Polypeptide, polynucleotide, microorganism of the genus Escherichia, method of production of O-acetylhomoserine and L-methionine.

Abstract

The present disclosure relates to a protein that has the activity of exporting O-acetyl-homoserine and an innovative modified protein thereof, a microorganism with the ability to produce O-acetyl-homoserine with enhanced protein expression, and a method for producing O-acetyl-homoserine using the microorganism.

Description

Descriptive Report of the Invention Patent for “POLYPEPTIDE, POLYNUCLEOTIDE, MICROORGANISM OF THE GENUS ESCHERICHIA, METHOD FOR PRODUCING O-ACETYL-HOMOSERIN AND L-METHIONINE” [FIELD OF TECHNIQUE]

[001] The present disclosure relates to a protein that has the activity of exporting O-acetyl-homoserine and an innovative modified protein thereof, a microorganism with the ability to produce O-acetyl-homoserine with enhanced protein expression and a method for producing O-acetyl-homoserine using the microorganism. [BACKGROUND OF THE TECHNIQUE]

[002] Methionine, which can be produced by chemical and biological synthesis, is used as a raw material for both the synthesis of infusions and medicines and for the synthesis of feed and food additives. Recently, a two-step process for the production of L-methionine from an L-methionine precursor, produced by fermentation, through an enzyme conversion reaction was disclosed (International Patent Publication WO 2008 / 013432). International Patent Publication WO 2008 / 013432 reveals that O-succinyl-homoserine and O-acetyl-homoserine can be used as a methionine precursor in the two-step process, and that it is very important to produce methionine precursors in high yield for economical large-scale methionine production.

[003] LeuE is known as a leucine export protein. As one of the proteins belonging to the homoserine / homoserine lactone efflux protein (RhtB) family, LeuE is a protein present in the inner membrane and is known to have the role of exporting leucine and its analogues as a putative uncharacterized transport protein.

[004] In the prior art relating to LeuE, it is known that a purine nucleoside or purine nucleotide can be produced by enhancing an amino acid sequence of the leuE gene (yeaS) or a sequence of Petition 870170093723, dated 01 / 12 / 2017, page 12 / 242 2 / 38 amino acids modified from the same, and the amount of amino acid production can be improved. Additionally, a modified leuE that has cysteine ​​exporting activity is known. [REVELATION] [TECHNICAL PROBLEM]

[005] Much effort has been made to improve the production of O-acetylhomoserine, and as a result, a protein that has the activity of exporting O-acetylhomoserine and a modified protein of the same have been discovered, which thus completes the present disclosure. [TECHNICAL SOLUTION]

[006] One objective of the present disclosure is to provide a polypeptide that has the activity of exporting O-acetyl-homoserine.

[007] Another objective of the present disclosure is to provide a polynucleotide that codes for the polypeptide.

[008] Yet another objective of the present disclosure is to provide a microorganism of the genus Escherichia that produces O-acetyl-homoserine, in which a polypeptide that has the activity of exporting O-acetyl-homoserine is included or overexpressed.

[009] Yet another objective of the present disclosure is to provide a method for producing O-acetyl-homoserine, which includes: cultivating a microorganism of the genus Escherichia that produces O-acetyl-homoserine in a medium; and recovering O-acetyl-homoserine from the cultured microorganism or from the cultured medium.

[010] Yet another objective of the present disclosure is to provide a method for producing L-methionine, which includes: cultivating the microorganism of the genus Escherichia that produces O-acetyl-homoserine in a medium; and converting the O-acetyl-homoserine to L-methionine by treating the cultured microorganism or the cultured medium or the O-acetyl-homoserine recovered from the cultured microorganism or the cultured medium with methyl mercaptan and an enzyme that converts methionine. [ADVANTAGEOUS EFFECTS OF THE INVENTION] Petition 870170093723, dated 01 / 12 / 2017, page 13 / 242 3 / 38

[011] The microorganism of the present disclosure, which includes a LeuE or a modified LeuE, which are inner membrane proteins, has enhanced activity in exporting O-acetyl-homoserine, and thus the efficiency of O-acetyl-homoserine production can be improved. Consequently, the microorganism of the present disclosure can be used for the efficient production of O-acetyl-homoserine. Additionally, O-acetyl-homoserine produced with high efficiency can be used for the economical, large-scale production of L-methionine. [Preferred Modality]

[012] To achieve the above objectives, one aspect of the present disclosure provides a polypeptide that has the activity of exporting O-acetyl-homoserine, wherein at least one amino acid selected from the group consisting of valine at position 1, leucine at position 30, phenylalanine at position 95 and phenylalanine at position 165 in the amino acid sequence of SEQ ID NO: 1 is replaced by another amino acid.

[013] As used in this document, the term “O-acetylhomoserine”, which is a specific intermediate material in the methionine biosynthesis pathway of microorganisms, refers to an acetyl derivative of L-homoserine. O-acetyl-homoserine is known to be produced by reacting homoserine and acetyl-CoA catalyzed by homoserine acetyltransferase, and has the formula C6H11NO4.

[014] As used in this document, the term “peptide that has the activity of exporting O-acetyl-homoserine” refers to a polypeptide that has the function of exporting O-acetyl-homoserine from a microorganism cell to the outside of the cell. Specifically, the peptide may refer to a LeuE protein that has the activity of exporting O-acetyl-homoserine and a modified protein thereof, but the peptide is not particularly limited to the same as long as it has the activity of exporting O-acetyl-homoserine.

[015] As used in this document, in relation to Petition 870170093723, dated 01 / 12 / 2017, page 14 / 242 4 / 38 amino acid transporters, the term “LeuE”, which is a protein belonging to the homoserine / homoserine lactone efflux protein family (RhtB), refers to a protein present in the inner membrane, but its exact function is unknown. In this respect, it has been confirmed that LeuE specifically exports O-acetyl-homoserine.

[016] LeuE may be a protein derived from a microorganism of the genus Escherichia, and specifically, LeuE derived from E. coli, but any LeuE that has the activity of exporting O-acetylmoserine may be included within the scope of the present disclosure without limitation as to the origin of the microorganism.

[017] Specifically, the peptide that has the activity of exporting O-acetylhomoserine may be a protein that has the amino acid sequence of SEQ ID NO: 1. Additionally, the peptide may be a protein that has an amino acid sequence that has the activity of exporting O-acetylhomoserine substantially equal to or equivalent to that of the amino acid sequence of SEQ ID NO: 1, while having a homology of at least 70%, specifically, at least 80%, and more specifically, at least 90% to the amino acid sequence of SEQ ID NO: 1. Alternatively, the peptide may be an amino acid sequence that has such homology when there is deletion, modification, substitution, or addition in part of the amino acid sequence that has the activity of exporting O-acetylhomoserine substantially equal to or equivalent to that of the amino acid sequence of SEQ ID NO: 1, and it is obvious that such a peptide also belongs to the scope of the present disclosure.

[018] As used in this document, the term “modified polypeptide” for the polypeptide that has O-acetylhomoserine exporting activity refers to a polypeptide that has enhanced O-acetylhomoserine exporting activity compared to that of the wild-type polypeptide or unmodified polypeptide. Specifically, the modified polypeptide is a peptide that has enhanced O-acetylhomoserine exporting activity. Petition 870170093723, dated 01 / 12 / 2017, page 15 / 242 5 / 38 homoserine compared to that of the polypeptide having the amino acid sequence SEQ ID NO: 1 due to a modification of at least one amino acid in the amino acid sequence of SEQ ID NO: 1.

[019] For example, the modified polypeptide may be a polypeptide in which at least one amino acid selected from the group consisting of valine at position 1, leucine at position 30, phenylalanine at position 95, and phenylalanine at position 165 in the amino acid sequence of SEQ ID NO: 1 is replaced by another amino acid. Specifically, the modified polypeptide may be a polypeptide in which valine at position 1 in the amino acid sequence of SEQ ID NO: 1 is replaced by methionine; phenylalanine at position 30 in the amino acid sequence of SEQ ID NO: 1 is replaced by any one selected from the group consisting of alanine, tryptophan, leucine, valine, glycine, serine, asparagine, aspartic acid, histidine, isoleucine, proline, tyrosine, glutamine, lysine, glutamic acid, cysteine, threonine, and arginine;Leucine at position 95 in the amino acid sequence of SEQ ID NO: 1 is replaced by any one selected from the group consisting of valine, phenylalanine, alanine, glycine, threonine, asparagine, aspartic acid, histidine, isoleucine, serine, proline, tyrosine, glutamine, lysine, glutamic acid, cysteine, tryptophan, and arginine; or phenylalanine at position 165 in the amino acid sequence of SEQ ID NO: 1 is replaced by any one selected from the group consisting of alanine, tryptophan, leucine, valine, glycine, serine, asparagine, aspartic acid, histidine, isoleucine, proline, tyrosine, glutamine, lysine, glutamic acid, cysteine, threonine, and arginine. More specifically, the modified polypeptide may be a polypeptide in which valine at position 1 in the amino acid sequence of SEQ ID NO: 1 is replaced by methionine;Phenylalanine at position 30 in the amino acid sequence of SEQ ID NO: 1 is replaced by any one selected from the group consisting of alanine, tryptophan, leucine, valine, glycine, serine, asparagine, aspartic acid, and histidine; leucine at position 95 in the amino acid sequence of SEQ ID NO: 1 is replaced by any one selected from the group consisting of; Petition 870170093723, dated 01 / 12 / 2017, p. 16 / 242 6 / 38 valine, phenylalanine, alanine, glycine, threonine, asparagine, aspartic acid, and histidine; or phenylalanine at position 165 in the amino acid sequence of SEQ ID NO: 1 is replaced by any one selected from the group consisting of alanine, tryptophan, leucine, valine, glycine, serine, asparagine, aspartic acid, and histidine. Even more specifically, the modified polypeptide may be a polypeptide in which valine at position 1 in the amino acid sequence of SEQ ID NO: 1 is replaced by methionine; and phenylalanine at position 30, leucine at position 95, and phenylalanine at position 165 in the amino acid sequence of SEQ ID NO: 1 is replaced by another amino acid. More specifically, the modified polypeptide may be a polypeptide consisting of an amino acid sequence with SEQ ID NO: 2, 133, 134, 137, 138, 141, or 142.Specifically, the modified polypeptide may be a protein that has an amino acid sequence that has enhanced O-acetyl-homoserine export activity substantially equal to or equivalent to that of the amino acid sequence of the modified polypeptide, while having at least 70% homology, specifically at least 80%, and more specifically at least 90% to the above amino acid sequences. Alternatively, in an amino acid sequence that has such homology and that has enhanced O-acetyl-homoserine export activity substantially equal to or equivalent to that of the amino acid sequence of the modified polypeptide, the amino acid sequence may be one where there is deletion, modification, substitution, or addition in part of the amino acid sequence.The polypeptide is an example of a modified polypeptide with enhanced O-acetyl-homoserine export activity compared to that of the wild-type polypeptide or unmodified polypeptide, but the polypeptide is not limited to the same. As used herein, the term “natural native state or unmodified state” refers to a state in which the introduction of the corresponding polypeptide or the introduction of activity modification in the present disclosure has not been achieved.

[020] As used herein, the term “homology” refers to. Petition 870170093723, dated 01 / 12 / 2017, page 17 / 242 7 / 38 refers to the degree of identity between nucleotides or amino acid residues of two amino acid sequences or nucleic acid sequences of a protein-coding gene, determined after aligning them to match each other as closely as possible to a particular comparison region. When homology is sufficiently high, the expression products of the corresponding gene may have the same or similar activity. The percentage of sequence identity can be determined using a known sequence comparison program (e.g., BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlign (DNASTAR Inc), etc.).

[021] One aspect of the present disclosure provides a polynucleotide encoding the polypeptide that has the activity of exporting O-acetyl-homoserine. In the present disclosure, the polypeptide that has the activity of exporting O-acetyl-homoserine is the same as that described above.

[022] For example, the polynucleotide may be one in which the initiation codon is replaced by ATG and may be the nucleotide sequence of SEQ ID NO: 4, 135, 136, 139, 140, 143 or 144, but the nucleotide sequence is not limited by the same. Additionally, with respect to the polynucleotide, the nucleotide sequence and modified nucleotide sequences of the same that encode the same amino acid sequence are also included in the present disclosure based on codon degeneracy. For example, the nucleotide sequence may be modified to have an optimized codon depending on the microorganism that is used.

[023] Specifically, the nucleotide sequence may be one that encodes an amino acid sequence that has O-acetylmosserine export activity substantially equal to or equivalent to that of the above nucleotide sequences, while having at least 70% homology, specifically at least 80%, and more specifically at least 90% to the above amino acid sequences. Alternatively, the nucleotide sequence may be a sequence with the ability to hybridize with a probe, which may be prepared from a known gene sequence. Petition 870170093723, dated 01 / 12 / 2017, p. 18 / 242 8 / 38 (for example, a sequence complementary to all or part of the nucleotide sequences above), under stringent conditions to encode a protein that has the activity of exporting O-acetyl-homoserine. As used in this document, the term “stringent condition” refers to a condition in which a so-called specific hybrid is formed while a non-specific hybrid is not formed.For example, a strict condition might include a condition where genes with high homology (e.g., 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 97% or more, and even more specifically 99% or more) can hybridize with each other, whereas genes with lower homology cannot hybridize with each other; or conditions for hybridization with conventional probes (i.e., conditions for washing once, and specifically, two or three more times under a saline concentration and temperature corresponding to 60 °C, 1 χ SSC, and 0.1% SDS; specifically below 60 °C, 0.1 χ SSC and 0.1% SDS, and more specifically below 68 °C, 0.1x SSC and 0.1% SDS) (Sambrook et al., Molecular Cloning: A Laboratory Handbook, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)).The probe used for hybridization can optionally be a part of the nucleotide sequence complementary to the nucleotide sequences above. Such a probe can be prepared by PCR using an oligonucleotide prepared based on a known sequence as a primer and a gene fragment containing that nucleotide sequence as a template. For example, a gene fragment of approximately 300 bp can be used as the probe. More specifically, when a gene fragment of approximately 300 bp is used as a probe, the conditions of 50 °C, 2x SSC, and 0.1% SDS are listed as washing conditions for hybridization.

[024] The genes used in the present disclosure, the protein sequences and the promoter sequences that they encode can be obtained from a known database (e.g., NCBI GenBank), but without limitation to the same. Petition 870170093723, dated 01 / 12 / 2017, p. 19 / 242 9 / 38

[025] One aspect of the present disclosure relates to a microorganism in which the polypeptide having the activity of exporting O-acetyl-homoserine or a modified polypeptide thereof is included or overexpressed. Specifically, the microorganism may be a microorganism that produces O-acetyl-homoserine or a modified polypeptide thereof, in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is included or overexpressed.

[026] The polypeptide that has the activity of exporting O-acetyl-homoserine and the modified polypeptide thereof are the same as those explained above.

[027] As used in this document, the term “O-acetyl-homoserine-producing microorganism” refers to a microorganism that has the ability to produce O-acetyl-homoserine within the microorganism and export it to a medium. The activity of producing O-acetyl-homoserine may be provided or enhanced by natural or artificial mutations or species improvement. Specifically, such O-acetyl-homoserine-producing microorganisms may be included regardless of their microbial origin, provided they are capable of producing O-acetyl-homoserine. In one embodiment, the microorganism may be one belonging to the genus Escherichia and, more specifically, Escherichia coli.

[028] Meanwhile, in the present disclosure, the microorganisms that produce O-acetyl-homoserine may be a modified microorganism in which a known modification is additionally introduced with respect to related mechanisms such as pathways related to homoserine biosynthesis and mechanisms related to O-acetyl-homoserine export, etc., in order to enhance the productivity of O-acetyl-homoserine off LeuE.

[029] Another specific embodiment of the present disclosure may relate to the microorganism that produces O-acetyl-homoserine in which, additionally, the cystathionine synthase activity is inactivated. Specifically, the microorganism may be one in which the gene encoding cystathionine synthase (metB) is suppressed or its expression is weakened compared to that of a micro Petition 870170093723, dated 01 / 12 / 2017, page 20 / 242 10 / 38 unmodified organism, however, without limitation to the same. The amino acid sequence of the metB gene can be obtained from a known database and any amino acid sequence that has cystathionine synthase activity can be included without limitation (e.g., a protein that has the amino acid sequence SEQ ID NO: 5). The protein that has the amino acid sequence SEQ ID NO: 5 can be a protein encoded by the nucleotide sequence SEQ ID NO: 6, however, without limitation to the same.

[030] Additionally, yet another specific embodiment of the present disclosure may refer to the microorganism that produces O-acetyl-homoserine in which, additionally, the homoserine kinase activity is inactivated. Specifically, the microorganism may be one in which the homoserine kinase activity is reduced compared to its endogenous activity from an unmodified microorganism or is removed.For example, the microorganism might be one in which the gene (thrB) encoding homoserine kinase is linked to a weaker promoter compared to a native promoter, or is modified or suppressed to have weak activity, but the promoter is not limited to the same. The amino acid sequence of the thrB gene can be obtained from a known database, and any amino acid sequence that has homoserine kinase activity can be included without limitation (e.g., a protein that has the amino acid sequence SEQ ID NO: 7). The protein that has the amino acid sequence SEQ ID NO: 7 could be a protein encoded by the nucleotide sequence SEQ ID NO: 8, however, without limitation to the same.

[031] As used in this document, the term “protein inactivation” refers to a case where the protein activity of a microorganism is reduced compared to the enzyme activity possessed by the microorganism in a native wild-type protein or unmodified protein; a case where the protein is not expressed at all; or a case where the protein is expressed but does not exhibit activity. Inactivation is a concept that includes a case where the activity of the protein itself Petition 870170093723, dated 01 / 12 / 2017, page 21 / 242 11 / 38 The enzyme is reduced or removed compared to the enzyme activity originally possessed by the microorganism due to modification, etc. of the gene encoding the enzyme; a case where the entire enzyme activity level in a cell is reduced or removed compared to the enzyme activity originally possessed by the wild-type strain of the microorganism due to inhibition of the expression or translation of the gene encoding the enzyme; a case where part or all of the gene is suppressed; and a combination thereof; but inactivation is not limited to these.

[032] Inactivation of an enzyme can be achieved by applying several well-known methods in the art. Examples of methods may include a method of replacing the gene encoding the enzyme on the chromosome with a gene modified to reduce the enzyme's activity, which includes the case where enzyme activity is removed; a method of introducing a modification in the expression control sequence of the gene encoding the enzyme on the chromosome; a method of replacing the expression control sequence of the gene encoding the enzyme with a sequence that has weak activity or no activity; a method of suppressing part or all of the gene encoding the enzyme on the chromosome; a method of introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to a transcript of the gene on the chromosome, thereby inhibiting translation from mRNA to the enzyme;A method of artificially incorporating a complementary sequence to the SD sequence upstream of the SD sequence of the gene encoding the enzyme, forming a secondary structure that makes ribosome attachment to it impossible; the method of incorporating a promoter to the 3' end of the open reading frame (ORF) to induce reverse transcription (reverse transcription engineering (RTE)), etc., and also a combination thereof, though the methods are not particularly limited to them.

[033] The method of modifying the expression control sequence can be performed by inducing a modification in the control sequence of Petition 870170093723, dated 01 / 12 / 2017, page 22 / 242 12 / 38 Expression by deletion, insertion, non-conservative or conservative substitution, or a combination thereof in the nucleic acid sequence of the expression control sequence in order to further weaken the activity of the expression control sequence, or by substituting it with a nucleic acid that has weaker activity. The expression control sequence may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome-binding region, and sequences that control transcription and translation termination.

[034] Furthermore, the gene sequence on the chromosome can be modified by inducing a sequence modification by deletion, insertion, non-conservative or conservative substitution, or a combination thereof in the gene sequence to further weaken enzyme activity; or by substituting with a gene sequence that has been enhanced to have weaker activity or a gene sequence that has been enhanced to have no activity, but the method is not limited to these.

[035] Additionally, the method of suppressing part or all of a gene encoding an enzyme can be performed by replacing a polynucleotide encoding the endogenous target protein within the chromosome with a polynucleotide or marker gene that has a partial deletion in the nucleic acid sequence, using a vector for chromosomal insertion within a bacterial strain. In an exemplary embodiment of the method of suppressing part or all of a gene, a method for gene suppression by homologous recombination can be used, but the method is not limited to it.

[036] As used in this document, the term “part” may vary depending on the types of polynucleotides, and it may also refer specifically to 1 to 300, more specifically 1 to 100, and even more specifically 1 to 50, but is not particularly limited to these.

[037] As used in this document, the term “recombination Petition 870170093723, dated 01 / 12 / 2017, page 23 / 242 "13 / 38 homologous" refers to genetic recombination that occurs through crossing over at loci in the genetic chain that have mutual homology.

[038] Furthermore, yet another specific embodiment of the present disclosure may relate to the microorganism that produces O-acetyl-homoserine in which, additionally, the homoserine acetyltransferase activity is enhanced compared to that of an unmodified microorganism. Specifically, the microorganism may be one in which the homoserine acetyltransferase activity is enhanced compared to that of an unmodified microorganism and, particularly, it may be one in which a modified metA gene encoding homoserine acetyltransferase with enhanced activity is introduced. The modified metA gene may be a gene encoding one in which the 111th amino acid of homoserine acetyltransferase is replaced by glutamic acid and the 112th amino acid of homoserine acetyltransferase is replaced by histidine, but without limitation thereto.The modified metA gene may include, without limitation, any amino acid sequence that has enhanced homoserine acetyltransferase activity compared to that of its wild type and, for example, may be a protein that has the amino acid sequence SEQ ID NO: 10. The embodiments of the preparation of the modified metA gene and its use, a strain that has enhanced homoserine acetyltransferase activity, etc., are disclosed in Patent No. KR 10-1335841, and the entire descriptive report of the patent may be incorporated herein by reference to the present disclosure.

[039] Additionally, yet another specific embodiment of the present disclosure may relate to a microorganism that produces O-acetylmoserine belonging to the genus Escherichia in which, moreover, the activity of aspartate semialdehyde dehydrogenase, pyridine nucleotide transhydrogenase or a combination thereof is enhanced compared to that of an unmodified microorganism.

[040] Additionally, yet another specific embodiment of the present disclosure may refer to a microorganism that produces O-acetyl Petition 870170093723, dated 01 / 12 / 2017, p. 24 / 242 14 / 38 homoserine in which, additionally, the activity of phosphoenolpyruvate carboxylase, aspartate aminotransferase, or a combination thereof is enhanced compared to that of an unmodified microorganism. As used herein, the term “enhancement” refers to the enhancement of the activity level of a protein possessed by a microorganism. The enhancement of a protein's activity is not limited as long as it can enhance the activity of each protein compared to that of the wild-type protein or unmodified protein, as in the enhancement of the activity of a target protein.Enhancement can be achieved by a method selected from the group consisting of i) a method of increasing the copy number of a polynucleotide encoding each protein, ii) a method of introducing a modification in the expression control sequence to increase the expression of the polynucleotide, iii) a method of modifying the polynucleotide sequence in the chromosome to enhance the activity of each protein, and iv) a combination thereof.Specifically, enhancement can be achieved by a method selected from the group consisting of a method of inserting a polynucleotide that includes a nucleotide sequence encoding each protein within the chromosome, a method of introducing the polynucleotide into a microorganism after introducing it into a vector system, a method of introducing a promoter with enhanced activity into a region upstream of the nucleotide sequence encoding each protein or introducing each protein with a modification in its promoter, a method of modifying the nucleotide sequence in the 5'-UTR region, and a method of introducing a modified nucleotide sequence into the nucleotide sequence encoding each protein, but enhancement methods are not limited to these.

[041] Yet another aspect of the present disclosure relates to a method for producing O-acetyl-homoserine which involves cultivating the microorganism of the genus Escherichia that produces O-acetyl-homoserine in a medium. Petition 870170093723, dated 01 / 12 / 2017, page 25 / 242 15 / 38

[042] Specifically, the above method refers to a method for producing O-acetyl-homoserine that includes cultivating the microorganism of the genus Escherichia that produces O-acetyl-homoserine in a medium, and recovering O-acetyl-homoserine from the cultured microorganism or from the cultured medium.

[043] As used in this document, the term “culture” refers to the growth of a microorganism in an appropriately adjusted environment. In the present disclosure, the culture process can be carried out using an appropriate medium and culture conditions well known in the art. The culture process can be easily adjusted for use by a person of ordinary skill in the art according to the strain that is selected. The culture can be carried out in a batch process, continuous culture, fed-batch culture, etc., known in the art, but is not particularly limited to the same. The medium and other culture conditions used for the cultivation of the microorganism of the present disclosure may not be particularly limited, but any medium conventionally used for the cultivation of microorganisms of the genus Escherichia can be used.Specifically, the microorganism of the present disclosure can be cultivated under aerobic conditions in a common medium containing appropriate sources of carbon, nitrogen, and phosphorus, inorganic compounds, amino acids and / or vitamins, etc., while adjusting the temperature, pH, etc.

[044] In the present disclosure, carbon sources may include carbohydrates such as glucose, antisense, sucrose, maltose, mannitol, sorbitol, etc.; alcohols such as sugar alcohol, glycerol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc., but the carbon sources are not limited to these. Additionally, natural organic nutrients such as hydrolyzed starch, molasses, blackstrap molasses, rice bran, cassava, sugarcane bagasse, corn steep liquor, etc. may be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reduced sugar) may be used. Petition 870170093723, dated 01 / 12 / 2017, page 26 / 242 16 / 38 and, additionally, various other carbon sources in an appropriate quantity may be used without limitation. These carbon sources may be used alone or in a combination of at least two types.

[045] Examples of nitrogen sources may include inorganic nitrogen sources (e.g., ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.); amino acids (glutamic acid, methionine, glutamine, etc.); and organic nitrogen sources (e.g., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc.). These nitrogen sources may be used alone or in a combination of at least two types, but are not limited to them.

[046] Examples of phosphorus sources may include monopotassium phosphate, dipotassium phosphate, and corresponding sodium-containing salts. Examples of inorganic compounds to be used may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. Additionally, amino acids, vitamins, and / or appropriate precursors may be included, but are not limited to them. These media or precursors may be added in a batch culture process or continuous culture process to a culture, however, are not limited to them.

[047] During the culture period in the present disclosure, the pH of a culture can be adjusted by adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in an appropriate manner. Additionally, during the culture period, an antifoaming agent, such as fatty acid polyglycol ester, can be added to prevent foaming. Furthermore, to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas can be injected into the culture, while to maintain the anaerobic states and Petition 870170093723, dated 01 / 12 / 2017, page 27 / 242 17 / 38 microaerobic cultures, nitrogen, hydrogen or carbon dioxide gases can be injected without air injection.

[048] The culture temperature can normally be from 27 °C to 37 °C and, specifically, from 30 °C to 35 °C, but is not limited to these. Additionally, the culture can be continued until the desired material (or materials) is obtained and, specifically, for 10 hours to 100 hours, however, without limitation to these.

[049] The recovery of O-acetyl-homoserine can be carried out using the method of cultivating a microorganism of the present disclosure. For example, the target O-acetyl-homoserine can be recovered from a culture using an appropriate method known in the art (e.g., batch culture, continuous culture or fed-batch culture, etc.). For example, methods such as centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc. can be used and, additionally, a combined method of appropriate methods known in the art can be used.

[050] The recovery process may include a separation process and / or a purification process.

[051] One aspect of the present disclosure relates to a method for producing L-methionine, which includes cultivating the microorganism of the genus Escherichia that produces O-acetyl-homoserine in a medium; and converting the O-acetyl-homoserine to L-methionine by treating the cultured microorganism or the cultured medium or the O-acetyl-homoserine of the cultured microorganism or the cultured medium with methyl mercaptan and an enzyme that converts methionine.

[052] For example, methionine can be produced from O-acetylhomoserine, which is recovered from a culture of a microorganism of the genus Escherichia that produces O-acetylhomoserine in a medium, by a two-step process (Patent no. KR 10-0905381).

[053] The two-step process involves a process of producing L-methionine and an organic acid by an enzyme reaction using Petition 870170093723, dated 01 / 12 / 2017, page 28 / 242 18 / 38 an enzyme that has the activity of converting O-acetyl-homoserine to methionine using O-acetyl-homoserine and methyl mercaptan as substrates or a strain that contains the enzyme.

[054] The enzyme that converts methionine includes all enzymes that convert O-acetyl-homoserine to methionine and, in particular, O-acetylhomoserine sulfhydrylase, but not limited to them.

[055] Specifically, the O-acetyl-homoserine sulfhydrylase to be used may be a derivative from microbial strains belonging to the genus Leptospira, the genus Chromobacterium and the genus Hyphomonas and, more specifically, a derivative from microbial strains belonging to the genera Leptospira meyeri, Pseudomonas aurogenosa, Hyphomonas neptunium and Chromobacterium violaceum.

[056] The reaction above is shown below: CH3SH + O-acetyl-L-homoserine <=> acetate + methionine

[057] Such an additional process for producing methionine is disclosed in Patent No. KR 10-0905381, and the entire descriptive report of the patent may be included by reference to the present disclosure. [DETAILED DESCRIPTION OF THE INVENTION]

[058] Hereafter, the present disclosure will be described in detail by means of exemplary examples. However, these exemplary examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure. REFERENCE EXAMPLE 1: PREPARATION OF strains that produce O-acetyl-homoserine 1-1. SUPPRESSION OF THE METB GENE IN WILD-TYPE E. COLI

[059] To produce strains that produce O-acetyl-homoserine, E. coli, which is a representative microorganism among microorganisms of the genus Escherichia, was used. For this purpose, E. coli K12 W3110 (ATCC 27325), wild-type E. coli, was obtained from the American Type Culture Collection. Petition 870170093723, dated 01 / 12 / 2017, p. 29 / 242 19 / 38 (ATCC) and used. A strain having defects in the metB gene (SEQ ID NO: 6) encoding cystathionine synthase gamma and in the thrB gene (SEQ ID NO: 8) encoding homoserine kinase in E. coli K12 strain W3110 was prepared. The strain prepared in this way that produces O-acetyl-homoserine was named W3-BT. An embodiment relating to the suppression of metB and thrB gene-suppressing strains is disclosed in Patent No. KR 10-0905381 or International Patent Publication WO 2008 / 013432 (see particularly, Examples 1-1 and 1-2 of Patent No. KR 10-0905381), and the entire descriptive report of the patent may be incorporated herein by reference to the present disclosure. 1-2. Preparation of an introduced strain with a modified META gene that has homoserine acetyltransferase activity.

[060] To enhance homoserine acetyltransferase activity in the strain obtained in Reference Example 1-1, an attempt was made to introduce the modified metA gene (SEQ ID NO: 10) encoding homoserine acetyltransferase which has enhanced activity within the strain. In an attempt to prepare such a strain, a pCL_Pcj1_metA (EH) plasmid was prepared by the method described in Examples 1 and 3 of Patent No. KR 10-1335841.

[061] Then, to prepare a surrogate cassette as a way to replace the modified metA gene prepared above by introducing it into the strain, PCR was performed using the pKD3 vector as a template together with primers SEQ ID NO: 23 and SEQ ID NO: 24. Specifically, PCR was repeatedly performed for a total of 30 cycles, in which denaturation was performed at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds and extension at 72 °C for 2 minutes.

[062] For the metA (EH) portion of the replacement cassette, PCR was performed using pCL-Pcj1-metA (EH) as the template along with primers with SEQ ID NO: 19 and SEQ ID NO: 20, while for the wild-type metA portion, primers with SEQ ID NO: 21 and SEQ ID NO: 22 were used, thus obtaining the PCR products. Based on 3 PCR products, the merA (EH) replacement cassette that Petition 870170093723, dated 01 / 12 / 2017, p. 30 / 242 20 / 38 containing a chloramphenicol marker was prepared using primers SEQ ID NO: 19 and SEQ ID NO: 22, and introduced by electroporation into the W3-BT strain, which was transformed with the pKD46 vector, prepared in Reference Example 1-1.

[063] The strains that were confirmed to have been introduced by the above process were again transformed with the pCP20 vector and grown in LB medium. The strain in which the chloramphenicol marker was removed and the metA gene was replaced by metA (EH) was named W3-BTA.

[064] An embodiment relating to the strain with enhanced homoserine acetyltransferase activity, etc. is disclosed in Patent No. KR 10-1335841 or International Patent Publication WO 2012 / 087039, and the entire descriptive report of the patent may be included herein by reference to the present disclosure. 1-3. Preparation of a strain that includes 2 copies of PPC, aspC, and asd genes.

[065] To increase the productivity of O-acetyl-homoserine from the W3BTA strain prepared in Reference Example 1-2, a known strategy of enhancing the biosynthetic pathway was introduced. An attempt was made to prepare strains in which the genes associated with phosphoenolpyruvate carboxylase involved in the biosynthesis of oxaloacetate from phosphoenolpyruvate, aspartate aminotransferase involved in the biosynthesis of aspartate from oxaloacetate, and aspartate semialdehyde dehydrogenase involved in the biosynthesis of homoserine from β-aspartyl phosphate were amplified to 2 copies, meaning that the ppc, aspC, and asd genes were amplified to 2 copies.

[066] For the preparation of the strains, the pSG-2ppc, pSG-2aspC and pSG-2asd plasmids were prepared by the method disclosed in Examples 1-1 to 1-3 of Patent No. KR 10-1117012, the above plasmids being introduced into the W3-BTA strain, and the strain in which the 3 different genes were sequentially amplified to 2 copies was prepared by the method described in Example 1-5 of the Korean patent. The strain prepared in this manner was named Petition 870170093723, dated 01 / 12 / 2017, p. 31 / 242 21 / 38 W3-BTA2PCD (= WCJM).

[067] An embodiment relating to the strain with enhanced activity of phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate semialdehyde dehydrogenase, etc., is disclosed in Patent No. KR 10-0905381 or International Patent Publication WO 2008 / 013432, and the entire descriptive report of the patent may be included herein by reference to the present disclosure. 1-4. Flask Culture Experiment

[068] To test the amount of O-acetyl-homoserine production in the strains prepared in Reference Examples 1-2 and 1-3, culture in an Erlenmeyer flask was performed. Strains W3110, W3-BTA, and WCJM were seeded in LB medium and cultured at 33 °C overnight. Loose colonies were seeded in 3 ml of LB medium and incubated at 33 °C for 5 hours, diluted 200 times in a 250 ml Erlenmeyer flask containing 25 ml of O-acetyl-homoserine production medium, and incubated again at 33 °C at 200 rpm for 30 hours, and the amount of O-acetyl-homoserine production was confirmed by HPLC analysis. The composition of the medium used is summarized in Table 1 below. [TABLE 1] COMPOSITION OF MEDIUM IN A BOTTLE THAT PRODUCES O-acetyl-homoserine Composition Concentration (per Liter) Glucose 40 g Ammonium Sulfate 17 g KH2PO4 1.0 g MgSO4 7H2O 0.5 g FeSO4 7H2O 5 mg MnSO4 8H2O 5 mg ZnSO4 5 mg Calcium Carbonate 30 g Yeast Extract 2 g Petition 870170093723, dated 01 / 12 / 2017, page 32 / 242 22 / 38 Methionine 0.15 g Threonine 0.15 g

[069] The amount of O-acetyl-homoserine produced was confirmed by HPLC analysis after cultivation for 30 hours using the above medium, and the results are summarized in Table 2 below. [TABLE 2] PRODUCTION OF O-ACETYLHOMOSERIN BY FRICTION 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

[070] As can be seen in Table 2 above, O-acetyl-homoserine was not produced at all in the wild-type strain W3110, however, the strain W3-BTA produced O-acetyl-homoserine (O-AH) at a concentration of 0.9 g / l, and the strain WCJM with an enhanced biosynthetic pathway produced O-acetyl-homoserine (O-AH) at a concentration of 1.2 g / l. EXAMPLE 1: SELECTION OF MEMBRANE PROTEINS THAT INCREASE THE PRODUCTIVITY OF O-acetyl-homoserine

[071] We tried to apply LeuE (SEQ ID NO: 1) derived from Escherichia coli, which was revealed as a membrane protein but was not revealed in relation to the activity of exporting O-acetyl-homoserine and producing O-acetyl-homoserine, for the production of O-acetyl-homoserine.

[072] In order to enhance the leuE gene in the strain, the leuE gene was cloned using an Sma I restriction site from the pCL vector.

[073] First, to prepare the leuE gene, PCR was performed for a total of 30 cycles using SEQ ID NOS primers: 11 and 12, in which denaturation was performed at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds, and extension at 68 °C for 1 minute. The PCR product Petition 870170093723, dated 01 / 12 / 2017, p. 33 / 242 The resulting 23 / 38 sample was subjected to electrophoresis on 1.0% agarose gel, and DNA was purified from the 800 bp band. The purified DNA was treated with the restriction enzyme Sma I at 37 °C overnight, and after further purification, the leuE gene and the pCL vector were cloned using T4 ligase. After transforming E. coli DH5 using the cloned plasmid, the transformed E. coli DH5 was selected in LB plate medium containing spectinomycin (50 pg / ml) to obtain the plasmid. The plasmid prepared in this way was introduced into the W3-BTA and WCJM strains, which are strains that produce O-acetyl-homoserine. These were named W3BTA / pCL-leuE and WCJM / pCL-leuE, respectively, and the vial evaluation of their O-acetyl-homoserine productivity was performed.

[074] Additionally, like the control groups, the empty vector pCL1920 was introduced into the W3-BTA and WCJM strains in the same method as described above, and named W3-BTA / pCL1920 and WCJM pCL1920, respectively, and the vial evaluation of its O-acetylmoserine productivity was performed.

[075] Specifically, each strain was electroplated on solid LB medium and cultured overnight in a 33 °C incubator. A single colony of the strain cultured overnight on LB plate medium was seeded onto 3 ml of LB medium and incubated at 33 °C for 5 hours, diluted 200 times in a 250 ml Erlenmeyer flask containing 25 ml of medium for O-acetylhomoserine production, and incubated again at 33 °C at 200 rpm for 30 hours, and the amount of O-acetylhomoserine production was confirmed by HPLC analysis. The results are summarized in Table 3 below. [TABLE 3] MEASUREMENT OF O-acetyl-homoserine PRODUCTION by culture in a flask OD (562 nm) Glycose Consumption (g / l) O-AH (g / l) W3-BTA / pCL1920 9.5 35 0.9 W3-BTA / pCL-leuE 8.2 36 1.0 Petition 870170093723, 01 / 12 / 2017, pág. 34 / 242 24 / 38 WCJM / pCL1920 9.6 35 1.2 WCJM / pCL-leuE 8.4 36 1.5

[076] As can be seen in Table 3 above, the WCJM strain introduced with leuE plasmid showed a lower OD compared to that of the control strain introduced with the empty vector, and the WCJM strain also showed higher glucose consumption. However, the WCJM strain produced O-acetylhomoserine at a concentration of 1.5 g / l, and this cannot confirm that the increased production of O-acetylhomoserine was due to the introduction of wild-type leuE. In any case, the results of being able to control OD and the increased glucose consumption rate confirmed the potential export activity of the strain. Consequently, an attempt was made to select modified strains that have enhanced O-acetylhomoserine export activity compared to that of the wild-type strain through structural modeling. EXAMPLE 2: PLASMID PREPARATION WITH LEUE START CODON MODIFICATION AND EVALUATION OF O-ACETYL homoserine PRODUCTIVITY

[077] The wild-type leuE start codon is known to be gtg, which codes for valine, an amino acid. To confirm the enhanced effect of leuE protein by changing the start codon to atg (i.e., a codon that codes for methionine), an experiment to change the start codon based on the plasmid prepared in Example 1 was performed. Specifically, the first amino acid in the amino acid sequence of SEQ ID NO: 1 was replaced with methionine to enhance the activity of exporting O-acetyl-homoserine. More specifically, a leuE(ATG) modification was prepared. To prepare the leuE(ATG) modification, primers from SEQ ID NO: 145 and SEQ ID NO: 146 were used, and a modified leuE(ATG) gene was prepared by site-specific mutagenesis (site-directed mutagenesis set, Stratagene, USA). The existing wild-type plasmid was named WT, and Petition 870170093723, dated 01 / 12 / 2017, page 35 / 242 25 / 38 The initiation codon variant plasmid was named WT_ATG, and the plasmid prepared in this way was introduced into the WCJM strain and the vial evaluation of its O-acetyl-homoserine productivity was performed.

[078] Specifically, each strain was electroplated on solid LB medium and cultured overnight in a 33 °C incubator. The strain cultured overnight on LB plate medium was seeded onto 25 ml of titer medium and incubated at 33 °C at 200 rpm for 40 hours. The results are summarized in Table 4 below. [TABLE 4] MEASUREMENT OF O-acetyl-homoserine PRODUCTION by culture in a flask 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

[079] As can be seen in Table 4 above, the strain introduced with the pCL-leuE WT(ATG) plasmid that has the start codon modification showed a lower OD compared to that of the wild-type strain but showed more rapid glucose consumption. The strain introduced with the pCL-leuE WT(ATG) plasmid that has the start codon modification produced O-acetyl-homoserine at a concentration of 2.6 g / l, which is an increase in productivity as much as 173% compared to the wild-type strain. EXAMPLE 3: PREPARATION OF A LEUE-MODIFIED PLASMID AND EVALUATION OF O-ACETYL-HOMOSERIA BUILDUP 3-1. Preparation of a plasmid modified with LEUE

[080] Experiments to prepare each of the three modified polypeptides that were expected to have stronger export activity than that of wild-type leuE based on the two types of plasmids, i.e., plasmidpCL-leuE WT and pCL-leuE WT(ATG) prepared in Examples 1 and 2, Petition 870170093723, dated 01 / 12 / 2017, page 36 / 242 26 / 38 were performed. Specifically, leuE modification positions were selected through structure modeling to enhance O-acetyl-homoserine export activity, and amino acids at positions 30, 95, and 165 in the amino acid sequence of SEQ ID NOS: 1 and 2 were substituted with different amino acids, respectively.

[081] More specifically, the L95V, F30A, and F165A modifications were prepared. For the preparation of the L95V modification, primers with SEQ ID NOS: 13 and 14 were used; for the F30A modification, primers with SEQ ID NOS: 25 and 26 were used; and for the F165A modification, primers with SEQ ID NOS: 27 and 28 were used. The leuE modified genes were prepared using the site-directed mutagenesis set (Stratagene, USA) together with each of the primer sets described above. Based on the existing wild-type plasmid WT, the modified plasmid L95V was named WT_M3; the modified plasmid F30A was named WT_M4 and the modified plasmid F165A was named WT_M6, respectively. Additionally, based on the plasmid with the start codon modification (i.e., WT(ATG)), the modified plasmid L95V was named WT(ATG)_M3, the modified plasmid F30A was named WT(ATG)_M4, and the modified plasmid F165A was named WT(ATG)_M6, respectively.The modified plasmids prepared in this way were introduced into the WCJM strain to evaluate the productivity of O-acetyl-homoserine in a flask.

[082] Specifically, each strain was electroplated on LB plate medium and cultured in a 33°C incubator overnight. The strain cultured overnight on solid LB medium was inoculated into 25 ml of the titer medium and then cultured in a 33°C incubator at 200 rpm for 40 hours. The results are shown in Table 5 below. [TABLE 5] MEASUREMENT OF O-acetyl-homoserine PRODUCTION by culture in a flask OD (562 nm) Glucose Consumption (g / l) O-AH (g / l) Petition 870170093723, dated 01 / 12 / 2017, page 37 / 242 27 / 38 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

[083] As can be seen in Table 5 above, all 3 strains introduced with the leuE-modified plasmid showed a decrease in OD compared to that of the wild type, but all 3 strains showed faster glucose consumption compared to that of the wild type and, in particular, the WT(ATG)_M6 strain was shown to produce O-acetylhomoserine at a concentration of 4.9 g / l, which thus shows the highest productivity of O-acetylhomoserine. Consequently, it was confirmed that all 3 modified strains of the present disclosure exhibited enhanced productivity of O-acetylhomoserine. Additionally, it was confirmed that when the amount of protein expression was increased by modifying the leuE start codon, the productivity of O-acetylhomoserine was further enhanced. 3-2. Preparation of Biosynthesis Pathway Genes and Modified Plasmids

[084] To maximize the productivity of O-acetyl-homoserine, a Petition 870170093723, dated 01 / 12 / 2017, p. 38 / 242 A 28 / 38 plasmid with the ability to enhance the biosynthetic pathway for homoserine was prepared. For cloning aspartate semaldehyde dehydrogenase, pyridine nucleotide transhydrogenase, and wild-type and modified LeuE into the pCL vector, the asd and pntAB genes were first introduced into the pCL vector.

[085] First, after obtaining the asd and pntAB genes, PCR was performed for a total of 30 cycles, in which denaturation was performed at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds and extension at 68 °C for 3 minutes, using SEQ ID NOS: 15 and 16 primers for the asd gene and SEQ ID NOS: 17 and 18 primers for the pntAB gene. The resulting PCR products were subjected to electrophoresis on 1.0% agarose gel and the DNAs obtained from strips sized respectively of 1.4 kb (asd) and 3 kb (pntAB) were purified.

[086] The two purified genes were ligated using stitching PCR (a technique in which the overlapping parts of two genes are first ligated without the use of any primer and then amplified using primers at both ends). The conditions for stitching PCR were to perform the PCR described above for 10 cycles and then perform the PCR for 20 cycles after adding primers with SEQ IDs NOS: 15 and 18. As a result, the combined asd-pntAB gene fragments were prepared and purified by electrophoresis. The purified fragments and the pCL vector were treated with SmaI at 37 °C overnight, further purified, and the pCL-asd-pntAB plasmid was prepared using T4 ligase.

[087] The leuE gene was cloned into the plasmid prepared in this way. In cloning, specifically, to obtain the leuE gene, PCR was performed for a total of 30 cycles, in which denaturation was performed at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds and extension at 68 °C for 1 minute, using primers with SEQ ID NOS: 29 and 30.

[088] The resulting PCR product was subjected to electrophoresis in 1.0% Petition 870170093723, dated 01 / 12 / 2017, p. 39 / 242 29 / 38 agarose gel and the DNA obtained from 800 bp was purified. The purified DNA and the pCL vector were treated with KpnI at 37 °C overnight, further purified, and the leuE gene and the pCL-asd-pntAB vector were cloned. The cloned plasmids were transformed into E. coli DH5α, and the transformed E. coli DH5α was selected from LB plate medium containing spectinomycin (50 pg / ml) and plasmids were obtained from it. The plasmids prepared in this way were introduced into the WCJM strain, which is a strain that produces O-acetyl-homoserine, and a vial evaluation was performed regarding its O-acetyl-homoserine productivity. The plasmids prepared in this way were a total of 4 types, and the wild type and 3 modified strains prepared in Example 2-1 were used.The 4 types of plasmids were introduced into the WCJM strain by electroporation and a vial evaluation was performed in the same manner as in Example 3-1. The results are shown in Table 6-1 below. [TABLE 6] MEASUREMENT OF O-acetyl-homoserine PRODUCTION by culture in a flask 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

[089] As can be seen in Table 6 above, as a result of simultaneously enhancing the biosynthesis pathway and leuE modification, the productivity of O-acetyl-homoserine was further improved. In particular, in the case of the strain in which the pCL-asd-pntAB-leuE WT_M6 plasmid was Petition 870170093723, dated 01 / 12 / 2017, page 40 / 242 30 / 38 introduced, the OD was decreased compared to that of the wild-type strain, but the strain showed faster glucose consumption and produced Oacetyl-homoserine at a concentration of 5.9 g / l, the highest among the strains. EXAMPLE 4: PREPARATION OF LEUE MODIFICATION BY SATURATED MUTAGENESIS AND EVALUATION OF O-acetyl-homoserine PRODUCTIVITY 4-1. Preparation of strains with LEUE modification by saturated mutagenesis and evaluation thereof.

[090] The modifications were prepared by saturated mutagenesis to produce different types of amino acid substitutions of the 3 leuE variants, which showed high productivity of O-acetyl-homoserine. The substituted amino acids were prepared using 17 types of M3 mutation, M4 mutation and M6 mutation, respectively, using the plasmids prepared in Example 2 as templates. Details are shown in Table 7 below. [TABLE 7] Modified Plasmid Substituted Amino Acid SEQ ID NO of M3 Primers L95F SEQ ID NOS: 31,32 L95A SEQ ID NOS: 33, 34 L95G SEQ ID NOS: 35, 36 L95T SEQ ID NOS: 37, 38 L95N SEQ ID NOS: 39, 40 L95D SEQ ID NOS: 41,42 L95H SEQ ID NOS: 43, 44 L95I SEQ ID NOS: 45, 46 L95S SEQ ID NOS: 47, 48 L95P SEQ ID NOS: 49, 50 L95Y SEQ ID NOS: 51,52 L95Q SEQ ID NOS: 53, 54 Petition 870170093723, dated 01 / 12 / 2017, page 41 / 242 31 / 38 L95K SEQ ID NOS: 55, 56 L95E SEQ ID NOS: 57, 58 L95C SEQ ID NOS: 59, 60 L95W SEQ ID NOS: 61,62 L95R SEQ ID NOS: 63, 64 M4 F30W SEQ ID NOS: 65, 66 F30L SEQ ID NOS: 67, 68 F30V SEQ ID NOS: 69, 70 F30G SEQ ID NOS: 71,72 F30S SEQ ID NOS: 73, 74 F30N SEQ ID NOS: 75, 76 F30D SEQ ID NOS: 77, 78 F30H SEQ ID NOS: 79, 80 F30I SEQ ID NOS: 81.82 F30P SEQ ID NOS: 83, 84 F30Y SEQ ID NOS: 85, 86 F30Q SEQ ID NOS: 87, 88 F30K SEQ ID NOS: 89, 90 F30E SEQ ID NOS: 91,92 F30C SEQ ID NOS: 93, 94 F30T SEQ ID NOS: 95, 96 F30R SEQ ID NOS: 97, 98 M6 F165W SEQ ID NOS: 99, 100 F165L SEQ ID NOS: 101,102 F165V SEQ ID NOS: 103, 104 F165G SEQ ID NOS: 105, 106 F165S SEQ ID NOS: 107, 108 F165N SEQ ID NOS: 109, 110 F165D SEQ ID NOS: 111, 112 F165H SEQ ID NOS: 113, 114 F165I SEQ ID NOS: 115, 116 Petition 870170093723, dated 01 / 12 / 2017, page 42 / 242 32 / 38 F165P SEQ ID NOS: 117, 118 F165Y SEQ ID NOS: 119, 120 F165Q SEQ ID NOS: 121,122 F165K SEQ ID NOS: 123, 124 F165E SEQ ID NOS: 125, 126 F165C SEQ ID NOS: 127, 128 F165T SEQ ID NOS: 129, 130 F165R SEQ ID NOS: 131,132

[091] Specifically, the modified leuE genes were prepared by performing a site-directed mutagenesis suite (Stratagene, USA) using the primers shown in Table 7 above. The plasmid was introduced into the WCJM strain and the vial was evaluated in the same manner as in Example 3-1. The results are shown in Table 8 below. [TABLE 8] MEASUREMENT OF O-acetyl-homoserine PRODUCTION by flask culture Strain Plasmid Modification 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 Modification 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 Petition 870170093723, dated 01 / 12 / 2017, page 43 / 242 33 / 38 Strain Plasmid Modification Location OD (562 nm) Glucose Consumption (g / l) O-AH (g / l) 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 Modification M4 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 Petition 870170093723, dated 01 / 12 / 2017, page 44 / 242 34 / 38 Strain Plasmid Modification Location OD (562 nm) Glucose Consumption (g / l) O-ah (g / l) F30T 8.9 36 3.0 F30R 8.6 38.5 2.9 M6 Modification 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

[092] As can be seen in Table 8 above, as a result of evaluating each of the modified strains, there was a slight difference in OD and glucose consumption rate. However, it was found that all the modified strains above had an enhanced amount of Oacetyl-homoserine production compared to the WCJM / pCL1920 and WCJM / pCL-leuE WT strains used as the control group. 4-2. PREPARATION OF a strain with enhanced LEUE modification into a strain with high O-acetyl-homoserine yield and Petition 870170093723, dated 01 / 12 / 2017, page 45 / 242 35 / 38 assessment of your O-acetyl-homoserine productivity

[093] A method for producing a strain that has the ability to produce O-acetyl-homoserine using a strain that has the ability to produce threonine by means of NTG mutation derived from wild-type W3110 is disclosed (International Patent Publication in WO 2012 / 087039). In particular, the strain prepared in this manner that produces O-acetyl-homoserine in high yield was deposited with the Korean Microorganism Conservation Center under Membership No. KCCM11146P.

[094] An attempt was made to determine whether the productivity of O-acetylmoserine can be further enhanced by introducing the leuE gene and modified strains thereof based on the strain above.

[095] Specifically, the leuE gene and 3 modified strains thereof were introduced by electroporation. The introduced strains were named KCCM11146P / pCL 1920, KCCM11146P / pCL-leuE WT, KCCM11146P / pCL-leuE M3, KCCM11146P / pCL-leuE M4 and KCCM11146P / pCL-leuE M6, respectively. To measure the O-acetyl-homoserine productivity of the leuE gene and the 3 modified strains thereof, a flask culture evaluation was performed. Specifically, LB medium was inoculated with 4 types from among the above strains and incubated overnight at 33 °C. Separate colonies were then inoculated into 3 ml of LB medium and cultured again at 33 °C for 5 hours, diluted 200 times in a 250 ml Erlenmeyer flask containing 25 ml of medium for O-acetyl-homoserine production, and incubated again at 33 °C at 200 rpm for 30 hours, and the amount of O-acetyl-homoserine production was confirmed by HPLC analysis. The results of the experiment are summarized in Table 9 below. [TABLE 9] MEASUREMENT OF O-acetyl-homoserine PRODUCTION by culture in a flask OD (562 nm) Glucose Consumption (g / l) O-ah (g / l) KCCM11146P / pCL192 0 18.3 40 14.2 Petition 870170093723, dated 01 / 12 / 2017, page 46 / 242 36 / 38 KCCM11146P / pCLleuE WT 17.9 40 16.3 18.8

[096] As can be seen in Table 9 above, it was confirmed that the strain prepared by introducing only pCL1920 into the KCCM11146P strain produced 14.2 g / l of O-acetyl-homoserine, and the leuE WT strain also showed an increase in the amount of O-acetyl-homoserine production compared to the original strain. Additionally, all 3 modified strains showed a decrease in OD, while strain M4 showed the highest yield of O-acetyl-homoserine production (19.2 g / l). Strains M4 and M6 showed an increase in the amount of O-acetyl-homoserine production.

[097] It was confirmed that the production of O-acetyl-homoserine was increased in the “M3, M4, and M6 KCCM11146P / pCL-leuE strains”, which are 3 leuE-modified strains of M3, M4, and M6 based on the KCCM11146P strain. As a result, the strains were named “CA05-4009”, “CA05-4010”, and “CA05-4011”, and were deposited with the KCCM on December 15, 2014, under Accessions in KCCM11645P, KCCM11646P, and KCCM11647P, respectively. EXAMPLE 5: PRODUCTION OF L-METHIONINE USING A CULTURE SOLUTION OF O-acetyl-homoserine produced and transferase

[098] An experiment was carried out to produce L-methionine using the O-acetyl-homoserine culture solution obtained in Example 4 and O-acetyl-homoserine sulfhydrylase, which is an enzyme that converts O-acetyl-homoserine to methionine.

[099] O-acetyl-homoserine sulfhydrylase, a converting enzyme, was Petition 870170093723, dated 01 / 12 / 2017, p. 47 / 242 37 / 38 prepared by the method provided in Example 1-2 of Patent No. KR 10-1250651, and the amount of L-methionine produced by a conversion reaction using the method provided in Example 3 of Patent No. KR 10-1250651 was measured. For the O-acetyl-homoserine used as the substrate, the KCCM11146P-pCL-leuE M4 culture solution (O-AH concentration; 19.2 g / l) obtained in Example 4 in the present disclosure was used, and the concentration of L-methionine produced therefrom is shown in Table 10 below. [TABLE 10] Time (min) 2 4 6 8 10 MetZ-rsp Methionine (g / l) 3.21 4.34 4.52 4.78 5.04 Conversion (%) 50% 68% 71% 75% 79%

[0100] As can be seen in Table 10 above, it was confirmed that the Oacetyl-homoserine contained in the culture solution of the KCCM11146P-pCLleuE M4 strain obtained in Example 4 was converted to methionine at a conversion rate of 79% within 10 minutes. From this result, it was confirmed that methionine can be successfully produced using the strain of the present disclosure.

[0101] Based on the foregoing, a person skilled in the art to whom this disclosure refers will be able to understand that this disclosure can be incorporated into other specific forms without modifying the technical concepts or essential characteristics of this disclosure. In this respect, the illustrative embodiments disclosed herein are for illustrative purposes only and should not be interpreted as limiting the scope of this disclosure. On the contrary, this disclosure is intended to encompass not only the illustrative embodiments, but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of this disclosure accordingly. Petition 870170093723, dated 01 / 12 / 2017, p. 48 / 242 38 / 38 defined by the attached claims.

Claims

1. Polypeptide having the activity of exporting O-acetyl-homoserine characterized by at least one amino acid selected from the group consisting of phenylalanine at position 30, leucine at position 95 and phenylalanine at position 165 in the amino acid sequence of SEQ ID NO: 1 being substituted by another amino acid.

2. Polypeptide, according to claim 1, characterized in that phenylalanine at position 30 in the amino acid sequence of SEQ ID NO: 1 is replaced by any one selected from the group consisting of alanine, tryptophan, leucine, valine, glycine, serine, asparagine, aspartic acid and histidine; leucine at position 95 in the amino acid sequence of SEQ ID NO: 1 is replaced by any one selected from the group consisting of valine, phenylalanine, alanine, glycine, threonine, asparagine, aspartic acid and histidine; or phenylalanine at position 165 in the amino acid sequence of SEQ ID NO: 1 is replaced by any one selected from the group consisting of alanine, tryptophan, leucine, valine, glycine, serine, asparagine, aspartic acid and histidine.

3. Polypeptide, according to claim 1, characterized in that valine at position 1 in the amino acid sequence of SEQ ID NO: 1 is further substituted by methionine.

4. Polypeptide, according to claim 1, wherein the polypeptide is characterized by being selected from the group consisting of amino acid sequences with SEQ ID NOS: 133, 134, 137, 138, 141 and 142.

5. A polynucleotide encoding the polypeptide characterized by having the activity of exporting O-acetyl-homoserine, as defined in claim 1, wherein at least one codon selected from the group consisting of nucleotide positions 88 to 90, 283 to 285 and 493 to 495 in the nucleotide sequence of SEQ ID NO: 3, or a degenerate sequence thereof, is replaced by a codon encoding another amino acid.

6. Polynucleotide, according to claim 5, wherein Petition 870260050260, dated 05 / 26 / 2026, page 13 / 15 2 / 3 polynucleotide is characterized by being selected from the group consisting of nucleic acid sequences with SEQ ID NOS: 135, 136, 139, 140, 143 and 144.

7. Microorganism of the genus Escherichia that produces O-acetylmoserine characterized by a modified polypeptide of the amino acid sequence of SEQ ID NO: 1 being comprised or overexpressed, wherein the modified polypeptide has substitutions of at least one amino acid selected from the group consisting of phenylalanine at position 30, leucine at position 95 and phenylalanine at position 165 in the amino acid sequence of SEQ ID NO:

1.

8. Microorganism, according to claim 7, characterized in that, with respect to the modified polypeptide, phenylalanine at position 30 in the amino acid sequence of SEQ ID NO: 1 is replaced by any one selected from the group consisting of alanine, tryptophan, leucine, valine, glycine, serine, asparagine, aspartic acid and histidine; leucine at position 95 in the amino acid sequence of SEQ ID NO: 1 is replaced by any one selected from the group consisting of valine, phenylalanine, alanine, glycine, threonine, asparagine, aspartic acid and histidine; or phenylalanine at position 165 in the amino acid sequence of SEQ ID NO: 1 is replaced by any one selected from the group consisting of alanine, tryptophan, leucine, valine, glycine, serine, asparagine, aspartic acid and histidine.

9. Microorganism, according to claim 7, characterized in that, with respect to the modified polypeptide, the valine at position 1 in the amino acid sequence of SEQ ID NO: 1 is further replaced by methionine.

10. Microorganism, according to claim 7, characterized in that the microorganism of the genus Escherichia is Escherichia coli.

11. Microorganism according to claim 7, characterized in that, additionally, the cystathionine synthase activity is inactivated.

12. Microorganism according to claim 7, characterized in that, additionally, the homoserine kinase activity is inactivated. Petition 870260050260, dated 05 / 26 / 2026, page 14 / 15 3 / 3 13. Microorganism according to claim 7, characterized in that, additionally, the homoserine acetyltransferase activity is enhanced compared to that of an unmodified microorganism.

14. Microorganism according to claim 7, characterized in that, additionally, the activities of aspartate semialdehyde dehydrogenase, pyridine nucleotide transhydrogenase or a combination thereof are enhanced compared to those of an unmodified microorganism.

15. Method for producing O-acetyl-homoserine characterized by comprising: cultivating the microorganism of the genus Escherichia that produces O-acetyl-homoserine, as defined in any one of claims 7 to 14, in a medium; and recovering O-acetyl-homoserine from the cultured microorganism or from the cultured medium.

16. Method for producing L-methionine characterized by comprising: cultivating the microorganism of the genus Escherichia that produces O-acetyl-homoserine, as defined in any one of claims 7 to 14, in a medium; and converting the O-acetyl-homoserine to L-methionine by treating the cultured microorganism or the cultured medium or the O-acetyl-homoserine recovered from the cultured microorganism or cultured medium with methyl mercaptan and a methionine-converting enzyme.

17. Method according to claim 16, characterized in that the enzyme that converts methionine is O-acetyl-homoserine sulfhydrylase.