KR101851452B1 - Microorganisms producing O-acetyl-homoserine and the method of producing O-Acetyl-homoserine using the same - Google Patents

Abstract

The present invention relates to a microorganism producing O-acetyl homoserine with high efficiency and a method of producing O-acetyl homoserine and L-methionine using the microorganism. The present invention proposes a method for producing O-acetyl homoserine-producing microorganisms and O-acetyl homoserine and L-methionine by enhancing the activity of proteins presumed to release O-acetyl homoserine.

Description

Microorganisms producing O-acetyl-homoserine and the method of producing O-Acetyl-homoserine using the same}

The present invention relates to a microorganism producing 0-acetyl homoserine with high efficiency and a method for producing O-acetyl homoserine using the microorganism.

O-acetyl homoserine acts as a precursor to methionine, a type of essential amino acid in vivo. Methionine is used not only as feed and food additives, but also as a synthetic raw material for infusions and pharmaceuticals.

Methionine is produced through chemical and biological synthesis. Recently, a two-step method of producing L-methionine from an L-methionine precursor produced through fermentation by an enzyme conversion reaction (International Publication No. WO/2008/013432) has also been known.

In the above two-step method, O-succinyl homoserine and O-acetyl homoserine are used as methionine precursors, and O-acetyl homoserine is used for economical mass production of methionine. It is very important to produce in high yield.

Under this background, the present inventors have made great efforts to increase the production of O-acetyl-homoserine, and as a result, have completed the present invention by discovering a protein having an activity of releasing O-acetyl-homoserine.

One object of the present invention is to provide a microorganism having an improved O-acetyl homoserine production ability.

Another object of the present invention is to provide a method for efficiently producing O-acetin homoserine using a microorganism having an improved O-acetyl homoserine production ability.

One aspect of the present invention includes a microorganism having an O-acetyl homoserine-producing ability in which the activity of the inner membrane protein YjeH is enhanced compared to the non-mutant microorganism.

The term "O-acetyl homoserine" used in the present invention refers to a specific intermediate material on the methionine biosynthesis pathway of microorganisms, and refers to an acetyl derivative of L-homoserine. It is known that homoserine and acetyl-CoA are produced by catalyzing the reaction by homoserine acetyl transferase, and has the formula of _{C 6} H ₁₁ NO _4.

The term used in the present invention, "a microorganism having the ability to produce O-acetyl homoserine" refers to a microorganism having the ability to produce O-acetyl homoserine in the organism and secrete it in the medium when the microorganism is cultured in a medium. do. O-acetyl homoserine production capacity can be conferred or enhanced by species improvement. Specifically, the microorganism having the ability to produce O-acetyl homoserine may be a microorganism of the genus Escherichia having the ability to produce O-acetyl homoserine, and more specifically, may be E. coli. For example, it may be E. coli that produces lysine, threonine, isoleucine, or methionine, but is not limited thereto. The term "YjeH" used in the present invention is known as a protein present in the inner membrane as one of the APC family for amino acid transporters, and is expected to act as an amino acid transporter, but its exact function is not known. Accordingly, the present inventors first confirmed that YjeH specifically releases O-acetyl homoserine.

Specifically, the YjeH may be derived from a microorganism of the genus Escherichia, and a more specific example may be YjeH derived from E. coli. In particular, it may be a protein having an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 70% or more, specifically 80% or more, and more specifically 90% or more homology thereto. In addition, as a sequence having homology, if an amino acid sequence having an activity of releasing O-acetyl homoserine is substantially the same as or corresponding to the amino acid sequence of SEQ ID NO: 1, some sequences have an amino acid sequence that has been deleted, modified, substituted or added. It is obvious that cases are also included in the scope of the present invention. In addition, nucleotide sequences encoding the same amino acid sequence and variants thereof due to genetic code degeneracy are also included in the present invention. As an example, it may be the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

The term "homology" used in the present invention refers to an amino acid or nucleotide sequence of a gene encoding a protein, after aligning the two sequences so as to be maximally identical in a specific comparison region, and then the same of bases or amino acid residues between sequences. Means degree. If the homology is sufficiently high, the expression product of the corresponding gene may have the same or similar activity. The percent of sequence identity may be determined using a known sequence comparison program, and examples include BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc), and the like.

In the present invention, the term "non-mutant microorganism" refers to a microorganism that has not introduced mutations in the activity of the corresponding protein, and refers to a base strain that introduces mutations in the activity of the corresponding protein. It can be natural or variant.

The term "enhancement" of protein activity as used in the present invention means improving the activity state of a protein possessed by microorganisms. The enhancement of the activity of the protein is not limited as long as the activity of each protein can be enhanced than that of the non-mutated microorganism, such as enhancement of the activity of the target protein. For example, i) increasing the number of copies of the polynucleotide encoding each protein, ii) modifying the expression control sequence to increase the expression of the polynucleotide, iii) modifying the polynucleotide sequence on the chromosome to enhance the activity of each protein. And iv) a combination thereof. Specifically, a method of inserting a polynucleotide containing a nucleotide sequence encoding each protein into a chromosome, a method of introducing the polynucleotide into a vector system and introducing it into a microorganism, improved activity upstream of the nucleotide sequence encoding each protein Selected from the group consisting of a method of introducing a promoter representing or introducing each protein that has mutated the promoter, a method of modifying the nucleotide sequence of the 5'-UTR region, and a method of introducing a variant of the nucleotide sequence encoding each protein. It may be performed by a method, but is not limited thereto.

In a specific embodiment of the present invention, the activity of YjeH may be enhanced than that of non-mutant microorganisms by increasing the copy number or enhancing the activity of the promoter. Specifically, it may be that a promoter exhibiting improved activity was introduced into the inner membrane protein YjeH to enhance its activity. As a specific example of the present invention, the promoter exhibiting the improved activity includes, without limitation, a promoter having increased activity compared to the yjeH autopromoter, and this includes a promoter of a gene having a higher activity than the gene expression inducing activity of the yjeH autopromoter, or yjeH self. It includes, without limitation, a mutant promoter whose activity is increased through genetic mutation such as a promoter. Specifically, the promoter exhibiting improved activity of the present invention may be selected from the group consisting of an icd promoter, a pro promoter, and a cysk promoter, and specifically, the icd promoter consists of the nucleotide sequence of SEQ ID NO: 51, and the pro promoter is It consists of the nucleotide sequence of SEQ ID NO: 52, and the cysk promoter may be composed of the nucleotide sequence of SEQ ID NO: 53, but each nucleotide sequence and 70% or more, specifically 80% or more, more specifically 90% or more phase It may be a base sequence having homology.

As a specific aspect of the present invention, the microorganism of the genus Escherichia having the ability to produce O-acetyl homoserine may additionally have a weakened or inactivated activity of cystathionine synthase. Specifically, the activity of the cystathionine synthase may be reduced or inactivated than that of the non-mutant microorganism, and in particular, the gene encoding cystathionine synthase (metB) may be defective, but is not limited thereto. Does not. The amino acid sequence of metB can be obtained from a known database, and the amino acid sequence having cystathionine synthase activity may be included without limitation, but for example, it may be a protein having the amino acid sequence of SEQ ID NO: 3. The protein having the amino acid sequence of SEQ ID NO: 3 may be a protein encoded by the nucleotide sequence of SEQ ID NO: 4, but is not limited thereto. In addition, as another aspect of the present invention, the microorganism of the genus Escherichia may additionally have weakened or inactivated the activity of homoserine kinase. Specifically, the activity of the homoserine kinase may be reduced or inactivated compared to the intrinsic activity of the non-mutant microorganism, and in particular, the gene encoding the homoserine kinase (thrB) may be deleted, but is not limited thereto. The amino acid sequence of thrB may be obtained from a known database, and the amino acid sequence having homoserine kinase activity may be included without limitation, but for example, it may be a protein having the amino acid sequence of SEQ ID NO: 5. The protein having the amino acid sequence of SEQ ID NO: 5 may be a protein encoded by the nucleotide sequence of SEQ ID NO: 6, but is not limited thereto.

In the present invention, the activity "weakening" of the protein means i) deletion of a part or all of the gene encoding each protein, ii) modification of the expression control sequence so that the expression of the gene is reduced, iii) so that the activity of the protein is weakened. Modification of the gene sequence on a chromosome and iv) may be performed by a method selected from the group consisting of a combination thereof, but is not limited thereto.

Specifically, the "reduced activity" of the protein of the present invention means that the activity of the enzyme is reduced when compared to the activity of the enzyme that the original microorganism has in the state of a natural or base strain. The attenuation is a case in which the activity of the enzyme itself is decreased compared to the activity of the enzyme of the original microorganism due to mutation of the gene encoding the enzyme, and the expression of the gene encoding the enzyme is inhibited or translation is inhibited. When the overall degree of enzyme activity is lower than that of the natural strain, the concept includes a combination thereof, but is not limited thereto.

The "inactivation" refers to a case in which the expression of the gene encoding the enzyme is not expressed at all compared to the natural strain, and even if it is expressed, there is no activity thereof.

Such attenuation or inactivation of enzyme activity can be achieved by application of various methods well known in the art. Examples of the method include a method of replacing a gene encoding the enzyme on a chromosome with a gene mutated to reduce the activity of the enzyme, including when the activity of the enzyme is removed; A method of introducing a mutation into an expression control sequence of a gene on a chromosome encoding the enzyme; A method of replacing the expression control sequence of the gene encoding the enzyme with a sequence having weak or no activity; A method of deleting all or part of a gene on a chromosome encoding the enzyme; A method of introducing an antisense oligonucleotide (eg, antisense RNA) that complements the transcript of the gene on the chromosome and inhibits translation from the mRNA into the enzyme; A method of making it impossible to attach a ribosome by artificially adding a sequence complementary to the SD sequence to the front end of the SD sequence of the gene encoding the enzyme to make it impossible to attach a ribosome, and of the ORF (open reading frame) of the sequence. There is a reverse transcription engineering (RTE) method of adding a promoter so as to be reverse transcribed at the 3'end, and can be achieved by a combination thereof, but is not particularly limited by the above example.

Specifically, the method of deleting some or all of the gene encoding the enzyme is to replace the polynucleotide encoding the intrinsic target protein in the chromosome with a polynucleotide or a marker gene in which some nucleic acid sequence has been deleted through a vector for chromosome insertion in bacteria. It can be done by doing. As an example of a method of deleting some or all of these genes, a method of deleting a gene by homologous recombination may be used, but is not limited thereto.

In the above, "some" may be different depending on the type of polynucleotide, specifically 1 to 300, preferably 1 to 100, more preferably 1 to 50, but is not particularly limited thereto.

In the above, "homologous recombination" refers to gene recombination that occurs through linkage exchange at loci of gene chains that are homologous to each other.

The method of modifying the expression control sequence is performed by inducing a mutation on the expression control sequence by deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence, or a combination thereof so as to further weaken the activity of the expression control sequence, or It can be carried out by replacing it with an active nucleic acid sequence. The expression control sequence includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence controlling termination of transcription and translation.

In addition, the method of modifying the gene sequence on the chromosome is performed by inducing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination of the gene sequence to further weaken the activity of the enzyme. It can be carried out by replacing with an improved gene sequence to have or an improved gene sequence so as not to have activity, but is not limited thereto.

In a specific embodiment of the present invention, the activity of each protein is deleted by using homologous recombination to delete the gene (metB) encoding the cystathionine synthase and/or the gene (thrB) encoding the homoserine kinase. Weakened.

In addition, as a specific embodiment of the present invention, the microorganisms of the genus Escherichia may have additionally enhanced activity of homoserine acetyltransferase compared to non-mutant microorganisms. Specifically, the activity of the homoserine acetyl transferase may be increased than that of the non-mutant microorganism, and in particular, a mutant metA gene encoding an enhanced homoserine acetyl transferase may be introduced. The mutant metA gene may be a gene encoding amino acid 111 of homoserine acetyl transferase substituted with glutamic acid and amino acid 112 with histidine. In particular, it may be composed of the nucleotide sequence of SEQ ID NO: 8. Not limited. The mutant metA may include, without limitation, an amino acid sequence in which the activity of homoserine acetyl transferase is enhanced than that of the wild type, but may be, for example, a protein having the amino acid sequence of SEQ ID NO: 7. An example of the preparation and utilization of the mutant metA gene, the homoserine acetyl transferase-enhanced strain, etc., is disclosed in Korean Patent Registration No. 10-1335841, and the entire specification of the patent is a reference material of the present invention. Can be included as.

In addition, in a specific aspect of the present invention, the microorganisms of the genus Escherichia may have additionally enhanced activity of aspartate kinase (EC 2.7.2.4) compared to non-mutant microorganisms. Specifically, the activity of the aspartate kinase may be increased than the intrinsic activity of the non-mutant microorganism, but is not limited thereto.

In a specific embodiment of the present invention, in order to maximize the production capacity of O-acetyl homoserine, the biosynthetic pathway of homoserine was additionally strengthened. Specifically, aspartate kinase and homoserine O-acetyltransferase were introduced using a plasmid, and YjeH was further strengthened in the strain strengthening the homoserine biosynthetic pathway as described above. Changes in the production capacity of O-acetyl homoserine were measured. As a result, as a result of enhancing the biosynthetic pathway and yjeH at the same time, the production capacity of O-acetyl homoserine was further improved.In particular, as a result of enhancing YjeH activity using the cysk promoter, about 93% (2.8 g/L -> 5.4 g/L) it was confirmed that the production capacity increased (Table 5).

In addition, in a specific embodiment of the present invention, by enhancing the activity of YjeH in the existing O-acetyl homoserine high-yield strain, it was confirmed whether the production capacity can be additionally increased. More specifically, based on KCCM11146P (international published application WO2012/087039), a strain that produces O-acetyl homoserine using a strain that has the ability to produce threonine through an NTG mutation derived from the wild-type W3110. O-acetyl homoserine production was confirmed by replacing the promoter of the endogenous yjeH gene with a promoter having high expression-inducing activity. As a result, it was confirmed that the production capacity can be further increased by enhancing the activity of YjeH in strains already having a high-yield O-acetyl homoserine-producing ability. In particular, as a result of enhancing YjeH activity using the cysk promoter, about It was confirmed that the production capacity increased by 14% (14.2 g/L -> 18.2 g/L) (Table 7).

Genes used in the present invention, protein sequences and promoter sequences that they encode can be obtained from known databases, for example, can be obtained from NCBI's GenBank, but are not limited thereto.

The gene encoding each protein and promoter of the present invention is not only the nucleotide sequence described in each of the above sequence numbers, but also 80% or more, specifically 90% or more, more specifically 95% or more, more specifically Examples include, without limitation, a nucleotide sequence that exhibits 98% or more, and most specifically, 99% or more homology, as long as it is a gene sequence encoding an enzyme that is substantially the same as each of the enzymes or exhibits a corresponding efficacy. In addition, in the case of a nucleotide sequence having such homology, it is obvious that a nucleotide sequence in which some sequences are deleted, modified, substituted or added are also included within the scope of the present invention.

In addition, the amino acid constituting each protein of the present invention is not only the amino acid sequence described in each of the above sequence numbers, but also a sequence having homology, and is substantially the same as each amino acid sequence or an amino acid sequence having the corresponding protein activity, It is obvious that some sequences are included in the scope of the present invention even if they have an amino acid sequence that has been deleted, modified, substituted or added.

As described above, by increasing the activity of YjeH based on microorganisms having various genetic backgrounds and various O-acetyl homoserine-producing ability, the effect of consistently increasing O-acetyl homoserine-producing ability was confirmed. This can happen through YjeH promoting the production of specific intermediates in the production process, but considering the characteristics of YjeH, which is an inner membrane protein and a family of amino acid transporters, it is the final product and the release of O-acetyl homoserine, a kind of amino acid. It is predicted to be achieved through increasing the ability to promote intracellular responses.

As another aspect of the present invention, the present invention comprises the step of obtaining a culture by culturing a microorganism of the genus Escheria having O-acetyl homoserine production ability according to the present invention, O-acetyl homoserine Provides the production method of

The medium and other culture conditions used for culturing the microorganisms of the present invention may be any medium without particular limitation as long as it is a medium used for culturing ordinary microorganisms of the genus Escherichia, but specifically, the microorganism of the present invention is used as a suitable carbon source and nitrogen source. , Personnel, inorganic compounds, amino acids and/or vitamins, etc., in a common medium, and can be cultured while controlling temperature, pH, etc. under aerobic conditions.

In the present invention, the carbon source includes carbohydrates such as glucose, fructose, sucrose, maltose, mannitol, sorbitol, and the like; Alcohols such as sugar alcohol, glycerol, pyruvic acid, lactic acid, citric acid, and the like; Amino acids such as organic acids, glutamic acid, methionine, and lysine may be included, but are not limited thereto. In addition, natural organic nutrients such as starch hydrolyzate, molasses, black strap molasses, rice winter, cassava, sugarcane residue and corn steep liquor can be used. Carbohydrates such as molasses) can be used, and other appropriate amounts of carbon sources can be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more.

Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, etc., organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, skim soybean cake or its degradation products, etc. Can be used. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

The personnel may include first potassium phosphate, second potassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like may be used, and amino acids, vitamins, and/or suitable precursors may be included. These media or precursors may be added to the culture in a batch or continuous manner, but are not limited thereto.

In the present invention, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in an appropriate manner during the culture of the microorganism. In addition, during cultivation, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen, or carbon dioxide gas may be injected without the injection of gas to maintain the anaerobic and microaerobic state.

The temperature of the culture may be 27°C to 37°C, and more specifically 30°C to 35°C, but is not limited thereto. The culture period may be continued until the production amount of the desired useful substance is obtained, and specifically, may be 10 to 100 hours, but is not limited thereto.

The production method of O-acetyl homoserine of the present invention may further include the step of recovering O-acetyl homoserine from the cultured microorganism or a culture thereof.

The step of recovering the O-acetyl homoserine may be performed using a suitable method known in the art according to the method of culturing the microorganism of the present invention, for example, a batch, continuous, or fed-batch culture method. Acetyl homoserine can be recovered.

The recovery step may include a purification process.

The thus recovered O-acetyl homoserine can produce methionine by a two-stage method (Korean Patent No. 10-0905381) developed by the present inventors, that is, a second-stage process.

The second step is an enzyme having O-acetyl homoserine sulfhydrylase activity or the enzyme using O-acetyl homoserine and methyl mercaptan produced by the L-methionine precursor producing strain as substrates. It includes a process of producing L-methionine and organic acid through an enzymatic reaction using the included strain.

More specifically, the present invention provides a method of producing L-methionine using an enzymatic reaction such as O-acetyl homoserine sulfhydrylase using O-acetyl homoserine accumulated by the above method as a substrate.

When O-acetylhomoserine is used as an L-methionine precursor in the two-step process, specifically, Leptospira sp., Chromobacterium sp., and Hyphomonas sp. Microbial strain belonging to, more specifically, Leptospira meyeri, Pseudomonas aurogenosa, Hyphomonas Neptunium, and a microorganism belonging to Chromobacterium Violaceum. O-acetyl homoserine sulfhydrylase derived from the strain can be used.

The reaction is as follows.

CH ₃ SH + O-acetyl-L-homoserine <=> acetate + methionine

Such an additional methionine production process is disclosed in Korean Patent Registration No. 10-0905381, and the entire specification of the patent may be included as reference material of the present invention.

The microorganism having enhanced activity of the inner membrane protein YjeH of the present invention improves the excretion ability of O-acetyl homoserine, thereby improving the production efficiency of O-acetyl homoserine, and the microorganism of the present invention is widely used to produce O-acetyl homoserine. I can.

1 is a diagram showing a cleavage diagram of a yjeH vector (pBAC-yjeH vector) according to the present invention.

The present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: O-acetyl homoserine Productivity Preparation of eggplant strain

1-1. Wild type In E. coli metB Gene defect

In order to produce the O-acetyl homoserine-producing strain, Escherichia coli, a representative microorganism among microorganisms in the genus Escherichia, was used. To this end, wild-type E. coli E. coli K12 W3110 (ATCC27325) was obtained from the American Type Culture Collection (ATCC) and used. First, in order to block the production pathway of O-succinyl-L-homoserine to cystathion, the metB gene (SEQ ID NO: 4) encoding cystathionine synthase was deleted.

Specifically, an FRT one step PCR deletion method was used for the deletion of the metB gene (Wanner BL., Proc. Natl. Acad. Sci. USA 97: 6640-6645, 2000). PCR reaction using the pKD3 vector (Wanner BL, Proc. Natl. Acad. Sci. USA 97: 6640-6645, 2000) as a template was performed using the primers TKd of SEQ ID NOs: 13 and 14 to prepare a deletion cassette. I did. At this time, the PCR reaction 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 subjected to electrophoresis on a 1.0% agarose gel, and then DNA was purified from a 1.2 kbp band. The recovered DNA fragment was previously transformed with a pKD46 vector (Wanner BL., Proc. Natl. Acad. Sci. USA 97: 6640-6645, 2000) in E. coli (K12) W3110 strain using electroporation using electroporation. Introduced. The W3110 strain transformed with pKD46 for electroporation was cultured at 30° C. _{until OD 600} = 0.6 using LB medium containing 100 ug/L ampicillin and 5 mM arabinose. Then, it was used after washing twice with sterile distilled water and once with 10% glycerol. At this time, electroporation was performed at 2500V. The recovered strain was plated on an LB plate medium containing 25 μg/L chloramphenichol and cultured overnight at 37° C., and then strains showing resistance were selected.

After performing PCR under the same conditions as above using the same primers (SEQ ID NOs: 13 and 14) using the strain selected therefrom as a template, it was confirmed that the size of the gene was observed at 1.2 Kb on a 1.0% agarose gel. The deletion of the metB gene was confirmed. The strain confirmed to be defective was again transformed with pCP20 vector (Wanner BL., Proc. Natl. Acad. Sci. USA 97: 6640-6645, 2000) and cultured in LB medium, and again 1.0% through PCR under the same conditions. After electrophoresis on an agarose gel, the final metB gene-deficient strain whose gene size was reduced to 150 bp was selected, and it was confirmed that the chromophhenicol marker was removed from the strain.

The strain in which the metB gene encoding cystathionine synthase was deleted was designated as W3-B.

1-2. thrB Gene defect

In order to increase the amount of O-succinyl homoserine synthesis from the W3-B strain obtained in Example 1-1, the thrB gene (SEQ ID NO: 6) encoding homoserine kinase was deleted in the strain. . In order to delete the thrB gene, the same FRT one step PCR deletion method as in the case of the deletion of the metB gene disclosed in Example 1-1 was used.

First, in order to prepare theB deletion cassette, a PCR reaction using a pKD4 vector (Wanner BL., Proc. Natl. Acad. Sci. USA 97: 6640-6645, 2000) as a template was performed to prepare a deletion cassette. I did. Specifically, using the primers of SEQ ID NOs: 15 and 16, the denaturation step was performed at 94°C for 30 seconds, the annealing step was performed at 55°C for 30 seconds, and the extension step was performed at 72°C for 1 minute. And, a PCR reaction was performed in which this was repeated 30 times.

The resulting PCR product was subjected to electrophoresis on a 1.0% agarose gel, and then DNA was purified from a 1.6 kbp band. The recovered DNA fragment was introduced into a W3-B strain previously transformed with pKD46 vector by electroporation. The recovered strain was plated on an LB plate medium containing 50 μg/L kanamycin and cultured overnight at 37° C., and then strains showing resistance were selected.

Using the strain selected through the above process as a direct template, PCR was performed under the same conditions as above using primers of SEQ ID NOs: 15 and 16, and then strains with a gene size of 1.6 Kb were selected on a 1.0% agarose gel. By doing so, the deletion of the thrB gene was confirmed. The identified strain was transformed again with the pCP20 vector and cultured in LB medium, and a final thrB gene deletion strain with a size of the gene reduced to 150 bp was produced on a 1.0% agarose gel through PCR under the same conditions, and a kanamycin marker It was confirmed that was removed. The strain in which the thrB gene encoding homoserine kinase was deleted, thus constructed and selected, was named W3-BT strain.

An example of the contents of the metB and thrB gene deletion strains is disclosed in Korean Patent Registration No. 10-0905381 or International Publication No. WO2008/013432, and the entire specification of the patent may be included as a reference material of the present invention. .

1-3. Homoserine acetyl Transferase Active Variant metA Production of transgenic strains

In order to enhance homoserine acetyltransferase activity in the strain obtained in Example 1-2, a mutant metA gene encoding homoserine acetyl transferase having enhanced activity in the strain (SEQ ID NO: 8) Was intended to be introduced.

To this end, in order to first prepare a mutant metA gene with enhanced activity, the metA gene was amplified and obtained through PCR using primers of SEQ ID NOs: 17 and 18 as a template using the chromosome of the wild-type strain W3110. The SEQ ID NO: 17 and a primer of SEQ ID NO: 18 used in the PCR is based on the E. coli chromosomal DNA sequence of NC_000913, which are registered with the US National Institutes of Health Gene Bank (NIH Gene Bank), the respective restriction enzymes EcoR Ⅴ site and a Hind Ⅲ site It was made to have.

The obtained PCR product and the pCL1920 plasmid containing pcj1 were cloned by treatment with restriction enzymes EcoR V and Hind III. After transforming E. coli DH5α with the cloned plasmid, it was cultured in an LB plate medium containing 50 μg/mL of spectinomycin, and the transformed E. The plasmid thus obtained was named pCL_Pcj1_metA.

Based on the obtained pCL_Pcj1_metA, a mutant metA gene was prepared using a site-directed mutagenesis kit (Stratagene, USA). Specifically, using the pCL_Pcj1_metA plasmid as a template, using the primers of SEQ ID NOs: 19 and 20, glycine (Gly), amino acid 111 of homoserine acetyl transferase, was substituted with glutamic acid (Glu) (G111E). The plasmid containing the thus prepared G111E metA gene was named pCL_Pcj1_metA(EL).

In addition, in order to substitute amino acid 111 of the homoserine acetyl transferase from glycine to glutamic acid, and to further substitute amino acid 112 from leucine to histidine, primers of SEQ ID NOs: 21 and 22 were used. Accordingly, a plasmid containing the metA gene in which amino acid 111 was substituted from glycine to glutamic acid and amino acid 112 from leucine to histidine was named pCL_Pcj1_metA(EH).

Next, in order to create a replacement cassette for introducing and replacing the mutant metA gene into the strain, the denaturation step using the pKD3 vector as a template and the primers of SEQ ID NOs: 27 and 28 is 30 at 94°C. Second, the annealing step was performed at 55° C. for 30 seconds, and the extension step was performed at 72° C. for 2 minutes, and a PCR reaction was carried out 30 times. For the metA (EH) portion of the replacement cassette, primers of SEQ ID NOs: 23 and 24 were used as a template with pCL-Pcj1-metA (EH) as a template, and primers of SEQ ID NOs: 25 and 26 were used for the metA wild-type portion to obtain respective PCR products. The W3-BT strain prepared in Example 1-2 in which three PCR products were previously transformed with a pKD46 vector to prepare a metA (EH) replacement cassette containing a chloramphenicol marker portion using primers of SEQ ID NOs: 23 and 26. Was introduced using electroporation. The strain confirmed to be introduced was transformed again with the pCP20 vector and cultured in LB medium, and the strain in which the chloramphenicol marker was removed and the metA gene was replaced with metA (EH) was named W3-BTA.

An example of the contents of such a homoserine acetyl transferase-enhanced strain, etc., is disclosed in Korean Patent Registration No. 10-1335841 or International Publication No. WO2012/087039, and the entire specification of the patent may be included as a reference material of the present invention. have.

1-4. ppc , aspC And asd Gene 2 copy Preparation of the containing strain

In order to increase the O-acetin homoserine production ability of the W3-BTA strain prepared in Example 1-3, a previously known biosynthetic pathway enhancement strategy was introduced. Phosphoenolpyruvate carboxylase involved in the biosynthesis of oxaloacetate from phosphoenolpyruvate, aspartate aminotransferase and β involved in the biosynthesis of aspartate from oxaloacetate -Aspartate-semialdehyde dehydrogenase, which is involved in the biosynthesis of homoserine from aspartyl phosphate, that is, the ppc gene, the aspC gene, and the asd gene were amplified by 2 copies.

Accordingly, the ppc gene was amplified to 2 copies using primers SEQ ID NOs: 29, 30, 31 and 32, and the aspC gene was amplified to 2 copies using primers SEQ ID NOs: 33 and 34, and the asd gene was SEQ ID NO: 35, Using primers 36, 37 and 38, each gene was amplified to 2 copies.

Through the above process, a strain having an enhanced O-acetyl homoserine biosynthetic pathway based on the W3-BTA strain was named W3-BTA2PCD (=WCJM).

Examples of the contents of such phosphoenolpyruvate carboxylase, aspartate aminotransferase, and aspartate semihaldehyde dehydrogenase-enhanced strains, etc. can be found in Korean Patent Registration No. 10-0905381 or International Publication WO2008 It is disclosed in /013432, and the entire specification of the patent may be included as a reference material of the present invention.

1-5. Flask culture experiment

In order to test the production of O-acetyl homoserine of the strains prepared in Examples 1-3 and 1-4, triangular plast culture was performed. After inoculating W3110, W3-BTA, and WCJM strains in LB medium and incubating overnight at 33° C., single colonies were inoculated in 3 mL LB medium and incubated at 33° C. for 5 hours, and then 25 mL O-acetyl homoserine was produced. It was diluted 200 times in a 250 mL Erlenmeyer flask containing a medium and incubated at 33° C. 200 rpm for 30 hours, and the production of O-acetyl homoserine was confirmed through HPLC analysis. The medium composition used for this is summarized in Table 1 below.

Composition of O-acetyl homoserine production flask medium Furtherance Concentration (per liter) glucose 40 g Ammonium sulfate 17 g KH ₂ PO ₄ 1.0 g MgSO ₄ 7H ₂ O 0.5 g FeSO ₄ 7H 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

The results of confirming the production amount of O-acetyl homoserine through HPLC analysis incubated for 30 hours using the medium are summarized in Table 2 below.

O-acetyl homoserine production through flask culture OD
(562 nm) Glucose consumption
(g/L) O-AH
(g/L) W3110 14.2 40 0 W3-bta 8.4 36 0.9 WCJM 9.6 35 1.2

As can be seen in Table 2, O-acetyl homoserine was not produced at all in the wild-type W3110, but 0.9 g/L of O-acetyl homoserine was produced in the W3-BTA strain, and 1.2 g in the WCJM strain with enhanced biosynthetic pathway. /L was created.

Example 2: O-acetyl homoserine Productivity Identification of increasing membrane proteins

The present inventors tried to apply yjeH (SEQ ID NO: 1), which has not disclosed a correlation between O-acetyl homoserine excretion ability and O-acetyl homoserine production ability.

In order to enhance the yjeH gene in the strain, the yjeH gene was cloned into a bac vector, and a Hind III restriction enzyme site in the bac vector was used. The used bac vector was Epicentre's copycontrol BAC Cloning kit (Cat. No. CCBAC1H- Hind III).

First, using the primers of SEQ ID NOs: 9 and 10 to obtain the yjeH gene, the denaturation step was at 94°C for 30 seconds, the annealing step was at 55°C for 30 seconds, and the extension step was at 68°C. The PCR reaction was carried out for 1 minute and performed 30 times. The resulting PCR product was subjected to electrophoresis on a 1.0% agarose gel, and then DNA was purified from a 1.2 kbp band. The purified DNA was treated with restriction enzyme Hind III overnight at 37[deg.] C., and after further purification, yjeH and BAC vectors were cloned using T4 ligase. After transforming E. coli DH5α using the cloned plasmid, a plasmid was obtained by selecting the transformed E. coli DH5α in an LB plate medium containing 50 μg/ml of chloramphenichol. The prepared plasmid was introduced into the O-acetyl homoserine producing strains W3-BTA and WCJM, and the flask was evaluated for the production ability of O-acetyl homoserine.

The resulting PCR product was subjected to electrophoresis on a 1.0% agarose gel, and then DNA was purified from a 1.2 kbp band. The purified DNA was treated with restriction enzyme Hind III overnight at 37°C, and after further purification, yjeH and BAC vectors were cloned using T4 ligase. After transforming E. coli DH5α using the cloned plasmid, a plasmid was obtained by selecting the transformed E. coli DH5α in an LB plate medium containing 50 μg/ml of chloramphenichol. The prepared plasmid was introduced into the O-acetyl homoserine producing strains W3-BTA and WCJM, and the flask was evaluated for the production ability of O-acetyl homoserine.

Specifically, each strain was plated on LB solid medium and then cultured overnight in a 33° C. incubator. After inoculating a single colony of the strain cultured overnight on LB plate medium into 3 ml LB medium, incubating it at 33° C. for 5 hours, and diluting 200-fold in a 250 ml Erlenmeyer flask containing 25 ml O-acetyl homoserine production medium. Then, it was incubated for 30 hours in an incubator at 33° C. and 200 rpm, and the production amount of O-acetyl homoserine was confirmed through HPLC analysis. The results are summarized in Table 3 below.

Measurement of O-acetyl homoserine production through flask culture OD
(562nm) Glucose consumption
(g/L) O-AH
(g/L) W3-BTA/pBAC 9.5 35 0.8 WCJM/pBAC 9.6 35 1.2 W3-BTA/ pBAC-yjeH 9.8 36 1.5 WCJM/pBAC-yjeH 10.1 37 2.3

As can be seen in Table 3, the introduction of yjeH plasmid into WCJM had a higher OD than the control strain containing the empty vector, and glucose consumption was also improved. It was confirmed that 2.3 g/L of O-acetyl homoserine was produced, and the production ability of O-acetyl homoserine was improved by introducing yjeH.

Example 3: yjeH Promoter enhanced plasmid construction and O-acetyl homoserine Productivity evaluation

3-1. yjeH Construction of promoter-enhanced plasmid

On the basis of the plasmid prepared in Example 2, an experiment was conducted to replace three promoters with stronger expression-inducing activity than the intrinsic yjeH promoter.

Specifically, a promoter-enhancing strain was prepared in PCL vector using pro, cysk or icd promoter. It was produced using the smaI restriction enzyme site of the PCL vector, the icd promoter (SEQ ID NO: 51) is SEQ ID NO: 39 and 40 primers, the pro promoter (SEQ ID NO: 52) is SEQ ID NOs: 41 and 42 primers, cysk promoter (SEQ ID NO: 53) Was prepared by amplifying by PCR using SEQ ID NOs: 43 and 44 primers. The prepared plasmid was introduced into WCJM, and the production ability of O-acetyl homoserine was evaluated in the flask.

Specifically, each strain was plated on an LB plate medium and then cultured overnight in an incubator at 33° C., the strain cultured overnight on an LB solid medium was inoculated into the 25 ml titer medium shown above, and then at 33° C. and 200 rpm. Incubated for 40 hours in an incubator, and the results are shown in Table 4.

Measurement of O-acetyl homoserine production through flask culture OD
(562nm) Glucose consumption
(g/L) O-AH
(g/L) WCJM/pCL1920 9.6 35 1.2 WCJM/pCL-yjeH 10.1 37 2.3 WCJM/pCL-Picd-yjeH 10.5 38 3.1 WCJM/pCL-Ppro-yjeH 10.7 38 3.5 WCJM/pCL-Pcysk-yjeH 9.4 39 4.4

As can be seen in Table 4, in the case of the strain into which the pCL-Pcysk-yjeH plasmid was introduced, OD decreased compared to the autogenous promoter, but showed a fast sugar consumption rate, and O-acetyl homoserine 4.4 g/L was produced, resulting in the highest production capacity. .

3-2. Production of biosynthetic pathway genes and promoter-enhanced plasmids

In order to maximize the production capacity of O-acetyl homoserine, a plasmid was constructed to enhance the biosynthetic pathway to homoserine. Aspartate kinase, homoserine O-acetyltransferase, and yjeH were cloned into a PCL vector using pCL-thrA-metX plasmid.

Specifically, in order to obtain the yjeH gene, the denaturation step was performed at 94°C for 30 seconds, the annealing step was at 55°C for 30 seconds, and the extension step was at 68°C using primers of SEQ ID NOs: 11 and 12. The PCR reaction was carried out for 1 minute and performed 30 times.

The resulting PCR product was subjected to electrophoresis on a 1.0% agarose gel, and then DNA was purified from a 1.2 kbp band. The purified DNA and plasmid were treated with restriction enzyme kpnI overnight at 37°C, and after further purification, yjeH and pCL vectors were cloned using T4 ligase. After transforming E. coli DH5α using the cloned plasmid, a plasmid was obtained by selecting the transformed E. coli DH5α in an LB plate medium containing 50 µg/mL spectinomycin. The prepared plasmid was introduced into WCJM, an O-acetyl homoserine producer, and the flask was evaluated for the production ability of O-acetyl homoserine. The prepared plasmids were all three types, and the three promoters prepared in Example 3-1 were used. Three kinds of plasmids were put into the WCJM strain, and flask evaluation was performed in the same manner as in Example 3-1, and the results are shown in Table 5 below.

Measurement of O-acetyl homoserine production through flask culture OD
(562nm) Glucose consumption
(g/L) O-AH
(g/L) WCJM/pC2 9.6 35 1.5 WCJM/pC2-yjeH 10.1 37 2.8 WCJM/pC2-Picd-yjeH 10.5 38 4.2 WCJM/pC2-Ppro-yjeH 10.7 38 4.5 WCJM/pC2-Pcysk-yjeH 9.4 39 5.4

As can be seen in Table 5, as a result of simultaneously enhancing the biosynthetic pathway and yjeH, the production capacity of O-acetyl homoserine was further improved, and the sequence was the same as previously described, in which the pC2-Pcysk-yjeH plasmid was introduced. In the case of the strain, OD decreased compared to the autologous promoter, but showed a fast sugar consumption rate, and 5.4 g/L of O-acetyl homoserine was produced, resulting in the highest production capacity.

Example 4: implicit yjeH Promoter-enhanced strain production and O-acetyl homoserine Productivity evaluation

4-1. Implicit yjeH Preparation and evaluation of fortified strains

In order to produce a strain that enhanced the intrinsic yjeH gene activity of WCJM, an O-acetyl homoserine-producing strain, an experiment was performed to replace the promoter. In the WCJM strain of the present invention, 1 copy of yjeH exists, and in order to enhance this gene, the strain was constructed by enhancing the promoter instead of increasing the number of copies of yjeH.

Specifically, as the promoter for replacement, the icd, cysK and pro promoters (Picd, Pcysk and Ppro) whose activity was confirmed in Example 3 were selected, and the FRT one step PCR deletion method specified above was used. (Wanner BL., Proc. Natl. Acad. Sci. USA 97: 6640-6645, 2000). The icd promoter was used as SEQ ID NOs: 45 and 46 primers, the cysk promoter was used as SEQ ID NOs: 47 and 48 primers, and the pro promoters were SEQ ID NOs: 49 and 50 primers. In the PCR, the denaturation step was performed at 94°C for 30 seconds, the annealing step was performed at 55°C for 30 seconds, and the extension step was performed at 72°C for 1 minute, and this was performed 30 times.

The PCR product obtained as a result of the above experiment was subjected to electrophoresis on a 1.0% agarose gel, and then DNA was purified from a band having a size of 2.5 kbp. The recovered DNA fragment was electroporated into a WCJM strain previously transformed with a pKD46 vector (Wanner BL., Proc. Natl. Acad. Sci. USA 97: 6640-6645, 2000). _{The WCJM strain transformed with pKD46 for electroporation was cultured at 30° C. to OD 600} = 0.6 using LB medium containing 100 μg/L ampicillin and 5 mM arabinose, and then sterilized. It was used after washing twice with distilled water and once with 10% glycerol. Electroporation was performed at 2500V. The recovered strain was plated on an LB plate medium containing 25 μg/L chloramphenichol, cultured overnight at 37° C., and then strains showing resistance were selected.

Using the selected strain as a template, PCR was performed under the same conditions as above using the primers of SEQ ID NOs: 45 and 46 for the icd promoter, the primers for SEQ ID NOs: 47 and 48 for the cysk promoter, and the primers for SEQ ID NOs: 49 and 50 for the pro promoter. Thereafter, it was confirmed that the yjeH intrinsic promoter was replaced with each foreign promoter by confirming that the size of the gene was observed at 2.5 Kb on a 1.0% agarose gel. The strain confirmed to be replaced was again transformed with pCP20 vector (PNAS (2000) vol97: P6640-6645) and cultured in LB medium, and the gene size was 1Kb on a 1.0% agarose gel through PCR under the same conditions. A small final promoter replacement strain was prepared, and it was confirmed that the kanamycin marker was removed. The thus-produced strain was named WCJM-PIY for the icd promoter replacement strain, WCJM-PCY for the cysk promoter replacement strain, and WCJM-PPY for the pro promoter replacement strain according to each promoter. Flask culture evaluation was performed to measure the O-acetyl homoserine production ability of the yjeH promoter replacement strain, and the results are shown in Table 6.

Measurement of O-acetyl homoserine production through flask culture OD
(562nm) Glucose consumption
(g/L) O-AH
(g/L) WCJM 9.6 35 1.2 WCJM-PIY 9.2 38 1.8 WCJM-PCY 10.5 38 3.1 WCJM-PPY 10.1 38 1.9

As can be seen in Table 6, the yjeH gene expression was enhanced by replacing the intrinsic yjeH promoter in the chromosome with a promoter with strong expression activity. Although the homoserine production ability did not show a rapid change, it was confirmed that the O-acetyl homoserine production ability of each strain was increased compared to the production strain WCJM.

4-2. O-acetyl homoserine in high yield strain yjeH Promoter enhanced strain production and O-acetyl homoserine Productivity evaluation

Conventionally, a method of producing a strain producing O-acetyl homoserine using a strain having the ability to produce threonine through NTG mutation derived from wild-type W3110 is known (International Publication No. WO2012/087039). The strain producing the high-yield O-acetyl homoserine produced at this time has been deposited with the Korean Microbial Conservation Center under the accession number KCCM11146P.

The accession number KCCM11146P strain exhausts 40 g/L of glucose when cultured in a flask and produces about 15 to 16 g/L of O-acetyl homoserine, and has a high yield of O-acetyl homoserine production capability. Accordingly, the present inventors have already confirmed whether the ability to produce O-acetyl homoserine can be further enhanced by enhancing the yjeH gene of the present invention based on the strain having a high production capacity of O-acetyl homoserine.

Specifically, it was observed by replacing the promoter of the yjeH gene with a promoter having high expression-inducing activity, and this was carried out using the same method as in Example 4-1. The yjeH promoter replacement strain of the KCCM11146P strain thus produced was named KCCM11146P-PIY for the icd promoter replacement strain, KCCM11146P-PCY for the cysk promoter replacement strain, and KCCM11146P-PPY for the pro promoter replacement strain according to each promoter.

Flask culture evaluation was performed to measure the O-acetyl homoserine production ability of the yjeH promoter replacement strain. Specifically, KCCM11146P, KCCM11146P-PIY, KCCM11146P-PCY, or KCCM11146P-PPY strain was inoculated in LB medium and cultured overnight at 33°C, and then single colonies were inoculated in 3 ml LB medium and then cultured at 33°C for 5 hours. Then, it was diluted 200 times in a 250 ml Erlenmeyer flask to which 25 ml O-acetyl homoserine production medium was added, and incubated at 33° C. 200 rpm for 30 hours, and the production of O-acetyl homoserine was confirmed through HPLC analysis. The results of the above experiment are summarized in Table 7 below.

Measurement of O-acetyl homoserine production through flask culture OD
(562nm) Glucose consumption
(g/L) O-AH
(g/L) KCCM11146P 18.3 40 14.2 KCCM11146P- PIY 16.2 40 16.3 KCCM11146P- PCY 19.2 40 18.2 KCCM11146P- PPY 18.8 40 16.2

As can be seen in Table 7 above, 14.2 g/L of O-acetyl homoserine was produced in the KCCM11146P strain, and the PCY was 18.2 g/L in the case of the promoter replacement strain, resulting in the highest production of O-acetyl homoserine, PIY and PPY. In the case of, the production of O-acetyl homoserine was also increased compared to the original strain.

The present inventors confirmed that the production of O-acetylhomoserine increased in the yjeH-enhanced strain based on the KCCM11146P strain, and named the strain'CA05-4008', and then under the Treaty of Budapest, the Korean Microbiological Conservation Center ( KCCM) was deposited with the accession number KCCM11484P.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

Korea Microorganism Conservation Center (overseas) KCCM11484P 20131122

<110> CJ CheilJedang Corporation <120> Microorganisms producing O-acetyl-homoserine and the method of producing O-Acetyl-homoserine using the same <130> PA131258/KR <160> 53 <170> KopatentIn 2.0 <210> 1 <211> 418 <212> PRT <213> E.coli yjeH <400> 1 Met Ser Gly Leu Lys Gln Glu Leu Gly Leu Ala Gln Gly Ile Gly Leu 1 5 10 15 Leu Ser Thr Ser Leu Leu Gly Thr Gly Val Phe Ala Val Pro Ala Leu 20 25 30 Ala Ala Leu Val Ala Gly Asn Asn Ser Leu Trp Ala Trp Pro Val Leu 35 40 45 Ile Ile Leu Val Phe Pro Ile Ala Ile Val Phe Ala Ile Leu Gly Arg 50 55 60 His Tyr Pro Ser Ala Gly Gly Val Ala His Phe Val Gly Met Ala Phe 65 70 75 80 Gly Ser Arg Leu Glu Arg Val Thr Gly Trp Leu Phe Leu Ser Val Ile 85 90 95 Pro Val Gly Leu Pro Ala Ala Leu Gln Ile Ala Ala Gly Phe Gly Gln 100 105 110 Ala Met Phe Gly Trp His Ser Trp Gln Leu Leu Leu Ala Glu Leu Gly 115 120 125 Thr Leu Ala Leu Val Trp Tyr Ile Gly Thr Arg Gly Ala Ser Ser Ser 130 135 140 Ala Asn Leu Gln Thr Val Ile Ala Gly Leu Ile Val Ala Leu Ile Val 145 150 155 160 Ala Ile Trp Trp Ala Gly Asp Ile Lys Pro Ala Asn Ile Pro Phe Pro 165 170 175 Ala Pro Gly Asn Ile Glu Leu Thr Gly Leu Phe Ala Ala Leu Ser Val 180 185 190 Met Phe Trp Cys Phe Val Gly Leu Glu Ala Phe Ala His Leu Ala Ser 195 200 205 Glu Phe Lys Asn Pro Glu Arg Asp Phe Pro Arg Ala Leu Met Ile Gly 210 215 220 Leu Leu Leu Ala Gly Leu Val Tyr Trp Gly Cys Thr Val Val Val Leu 225 230 235 240 His Phe Asp Ala Tyr Gly Glu Lys Met Ala Ala Ala Ala Ser Leu Pro 245 250 255 Lys Ile Val Val Gln Leu Phe Gly Val Gly Ala Leu Trp Ile Ala Cys 260 265 270 Val Ile Gly Tyr Leu Ala Cys Phe Ala Ser Leu Asn Ile Tyr Ile Gln 275 280 285 Ser Phe Ala Arg Leu Val Trp Ser Gln Ala Gln His Asn Pro Asp His 290 295 300 Tyr Leu Ala Arg Leu Ser Ser Arg His Ile Pro Asn Asn Ala Leu Asn 305 310 315 320 Ala Val Leu Gly Cys Cys Val Val Ser Thr Leu Val Ile His Ala Leu 325 330 335 Glu Ile Asn Leu Asp Ala Leu Ile Ile Tyr Ala Asn Gly Ile Phe Ile 340 345 350 Met Ile Tyr Leu Leu Cys Met Leu Ala Gly Cys Lys Leu Leu Gln Gly 355 360 365 Arg Tyr Arg Leu Leu Ala Val Val Gly Gly Leu Leu Cys Val Leu Leu 370 375 380 Leu Ala Met Val Gly Trp Lys Ser Leu Tyr Ala Leu Ile Met Leu Ala 385 390 395 400 Gly Leu Trp Leu Leu Leu Pro Lys Arg Lys Thr Pro Glu Asn Gly Ile 405 410 415 Thr Thr <210> 2 <211> 1257 <212> DNA <213> E.coli yjeH <400> 2 atgagtggac tcaaacaaga actggggctg gcccagggca ttggcctgct atcgacgtca 60 ttattaggca ctggcgtgtt tgccgttcct gcgttagctg cgctggtagc gggcaataac 120 agcctgtggg cgtggcccgt tttgattatc ttagtgttcc cgattgcgat tgtgtttgcg 180 attctgggtc gccactatcc cagcgcaggc ggcgtcgcgc acttcgtcgg tatggcgttt 240 ggttcgcggc ttgagcgagt caccggctgg ctgtttttat cggtcattcc cgtgggtttg 300 cctgccgcac tacaaattgc cgccgggttc ggccaggcga tgtttggctg gcatagctgg 360 caactgttgt tggcagaact cggtacgctg gcgctggtgt ggtatatcgg tactcgcggt 420 gccagttcca gtgctaatct acaaaccgtt attgccggac ttatcgtcgc gctgattgtc 480 gctatctggt gggcgggcga tatcaaacct gcgaatatcc cctttccggc acctggtaat 540 atcgaactta ccgggttatt tgctgcgtta tcagtgatgt tctggtgttt tgtcggtctg 600 gaggcatttg cccatctcgc ctcggaattt aaaaatccag agcgtgattt tcctcgtgct 660 ttgatgattg gtctgctgct ggcaggatta gtctactggg gctgtacggt agtcgtctta 720 cacttcgacg cctatggtga aaaaatggcg gcggcagcat cgcttccaaa aattgtagtg 780 cagttgttcg gtgtaggagc gttatggatt gcctgcgtga ttggctatct ggcctgcttt 840 gccagtctca acatttatat acagagcttc gcccgcctgg tctggtcgca ggcgcaacat 900 aatcctgacc actacctggc acgcctctct tctcgccata tcccgaataa tgccctcaat 960 gcggtgctcg gctgctgtgt ggtgagcact ttggtgattc atgctttaga gatcaatctg 1020 gacgctctta ttatttatgc caatggcatc tttattatga tttatctgtt atgcatgctg 1080 gcaggctgta aattattgca aggacgttat cgactactgg cggtggttgg cgggctgtta 1140 tgcgttctgt tactggcaat ggtcggctgg aaaagtctct atgcgctgat catgctggcg 1200 gggttatggc tgttgctgcc aaaacgaaaa acgccggaaa atggcataac cacataa 1257 <210> 3 <211> 386 <212> PRT <213> E.coli metB <400> 3 Met Thr Arg Lys Gln Ala Thr Ile Ala Val Arg Ser Gly Leu Asn Asp 1 5 10 15 Asp Glu Gln Tyr Gly Cys Val Val Pro Pro Ile His Leu Ser Ser Thr 20 25 30 Tyr Asn Phe Thr Gly Phe Asn Glu Pro Arg Ala His Asp Tyr Ser Arg 35 40 45 Arg Gly Asn Pro Thr Arg Asp Val Val Gln Arg Ala Leu Ala Glu Leu 50 55 60 Glu Gly Gly Ala Gly Ala Val Leu Thr Asn Thr Gly Met Ser Ala Ile 65 70 75 80 His Leu Val Thr Thr Val Phe Leu Lys Pro Gly Asp Leu Leu Val Ala 85 90 95 Pro His Asp Cys Tyr Gly Gly Ser Tyr Arg Leu Phe Asp Ser Leu Ala 100 105 110 Lys Arg Gly Cys Tyr Arg Val Leu Phe Val Asp Gln Gly Asp Glu Gln 115 120 125 Ala Leu Arg Ala Ala Leu Ala Glu Lys Pro Lys Leu Val Leu Val Glu 130 135 140 Ser Pro Ser Asn Pro Leu Leu Arg Val Val Asp Ile Ala Lys Ile Cys 145 150 155 160 His Leu Ala Arg Glu Val Gly Ala Val Ser Val Val Asp Asn Thr Phe 165 170 175 Leu Ser Pro Ala Leu Gln Asn Pro Leu Ala Leu Gly Ala Asp Leu Val 180 185 190 Leu His Ser Cys Thr Lys Tyr Leu Asn Gly His Ser Asp Val Val Ala 195 200 205 Gly Val Val Ile Ala Lys Asp Pro Asp Val Val Thr Glu Leu Ala Trp 210 215 220 Trp Ala Asn Asn Ile Gly Val Thr Gly Gly Ala Phe Asp Ser Tyr Leu 225 230 235 240 Leu Leu Arg Gly Leu Arg Thr Leu Val Pro Arg Met Glu Leu Ala Gln 245 250 255 Arg Asn Ala Gln Ala Ile Val Lys Tyr Leu Gln Thr Gln Pro Leu Val 260 265 270 Lys Lys Leu Tyr His Pro Ser Leu Pro Glu Asn Gln Gly His Glu Ile 275 280 285 Ala Ala Arg Gln Gln Lys Gly Phe Gly Ala Met Leu Ser Phe Glu Leu 290 295 300 Asp Gly Asp Glu Gln Thr Leu Arg Arg Phe Leu Gly Gly Leu Ser Leu 305 310 315 320 Phe Thr Leu Ala Glu Ser Leu Gly Gly Val Glu Ser Leu Ile Ser His 325 330 335 Ala Ala Thr Met Thr His Ala Gly Met Ala Pro Glu Ala Arg Ala Ala 340 345 350 Ala Gly Ile Ser Glu Thr Leu Leu Arg Ile Ser Thr Gly Ile Glu Asp 355 360 365 Gly Glu Asp Leu Ile Ala Asp Leu Glu Asn Gly Phe Arg Ala Ala Asn 370 375 380 Lys Gly 385 <210> 4 <211> 1161 <212> DNA <213> E.coli metB <400> 4 atgacgcgta aacaggccac catcgcagtg cgtagcgggt taaatgacga cgaacagtat 60 ggttgcgttg tcccaccgat ccatcctcc agcacctata actttaccgg atttaatgaa 120 ccgcgcgcgc atgattactc gcgtcgcggc aacccaacgc gcgatgtggt tcagcgtgcg 180 ctggcagaac tggaaggtgg tgctggtgca gtacttacta ataccggcat gtccgcgatt 240 cacctggtaa cgaccgtctt tttgaaacct ggcgatctgc tggttgcgcc gcacgactgc 300 tacggcggta gctatcgcct gttcgacagt ctggcgaaac gcggttgcta tcgcgtgttg 360 tttgttgatc aaggcgatga acaggcatta cgggcagcgc tggcagaaaa acccaaactg 420 gtactggtag aaagcccaag taatccattg ttacgcgtcg tggatattgc gaaaatctgc 480 catctggcaa gggaagtcgg ggcggtgagc gtggtggata acaccttctt aagcccggca 540 ttacaaaatc cgctggcatt aggtgccgat ctggtgttgc attcatgcac gaaatatctg 600 aacggtcact cagacgtagt ggccggcgtg gtgattgcta aagacccgga cgttgtcact 660 gaactggcct ggtgggcaaa caatattggc gtgacgggcg gcgcgtttga cagctatctg 720 ctgctacgtg ggttgcgaac gctggtgccg cgtatggagc tggcgcagcg caacgcgcag 780 gcgattgtga aatacctgca aacccagccg ttggtgaaaa aactgtatca cccgtcgttg 840 ccggaaaatc aggggcatga aattgccgcg cgccagcaaa aaggctttgg cgcaatgttg 900 agttttgaac tggatggcga tgagcagacg ctgcgtcgtt tcctgggcgg gctgtcgttg 960 tttacgctgg cggaatcatt agggggagtg gaaagtttaa tctctcacgc cgcaaccatg 1020 acacatgcag gcatggcacc agaagcgcgt gctgccgccg ggatctccga gacgctgctg 1080 cgtatctcca ccggtattga agatggcgaa gatttaattg ccgacctgga aaatggcttc 1140 cgggctgcaa acaaggggta a 1161 <210> 5 <211> 310 <212> PRT <213> E.coli thrB <400> 5 Met Val Lys Val Tyr Ala Pro Ala Ser Ser Ala Asn Met Ser Val Gly 1 5 10 15 Phe Asp Val Leu Gly Ala Ala Val Thr Pro Val Asp Gly Ala Leu Leu 20 25 30 Gly Asp Val Val Thr Val Glu Ala Ala Glu Thr Phe Ser Leu Asn Asn 35 40 45 Leu Gly Arg Phe Ala Asp Lys Leu Pro Ser Glu Pro Arg Glu Asn Ile 50 55 60 Val Tyr Gln Cys Trp Glu Arg Phe Cys Gln Glu Leu Gly Lys Gln Ile 65 70 75 80 Pro Val Ala Met Thr Leu Glu Lys Asn Met Pro Ile Gly Ser Gly Leu 85 90 95 Gly Ser Ser Ala Cys Ser Val Val Ala Ala Leu Met Ala Met Asn Glu 100 105 110 His Cys Gly Lys Pro Leu Asn Asp Thr Arg Leu Leu Ala Leu Met Gly 115 120 125 Glu Leu Glu Gly Arg Ile Ser Gly Ser Ile His Tyr Asp Asn Val Ala 130 135 140 Pro Cys Phe Leu Gly Gly Met Gln Leu Met Ile Glu Glu Asn Asp Ile 145 150 155 160 Ile Ser Gln Gln Val Pro Gly Phe Asp Glu Trp Leu Trp Val Leu Ala 165 170 175 Tyr Pro Gly Ile Lys Val Ser Thr Ala Glu Ala Arg Ala Ile Leu Pro 180 185 190 Ala Gln Tyr Arg Arg Gln Asp Cys Ile Ala His Gly Arg His Leu Ala 195 200 205 Gly Phe Ile His Ala Cys Tyr Ser Arg Gln Pro Glu Leu Ala Ala Lys 210 215 220 Leu Met Lys Asp Val Ile Ala Glu Pro Tyr Arg Glu Arg Leu Leu Pro 225 230 235 240 Gly Phe Arg Gln Ala Arg Gln Ala Val Ala Glu Ile Gly Ala Val Ala 245 250 255 Ser Gly Ile Ser Gly Ser Gly Pro Thr Leu Phe Ala Leu Cys Asp Lys 260 265 270 Pro Glu Thr Ala Gln Arg Val Ala Asp Trp Leu Gly Lys Asn Tyr Leu 275 280 285 Gln Asn Gln Glu Gly Phe Val His Ile Cys Arg Leu Asp Thr Ala Gly 290 295 300 Ala Arg Val Leu Glu Asn 305 310 <210> 6 <211> 933 <212> DNA <213> E,coli thrB <400> 6 atggttaaag tttatgcccc ggcttccagt gccaatatga gcgtcgggtt tgatgtgctc 60 ggggcggcgg tgacacctgt tgatggtgca ttgctcggag atgtagtcac ggttgaggcg 120 gcagagacat tcagtctcaa caacctcgga cgctttgccg ataagctgcc gtcagaacca 180 cgggaaaata tcgtttatca gtgctgggag cgtttttgcc aggaactggg taagcaaatt 240 ccagtggcga tgaccctgga aaagaatatg ccgatcggtt cgggcttagg ctccagtgcc 300 tgttcggtgg tcgcggcgct gatggcgatg aatgaacact gcggcaagcc gcttaatgac 360 actcgtttgc tggctttgat gggcgagctg gaaggccgta tctccggcag cattcattac 420 gacaacgtgg caccgtgttt tctcggtggt atgcagttga tgatcgaaga aaacgacatc 480 atcagccagc aagtgccagg gtttgatgag tggctgtggg tgctggcgta tccggggatt 540 aaagtctcga cggcagaagc cagggctatt ttaccggcgc agtatcgccg ccaggattgc 600 attgcgcacg ggcgacatct ggcaggcttc attcacgcct gctattcccg tcagcctgag 660 cttgccgcga agctgatgaa agatgttatc gctgaaccct accgtgaacg gttactgcca 720 ggcttccggc aggcgcggca ggcggtcgcg gaaatcggcg cggtagcgag cggtatctcc 780 ggctccggcc cgaccttgtt cgctctgtgt gacaagccgg aaaccgccca gcgcgttgcc 840 gactggttgg gtaagaacta cctgcaaaat caggaaggtt ttgttcatat ttgccggctg 900 gatacggcgg gcgcacgagt actggaaaac taa 933 <210> 7 <211> 309 <212> PRT <213> Artificial Sequence <220> <223> metA <400> 7 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 <210> 8 <211> 933 <212> DNA <213> Artificial Sequence <220> <223> metA <400> 8 atggttaaag tttatgcccc ggcttccagt gccaatatga gcgtcgggtt tgatgtgctc 60 ggggcggcgg tgacacctgt tgatggtgca ttgctcggag atgtagtcac ggttgaggcg 120 gcagagacat tcagtctcaa caacctcgga cgctttgccg ataagctgcc gtcagaacca 180 cgggaaaata tcgtttatca gtgctgggag cgtttttgcc aggaactggg taagcaaatt 240 ccagtggcga tgaccctgga aaagaatatg ccgatcggtt cgggcttagg ctccagtgcc 300 tgttcggtgg tcgcggcgct gatggcgatg aatgaacact gcggcaagcc gcttaatgac 360 actcgtttgc tggctttgat gggcgagctg gaaggccgta tctccggcag cattcattac 420 gacaacgtgg caccgtgttt tctcggtggt atgcagttga tgatcgaaga aaacgacatc 480 atcagccagc aagtgccagg gtttgatgag tggctgtggg tgctggcgta tccggggatt 540 aaagtctcga cggcagaagc cagggctatt ttaccggcgc agtatcgccg ccaggattgc 600 attgcgcacg ggcgacatct ggcaggcttc attcacgcct gctattcccg tcagcctgag 660 cttgccgcga agctgatgaa agatgttatc gctgaaccct accgtgaacg gttactgcca 720 ggcttccggc aggcgcggca ggcggtcgcg gaaatcggcg cggtagcgag cggtatctcc 780 ggctccggcc cgaccttgtt cgctctgtgt gacaagccgg aaaccgccca gcgcgttgcc 840 gactggttgg gtaagaacta cctgcaaaat caggaaggtt ttgttcatat ttgccggctg 900 gatacggcgg gcgcacgagt actggaaaac taa 933 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> yjeH primer <400> 9 aagctttgat aactctcctt tg 22 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> yjeH primer <400> 10 aagctttatg tggttatgcc at 22 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> yjeH gene primer <400> 11 ggtaccaggt cgactctaga ggatcccc 28 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> yjeH gene primer <400> 12 ggtaccttat gtggttatgc cat 23 <210> 13 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> MetB primer <400> 13 ttaccccttg tttgcagccc ggaagccatt ttccaggtcg gcaattaaat catatgaata 60 tcctccttag 70 <210> 14 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> metB primer <400> 14 ttactctggt gcctgacatt tcaccgacaa agcccaggga acttcatcac gtgtaggctg 60 gagctgcttc 70 <210> 15 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> thrB primer <400> 15 aaagaatatg ccgatcggtt cgggcttagg ctccagtgcc tgttcggtgg gtgtaggctg 60 gagctgcttc 70 <210> 16 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> thrB primer <400> 16 agacaaccga catcgctttc aacattggcg accggagccg ggaaggcaaa catatgaata 60 tcctccttag 70 <210> 17 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> metA primer <400> 17 aattgatatc atgccgattc gtgtgccgg 29 <210> 18 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> metA primer <400> 18 aattaagctt ttaatccagc gttggattca tgtg 34 <210> 19 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> metA G111E mutagenesis primer <400> 19 ttgtaactgg tgcgccgctg gaactggtgg ggtttaatga tgtc 44 <210> 20 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> metA G111E mutagenesis primer <400> 20 gacatcatta aaccccacca gttccagcgg cgcaccagtt acaa 44 <210> 21 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> metA EH mutagenesis primer <400> 21 tgtaactggt gcgccgctgg aacatgtggg gtttaatgat gtcg 44 <210> 22 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> metA EH mutagenesis primer <400> 22 cgacatcatt aaaccccaca tgttccagcg gcgcaccagt taca 44 <210> 23 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> metA EH primer <400> 23 aattgatatc atgccgattc gtgtgccgg 29 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> metA EH primer <400> 24 aattaagcct gctgaggtac gtttcgg 27 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> metA WT primer <400> 25 cagcaggtga ataaatttta ttc 23 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> metA WT primer <400> 26 cgcgaatgga agctgtttcc 20 <210> 27 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> pKD3 primer <400> 27 tttccgaaac gtacctcagc aggtgtaggc tggagctgct tc 42 <210> 28 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> pKD3 primer <400> 28 gaataaaatt tattcacctg ctgcatatga atatcctcct tag 43 <210> 29 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> ppc 2 copy primer <400> 29 gccggaattc tgtcggatgc gatacttgcg c 31 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> ppc 2 copy primer <400> 30 gaaggagctc agaaaaccct cgcgcaaaag 30 <210> 31 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> ppc 2 copy primer <400> 31 gccggagctc tgtcggatgc gatacttgcg c 31 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> ppc 2 copy primer <400> 32 gaagggtacc agaaaaccct cgcgcaaaag 30 <210> 33 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> aspC 2 copy primer <400> 33 tccgagctca taagcgtagc gcatcaggca 30 <210> 34 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> aspC 2 copy primer <400> 34 tccgagctcg tccacctatg ttgactacat 30 <210> 35 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> asd 2 copy primer <400> 35 ccggaattcc caggagagca ataagca 27 <210> 36 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> asd 2 copy primer <400> 36 ctagtctaga tgctctattt aactcccg 28 <210> 37 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> asd 2 copy primer <400> 37 ctagtctaga ccaggagagc aataagca 28 <210> 38 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> asd 2 copy primer <400> 38 ccggaattct gctctattta actcccg 27 <210> 39 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> icd promoter primer <400> 39 cccggggtat tttcagagat tat 23 <210> 40 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> icd promoter primer <400> 40 cccgggatcc tccttcgagc gctactggtt 30 <210> 41 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> pro promoter primer <400> 41 cccggggatc ctctgtgtgg aattctcgga ca 32 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> pro promoter primer <400> 42 cccgggaatt cattaaagag gagaaaggat 30 <210> 43 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> cysk promoter primer <400> 43 cccgggagct tccagcctgt ttacgatga 29 <210> 44 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> cysk promoter primer <400> 44 cccgggtccc aatttcatac agttaagga 29 <210> 45 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> icd promoter primer <400> 45 gaaaaagaaa aaaaattctg gcgattgtgt aggctggagc tgct 44 <210> 46 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> icd promoter primer <400> 46 cggcaattca taatctctga aaatacgcca tggtccatat gaatatcc 48 <210> 47 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> cysk promoter primer <400> 47 gaaaaagaaa aaaaattctg gcgattgtgt aggctggagc tgct 44 <210> 48 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> cysk promoter primer <400> 48 gcgggatcat cgtaaacagg ctgccatggt ccatatgaat atcc 44 <210> 49 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> pro promoter primer <400> 49 gaaaaagaaa aaaaattctg gcgattgtgt aggctggagc tgct 44 <210> 50 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> pro promoter primer <400> 50 tgtccgagaa ttccacacag aggatcgcca tggtccatat gaatatcc 48 <210> 51 <211> 197 <212> DNA <213> Artificial Sequence <220> <223> icd promoter <400> 51 gtattttcag agattatgaa ttgccgcatt atagcctaat aacgcgcatc tttcatgacg 60 gcaaacaata gggtagtatt gacaagccaa ttacaaatca ttaacaaaaa attgctctaa 120 agcatccgta tcgcaggacg caaacgcata tgcaacgtgg tggcagacga gcaaaccagt 180 agcgctcgaa ggaggat 197 <210> 52 <211> 197 <212> DNA <213> Artificial Sequence <220> <223> pro promoter <400> 52 gtattttcag agattatgaa ttgccgcatt atagcctaat aacgcgcatc tttcatgacg 60 gcaaacaata gggtagtatt gacaagccaa ttacaaatca ttaacaaaaa attgctctaa 120 agcatccgta tcgcaggacg caaacgcata tgcaacgtgg tggcagacga gcaaaccagt 180 agcgctcgaa ggaggat 197 <210> 53 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> cysk promoter <400> 53 agcttccagc ctgtttacga tgatcccgct gcttaatctg ttcatcatgc ccgttgccgt 60 ttgtggcgcg acggcgatgt gggtcgattg ctatcgcgat aaacacgcga tgtggcggta 120 acaatctacc ggttattttg taaaccgttt gtgtgaaaca ggggtggctt atgccgcccc 180 ttattccatc ttgcatgtca ttatttccct tctgtatata gatatgctaa atccttactt 240 ccgcatattc tctgagcggg tatgctacct gttgtatccc aatttcatac agttaagga 299

Claims (9)

The activity of the protein comprising the amino acid sequence of SEQ ID NO: 1 is enhanced as compared to that of the non-mutant microorganism, and the activity of cystathionine synthase is weakened or inactivated compared with that of the non-mutant microorganism, Culturing an Escherichia genus microorganism having the ability to produce O-acetyl homoserine; And producing L-methionine through an enzyme reaction with the O-acetyl homoserine as a substrate.
2. The method according to claim 1, wherein the step of producing O-acetyl homoserine further comprises recovering O-acetyl homoserine from the cultured microorganism or a culture thereof. .
The method of claim 1, wherein the step of producing L-methionine further comprises using methyl mercaptan as a substrate.
The method for producing L-methionine according to claim 1, wherein the enzyme reaction is performed using an enzyme having an O-acetyl homoserine sulfhydrylase activity or a strain containing the enzyme.
The method for producing L-methionine according to claim 1, wherein the Escherichia genus is Escherichia coli .
The method according to claim 1, wherein the Escherichia genus microorganism further has a weakened or inactivated homoserine kinase activity.
The method according to claim 1, wherein the Escherichia genus microorganism is further enhanced in the activity of homoserine acetyltransferase as compared to the non-mutant microorganism.
The method according to claim 1, wherein the Escherichia genus is selected from the group consisting of phosphoenolpyruvate carboxylases, aspartate aminotransferase and aspartate semialdehyde wherein the enzyme activity is further enhanced by at least one enzyme activity selected from the group consisting of dehydrogenase.
The method according to claim 1, wherein the Escherichia genus microorganism is further enhanced in activity of aspartate kinase as compared to the non-mutant microorganism.

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