[1]Herein to which a protein O- acetyl homoserine can be discharged and new variants thereof, the microorganism having the expression is enhanced O- acetyl homoserine-producing ability of the protein and use them to a method of producing O- acetyl-homoserine will be.
[2]
BACKGROUND
[3]
Methionine can be produced by chemical synthesis and biological synthesis, as well as feed and food additives are used as starting materials for synthesis of suaekje, and pharmaceuticals. In recent years, there is a two-stage method of producing L- methionine by enzyme conversion reaction from a L- methionine precursor production through fermentation bar known (International Patent Publication No. WO2008 / 013432). In the above two-stage method in methionine precursor O- succinyl homoserine (O-succinyl homoserine) and O- acetyl-homoserine (O-acetyl homoserine) it was disclosed that can be used, methionine precursor for the economic mass production of methionine it is very important to produce a high yield.
[4]
[5]
LeuE is leucine exhaust protein was known as (leucine export protein), and homoserine / homoserine lactone exhaust protein (Homoserine / homoserine lactone efflux protein, RhtB) protein present in one of the family of inner film, leucine and its by Putative uncharacterized transport protein it is known that exhaust analog (analogue).
[6]
It is possible to produce the prior art leuE (yeaS) purine nucleoside (purine nucleoside) with the amino acid sequence or enhanced variants thereof of a gene or a purine nucleotide (purine nucleotide) related LeuE, been known that the amino acid production can be improved . In addition, it is known that leuE variants with the discharge function of cysteine.
[7]
Detailed Description of the Invention
SUMMARY
[8]
The inventors of the present application are O- acetyl- and completed the present excavation by the protein, and variants thereof which is active for discharging the homoserine-result sought example, O- acetylation to increase the production of homoserine.
[9]
Problem solving means
[10]
One object of the present application is to provide a polypeptide having a O- acetyl homoserine emptying.
[11]
It is another object of the present application is to provide a polynucleotide encoding the polypeptide.
[12]
It is another object of the present application is Escherichia (to produce containing the polypeptide having a O- acetyl homoserine emptying or over-expression, O- acetyl homoserine Escherichia to provide a) spp.
[13]
It is another object of the present application provides a method comprising culturing a microorganism of the genus Escherichia which produce said O- acetyl homoserine in a culture medium; And
[14]
To provide a recovering the O- acetyl-homoserine in the medium or the microorganism cultured in the culturing step, O- acetyl homoserine production method.
[15]
It is another object of the present application provides a method comprising culturing a microorganism of the genus Escherichia which produce said O- acetyl homoserine in a culture medium; And
[16]
The microorganism or the culture medium in the incubation phase or the O- acetyl-homoserine by treating the methyl mercaptan and a methionine-converting enzyme O- acetyl homoserine recovered from the microorganism or the culture medium in the culture step into L- methionine comprising the step of switching, to provide a method for producing L- methionine.
[17]
Effects of the Invention
[18]
Microorganism having an inner film or LeuE LeuE protein variants of the present application is O- acetyl-homoserine is promoting ability discharge bar, the microorganism of the present application that the production efficiency is improved of O- acetyl-homoserine can be efficiently used for the production of O- acetyl-homoserine have. Also can be mass-produced to L- methionine and economically by using the O- acetyl-homoserine produced with high efficiency.
[19]
Best Mode for Carrying Out the Invention
[20]
In order to achieve the above object, one aspect of the present application is the first amino acid Val, the 30th amino acid leucine, the 95th amino acid phenylalanine or 165th at least one amino acid selected from the group consisting of the amino acid phenylalanine another amino acid in the amino acid sequence of SEQ ID NO: 1 the present invention relates to a polypeptide having an O- acetyl homoserine emptying substituted.
[21]
[22]
As used herein, the term, "O- acetyl homoserine" is a specific intermediate materials on the methionine biosynthesis pathway of microorganisms, it means the L- homoserine acetyl derivative. This is known to be produced and the homoserine acetyl -CoA the reaction by homoserine acetyltransferase a catalyst, C 6 H 11 NO 4 has the formula.
[23]
[24]
As used herein, the term "polypeptide having a O- acetyl homoserine emptying" means a polypeptide having a function for discharging out of the O- acetyl-homoserine in the microbial cells. More specifically, and can mean the LeuE proteins and variants thereof having the O- acetyl homoserine emptying, it is not particularly limited as long as having a O- acetyl homoserine emptying.
[25]
[26]
As used herein, the term, "LeuE" is an amino acid transporter (amino acid transporter) homoserine / homoserine lactone exhaust protein (Homoserine / homoserine lactone efflux protein, RhtB), but is known as a protein present in one inner film of the family for the exact function is not known. Thus the present inventors have first confirmed that LeuE ejects the O- acetyl homoserine specifically.
[27]
The LeuE is S may be derived from a microorganism of the genus Escherichia, may be a specific example LeuE derived from E. coli, can be included herein without limitation as long as the microorganism-derived that has the ability to drain the O- acetyl homoserine.
[28]
Specifically, the polypeptide having a O- acetyl homoserine emptying may be a protein having the amino acid sequence of SEQ ID NO: 1. In addition, more specifically, the amino acid sequence of SEQ ID NO: 1 and less than 70%, specifically at least 80%, has a greater than 90% homologous, substantially the same as or corresponding to the amino acid sequence of SEQ ID NO: 1 of O- acetyl-homoserine It may be a protein having an amino acid sequence having an emission activity. Or the amino acid sequence having substantially the amino acid sequence SEQ ID NO: 1 and active to the same or equivalent to the discharge O- acetyl homoserine in a sequence having a homology of these, some of the sequences are deleted, modified, substituted or added in the amino acid sequence one number, and is also included in the scope of the present is self-evident if thereof.
[29]
As used herein, the term, O- "variant" of a polypeptide having an acetyl homoserine emptying is a polypeptide having improved ability to natural or bibyeon natural discharge of O- acetyl-homoserine in comparison with the polypeptide in the state discharged neunge It refers. Specifically, a polypeptide having one or more amino acid mutations in the amino acid sequence SEQ ID NO: 1 feature a polypeptide having improved O- acetyl homoserine discharge due to the amino acid sequence of SEQ ID NO: 1.
[30]
For example, it can be in the amino acid sequence of SEQ ID No. 1 the first amino acid Val, the 30th amino acid leucine, the 95th amino acid phenylalanine or 165th amino acid is one or more selected from the group consisting of the amino acid phenylalanine polypeptide substituted with another amino acid. Substituted with one particular amino acid methionine is valine in the amino acid sequence of SEQ ID NO: 1 or; To the 30th amino acid phenylalanine is selected from alanine, tryptophan, leucine, valine, glycine, serine, asparagine, aspartic acid, histidine, isoleucine, proline, tyrosine, glutamine, lysine, glutamic acid, cysteine, threonine, and the group consisting of arginine optionally substituted; The 95th amino acid leucine and the valine, phenylalanine, alanine, glycine, threonine, asparagine, aspartic acid, histidine, isoleucine, serine, proline, tyrosine, glutamine, lysine, glutamic acid, cysteine, tryptophan and substituted as selected from the group consisting of arginine .; Or 165 amino acid phenylalanine is selected from alanine, tryptophan, leucine, valine, glycine, serine, asparagine, aspartic acid, histidine, isoleucine, proline, tyrosine, glutamine, lysine, glutamic acid, cysteine, threonine, and the group consisting of arginine that may be substituted. More specifically, the 1st amino acid methionine is substituted with valine in the amino acid sequence of SEQ ID NO: 1; The 30th amino acid phenylalanine, alanine, tryptophan, leucine, valine, glycine, serine, asparagine, or substituted by selected from the group consisting of aspartic acid and histidine; The 95th amino acid leucine and the valine, phenylalanine, alanine, glycine, threonine, asparagine, or substituted by selected from the group consisting of aspartic acid and histidine; Or 165 amino acid phenylalanine, this may be one selected from substituted by alanine, tryptophan, leucine, valine, glycine, serine, asparagine, aspartic acid, and the group consisting of histidine. More specifically, there is a first amino acid is substituted with valine is methionine, the 30th amino acid phenylalanine, 95th amino acid leucine or phenylalanine amino acid 165 in the amino acid sequence of SEQ ID NO: 1 can be substituted with another amino acid. More may be more specifically SEQ ID NO: 2, 133, 134, 137, 138, 141, or a polypeptide consisting of an amino acid sequence of 142. Specifically, the sequence that is 70% or more, for example at least 80%, more specifically, has a greater than 90% homologous, substantially the same as or correspond to the amino acid sequence of the variant O- acetyl homoserine discharge capability enhanced amino acid It may be a protein comprising the sequence. Or substantially equal to or corresponds to amino acid sequence variants of the sequence as having such homology O- acetyl-homoserine in the enhanced discharge ability amino acid sequence. Some sequences may be deleted, modified, substituted or added in the amino acid sequence. Wherein the polypeptide is an example of the ability of a natural or nature bibyeon the discharge O- acetyl homoserine, compared to the polypeptide in a state increased polypeptide variants, and the like. The term, herein, the term "natural state or bibyeon natural state" means the state has not introduced a variation of the introduction of the polypeptide of the present or active.
[31]
[32]
As used herein, the term "homology" is, in the amino acid sequence or nucleic acid sequence of the gene encoding the protein, so that the best possible match between the positive sequence in a specific comparison region alignment (align) the same base or amino acid residues between which after sequence It refers to a degree. If homology is sufficiently high expression product of the gene it may have the same or similar activity. % Of the sequence identity may be determined using the sequence comparison program known, there may be mentioned BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc), etc., for example.
[33]
[34]
One aspect of the present application is directed to a polynucleotide encoding the polypeptide having the above-O- acetyl homoserine emptying. Herein a polypeptide having the O- acetyl homoserine emptying are the same as previously described.
[35]
For example, the polynucleotide is the initiation codon may be replaced with an ATG, SEQ ID No. 4, 135, 136, 139, 140 may be a 143, or a nucleotide sequence of 144, but is not limited thereto. In addition, the polynucleotides due to the degeneracy (genetic code degeneracy) of the genetic code are included in the base sequence and also variants thereof encoding the same amino acid sequence herein. For example, it may be, which is modified to have the optimum codon depending on the microorganism used.
[36]
Specifically, the nucleotide sequence with 70% or more, for example 80% or more, more specifically, has an activity of at least 90% homologous, substantially the same as or corresponds to the base sequence discharging O- acetyl-homoserine having the amino acid sequence may be a nucleotide sequence encoding. Or which it can be prepared from a known gene sequence probe, e.g., by screen hydride under stringent conditions with the sequence complementary to the entire or part of the nucleotide sequence, coding for a protein having the activity to discharge O- acetyl-homoserine sequence may be. And forming the "stringent conditions" refers to a so-called specific hybrid, refers to a condition non-specific hybrid is not formed. For example the expressed, homology between high gene, for example, 80% or more, specifically, more specifically at least 90%, at least 95%, more specifically 97% or more, particularly Specifically, more than 99% of the hybrid gene having the same sex between shoes, rather homologous conditions do not hybridize low gene together, or the 60 ℃ washing condition of an ordinary Southern hybridization, and 1 × SSC, 0.1% SDS, specifically 60 ℃, 0.1 × SSC, with 0.1% SDS, more specifically, in the salt concentration and temperature corresponding to 68 ℃, 0.1 × SSC, 0.1% SDS, 1 time, and specifically may be exemplified by conditions under which washing twice or three times ( Sambrook et al, Molecular Cloning:.. A Laboratory Manual, 3rd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)).
[37]
The probe used for the hybridization may be a part of the complementary sequence of the base sequence. These probes, with the oligonucleotides constructed based on the known sequences as primers, can be constructed by PCR, a gene fragment containing these sequences as the template. For example, as the probe, it may be a gene fragment of 300bp long. More specifically, as a probe, in the case of using a gene fragment of about 300bp length, as the washing conditions of hybridization, 50 ℃, are exemplified a 2 × SSC and 0.1% SDS.
[38]
[39]
Gene, as used herein, and they are coded protein sequence and the promoter sequence can be obtained from a known database. Examples, but can be obtained in the NCBI GenBank etc., not limited to this.
[40]
[41]
One aspect of the present application relates to a microorganism producing the polypeptide containing the polypeptide or variant thereof having the above-O- acetyl homoserine emptying or over-expression, O- acetyl homoserine. Specifically, the microorganism may be a polypeptide that contains a polypeptide or variant thereof consisting of the amino acid sequence of SEQ ID NO: 1 or over-expression, the microorganism to produce O- acetyl homoserine.
[42]
The O- acetyl homoserine polypeptides and variant polypeptides having a discharge function are as described above.
[43]
When the term, "O- acetyl-homoserine producing microorganism" as used herein means microorganisms are cultured in a culture medium, to produce O- acetyl-homoserine in the organism means a microorganism having the ability to secrete them into the medium. O- acetyl homoserine production capacity can be enhanced or imparted by the natural or artificial mutants or species improved. Specifically, microorganisms that produce O- acetyl homoserine may be included regardless the one derived from a microorganism having an O- acetyl homoserine-producing ability. For example Escherichia ( Escherichia can lie) and, more specifically, E. coli ( Escherichia coli may be).
[44]
On the other hand, microorganisms that produce O- acetyl-homoserine in the present application for the associated mechanism, such as the separate and LeuE homoserine biosynthesis to promote O- acetyl homoserine-producing ability to the associated path or O- acetyl homoserine source strength related mechanisms It may be a variation of an existing well-known additionally introduced mutant microorganisms.
[45]
[46]
Other specifically one aspect of the present application may be activated for further active fire system tachioh non synthase (cystathionine synthase) the microorganisms producing the O- acetyl homoserine. Specifically, the sheath tachioh non synthase gene defect (metB) encoding the kinase, or, but may have been weakened as compared to the expression bibyeon this microorganism is not limited to this. Amino acid sequence having an amino acid sequence of the metB may be obtained from a known database, cis tachioh non synthase activity may be included, without limitation, the example may be a protein having an amino acid sequence of SEQ ID NO: 5. A protein comprising the amino acid sequence of SEQ ID NO: 5, may be a protein which the nucleotide sequence of SEQ ID NO: 6 coding, but is not limited thereto.
[47]
Further, another specific aspect of the present application may be activated for further active fire homoserine kinase (homoserine kinase) a microorganism producing the O- acetyl homoserine. Specifically, the activity of the homoserine kinase can be those bibyeon is reduced or removed than the endogenous activity of the microorganisms. By way of example, the gene (thrB) coding for homoserine kinase, but may be attached to a weak promoter or to variation of the weak activity or defects than the natural promoter, and is not limited to this. Amino Acid sequence of the thrB may be obtained from a known database, amino acid sequences having a homoserine kinase activity may be included, without limitation, the example may be a protein having an amino acid sequence of SEQ ID NO: 7. A protein comprising the amino acid sequence of SEQ ID NO: 7 may be a protein which the nucleotide sequence of SEQ ID NO: 8 encoding, but is not limited thereto.
[48]
The term herein, the term "deactivation" of the protein has the original microorganisms when compared with the activity of the enzyme in the bibyeon natural or said protein has in the state, its activity is reduced, if not at all expressed and expression even if means the absence of its activity. The inactivation and if reduced compared to the activity of the enzyme with the enzyme itself, the active has the original microorganisms such as variation of the gene encoding the enzyme or eliminate, inhibit expression of the gene encoding it, or translated (translation) inhibition, etc. If the overall degree of enzymatic activity in the cell is low, or the removal compared to wild-type strain, in the case where the gene is deleted a part or a whole part, and a concept including combinations of these, too, are not limited.
[49]
Inactivation of this enzyme activity may be achieved by application of various methods well-known in the art. An example of the method, the method that a mutant gene such that the activity of the enzyme reduced, including a case in which the activity of the enzyme has been removed, a replacement gene encoding the enzyme on a chromosome; A method of introducing a mutation into an expression control sequence of the gene on the chromosome encoding the enzyme; How to replace the expression control sequence of the gene coding for the enzyme with low or no activity sequences; A method of deleting the entire or part of the gene on the chromosome encoding the enzyme; To antisense oligonucleotide to bind to complementary to the transcript of the gene on the chromosome inhibit translation of the enzyme from the mRNA introducing a nucleotide (e.g., antisense RNA); Of the artificially added in 2 ORF (open reading frame) of the method and the corresponding sequence attached that make this possible in order to form a structure ribosomes (ribosome) the SD sequence and the sequence complementary to the front end of SD sequence of the gene coding for the enzyme 3 'and the like, RTE (reverse transcription engineering) method of adding the promoter to reverse transcription at the terminal, a combination of the two can also be achieved, but is not particularly limited by the examples.
[50]
[51]
Method of modifying the expression control sequence is performed by inducing a mutation on the controlled expression of a nucleic acid sequence to further weaken the activity of the expression control sequence by deletion, insertion, Vivo wholly or conservative substitution or a combination of these sequences or more weak It can be carried out by replacing a nucleic acid sequence having activity. The expression control sequence is one containing the sequence for controlling the termination of the sequence, and a transcription and decryption encoding a promoter, operator sequence, a ribosome binding site, and the like.
[52]
In addition, the method of modifying the gene sequence on the chromosome has deletion of the gene sequence to further weaken the activity of the enzyme, inserted vivo wholly or conservative substitutions or performed by inducing a mutation on the sequence, or more weakly activated by a combination of both have to be done by replacing the improved so that the improved gene sequence or active gene sequence, but is not limited to this.
[53]
In addition, a method for deleting a part or all of the gene encoding the enzyme, by replacing the polynucleotide encoding my intrinsic target protein chromosome through a bacterial vector for in chromosome insert as part of the nucleic acid sequence is deleted polynucleotides or marker gene It can be carried out. In one example of a method for deleting a part or all of these genes can be used a method of deleting a gene by homologous recombination, but it is not limited to this.
[54]
Means "part" in the poly it differs according to the type of the nucleotide, but, specifically, is 1 to 300, and more specifically 1100 to, still more specifically, but can clear up 1 to 50, limited to a specific no.
[55]
"Homologous recombination (homologous recombination)" in the above refers to a genetic recombination takes place through the exchange connection in the gene locus of the chain with a homology to each other.
[56]
[57]
In addition, another specific aspect of the present application may be one, the activity of additional homoserine acetyltransferase (homoserine acetyltransferase) a microorganism producing the O- acetyl-homoserine The bibyeon enhanced as compared to microorganisms. Specifically, and the activity of the homoserine acetyltransferase be those bibyeon the increased activity of the microorganism, in particular it may be a variant metA gene encoding the activity of homoserine acetyl transferase are introduced enhanced. The variant metA genes and replacing the 111 amino acids of the homoserine acetyl transferase as glutamic acid, may be a gene encoding the 112 amino acids are substituted with histidine, but is not limited thereto. The variant metA homoserine is the amino acid sequence than wild-type activity-enhanced activity of the acetyltransferase may be included are, without limitation, may be a Examples of the protein having the amino acid sequence of SEQ ID NO: 10. The advantage of producing the same and its variants metA gene, an example of the information, etc. The homoserine acetyltransferase enhanced strain is disclosed in Korea Patent Registration No. 10-1335841, the specification of the patent a whole by reference herein It may be included.
[58]
[59]
It is another aspect of the present application is further a microorganism producing the O- acetyl-homoserine of aspartate semi-aldehyde dehydrogenase (aspartate semialdehyde dehydrogenase), pyridine nucleotide trans dehydrogenase kinase (pyridine nucleotide transhydrogenase), or a combination thereof the activity may be a microorganism of the genus Escherichia which bibyeon the production cost, O- acetyl homoserine enhanced as compared to microorganisms.
[60]
In addition, other specific embodiment is wherein the acetyl-O- play in addition phosphonium microorganisms producing homoserine pyruvate kinase carboxyl La (phosphoenolpyruvate carboxylase), aspartate aminotransferase activity or a combination thereof of the present bibyeon It can be enhanced as compared to microbes. "Strengthening" of the term protein activity as used herein, means to hold the micro-organisms and improve the active state of the protein. Enhance the activity of the protein is not as long as the activity of each protein, such as strengthening of the activity of the protein of interest can enhance bibyeon than the natural state of the protein in its natural state or limiting. For example, i) increasing the copy number of the polynucleotide encoding the respective proteins, ii) transformation of the expression control sequences to increase the expression of the polynucleotide, iii) modification of the polynucleotide sequence on the chromosome so that enhance the activity of each protein and iv) it may be performed by a method selected from the group consisting of a combination thereof. Specifically, the active addition to upstream of the nucleotide sequence encoding the nucleotide method a polynucleotide containing the sequence to insert into a chromosome, a method of introducing microorganisms by introducing the polynucleotide in a vector system, each protein encoding the respective protein introducing a promoter selected from or representing the method for introducing the respective protein gave a mutation to a promoter, the method and the group consisting of a method of introducing a variant of the nucleotide sequence encoding the respective protein of modifying the base sequence of the 5'-UTR region It may be performed by a method, but is not limited to this.
[61]
[62]
One aspect of the present application is directed to O- acetyl homoserine production method comprising the step of culturing a microorganism of the genus Escherichia which produce said O- acetyl homoserine in a culture medium.
[63]
More specifically, the method comprising culturing a microorganism of the genus Escherichia which produce said O- acetyl homoserine in a culture medium; And to a production method for recovering the O- acetyl-homoserine in the medium or the microorganism cultured in the culture step.
[64]
The term herein, the term "culture" is meant that the growth in the environmental conditions of suitably adjusting the microorganism. Culturing process of the present application may be made in accordance with the appropriate culture medium and culture conditions known in the art. This culturing process may be used by those skilled in the art easily adjusted according to the selected strain. In the method, the method comprising culturing the microorganism, and may be particularly, but not limited thereto, carried out by the known batch culture method, the continuous culture method, a fed-batch culture method. Medium, and other cultures used for the culture of microorganisms conditions of the present application, if the medium used in a conventional S-cultivation of Escherichia spp, but can whichever used without particular limitations, specifically, a suitable carbon source, a nitrogen source, persons of microorganisms of the present , in a conventional medium containing inorganic compounds, amino acids and / or vitamins, etc. while controlling the temperature, pH, etc. under aerobic conditions, it can be cultured.
[65]
Carbohydrates such herein as the carbon source are glucose, fructose, sucrose, maltose, mannitol, sorbitol; Alcohols such as sugar alcohols, glycerol, pyruvic acid, lactic acid, citric acid; It may include amino acids such as organic acids, glutamic acid, methionine, lysine, but not limited to this. In addition, starch hydrolyzate, molasses, black strap molasses, rice winter, Casa member, sugar cane waste, and corn steep liquor can be used an organic nutrient source of such natural, specifically, the switch to glucose and sterilized pretreated molasses (that is, a reducing sugar the molasses), and the carbohydrate can be used as such, can be variously used for any other suitable amount of carbon sources, without limitation. These carbon sources can be used alone or in combination of two or more.
[66]
The inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, carbonate facial titanium, ammonium nitrate wish; Organic nitrogen sources such as amino acids, such as glutamic acid, methionine, glutamine, peptone, NZ- amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation product, defatted soybean cake or its degradation products It can be used. These nitrogen sources can be used alone or two or more of them, but this can be used in combination, but is not limited thereto.
[67]
The personnel in the phosphoric acid first, potassium phosphate, potassium 2, or its corresponding sodium-containing salts can be included such as. Inorganic compounds include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, may be used such as calcium carbonate and more than one amino acid, may be included are vitamins and / or appropriate precursors, but is not limited thereto. These media or precursors may be added batchwise or continuously to the culture, but is not limited thereto.
[68]
As used herein, by a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid was added in an appropriate manner in the culture during the culturing of the microorganism, it is possible to adjust the pH of water culture. In addition, during the culture it is possible to suppress the air bubbles generated by using antifoaming agents such as fatty acid polyglycol ester. Furthermore, it can be injected without injection of the gas or of nitrogen, hydrogen or carbon dioxide gas in order to maintain the culture, oxygen or oxygen-containing gas injected into or anaerobic conditions and Miho group into the water in order to maintain aerobic condition of water culture.
[69]
Incubation temperature of water may be a 27 ℃ to 37 ℃, more specifically, be a 30 ℃ to 35 ℃, but is not limited thereto. The incubation period may be continued until it is obtained the production of the desired useful substance, specifically, it may be a 10 hours to 100 hours but not limited thereto.
[70]
Recovering the O- acetyl homoserine method of culture of the present microorganism, for example, batch, continuous or fed-batch culture method according to the art using suitable methods known in the art O- acetyl desired from the culture broth It can be recovered homoserine. For example, this can be used centrifugation, filtration, anion exchange chromatography, crystallization and HPLC, etc., it may also be used in combination of a suitable method known in the art.
[71]
The recovering step may include a separation step and / or purification step.
[72]
[73]
One aspect of the present application includes the steps of: culturing a microorganism of the genus Escherichia to produce O- acetyl homoserine in a culture medium of the present application; And the culture medium or the microorganism cultured in the step, or the culture by a O- acetyl homoserine recovered from the microorganism or the culture medium in step processes the methylmercaptan and methionine-converting enzyme L- methionine of O- acetyl-homoserine It relates to L- methionine production comprising the step of switching to.
[74]
For example, O- acetyl-homo Escherichia spp the step of O- acetyl-homoserine The cultured medium was recovered from the second method of producing a serine (Republic of Korea Patent No. 10-0905381 Ho) to produce the methionine using have.
[75]
The two-step process is O- acetyl-homoserine and methyl mercaptan to use as a substrate O- acetyl homoserine through the enzyme reaction using the strain containing the enzyme or the enzyme has an activity to convert the methionine L- methionine and organic acid of a step of production.
[76]
The methionine-converting enzyme comprises all of the enzyme to convert O- acetyl-homoserine to methionine, number of days, especially O- acetyl homoserine sulfinyl deurilra claim (Oacetylhomoserine sulfhydrylase), but is not limited thereto.
[77]
[78]
Specifically, the O- acetyl homoserine sulfinyl deurilra agent in Leptospira (Leptospira sp.), Chromotherapy tumefaciens in (Chromobacterium sp.), Microbial strain belonging to the genus Pseudomonas Scientific (Hyphomonas sp.), More specifically, Leptospira methoxy Eri (Leptospira meyeri), Pseudomonas Ke rugi labor (Pseudomonas aurogenosa), hypo Pseudomonas Yep Tunisia Titanium (Hyphomonas Neptunium), chromotherapy tumefaciens non come syum of O- acetyl-homoserine sulfinyl deurilra derived from a microorganism strain belonging to the (Chromobacterium Violaceum) there dehydratase can be used.
[79]
The reaction is as follows.
[80]
[81]
CH 3 SH + O- acetyl -L- homoserine <=> acetate + Methionine
[82]
[83]
As for the additional methionine production process, such is disclosed in Republic of Korea Patent No. 10-0905381 number, the specification of said patent whole may be included by reference in the present application.
[84]
Mode for the Invention
[85]
To the following embodiments will be described in more detail in this context. However, these examples are for explaining the present application by way of example, it is not the scope of the present application limited to these Examples.
[86]
[87]
Reference Example 1: O- acetyl-homoserine producing ability production of strains having
[88]
[89]
1-1. Wild type of E. coli metB genetic defects
[90]
In order to produce O- acetyl homoserine-producing strain was used as the representative microorganism of Escherichia coli in Escherichia spp. To this end, the wild-type E. coli, the E. coli to K12 W3110 (ATCC27325) was used to obtain from the US Biological Resource Center (American Type Culture Collection, ATCC) . The E. coli W3110 strain in the cis tachioh to prepare a non-gamma-synthase metB gene (SEQ ID NO: 6) and the thrB gene (SEQ ID NO: 8) is a deficient strain encoding the homoserine kinase coding for dehydratase. The thus fabricated O- acetyl homoserine-producing strain was named W3-BT. One example of such information or the like metB and thrB gene deletion strains are disclosed in the Republic of Korea Patent No. 10-0905381, or International Publication No. WO2008 / 013432 and No. (in particular, the Republic of Korea Patent No. 10-0905381 No. Example 1-1 1-2 and reference), the specification of said patent whole may be included by reference in the present application.
[91]
[92]
1-2. Homoserine acetyl transferase variant which is active metA making transgenic strains
[93]
To enhance the homoserine acetyl transferase (homoserine acetyltransferase) activity in the strains obtained in the above Reference Example 1-1, variant metA gene encoding homoserine acetyltransferase has an enhanced activity on the strains (SEQ ID NO: 10) We were introduced to.
[94]
Strains produced were produced pCL_Pcj1_metA (EH) plasmid by the method disclosed in the Republic of Korea Registered Patent No. 10-1335841 exemplary arc Examples 1 and 3.
[95]
Next, the production a variant metA gene as a template for pKD3 vector to produce a replacement cassette as a way of substitution was introduced into the strain was amplified and obtained by PCR using primers of SEQ ID NO: 23 and SEQ ID NO: 24 . Specifically, using primers of SEQ ID NOs: 23 and 24 modified (denaturation) step is 30 seconds at 94 ℃, annealing (annealing) step is from 55 ℃ 30 seconds, and extension (extension) step is carried out for 2 minutes at 72 ℃ , which was the PCR reaction proceeds to perform this repeated 30 times.
[96]
metA (EH) portion of the replacement cassette is pCL-Pcj1-metA the (EH) as a template was performed using the SEQ ID NO: 19 and SEQ ID NO: 20 primers, metA wild-type section respectively of PCR using SEQ ID NO: 21 and SEQ ID NO: 22 Primer to give the product. The three PCR products SEQ ID NO: 19 and SEQ ID NO: 22 using the primer to produce a metA (EH) replacement cassette containing a chloramphenicol marker part, the W3- produced in Reference Example 1-1 was transformed with pKD46 vector preview It was introduced by the electroporation in BT strains.
[97]
The strains introduced is confirmed through the process by re-transformed with pCP20 vector and cultured in LB medium to remove the chloramphenicol marker is named LA metA gene metA (EH) W3-BTA in the replacement strains.
[98]
In this example of the information or the like, such as homoserine acetyltransferase enhanced strain are disclosed in the Republic of Korea Patent No. 10-1335841, or International Publication No. WO2012 / 087039 call, the specification of said patent whole may be included by reference in the present application .
[99]
[100]
1-3. ppc , aspC and asd production of strains containing the gene copy 2
[101]
In order to increase the Oh O- paroxetine homoserine-producing ability of a strain W3-BTA produced in Reference Example 1-2 it was introduced into the biosynthetic pathways enhance the known strategies. Phosphorylation play pie on phosphodiesterase involved in the biosynthesis of oxaloacetic acetate from play pyruvate to the base bait carboxyl la kinase (phosphoenolpyruvate carboxylase), octanoic aspartate aminotransferase and β which is involved in the biosynthesis of the aspartate from Salo acetate - aspartate to semi-aldehyde dehydrogenase (aspartate-semialdehyde dehydrogenase), that is, which is involved in the biosynthesis of homoserine from aspartyl phosphate ppc gene, aspC gene, and asd was to produce a strain in which amplify the gene in 2 copy.
[102]
Strains produced are the Republic of Korea Patent No. 10-1117012 favor Examples 1-1 to 1-3 of the plasmid in a way pSG-2ppc, pSG-2aspC, pSG-2asd plasmid and, W3-BTA strain disclosed in introduced and was produced 2copy amplified strains of the three genes in sequence by the method of example 1-5 of the patent. The thus produced strain was named W3-BTA2PCD (= WCJM).
[103]
In the phosphorylation play pie base of the bait carboxyl la kinase, aspartate aminotransferase and aspartate semi example of the information or the like to formaldehyde dehydrogenase enhanced strain Republic of Korea Patent No. 10-0905381, or International Publication No. WO2008 / 013 432 is disclosed a call, the context of the whole patent may be incorporated by reference herein.
[104]
[105]
1-4. Flask culture experiments
[106]
In order to test the O- acetyl homoserine productivity of the strains prepared in the above Reference Examples 1-2 and 1-3 was subjected to triangular plast culture. After inoculated W3110, W3-BTA, WCJM strain in LB medium overnight culture at 33 ℃, then inoculated with a single colony on a 3 mL LB medium culture for 5 hours at 33 ℃, and again 25 mL O- acetyl homoserine production 200 fold diluted in 250 mL Erlenmeyer flask containing a culture medium by 30 hours incubation at 33 200 rpm by ℃ was confirmed O- acetyl-homoserine produced by the HPLC analysis. The medium composition used is summarized in Table 1 below.
[107]
[108]
[Table 1] Composition O- acetyl homoserine production medium flasks
Furtherance Concentration (per liter)
glucose 40 g
Ammonium sulfate 17 g
Kh 2 the 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

[109]
Using the medium by 30 hours the culture was analyzed with HPLC to confirm the O- acetyl homoserine production is summarized in Table 2 below.
[110]
[111]
[Table 2] O- acetyl homoserine production by 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

[112]
The As can be seen from Table 2, the wild-type W3110 O- acetyl-homoserine is not generated at all, O- acetyl homoserine (O-AH) is 0.9 g / L was produced in the W3-BTA strain, the biosynthetic pathway is enhanced the 1.2 g / L was produced in the WCJM strain.
[113]
[114]
Example 1: O- acetyl-homoserine producing ability screening of the membrane protein to increase
[115]
[116]
The inventors of the present application are seen to apply a film, but discloses a protein O- acetyl homoserine emptying and O- acetyl homoserine-producing ability no disclosure associated with the E. coli-derived leuE (SEQ ID NO: 1), to O- acetyl homoserine production .
[117]
LeuE the gene to enhance leuE gene in strains was performed for cloning using the restriction enzyme smaⅠ place of pCL vector.
[118]
First, in order to obtain leuE gene using the primers of SEQ ID NOS: 11 and 12 modified (denaturation) step is 30 seconds at 94 ℃, annealing (annealing) step is from 55 ℃ 30 seconds, and extension (extension) step at 68 ℃ carried out for 1 minute, followed by a PCR reaction proceeds to do this 30 times. As a result, after the thus obtained PCR product was electrophoresed in 1.0% agarose gel, DNA was purified from a band of 800bp in size. After overnight at 37 ℃ process gave the purified DNA with restriction enzymes smaⅠ, then purified once more, T4 Riga using the Kinase (ligase) was cloned leuE and pCL vector. By using a cloning plasmid transformed E. coli DH5α and then, by selecting the spectinomycin (spectinomycin) the transformed E. coli DH5α on LB plate medium containing 50 ㎍ / mL to obtain a plasmid. A production plasmid O- acetyl homoserine production master W3-BTA, and the introduction of strain in WCJM, each W3-BTA / pCL-leuE and WCJM / pCL-leuE was termed, O- flask for the feature in the production of acetyl-homoserine It conducted an evaluation.
[119]
In addition, by introducing the vector pCL1920 ball in the same manner as the strain in the control group, and W3-BTA WCJM were respectively named W3-BTA / pCL1920 and WCJM / pCL1920, the flask was evaluated for the feature of the O- acetyl homoserine production It was in progress.
[120]
[121]
After specifically, each strain plated on LB solid medium and cultured overnight at 33 ℃ incubator. A single colony of the LB plate for overnight culture a strain to the medium in 3 ml LB culture medium after inoculation, this incubated at 33 ℃ 5 hours, and 200-fold in 250 ml Erlenmeyer flask containing 25 ml re-O- acetyl homoserine production medium diluted and was 30 hours incubation at 33 ℃, an incubator of 200 rpm, by the HPLC analysis was confirmed O- acetyl homoserine productivity. Summarizing the results thereof to shown in Table 3.
[122]
[123]
[Table 3] O- acetyl homoserine production measured by flask culture
OD (562nm) Glucose consumption (g / L) O-AH (g / L)
W3-BTA/pCL1920 9.5 35 0.9
W3-BTA/pCL-leuE 8.2 36 1.0
WCJM/pCL1920 9.6 35 1.2
WCJM/pCL-leuE 8.4 36 1.5

[124]
As can be seen in Table 3, the introduction of a plasmid leuE WCJM OD is no lower than the control strain containing the empty vector, it was also enhanced glucose consumption. However, the possibility of O- acetyl-homoserine is 1.5 g / L are produced eoteuna O- acetyl-homoserine by the introduction of wild-type leuE is difficult to obtain the result that the production enhancement capability, when viewed in the enhanced consumption rate per OD is controlled and emptying a was confirmed, screening was to a mutation which may be enhanced ability of O- acetyl-homoserine through the emission than wild-type structural modeling.
[125]
[126]
Example 2: leuE initiation codon changes plasmid production and O-acetyl homoserine -producing ability evaluation
[127]
[128]
Initiation codon of the wild-type leuE is known as the valine amino acid coded by gtg. A plasmid prepared in Example 1 to change the start codon in order to confirm the reinforcing effect of the protein leuE methionine amino acid codon atg to as the experiment was performed to change the initiation codon to the base. Specifically, the first amino acid in the amino acid sequence of SEQ ID NO: 1 to strengthen the O-acetyl homoserine emptying was substituted with methionine.
[129]
More specifically, to prepare a leuE (ATG) mutation. To produce a leuE (ATG) mutations were used for SEQ ID NO: 145 and SEQ ID NO: 146 Primer position - using a specific mutation kit (site-directed mutagenesis kit, stratagene, USA) was prepared variant leuE (ATG) gene . The old wild-type plasmid WT, initiation codon mutant plasmid and named WT_ATG and introducing the produced plasmid WCJM evaluate the production performance of O- acetyl-homoserine in the flask.
[130]
After specifically, each strain plated on LB plate medium and incubated overnight at 33 ℃ incubator. Overnight inoculate 25 ㎖ titer medium shown in the cultured strain in the LB solid medium, and then it was incubated 40 hours at 33 ℃, an incubator of 200 rpm, showed its results are shown in Table 4.
[131]
[132]
[Table 4] O- acetyl homoserine production measured by flask culture
OD(562 nm) Glucose consumption (g / L) O-AH (g / L)
WCJM/pCL-leuE WT 8.4 36 1.5
WCJM/pCL-leuE WT(ATG) 7.6 39 2.6

[133]
As can be seen from Table 4, pCL-leuE WT (ATG) start codon, but the mutant plasmid is introduced strain OD is lower than the wild type, showed a consumption rate per fast, O- acetyl homoserine is 2.6 g / L are produced compared to wild-type was a whopping 173% increase in production capability.
[134]
[135]
Example 3: leuE mutant plasmid production and O- acetyl homoserine -producing ability evaluation
[136]
[137]
3-1. leuE mutant plasmid production
[138]
Example 1 and the plasmid pCL-leuE WT and pCL-leuE WT prepared in 2 (ATG) 3 of variations expected of two or with the primary discharge stronger ability than wild-type leuE was conducted an experiment to produce respectively. Specifically, the O- acetyl homoserine discharge were selected for mutation leuE position through structural modeling to enhance performance, the 30th amino acid in the amino acid sequence of SEQ ID NO: 1 and 2, the 95th amino acid and 165 amino acid by another amino acid It was substituted.
[139]
More specifically, to prepare a L95V, F30A, or F165A mutation. To produce the L95V mutation was used primers of SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 25 is F30A, and SEQ ID NO: 26 primers, F165A were used to SEQ ID NO: 27 and SEQ ID NO: 28 primer. Position with the respective primer was prepared variant leuE gene by a specific mutation kit (site-directed mutagenesis kit, stratagene, USA). L95V variants plasmid which is based on the existing wild type plasmid WT is WT_M3, F30A is WT_M4, F165A was named WT_M6. Further variants plasmid L95V based plasmid WT (ATG) is the initiation codon is changed WT (ATG) _M3, F30A is WT (ATG) _M4, F165A was named WT (ATG) _M6. And introducing the produced mutant plasmid were evaluated WCJM producing ability of O- acetyl-homoserine in the flask.
[140]
After specifically, each strain plated on LB plate medium and incubated overnight at 33 ℃ incubator. Overnight inoculate 25 ㎖ titer medium shown in the cultured strain in the LB solid medium, and then it was incubated 40 hours at 33 ℃, an incubator of 200 rpm, showed its results are shown in Table 5.
[141]
[142]
[Table 5] O- acetyl homoserine production measured by flask culture
OD(562 nm) Glucose consumption (g / L) O-AH (g / L)
WCJM/pCL1920 9.6 35 1.3
WCJM/pCL-leuE WT 8.4 36 1.5
WCJM/pCL-leuE WT_M3 8.2 38 2.3
WCJM/pCL-leuE WT_M4 7.9 38 3.7
WCJM/pCL-leuE WT_M6 8.0 39 4.8
WCJM/pCL-leuE WT(ATG) 7.6 39 2.6
WCJM/pCL-leuE WT(ATG)_M3 7.5 40 3.1
WCJM/pCL-leuE WT(ATG)_M4 7.3 39 3.6
WCJM/pCL-leuE WT(ATG)_M6 7.5 40 4.9

[143]
As can be seen from Table 5, leuE mutant plasmid is introduced, but for all three mutant strains OD is lower than the wild type, it showed a consumption rate per fast, especially for leuE WT (ATG) _M6 O- acetyl-homoserine 4.9 g / L has been producing the highest production capability. So that the present application were all variants three kinds of verify the increased production capability of O- acetyl-homoserine, and can be further increased ability O-acetyl-homoserine production was increased when the protein expression level by changing the start codon leuE He was doing OK.
[144]
[145]
3-2. Biosynthetic pathway genes and variants produced plasmid
[146]
In order to maximize the production capability of O- acetyl-homoserine to prepare a plasmid for enhancing the biosynthetic pathway to homoserine. Aspartate semi-aldehyde dehydrogenase (aspartate semialdehyde dehydrogenase) and pyridine nucleotide trans dehydrogenase kinase (pyridine nucleotide transhydrogenase) and LeuE was introduced and wild-type asd gene and pntAB the first pCL vector for cloning a mutant in pCL vector.
[147]
First, in order to obtain the asd and pntAB gene, using the SEQ ID NO: 15 and SEQ ID NO: 16 primer, SEQ ID NO: 17 and a primer of SEQ ID NO: 18 for pntAB for asd denaturation (denaturation) step is 30 seconds at 94 ℃, annealing (annealing) step is from 55 ℃ 30 seconds, and extension (extension) step was conducted a PCR reaction performed at 68 ℃ for 3 minutes, and do this 30 times. The result was purified after each post the PCR products in 1.0% agarose gel electrophoresis, 1.4kb (asd) and 3kb DNA from the band of (pntAB) size obtained.
[148]
The two genes purified sewing PCR was connected through a (in the first step PCR technique used to amplify and then connected using the overlap portion of the two genes without primers, primers of both ends). Sewing PCR conditions were carried out after performing 10 times, adding primers of SEQ ID NO: 15 and SEQ ID NO: 18 after the PCR reaction 20 times in the PCR reaction described above for the first step. Was it produced the asd-pntAB gene combinations fragments through, which was then purified from gel electrophoresis, and then given to process the restriction enzyme samI overnight at 37 ℃ with pCL vector, using a time after further purification, T4 ligase (ligase) the pCL-asd-pntAB plasmid was produced.
[149]
In the thus produced plasmid it was cloned leuE gene. The cloning is specifically, by using the SEQ ID NO: 29 and SEQ ID NO: 30 primer modified (denaturation) step is 30 seconds at 94 ℃, annealing (annealing) step is 30 seconds at 55 ℃ to obtain the leuE gene, extend (extension) step was performed a PCR reaction carried out for 1 minute at 68 ℃, and do this 30 times.
[150]
As a result, after the thus obtained PCR product was electrophoresed in 1.0% agarose gel, DNA was purified from a band of 800bp in size. After the process gave the purified plasmid DNA with the restriction enzymes overnight at 37 ℃ kpnI, it was cloned once after further purification, T4 Riga leuE and pCL-asd-pntAB vector using the Kinase (ligase). By using a cloning plasmid transformed into E. coli DH5α and then, by selecting a transformant E. coli DH5α on LB plate medium containing spectinomycin (spectinomycin) 50㎍ / mL to obtain a plasmid. By introducing a plasmid in the production O- acetyl homoserine production master WCJM conducted a flask evaluation for the feature production of O- acetyl-homoserine. The plasmid was produced using both the wild type and mutant 3 kinds manufactured in Example 2-1 to 4 kinds. 4 kinds of plasmids was in progress the flask was evaluated in the same manner as in Example 3-1 was introduced using electroporation to WCJM strain, and the results are shown below in Table 6.
[151]
[152]
[Table 6] O- acetyl homoserine production measured by flask culture
OD(562 nm) Glucose consumption (g / L) O-AH (g / L)
WCJM/pCL-asd-pntAB 9.8 36 1.8
WCJM/pCL-asd-pntAB-leuE WT 9.5 37 2.0
WCJM/pCL-asd-pntAB-leuE WT_M3 8.2 38 3.0
WCJM/pCL-asd-pntAB-leuE WT_M4 7.5 38 4.2
WCJM/pCL-asd-pntAB-leuE WT_M6 7.8 38 5.9

[153]
As can be seen from Table 6, the results gave enhance the biosynthetic pathway and leuE mutations at the same time, has further improved capability of production of O- acetyl-homoserine. In particular, the pCL-asd-pntAB-leuE WT_M6 plasmid is introduced strain, but OD is lower than the wild type, showed a consumption rate per fast, O- acetyl homoserine is 5.9 g / L are produced the highest production ability.
[154]
[155]
Example 4: saturated the mutagenesis through leuE variations produced and O- acetyl homoserine -producing ability evaluation
[156]
[157]
4-1. It saturated the mutagenesis through leuE mutant Fabrication and Evaluation
[158]
Production ability O- acetyl-homoserine to produce another type of amino acid substitution mutation was high leuE three kinds was produced by the mutation saturated mutagenesis. Amino acid substitutions were made in the plasmid produced 17 kinds of mutation, respectively M3, M4 mutant, and M6 mutations in Example 2 as a template. The list is shown in the following Table 7.
[159]
[160]
Table 7
Variations plasmid Substituted amino acid Primer SEQ ID NO:
M3 L95F SEQ ID NO: 31, 32
L95A SEQ ID NO: 33, 34
L95G SEQ ID NO: 35, 36
L95T SEQ ID NO: 37, 38
L95N SEQ ID NO: 39, 40
L95D SEQ ID NO: 41, 42
L95H SEQ ID NO: 43, 44
L95I SEQ ID NO: 45, 46
L95S SEQ ID NO: 47, 48
L95P SEQ ID NO: 49, 50
L95Y SEQ ID NO: 51, 52
L95Q SEQ ID NO: 53, 54
L95K SEQ ID NO: 55, 56
L95E SEQ ID NO: 57, 58
L95C SEQ ID NO: 59, 60
L95W SEQ ID NO: 61, 62
L95R SEQ ID NO: 63, 64
M4 F30W SEQ ID NO: 65, 66
F30L SEQ ID NOs: 67, 68
F30V SEQ ID NO: 69, 70
F30G SEQ ID NO: 71, 72
F30S SEQ ID NO: 73, 74
F30N SEQ ID NO: 75 and 76
F30D SEQ ID NO: 77, 78
F30H SEQ ID NO: 79, 80
F30I SEQ ID NO: 81, 82
F30P SEQ ID NO: 83, 84
F30Y SEQ ID NO: 85 and 86
F30Q SEQ ID NO: 87 and 88
F30K SEQ ID NO: 89, 90
F30E SEQ ID NO: 91, 92
F30C SEQ ID NO: 93, 94
F30T SEQ ID NO: 95, 96
F30R SEQ ID NO: 97, 98,
M6 F165W SEQ ID NO: 99, 100
F165L SEQ ID NO: 101, 102
F165V SEQ ID NO: 103, 104
F165G SEQ ID NO: 105, 106
F165S SEQ ID NO: 107, 108
F165N SEQ ID NO: 109, 110
F165D SEQ ID NO: 111, 112
F165H SEQ ID NO: 113, 114
F165I SEQ ID NO: 115, 116
F165P SEQ ID NO: 117, 118
F165Y SEQ ID NO: 119, 120
F165Q SEQ ID NO: 121, 122
F165K SEQ ID NO: 123, 124
F165E SEQ ID NO: 125, 126
F165C SEQ ID NO: 127, 128
F165T SEQ ID NO: 129, 130
F165R SEQ ID NO: 131, 132

[161]
Specifically, using primers presented in Table 7, where - by performing specific mutation kit (site-directed mutagenesis kit, Stratagene, USA) was prepared variant leuE gene. The produced mutant plasmid was carried flask evaluated in the same manner as in Example 3-1, and the strain introduced into the WCJM. The results are given in Table 8.
[162]
[163]
[Table 8] O- acetyl homoserine production measured by flask culture
Strain Plasmid Variations Location OD(562 nm) Glucose consumption (g / L) O-AH (g / L)
WCJM pCL1920 9.6 35 1.3
pCL-leuE WT 8.4 36 1.5
pCL-leuE WT_M3 L95V 8.2 38 2.3
pCL-leuE WT_M4 F30A 7.9 38 3.7
pCL-leuE WT_M6 F165A 8.0 39 4.8
M3 mutation L95F 8.6 38 2.3
L95A 8.3 38 2.2
L95G 9.2 37 2.1
L95T 9.4 37.5 2.3
L95N 8.8 38 2.4
L95D 8.7 36 2.2
L95H 9.5 35 2.3
L95I 9.5 37.5 2.2
L95S 9.3 37 2.5
L95P 9.2 36 2.5
L95Y 8.9 35 2.2
L95Q 9.4 38 3.1
L95K 9.2 38.5 2.2
L95E 8.6 37 2.6
L95C 8.9 37.5 2.4
L95W 9.9 38 2.1
L95R 9.3 38 2.3
M4 variants F30W 7.5 38 3.2
F30L 7.2 36 3.1
F30V 7.3 35 2.6
F30G 8.3 36 3.4
F30S 7.9 35 3.6
F30N 8.2 37 3.5
F30D 8.6 38 3.0
F30H 8.8 34 2.9
F30I 8.3 35 3.5
F30P 8.6 35.5 3.1
F30Y 7.9 34 2.9
F30Q 8.6 34 2.8
F30K 8.8 35 3.1
F30E 7.6 35.5 2.5
F30C 7.9 35 2.4
F30T 8.9 36 3.0
F30R 8.6 38.5 2.9
M6 mutations F165W 8.2 39 4.2
F165L 8.3 38 4.5
F165V 8.4 38 4.1
F165G 8.0 39 4.6
F165S 7.9 37 4.7
F165N 8.8 39 4.7
F165D 7.8 38 4.5
F165H 7.9 38 4.5
F165I 7.8 37 4.1
F165P 7.7 37.5 4.2
F165Y 8.2 38 4.6
F165Q 8.4 38 3.9
F165K 7.6 39 4.0
F165E 7.7 36.5 4.2
F165C 7.6 36.5 4.3
F165T 8.5 34 3.7
F165R 8.3 38 3.9

[164]
The evaluation of each variant As shown in Table 8, but show slightly differences in the OD or long sugar consumption rate, all the mutant strains WCJM / pCL1920 and WCJM / pCL-O- leuE WT than used as a control it was confirmed that the increase production of acetyl-homoserine.
[165]
[166]
4-2. O- acetyl homoserine in high yield of the strain leuE enhanced mutant strain produced and O- acetyl homoserine -producing ability evaluation
[167]
A method of using a strain having the ability to produce non-Leo used NTG through the mutation of the wild-type W3110 strain derived from a conventional production to produce O- acetyl-homoserine is known (International Patent Publication No. WO2012 / 087039). The strain producing O- acetyl homoserine in high yield in the production is deposited as accession No. KCCM11146P in Korea Culture Center of Microorganisms.
[168]
Introducing leuE gene and its mutant of the present application on the basis of the strain seen by checking whether to further promote O- acetyl homoserine-producing ability.
[169]
Specifically, the leuE 3 variant genes and was introduced by the electroporation method with the plasmid form. The introduced strain was named KCCM11146P / pCL1920, KCCM11146P / pCL-leuE WT, KCCM11146P- pCL-leuE M3, KCCM11146P- pCL-leuE M4 or KCCM11146P / pCL-leuE M6. The O- acetyl homoserine-producing ability of the mutant gene and leuE flask culture was carried out for evaluation to measure. More specifically, LB and then to the medium inoculated with the four kinds of the strains was cultured overnight at 33 ℃, then inoculated with a single colony in 3 ml LB medium, and 5 hours of incubation at 33 ℃, again 25 ml O- acetyl homoserine production the O- acetyl homoserine productivity was confirmed by HPLC analysis by 200-fold dilution in 250 ml Erlenmeyer flask, the medium is added and incubated for 30 hours at 33 ℃ 200 rpm. In summary the above experimental results to given in Table 9.
[170]
[171]
[Table 9] O- acetyl homoserine production measured by flask culture
OD (562nm) Glucose consumption (g / L) O-AH (g / L)
KCCM11146P/pCL1920 18.3 40 14.2
KCCM11146P/pCL-leuE WT 17.9 40 16.3
KCCM11146P/pCL-leuE M3 17.5 40 16.9
KCCM11146P/pCL-leuE M4 16.8 40 19.2
KCCM11146P/pCL-leuE M6 17.2 40 18.8

[172]
As can be seen from Table 9, the strain introduced into the strain KCCM11146P only pCL1920 O- acetyl homoserine is 14.2 g / L was produced, in the case of FIG was leuE WT than the increase in the yield of O- acetyl-homoserine original strain. In addition, the variation was 3 species all OD is reduced, in the case of M4 there was a production of O- acetyl-homoserine highest, 19.2 g / L, even if the rest of M3, M6 mutations increase the yield of O- acetyl-homoserine confirmed.
[173]
The inventors of the present application are based KCCM11146P strain leuE 3 jong mutant M3, M4, M6 mutations enhance strain 'KCCM11146P / pCL-leuE M3, M4, M6' from O- acetyl homoserine confirmed that the production is increased and the strains' CA05 -4009, 'CA05-4010,' accession to the CA05-4011 'naming Korea Culture Center of microorganisms (KCCM) on December 15th 2014 under the Budapest Treaty and then were given an accession number KCCM11645P, KCCM11646P, KCCM11647P.
[174]
[175]
Example 5: Production of O- acetyl-homoserine producing L- methionine using the culture medium and converting enzyme
[176]
[177]
Using the Example 4 obtained in a culture medium, an enzyme with this activity and the transition to methionine of O- acetyl-homoserine O- acetyl homoserine sulfinyl deurilra claim was performed an experiment to produce L- methionine.
[178]
Method set forth in the Republic of Korea Registered Patent No. 10-1250651 embodiment favors Example 1-2 to a conversion enzyme O- acetyl homoserine was produced sulfinyl deurilra claim, L- generated by performing the conversion in a manner set forth in Example 3 of Patent a methionine was measured. O- acetyl-homo-serine is used as a substrate, the above embodiment was proceeds to KCCM11146P- pCL-leuE culture (O-AH concentration 19.2 g / L) obtained in 4 of the M4, the concentration of the produced L- methionine are present in the following Table 10 below.
[179]
[180]
[Table 10]
Time (min)
2 4 6 8 10
MetZ-rsp 메치오닌 (g / L) 3.21 4.34 4.52 4.78 5.04
Conversion Rate (%) 50 % 68 % 71 % 75 % 79 %

[181]
As can be seen from Table 10, it was confirmed that the O- acetyl homoserine included in the culture medium of a KCCM11146P-pCL-leuE M4 strain obtained in Example 4 is converted to methionine in a 79% conversion rate for 10 minutes. Thereby, it was confirmed that can successfully produce the methionine using the strain of the present application.
[182]
[183]
From the above description, those skilled in the present application will appreciate that the present application without changing the technical spirit or essential features may be embodied in other specific forms. The embodiments described above In this regard, the examples should be understood as illustrative and not be limiting in all aspects. Scope of the present application is to be the meaning and scope, and all such modifications as derived from the equivalent concepts of the claims to be described later, rather than the foregoing description be construed as included within the scope of the present application.
[184]
[185]
[186]
[187]

Claims

[Claim 1]The first amino acid Val, the 30th amino acid phenylalanine, 95th amino acid leucine or 165th amino acid is one or more selected from the group consisting of the amino acid phenylalanine is substituted with another amino acid, O- acetyl homoserine emptying in the amino acid sequence of SEQ ID NO: 1 with the polypeptide.
[Claim 2]
The method of claim 1, wherein the polypeptide or amino acid valine is substituted with one methionine in the amino acid sequence of SEQ ID NO: 1; The 30th amino acid phenylalanine, alanine, tryptophan, leucine, valine, glycine, serine, asparagine, or substituted by selected from the group consisting of aspartic acid and histidine; The 95th amino acid leucine and the valine, phenylalanine, alanine, glycine, threonine, asparagine, or substituted by selected from the group consisting of aspartic acid and histidine; Or 165 amino acid phenylalanine, alanine, tryptophan, leucine, valine, is substituted to be selected from glycine, serine, asparagine, aspartic acid, and the group consisting of histidine, O- acetyl homoserine polypeptide having a discharge function.
[Claim 3]
The method of claim 1, wherein the polypeptide is substituted with one amino acid valine, the methionine in the amino acid sequence of SEQ ID NO: 1, the 30th amino acid phenylalanine, 95th amino acid leucine or 165 th amino acid phenylalanine is substituted with another amino acid, acetyl-O- homoserine polypeptide having a discharge function.
[Claim 4]
The method of claim 1, wherein the polypeptide is SEQ ID NO: 2,133, 134, 137, 138, 141 and 142 amino acid sequence selected from the group consisting of, O- acetyl homoserine polypeptide having a discharge function.
[Claim 5]
Claim 1 O- acetyl homoserine polynucleotide encoding a polypeptide having a discharge function.
[Claim 6]
The method of claim 5 wherein the the initiation codon of said polynucleotide further substituted by ATG, the polynucleotide.
[Claim 7]
According to claim 5, wherein the polynucleotide is SEQ ID NO: 4, 135, 136, 139, 140, 143 and 144 selected from the group consisting of the nucleotide sequence of the polynucleotide.
[Claim 8]
Containing a polypeptide or variant thereof, the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or over-expression, O- acetyl Escherichia (homoserine producing Escherichia ) spp.
[Claim 9]
The method of claim 8, wherein the variant polypeptide is substituted with one or more amino acids selected from the group consisting of the 1st amino acid Val, the 30th amino acid leucine, the 95th amino acid phenylalanine, or 165 amino acid phenylalanine in the amino acid sequence of SEQ ID NO: 1 with other amino acids a, O- acetyl Escherichia (homoserine producing Escherichia ) spp.
[Claim 10]
The method of claim 8, wherein the variant polypeptide is substituted with one amino acid valine, the methionine in the amino acid sequence of SEQ ID NO: 1; The 30th amino acid phenylalanine, alanine, tryptophan, leucine, valine, glycine, serine, asparagine, or substituted by selected from the group consisting of aspartic acid and histidine; The 95th amino acid leucine and the valine, phenylalanine, alanine, glycine, threonine, asparagine, or substituted by selected from the group consisting of aspartic acid and histidine; Or 165 amino acid phenylalanine, alanine, tryptophan, leucine, valine, glycine, serine, asparagine, aspartic acid and, O- acetyl Escherichia spp producing homoserine to be substituted selected from the group consisting of histidine.
[Claim 11]
10. The method of claim 9, wherein the mutant polypeptide is substituted with one amino acid valine, the methionine in the amino acid sequence of SEQ ID NO: 1, the 30th amino acid phenylalanine, 95th amino acid leucine or phenylalanine amino acid 165 are substituted with other amino acids, O- acetyl homoserine polypeptide having a discharge function.
[Claim 12]
The method of claim 8, wherein said microorganism of the genus Escherichia is Escherichia coli ( Escherichia coli ) which, Escherichia spp to produce O- acetyl homoserine.
[Claim 13]
The method of claim 8, wherein said microorganism of the genus Escherichia is additionally cis tachioh non synthase is inactivated, the microorganism of the genus Escherichia to produce O- acetyl homoserine activity (cystathionine synthase).
[Claim 14]
The method of claim 8, wherein said microorganism of the genus Escherichia is a microorganism of the genus Escherichia to produce an additional homoserine kinase inactivating activity (homoserine kinase), O- acetyl homoserine.
[Claim 15]
The method of claim 8, wherein said microorganism of the genus Escherichia is further homoserine acetyltransferase activity of the (homoserine acetyltransferase) the bibyeon enhanced compared to the microorganism, O- acetyl Escherichia spp producing homoserine.
[Claim 16]
The method of claim 8, wherein the Escherichia genus microorganism further aspartate semi-aldehyde dehydrogenase (aspartate semialdehyde dehydrogenase), pyridine nucleotide trans dehydrogenase kinase (pyridine nucleotide transhydrogenase) or activity of a combination of the bibyeon microorganisms for enhanced contrast, O- acetyl homoserine producing Escherichia spp.
[Claim 17]
Claim 8 to 16 the method comprising culturing in a medium a microorganism of the genus Escherichia which produces any one of O- acetyl-homoserine of wherein; And, O- acetyl homoserine production method comprising the step of recovering O- acetyl-homoserine in the medium or the microorganism cultured in the culture step.
[Claim 18]
Claim 8 to 16 the method comprising culturing in a medium a microorganism of the genus Escherichia which produces any one of O- acetyl-homoserine of wherein; And the culture medium or the microorganism cultured in the step, or the culture by a O- acetyl homoserine recovered from the microorganism or the culture medium in step processes the methylmercaptan and methionine-converting enzyme L- methionine of O- acetyl-homoserine comprising the step of switching, the method of producing L- methionine.
[Claim 19]
The method of claim 18 wherein the methionine-converting enzyme is O- acetyl homoserine sulfinyl deurilra agent, L- methionine production method.