO: 29 and has a mutation at amino acid position 345. In addition, wild-type thrA gene of E. coli W3110 strain was PCR amplified in the same manner as described above and cloned into a vector named pCJ-thrA-CL. These vectors were each electroporated with W3BTJ-A11 and the O-succinylhomoserine production of each strain was assayed by the flask culture described in Example 2-1. As a result, the strain transformed with the vector with the thrA gene in the mutant form accumulated O-succinyl homoserine at very high levels.

O-succinyl homoserine production in the presence of thrA expression vector Plasmid OSH Production (%) W3BTJA11 pCL1920 100 pCJ-thrA-CL 119 pCJ-thrA (M) -CL 153

<1-7> Conversion of L-threonine Producing Strains

O-succinyl homoserine producing strains were prepared in the same manner as in 1-1 to 1-5 using E. coli CJM002 (KCCM-10568), which is an L-threonine producing strain whose methionine requirement was released. Strains containing the mutant metA gene on the chromosome were named CJM-A10, CJM-A11, and CJM-A32. In addition, using the L-threonine production strain E. coli FTR2533 (KCCM10541) described in PCT / KR2005 / 00344 to produce a methionine precursor production strain in the same manner as 1-1 to 1-5 and named it CJM-A11. In addition, each strain was transformed with a thrA expression vector as described in Examples 1-6 and the methionine precursor production of these strains was measured by flask culture described in Example 2-1. As a result, CJM-A11 containing pCJ-thrA (m) -CL plasmid showed very high O-succinylhomoserine production.

Methionine precursor (O-succinyl homoserine) production by flask culture O-succinyl homoserine (%) CJM-A10 100 CJM-A11 113 CJM-A32 80 CJM2-A11 94 CJM-A11 (pCJ-thrA (m) -CL) 127

Example  2: Fermentation for the Production of L-Methionine Precursor

<2-1> Flask culture experiment

Erlenmeyer flask culture was performed to test the methionine precursor production of the strain prepared in Example 1. Each strain was inoculated into plate LB medium and incubated overnight at 31 ° C. Single colonies were inoculated in 3 ml LB medium and incubated at 31 ° C. for 5 hours, then diluted 200-fold in 250 ml Erlenmeyer flasks containing 25 ml methionine precursor production medium and incubated at 31 ° C. at 200 rpm for 64 hours to produce methionine precursor through HPLC analysis. Was compared. As a result, the methionine production capacity showed a significant increase in methionine precursor producing strains prepared from L-methionine producing strains free from methionine requirement.

Flask medium composition for methionine precursor production Furtherance Concentration (/ L) Glucose 40 g Ammonium sulfate 17 g KH ₂ PO ₄ 1.0 g MgSO ₄ ㆍ 7H ₂ O 0.5 g FeSO _{4 7} H ₂ O 5 mg MnSO ₄ 8H ₂ O 5 mg ZnSO ₄ 5 mg Calcium carbonate 30 g Yeast extract 2 g Methionine 0.15 g Threonine 0.15 g

<2-2> large capacity fermentation

For mass production of O-succinyl homoserine, several strains showing O-succinyl homoserine production capacity were selected and cultured in 5 L fermenters. Each strain was inoculated in plate LB medium containing antibiotic spectinomycin and incubated overnight at 31 ° C. Single colonies were then inoculated in 10 ml of LB medium containing spectinomycin and incubated at 31 ° C. for 5 hours. The culture solution was diluted 100-fold in a 1000 ml Erlenmeyer flask containing 200 ml of methionine precursor seed medium, incubated for 3-10 hours at 31 ° C and 200 rpm, and then inoculated into a 5L fermenter and fed by a fed batch fermentation method. Incubated for ~ 100 hours. The results of analyzing the methionine precursor concentration in HPLC in the cultured fermentation broth were as shown in Table 7.

Fermentor medium composition for methionine precursor production Furtherance Seed media Main media Feed media Glucose (g / L) 10.1 40 600 MgSO ₄ 7H ₂ 0 (g / L) 0.5 4.2 Yeast extract (g / L) 10 3.2 KH ₂ PO ₄ 3 3 8 Ammonium sulfate (g / L) 6.3 NH ₄ Cl (g / L) One NaCl (g / L) 0.5 Na ₂ HPO ₄ 12H ₂ O (g / L) 5.07 DL-Methionine (g / L) 0.5 0.5 L-Isoleucine (g / L) 0.05 0.5 0.5 L-Threonine (g / L) 0.5 0.5

Methionine Precursor Production in Fermenter O-succinylhomoserine (g / L) CJM-BTJ / pCJ-MetA-CL > 80 CJM-A11 / pCJ-thrA (M) -CL > 100 CJM2-A11 / pCJ-thrA (M) -CL > 90

1 is a diagram illustrating the genetic manipulation of methionine precursor producing strains.

Figure 2 is a diagram illustrating the chemical structure of the two-step process for the production of methionine.

3 shows a cleavage map of pMetA-CL for expression of feedback resistant metA gene.

<110> CJ Jeiljedang Corporation <120> MICROORGANISM PRODUCING L-METHIONINE PRECURSOR AND THE METHOD OF          PRODUCING L-METHIONINE PRECURSOR USING THE MICROORGANISM <130> PA090118 / KR <150> US12 / 062,927 <151> 2008-04-04 <160> 30 <170> KopatentIn 1.71 <210> 1 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of Chloramphenichol <400> 1 ttactctggt gcctgacatt tcaccgacaa agcccaggga acttcatcac gtgtaggctg 60 gagctgcttc 70 <210> 2 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of Chloramphenichol <400> 2 ttaccccttg tttgcagccc ggaagccatt ttccaggtcg gcaattaaat catatgaata 60 tcctccttag 70 <210> 3 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of kanamycin <400> 3 aaagaatatg ccgatcggtt cgggcttagg ctccagtgcc tgttcggtgg gtgtaggctg 60 gagctgcttc 70 <210> 4 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of kanamycin <400> 4 agacaaccga catcgctttc aacattggcg accggagccg ggaaggcaaa catatgaata 60 tcctccttag 70 <210> 5 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of Chloramphenichol <400> 5 atggctgaat ggagcggcga atatatcagc ccatacgctg agcacggcaa ggtgtaggct 60 ggagctgctt c 71 <210> 6 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of Chloramphenichol <400> 6 gtattcccac gtctccgggt taatccccat ctcacgcatg atctccatat gaatatcctc 60 cttag 65 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of metJ <400> 7 gggctttgtc ggtgaaatg 19 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of metJ <400> 8 actttgcgat gagcgagag 19 <210> 9 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> primer for deletion of metA <400> 9 caatttcttg cgtgaagaaa acgtctttgt gatgacaact tctcgtgcgt gtgtaggctg 60 gagctgcttc 70 <210> 10 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> primer for deletion of metA <400> 10 aatccagcgt tggattcatg tgccgtagat cgtatggcgt gatctggtag catatgaata 60 tcctccttag 70 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of metA <400> 11 aatggatcct gccgtgagcg gcgaatac 28 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of metA <400> 12 agctctagac tgctgaggta cgtttcgg 28 <210> 13 <211> 930 <212> DNA <213> Escherichia coli <400> 13 atgccgattc gtgtgccgga cgagctaccc gccgtcaatt tcttgcgtga agaaaacgtc 60 tttgtgatga caacttctcg tgcgtctggt caggaaattc gtccgcttaa ggttctgatc 120 cttaacctga tgccgaagaa gattgaaact gaaaatcagt ttctgcgcct gctttcaaac 180 tcacctttgc aggtcgatat tcagctgttg cgcatcgatt cccgtgaatc gcgcaacacg 240 cccgcagagc atctgaacaa cttctactgt aactttgaag atattcagga tcagaacttt 300 gacggtttga ttgtaactgg tgcgccgctg ggcctggtgg agtttaatga tgtcgcttac 360 tggccgcaga tcaaacaggt gctggagtgg tcgaaagatc acgtcacctc gacgctgttt 420 gtctgctggg cggtacaggc cgcgctcaat atcctctacg gcattcctaa gcaaactcgc 480 accgaacaac tctctggcgt ttacgagcat catattctcc atcctcatgc gcttctgacg 540 cgtggctttg atgattcatt cctggcaccg cattcgcgct atgctgactt tccggctgcg 600 ttgattcgtg attacaccga tctggaaatt ctggcagaga cggaagaagg ggatgcatat 660 ctgttagcca gtaaagataa gcgcattgcc tttgtgacgg gccatcccga atatgatgcg 720 caaacgctgg cgcaggaatt tttccgcgat gtggaagccg gactagaccc ggatgtaccg 780 tataactatt tcccgcacaa tgatccgcaa aatacaccgc gagcgagctg gcgtagtcac 840 ggtaatttac tgtttaccaa ctggctcaac tattacgtct accggatcac gccatacgat 900 ctacggcaca tgaatccaac gctggattaa 930 <210> 14 <211> 309 <212> PRT <213> Escherichia coli <400> 14 Met Pro Ile Arg Val Pro Asp Glu Leu Pro Ala Val Asn Phe Leu Arg   1 5 10 15 Glu Glu Asn Val Phe Val Met Thr Thr Ser Arg Ala Ser Gly Gln Glu              20 25 30 Ile Arg Pro Leu Lys Val Leu Ile Leu Asn Leu Met Pro Lys Lys Ile          35 40 45 Glu Thr Glu Asn Gln Phe Leu Arg Leu Leu Ser Asn Ser Pro Leu Gln      50 55 60 Val Asp Ile Gln Leu Leu Arg Ile Asp Ser Arg Glu Ser Arg Asn Thr  65 70 75 80 Pro Ala Glu His Leu Asn Asn Phe Tyr Cys Asn Phe Glu Asp Ile Gln                  85 90 95 Asp Gln Asn Phe Asp Gly Leu Ile Val Thr Gly Ala Pro Leu Gly Leu             100 105 110 Val Glu Phe Asn Asp Val Ala Tyr Trp Pro Gln Ile Lys Gln Val Leu         115 120 125 Glu Trp Ser Lys Asp His Val Thr Ser Thr Leu Phe Val Cys Trp Ala     130 135 140 Val Gln Ala Ala Leu Asn Ile Leu Tyr Gly Ile Pro Lys Gln Thr Arg 145 150 155 160 Thr Glu Gln Leu Ser Gly Val Tyr Glu His His Ile Leu His Pro His                 165 170 175 Ala Leu Leu Thr Arg Gly Phe Asp Asp Ser Phe Leu Ala Pro His Ser             180 185 190 Arg Tyr Ala Asp Phe Pro Ala Ala Leu Ile Arg Asp Tyr Thr Asp Leu         195 200 205 Glu Ile Leu Ala Glu Thr Glu Glu Gly Asp Ala Tyr Leu Leu Ala Ser     210 215 220 Lys Asp Lys Arg Ile Ala Phe Val Thr Gly His Pro Glu Tyr Asp Ala 225 230 235 240 Gln Thr Leu Ala Gln Glu Phe Phe Arg Asp Val Glu Ala Gly Leu Asp                 245 250 255 Pro Asp Val Pro Tyr Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr             260 265 270 Pro Arg Ala Ser Trp Arg Ser His Gly Asn Leu Leu Phe Thr Asn Trp         275 280 285 Leu Asn Tyr Tyr Val Tyr Arg Ile Thr Pro Tyr Asp Leu Arg His Met     290 295 300 Asn Pro Thr Leu Asp 305 <210> 15 <211> 930 <212> DNA <213> Escherichia coli <400> 15 atgccgattc gtgtgccgga cgagctaccc gccgtcaatt tcttgcgtga agaaaacgtc 60 tttgtgatgt caacttctcg tgcgtctggt caggaaattc gtccacttaa ggttctgatc 120 cttaacctga tgccgaagaa gattgaaact gaaaatcagt ttctgcgcct gctttcaaac 180 tcacctttgc aggtcgatat tcagctgttg cgcatcgatt ctcgtgaatc gcgcaacacg 240 cccgcagagc atctgaacaa cttctactgt aactttgaag atattcagga tcagaacttt 300 gacggtttga ttgtaactgg tgcgccgctg ggcctggtgg agtttaatga tgtcgcttac 360 tggccgcaga tcaaacaggt gctggagtgg tcgaaagatc acgtcacctc gacgctgttt 420 gtctgctggg cggtacaggc cgcgctcaat atcctctacg gcattcctaa gcaaactcgc 480 accgaaaaac tctctggcgt ttacgagcat catattctcc atcctcatgc gcttctgacg 540 cgtggctttg atgattcatt cctggcaccg cattcgcgct atgctgactt tccggcagcg 600 ttgattcgtg attacaccga tctggaaatt ctggcagaga cggaagaagg ggatgcatat 660 ctgtttgcca gtaaagataa gcgcattgcc tttgtgacgg gccatcccga atatgatgcg 720 caaacgctgg cgcaggaatt tttccgcgat gtggaagccg gactagaccc ggatgtaccg 780 tataactatt tcccgcacaa tgatccgcaa aatacaccgc gagagagctg gcgtagtcac 840 ggtaatttac tgtttaccaa ctggctcaac tattacgtct accagatcgc gccatacgat 900 ctacggcaca tgtatccaac gctggattaa 930 <210> 16 <211> 309 <212> PRT <213> Escherichia coli <400> 16 Met Pro Ile Arg Val Pro Asp Glu Leu Pro Ala Val Asn Phe Leu Arg   1 5 10 15 Glu Glu Asn Val Phe Val Met Ser Thr Ser Arg Ala Ser Gly Gln Glu              20 25 30 Ile Arg Pro Leu Lys Val Leu Ile Leu Asn Leu Met Pro Lys Lys Ile          35 40 45 Glu Thr Glu Asn Gln Phe Leu Arg Leu Leu Ser Asn Ser Pro Leu Gln      50 55 60 Val Asp Ile Gln Leu Leu Arg Ile Asp Ser Arg Glu Ser Arg Asn Thr  65 70 75 80 Pro Ala Glu His Leu Asn Asn Phe Tyr Cys Asn Phe Glu Asp Ile Gln                  85 90 95 Asp Gln Asn Phe Asp Gly Leu Ile Val Thr Gly Ala Pro Leu Gly Leu             100 105 110 Val Glu Phe Asn Asp Val Ala Tyr Trp Pro Gln Ile Lys Gln Val Leu         115 120 125 Glu Trp Ser Lys Asp His Val Thr Ser Thr Leu Phe Val Cys Trp Ala     130 135 140 Val Gln Ala Ala Leu Asn Ile Leu Tyr Gly Ile Pro Lys Gln Thr Arg 145 150 155 160 Thr Glu Lys Leu Ser Gly Val Tyr Glu His His Ile Leu His Pro His                 165 170 175 Ala Leu Leu Thr Arg Gly Phe Asp Asp Ser Phe Leu Ala Pro His Ser             180 185 190 Arg Tyr Ala Asp Phe Pro Ala Ala Leu Ile Arg Asp Tyr Thr Asp Leu         195 200 205 Glu Ile Leu Ala Glu Thr Glu Glu Gly Asp Ala Tyr Leu Phe Ala Ser     210 215 220 Lys Asp Lys Arg Ile Ala Phe Val Thr Gly His Pro Glu Tyr Asp Ala 225 230 235 240 Gln Thr Leu Ala Gln Glu Phe Phe Arg Asp Val Glu Ala Gly Leu Asp                 245 250 255 Pro Asp Val Pro Tyr Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr             260 265 270 Pro Arg Glu Ser Trp Arg Ser His Gly Asn Leu Leu Phe Thr Asn Trp         275 280 285 Leu Asn Tyr Tyr Val Tyr Gln Ile Ala Pro Tyr Asp Leu Arg His Met     290 295 300 Tyr Pro Thr Leu Asp 305 <210> 17 <211> 930 <212> DNA <213> Escherichia coli <400> 17 atgccgattc gtgtgccgga cgagctaccc gccgtcaatt tcttgcgtga agaaaacgtc 60 tttgtgatga caacttctcg tgcgtctggt caggaaattc gtccacttaa ggttctgatc 120 cttaacctga tgccgaagaa gattgaaact gaaaatcagt ttctgcgcct gctttcaaac 180 tcacctttgc aggtcgatat tcagctgttg cgcatcgatt cccgtgaatc gcgcaacacg 240 cccgcagagc atctgaacaa cttctactgt aactttgaag atattcagga tcagaacttt 300 gacggtttga ttgtaactgg tgcgccgctg ggcctggtgg agtttaatga tgtcgcttac 360 tggccgcaga tcaaacaggt gctggagtgg tcgaaagatc acgtcacctc gacgctgttt 420 gtctgctggg cggtacaggc cgcgctcaac atcctctacg gcattcctaa gcaaactcgc 480 accgaaaaac tctctggcgt ttacgagcat catattctcc atcctcatgc gcttctgacg 540 cgtggctttg atgattcatt cctggcaccg cattcgcgct atgctgactt tccggcagcg 600 ttgattcgtg attacaccga tctggaaatt ctggcagaga cggaagaagg ggatgcatat 660 ctgtttgcca gtaaagataa gcgcattgcc tttgtgacgg gccatcccga atatgatgcg 720 caaacgctgg cgcaggaatt tttccgcgat gtggaagccg gactagaccc ggatgtaccg 780 tataactatt tcccgcacaa tgatccgcaa aatacaccgc gagcgagctg gcgtagtcac 840 ggtaatttac tgtttaccaa ctggctccac tattacgtct accagatcac gccatacgat 900 ctacggcaca tgaatccaac gctggattaa 930 <210> 18 <211> 309 <212> PRT <213> Escherichia coli <400> 18 Met Pro Ile Arg Val Pro Asp Glu Leu Pro Ala Val Asn Phe Leu Arg   1 5 10 15 Glu Glu Asn Val Phe Val Met Thr Thr Ser Arg Ala Ser Gly Gln Glu              20 25 30 Ile Arg Pro Leu Lys Val Leu Ile Leu Asn Leu Met Pro Lys Lys Ile          35 40 45 Glu Thr Glu Asn Gln Phe Leu Arg Leu Leu Ser Asn Ser Pro Leu Gln      50 55 60 Val Asp Ile Gln Leu Leu Arg Ile Asp Ser Arg Glu Ser Arg Asn Thr  65 70 75 80 Pro Ala Glu His Leu Asn Asn Phe Tyr Cys Asn Phe Glu Asp Ile Gln                  85 90 95 Asp Gln Asn Phe Asp Gly Leu Ile Val Thr Gly Ala Pro Leu Gly Leu             100 105 110 Val Glu Phe Asn Asp Val Ala Tyr Trp Pro Gln Ile Lys Gln Val Leu         115 120 125 Glu Trp Ser Lys Asp His Val Thr Ser Thr Leu Phe Val Cys Trp Ala     130 135 140 Val Gln Ala Ala Leu Asn Ile Leu Tyr Gly Ile Pro Lys Gln Thr Arg 145 150 155 160 Thr Glu Lys Leu Ser Gly Val Tyr Glu His His Ile Leu His Pro His                 165 170 175 Ala Leu Leu Thr Arg Gly Phe Asp Asp Ser Phe Leu Ala Pro His Ser             180 185 190 Arg Tyr Ala Asp Phe Pro Ala Ala Leu Ile Arg Asp Tyr Thr Asp Leu         195 200 205 Glu Ile Leu Ala Glu Thr Glu Glu Gly Asp Ala Tyr Leu Phe Ala Ser     210 215 220 Lys Asp Lys Arg Ile Ala Phe Val Thr Gly His Pro Glu Tyr Asp Ala 225 230 235 240 Gln Thr Leu Ala Gln Glu Phe Phe Arg Asp Val Glu Ala Gly Leu Asp                 245 250 255 Pro Asp Val Pro Tyr Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr             260 265 270 Pro Arg Ala Ser Trp Arg Ser His Gly Asn Leu Leu Phe Thr Asn Trp         275 280 285 Leu His Tyr Tyr Val Tyr Gln Ile Thr Pro Tyr Asp Leu Arg His Met     290 295 300 Asn Pro Thr Leu Asp 305 <210> 19 <211> 930 <212> DNA <213> Escherichia coli <400> 19 atgccgattc gtgtgccgga cgagctaccc gccgtcaatt tcttgcgtga agaaaacgtc 60 tttgtgatga caacttctcg tgcgcctggt caggaaattc gtccacttaa ggttctgatc 120 cttaacctga tgccgaagaa gattgaaact gaaaatcagt ttctgcgcct gctttcaaac 180 tcacctttgc aggtcgatat tcagctgttg cgcatcgatt cccgtgaatc gcgcaacacg 240 cccgcagagc atctgaacaa cttctactgt aactttgaag atattcagga tcagaacttt 300 gacggtttga ttgtaactgg tgcgccgctg ggcctggtgg ggtttaatga tgtcgcttac 360 tggccgcaga tcaaacaggt gctggagtgg tcgaaagatc acgtcacctc gacgctgtct 420 gtctgctggg cggtacaggc cgcgctcaat atcctctacg gcattcctaa gcaaactcgc 480 accgaaaaac tctctggcgt ttacgagcat catattctcc atcctcatgc gcttctgacg 540 cgtggctttg atgattcatt cctggcaccg cattcgcgct atgctgactt tccggcagcg 600 ttgattcgtg attacaccga tctggaaatt ctggcagaga cggaagaagg ggatgcatat 660 ctgtttgcca gtaaagataa gcgcattgcc tttgtgacgg gccatcccga atatgatgcg 720 caaacgctgg cgcaggaatt tttccgcgat gtggaagccg gactagaccc ggatgtaccg 780 tataactatt tcccgcacaa tgatccgcaa aatacaccgc gagcgagctg gcgtagtcac 840 ggtaatttac tgtttaccaa ctggctcaac tattacgtct accagatcac gccatacgat 900 ctacggcaca tgaacccaac gctggattaa 930 <210> 20 <211> 309 <212> PRT <213> Escherichia coli <400> 20 Met Pro Ile Arg Val Pro Asp Glu Leu Pro Ala Val Asn Phe Leu Arg   1 5 10 15 Glu Glu Asn Val Phe Val Met Thr Thr Ser Arg Ala Pro Gly Gln Glu              20 25 30 Ile Arg Pro Leu Lys Val Leu Ile Leu Asn Leu Met Pro Lys Lys Ile          35 40 45 Glu Thr Glu Asn Gln Phe Leu Arg Leu Leu Ser Asn Ser Pro Leu Gln      50 55 60 Val Asp Ile Gln Leu Leu Arg Ile Asp Ser Arg Glu Ser Arg Asn Thr  65 70 75 80 Pro Ala Glu His Leu Asn Asn Phe Tyr Cys Asn Phe Glu Asp Ile Gln                  85 90 95 Asp Gln Asn Phe Asp Gly Leu Ile Val Thr Gly Ala Pro Leu Gly Leu             100 105 110 Val Gly Phe Asn Asp Val Ala Tyr Trp Pro Gln Ile Lys Gln Val Leu         115 120 125 Glu Trp Ser Lys Asp His Val Thr Ser Thr Leu Ser Val Cys Trp Ala     130 135 140 Val Gln Ala Ala Leu Asn Ile Leu Tyr Gly Ile Pro Lys Gln Thr Arg 145 150 155 160 Thr Glu Lys Leu Ser Gly Val Tyr Glu His His Ile Leu His Pro His                 165 170 175 Ala Leu Leu Thr Arg Gly Phe Asp Asp Ser Phe Leu Ala Pro His Ser             180 185 190 Arg Tyr Ala Asp Phe Pro Ala Ala Leu Ile Arg Asp Tyr Thr Asp Leu         195 200 205 Glu Ile Leu Ala Glu Thr Glu Glu Gly Asp Ala Tyr Leu Phe Ala Ser     210 215 220 Lys Asp Lys Arg Ile Ala Phe Val Thr Gly His Pro Glu Tyr Asp Ala 225 230 235 240 Gln Thr Leu Ala Gln Glu Phe Phe Arg Asp Val Glu Ala Gly Leu Asp                 245 250 255 Pro Asp Val Pro Tyr Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr             260 265 270 Pro Arg Ala Ser Trp Arg Ser His Gly Asn Leu Leu Phe Thr Asn Trp         275 280 285 Leu Asn Tyr Tyr Val Tyr Gln Ile Thr Pro Tyr Asp Leu Arg His Met     290 295 300 Asn Pro Thr Leu Asp 305 <210> 21 <211> 930 <212> DNA <213> Escherichia coli <400> 21 atgccgattc gtgtgccgga cgagctaccc gccgtcaatt tcttgcgtga agaaaacgtc 60 tttgtgatga caacttctcg tgcgtctggt caggaaattc gtccacttaa ggttctgatc 120 cttaacctga tgccgaagaa gattgaaact gaaaatcagt ttctgcgcct gctttcaaac 180 tcacctttgc aggtcgatat tcagctgttg cgcatcgatt cccgtgaatc gcgcagcacg 240 cccgcagagc atctgaacaa cttctactgt aactttgaag atattcagga tcagaacttt 300 gacggtttga ttgtaactgg tgcgccgctg ggcctggtgg agtttaatga tgtcgcttac 360 tggccgcaga tcaaacaggt gctggagtgg tcgaaagatc acgtcacctc gacgctgatt 420 gtctgctggg cggtacaggc cgcgctcaat atcctctacg gcattcctaa gcaaactcgc 480 accgaaaaac tctctggcgt ttacgagcat catattctcc atcctcatgc gcttctgacg 540 cgtggctttg atgattcatt cctggcaccg cactcgcgct atgctgactt tccggcagcg 600 ttgattcgtg attacaccga tctggaaatt ctggcagaga cggaagaagg ggatgcatat 660 ctgtttgcca gtaaagataa gcgcattgcc tttgtgacgg gccatcccga atatgatgcg 720 caaacgctgg cgcaggaatt tttccgcgat gtggaagccg gactagaccc ggatgtaccg 780 tataactatt tcccgcacaa tgatccgcaa aatacaccgc gagcgagctg gcgtagtcac 840 ggtaatttac tgtttaccaa ctggctcaac aattacgtct accagatcac gccatacgat 900 ctacggcact tgaatccaac gctggattaa 930 <210> 22 <211> 309 <212> PRT <213> Escherichia coli <400> 22 Met Pro Ile Arg Val Pro Asp Glu Leu Pro Ala Val Asn Phe Leu Arg   1 5 10 15 Glu Glu Asn Val Phe Val Met Thr Thr Ser Arg Ala Ser Gly Gln Glu              20 25 30 Ile Arg Pro Leu Lys Val Leu Ile Leu Asn Leu Met Pro Lys Lys Ile          35 40 45 Glu Thr Glu Asn Gln Phe Leu Arg Leu Leu Ser Asn Ser Pro Leu Gln      50 55 60 Val Asp Ile Gln Leu Leu Arg Ile Asp Ser Arg Glu Ser Arg Ser Thr  65 70 75 80 Pro Ala Glu His Leu Asn Asn Phe Tyr Cys Asn Phe Glu Asp Ile Gln                  85 90 95 Asp Gln Asn Phe Asp Gly Leu Ile Val Thr Gly Ala Pro Leu Gly Leu             100 105 110 Val Glu Phe Asn Asp Val Ala Tyr Trp Pro Gln Ile Lys Gln Val Leu         115 120 125 Glu Trp Ser Lys Asp His Val Thr Ser Thr Leu Ile Val Cys Trp Ala     130 135 140 Val Gln Ala Ala Leu Asn Ile Leu Tyr Gly Ile Pro Lys Gln Thr Arg 145 150 155 160 Thr Glu Lys Leu Ser Gly Val Tyr Glu His His Ile Leu His Pro His                 165 170 175 Ala Leu Leu Thr Arg Gly Phe Asp Asp Ser Phe Leu Ala Pro His Ser             180 185 190 Arg Tyr Ala Asp Phe Pro Ala Ala Leu Ile Arg Asp Tyr Thr Asp Leu         195 200 205 Glu Ile Leu Ala Glu Thr Glu Glu Gly Asp Ala Tyr Leu Phe Ala Ser     210 215 220 Lys Asp Lys Arg Ile Ala Phe Val Thr Gly His Pro Glu Tyr Asp Ala 225 230 235 240 Gln Thr Leu Ala Gln Glu Phe Phe Arg Asp Val Glu Ala Gly Leu Asp                 245 250 255 Pro Asp Val Pro Tyr Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr             260 265 270 Pro Arg Ala Ser Trp Arg Ser His Gly Asn Leu Leu Phe Thr Asn Trp         275 280 285 Leu Asn Asn Tyr Val Tyr Gln Ile Thr Pro Tyr Asp Leu Arg His Leu     290 295 300 Asn Pro Thr Leu Asp 305 <210> 23 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer for integration of metA <400> 23 tttccgaaac gtacctcagc aggtgtaggc tggagctgct tc 42 <210> 24 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> primer for integration of metA <400> 24 gaataaaatt tattcacctg ctgcatatga atatcctcct tag 43 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer for integration of metA <400> 25 cagcaggtga ataaatttta ttc 23 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for integration of metA <400> 26 cgcgaatgga agctgtttcc 20 <210> 27 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer for ampilification thr A <400> 27 ctggcaaagc tttcaaagga aaactccttc gt 32 <210> 28 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer for ampilification of thrA <400> 28 agtcgtgata tcatgcgagt gttgaagttc gg 32 <210> 29 <211> 820 <212> PRT <213> Escherichia coli <220> <221> PEPTIDE (222) (1) .. (820) <223> homoserine dehydrogenase fused with aspartate kinase <400> 29 Met Arg Val Leu Lys Phe Gly Gly Thr Ser Val Ala Asn Ala Glu Arg   1 5 10 15 Phe Leu Arg Val Ala Asp Ile Leu Glu Ser Asn Ala Arg Gln Gly Gln              20 25 30 Val Ala Thr Val Leu Ser Ala Pro Ala Lys Ile Thr Asn His Leu Val          35 40 45 Ala Met Ile Glu Lys Thr Ile Ser Gly Gln Asp Ala Leu Pro Asn Ile      50 55 60 Ser Asp Ala Glu Arg Ile Phe Ala Glu Leu Leu Thr Gly Leu Ala Ala  65 70 75 80 Ala Gln Pro Gly Phe Pro Leu Ala Gln Leu Lys Thr Phe Val Asp Gln                  85 90 95 Glu Phe Ala Gln Ile Lys His Val Leu His Gly Ile Ser Leu Leu Gly             100 105 110 Gln Cys Pro Asp Ser Ile Asn Ala Ala Leu Ile Cys Arg Gly Glu Lys         115 120 125 Met Ser Ile Ala Ile Met Ala Gly Val Leu Glu Ala Arg Gly His Asn     130 135 140 Val Thr Val Ile Asp Pro Val Glu Lys Leu Leu Ala Val Gly His Tyr 145 150 155 160 Leu Glu Ser Thr Val Asp Ile Ala Glu Ser Thr Arg Arg Ile Ala Ala                 165 170 175 Ser Arg Ile Pro Ala Asp His Met Val Leu Met Ala Gly Phe Thr Ala             180 185 190 Gly Asn Glu Lys Gly Glu Leu Val Val Leu Gly Arg Asn Gly Ser Asp         195 200 205 Tyr Ser Ala Ala Val Leu Ala Ala Cys Leu Arg Ala Asp Cys Cys Glu     210 215 220 Ile Trp Thr Asp Val Asp Gly Val Tyr Thr Cys Asp Pro Arg Gln Val 225 230 235 240 Pro Asp Ala Arg Leu Leu Lys Ser Met Ser Tyr Gln Glu Ala Met Glu                 245 250 255 Leu Ser Tyr Phe Gly Ala Lys Val Leu His Pro Arg Thr Ile Thr Pro             260 265 270 Ile Ala Gln Phe Gln Ile Pro Cys Leu Ile Lys Asn Thr Gly Asn Pro         275 280 285 Gln Ala Pro Gly Thr Leu Ile Gly Ala Ser Arg Asp Glu Asp Glu Leu     290 295 300 Pro Val Lys Gly Ile Ser Asn Leu Asn Asn Met Ala Met Phe Ser Val 305 310 315 320 Ser Gly Pro Gly Met Lys Gly Met Val Gly Met Ala Ala Arg Val Phe                 325 330 335 Ala Ala Met Ser Arg Ala Arg Ile Phe Val Val Leu Ile Thr Gln Ser             340 345 350 Ser Ser Glu Tyr Ser Ile Ser Phe Cys Val Pro Gln Ser Asp Cys Val         355 360 365 Arg Ala Glu Arg Ala Met Gln Glu Glu Phe Tyr Leu Glu Leu Lys Glu     370 375 380 Gly Leu Leu Glu Pro Leu Ala Val Thr Glu Arg Leu Ala Ile Ile Ser 385 390 395 400 Val Val Gly Asp Gly Met Arg Thr Leu Arg Gly Ile Ser Ala Lys Phe                 405 410 415 Phe Ala Ala Leu Ala Arg Ala Asn Ile Asn Ile Val Ala Ile Ala Gln             420 425 430 Gly Ser Ser Glu Arg Ser Ile Ser Val Val Val Asn Asn Asp Asp Ala         435 440 445 Thr Thr Gly Val Arg Val Thr His Gln Met Leu Phe Asn Thr Asp Gln     450 455 460 Val Ile Glu Val Phe Val Ile Gly Val Gly Gly Val Gly Gly Ala Leu 465 470 475 480 Leu Glu Gln Leu Lys Arg Gln Gln Ser Trp Leu Lys Asn Lys His Ile                 485 490 495 Asp Leu Arg Val Cys Gly Val Ala Asn Ser Lys Ala Leu Leu Thr Asn             500 505 510 Val His Gly Leu Asn Leu Glu Asn Trp Gln Glu Glu Leu Ala Gln Ala         515 520 525 Lys Glu Pro Phe Asn Leu Gly Arg Leu Ile Arg Leu Val Lys Glu Tyr     530 535 540 His Leu Leu Asn Pro Val Ile Val Asp Cys Thr Ser Ser Gln Ala Val 545 550 555 560 Ala Asp Gln Tyr Ala Asp Phe Leu Arg Glu Gly Phe His Val Val Thr                 565 570 575 Pro Asn Lys Lys Ala Asn Thr Ser Ser Met Asp Tyr Tyr His Gln Leu             580 585 590 Arg Tyr Ala Ala Glu Lys Ser Arg Arg Lys Phe Leu Tyr Asp Thr Asn         595 600 605 Val Gly Ala Gly Leu Pro Val Ile Glu Asn Leu Gln Asn Leu Leu Asn     610 615 620 Ala Gly Asp Glu Leu Met Lys Phe Ser Gly Ile Leu Ser Gly Ser Leu 625 630 635 640 Ser Tyr Ile Phe Gly Lys Leu Asp Glu Gly Met Ser Phe Ser Glu Ala                 645 650 655 Thr Thr Leu Ala Arg Glu Met Gly Tyr Thr Glu Pro Asp Pro Arg Asp             660 665 670 Asp Leu Ser Gly Met Asp Val Ala Arg Lys Leu Leu Ile Leu Ala Arg         675 680 685 Glu Thr Gly Arg Glu Leu Glu Leu Ala Asp Ile Glu Ile Glu Pro Val     690 695 700 Leu Pro Ala Glu Phe Asn Ala Glu Gly Asp Val Ala Ala Phe Met Ala 705 710 715 720 Asn Leu Ser Gln Leu Asp Asp Leu Phe Ala Ala Arg Val Ala Lys Ala                 725 730 735 Arg Asp Glu Gly Lys Val Leu Arg Tyr Val Gly Asn Ile Asp Glu Asp             740 745 750 Gly Val Cys Arg Val Lys Ile Ala Glu Val Asp Gly Asn Asp Pro Leu         755 760 765 Phe Lys Val Lys Asn Gly Glu Asn Ala Leu Ala Phe Tyr Ser His Tyr     770 775 780 Tyr Gln Pro Leu Pro Leu Val Leu Arg Gly Tyr Gly Ala Gly Asn Asp 785 790 795 800 Val Thr Ala Ala Gly Val Phe Ala Asp Leu Leu Arg Thr Leu Ser Trp                 805 810 815 Lys Leu Gly Val             820 <210> 30 <211> 309 <212> PRT <213> Escherichia coli <220> <221> PEPTIDE (222) (1) .. (309) <223> homoserine O-succinyltransferase with GenBank Accession No. AP          004514 <400> 30 Met Pro Ile Arg Val Pro Asp Glu Leu Pro Ala Val Asn Phe Leu Arg   1 5 10 15 Glu Glu Asn Val Phe Val Met Thr Thr Ser Arg Ala Ser Gly Gln Glu              20 25 30 Ile Arg Pro Leu Lys Val Leu Ile Leu Asn Leu Met Pro Lys Lys Ile          35 40 45 Glu Thr Glu Asn Gln Phe Leu Arg Leu Leu Ser Asn Ser Pro Leu Gln      50 55 60 Val Asp Ile Gln Leu Leu Arg Ile Asp Ser Arg Glu Ser Arg Asn Thr  65 70 75 80 Pro Ala Glu His Leu Asn Asn Phe Tyr Cys Asn Phe Glu Asp Ile Gln                  85 90 95 Asp Gln Asn Phe Asp Gly Leu Ile Val Thr Gly Ala Pro Leu Gly Leu             100 105 110 Val Glu Phe Asn Asp Val Ala Tyr Trp Pro Gln Ile Lys Gln Val Leu         115 120 125 Glu Trp Ser Lys Asp His Val Thr Ser Thr Leu Phe Val Cys Trp Ala     130 135 140 Val Gln Ala Ala Leu Asn Ile Leu Tyr Gly Ile Pro Lys Gln Thr Arg 145 150 155 160 Thr Glu Lys Leu Ser Gly Val Tyr Glu His His Ile Leu His Pro His                 165 170 175 Ala Leu Leu Thr Arg Gly Phe Asp Asp Ser Phe Leu Ala Pro His Ser             180 185 190 Arg Tyr Ala Asp Phe Pro Ala Ala Leu Ile Arg Asp Tyr Thr Asp Leu         195 200 205 Glu Ile Leu Ala Glu Thr Glu Glu Gly Asp Ala Tyr Leu Phe Ala Ser     210 215 220 Lys Asp Lys Arg Ile Ala Phe Val Thr Gly His Pro Glu Tyr Asp Ala 225 230 235 240 Gln Thr Leu Ala Gln Glu Phe Phe Arg Asp Val Glu Ala Gly Leu Asp                 245 250 255 Pro Asp Val Pro Tyr Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr             260 265 270 Pro Arg Ala Ser Trp Arg Ser His Gly Asn Leu Leu Phe Thr Asn Trp         275 280 285 Leu Asn Tyr Tyr Val Tyr Gln Ile Thr Pro Tyr Asp Leu Arg His Met     290 295 300 Asn Pro Thr Leu Asp 305  

Claims (17)

1) homoserine O-succinyltransferase activity (EC2.3.1.46) is introduced and enhanced, said homoserine O-succinyltransferase has feedback resistance to methionine; And 2) from the genus Escherichia sp., Which can produce O-succinyl homoserine with enhanced aspartokinase or homoserine dehydrogenase activity (EC2.7.2.4 or 1.1.1.3). Strain. The method of claim 1, wherein the homoserine O-succinyltransferase is 24, 29, 79, 114, 140, 163, 222, 275, 290, 291, 295, 297, 304 or A strain having a mutation at one or more of positions 305. The strain of claim 1, wherein the homoserine O-succinyltransferase has an amino acid sequence of SEQ ID NO: 14, 16, 18, 20, or 22. The strain of claim 1, wherein the aspartokinase or homoserine dehydrogenase has a mutation at position 345 in the amino acid sequence. The strain of claim 1, wherein the aspartokinase or homoserine dehydrogenase has an amino acid sequence of SEQ ID NO. The strain of claim 1, wherein the strain is derived from L-threonine, L-isoleucine or L-lysine producing strain. The strain of claim 1, wherein the strain is Escherichia coli . The strain according to claim 1, wherein the strain is attenuated or inactivated cystathionine gamma synthase, O-succinyl homoserine sulfhydrylase or O-acetylhomoserine sulfhydrylase. The strain of claim 1, wherein the strain is attenuated or inactivated homoserine kinase. The strain of claim 1, wherein the strain is attenuated or inactivated by a transcriptional regulator of the methionine synthesis pathway. The strain according to claim 1, wherein the strain has weakened or inactivated intact homoserine O-acetyltransferase activity or intact homoserine O-succinyltransferase activity. The strain according to claim 1, wherein the strain is a metB gene encoding cystathionine gamma synthase, a thrB gene encoding homoserine kinase, and a metJ gene, which is a transcriptional regulator of metA gene, is weakened or inactivated. The method of claim 1, wherein the strain is E. coli MF001 Escherichia , which is a threonine producing strain free from L-methionine-dependent coli MF001, Accession No. KCCM 10568). The strain of claim 1, wherein the strain is derived from Escherichia coli FTR2533 (Accession No. KCCM 10541), which is a threonine producing strain. The strain of claim 1, wherein the strain is represented by accession number KCCM 10922P. The strain of claim 1, wherein the strain is represented by accession number KCCM 10924P. 1) fermenting the strain according to any one of claims 1 to 16; 2) culturing O-succinylhomoserine in medium or strains; And 3) isolating O-succinyl homoserine.

Images