RU2804941C1 - Variant of inner membrane protein and method for obtaining target product using it - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is related in particular to a variant of the YjeH protein, which is an inner membrane protein, to a microorganism that includes it, and to a method for obtaining the target product using it. The microorganism can be used to produce O-acetylhomoserine, homoserine, methionine or glufosinate.

EFFECT: invention makes it possible to obtain O-acetylhomoserine, homoserine, methionine or glufosinate with a high degree of efficiency.

15 cl, 1 dwg, 14 tbl, 5 ex

Description

Field of technology to which the invention relates

The present invention relates to a variant of the YjeH protein, which is an inner membrane protein, and to a method for obtaining the desired product using it.

Prior Art

O-Acetylhomoserine acts as a precursor to methionine, which is one of the essential amino acids in the human body. Homoserine, which is a precursor to both methionine and threonine, is known to be converted to threonine via phosphohomoserine and converted to methionine via acetylhomoserine.

Methionine is used not only as a feed and food additive, but also as a raw ingredient for the preparation of infusion solutions and pharmaceuticals and is obtained through chemical and biological synthesis. A method for producing L-methionine in two steps from a fermentation-derived L-methionine precursor by enzymatic conversion has been published (International Publication WO 2008/013432).

In this two-step process, O-succinylhomoserine and O-acetylhomoserine are used as methionine precursors, and it is very important to obtain O-acetylhomoserine in high yield for economical mass production.

Disclosure of the Invention

Technical problem

As a result of intensive efforts to enhance the production of O-acetylhomoserine or homoserine, the present inventors discovered a variant of the inner membrane protein having enhanced O-acetylhomoserine or homoserine export activity, whereby the present invention was carried out.

Technical solution

The present invention provides a variant of the YjeH protein, which is an inner membrane protein, comprising i) replacement of the amino acid corresponding to position 92 with asparagine, ii) replacement of the amino acid corresponding to position 351 with leucine, or iii) replacement at both positions i ) and ii), in the amino acid sequence of SEQ ID NO: 1, and having 95% or more but less than 100% homology with SEQ ID NO: 1.

The present invention provides a polynucleotide encoding this variant.

The present invention provides a vector comprising this polynucleotide.

The present invention provides a microorganism comprising at least one of: a variant of the inner membrane protein YjeH comprising i) replacing the amino acid corresponding to position 92 with asparagine, ii) replacing the amino acid corresponding to position 351 with leucine, or iii) replacing at both positions i) and ii), in the amino acid sequence of SEQ ID NO: 1, and having 95% or more but less than 100% homology with SEQ ID NO: 1; a polynucleotide encoding this variant; and a vector encoding this polynucleotide.

The present invention provides a method for obtaining the target product, comprising cultivating a microorganism in a culture medium, where the microorganism includes at least one of: a variant of the inner membrane protein YjeH, including i) replacement of the amino acid corresponding to the 92nd position with asparagine, ii) replacement of the amino acid, corresponding to position 351, leucine, or iii) a substitution at both positions i) and ii) in the amino acid sequence of SEQ ID NO: 1, and having 95% or more but less than 100% homology with SEQ ID NO: 1; a polynucleotide encoding this variant; and a vector encoding this polynucleotide.

The present invention provides a method for producing methionine, comprising producing O-acetylhomoserine by cultivating a microorganism in a culture medium and converting O-acetylhomoserine to methionine by reacting O-acetylhomoserine with a sulfide, wherein the microorganism comprises at least one of: a variant of the inner membrane protein YjeH, comprising i) replacing the amino acid corresponding to position 92 with asparagine, ii) replacing the amino acid corresponding to position 351 with leucine, or iii) replacing at both positions i) and ii), in the amino acid sequence SEQ ID NO: 1, and having homology of 95% or more but less than 100% with SEQ ID NO: 1; a polynucleotide encoding this variant; and a vector encoding this polynucleotide.

The present invention provides a method for producing glufosinate, comprising producing O-acetylhomoserine or homoserine by cultivating a microorganism in a culture medium and converting O-acetylhomoserine or homoserine to glufosinate, wherein the microorganism comprises at least one of: a variant of the inner membrane protein YjeH, comprising i) a substitution the amino acid corresponding to position 92 with asparagine, ii) substitution of the amino acid corresponding to position 351 with leucine, or iii) substitution at both positions i) and ii), in the amino acid sequence of SEQ ID NO: 1, and having 95% homology or more but less than 100% with SEQ ID NO: 1; a polynucleotide encoding this variant; and a vector encoding this polynucleotide.

The present invention provides a composition for producing homoserine, comprising a microorganism or a culture of a microorganism.

The present invention provides a composition for the production of O-acetylhomoserine, comprising a microorganism or a culture of a microorganism.

The present invention provides the use of a variant of the inner membrane protein YjeH to produce O-acetylcysteine or homoserine.

The present invention provides the use of a microorganism for the production of O-acetylcysteine or homoserine.

Beneficial effects of the invention

O-Acetylhomoserine or homoserine can be produced in high yields by cultivating a microorganism having the ability to produce O-acetylhomoserine or homoserine modified with an inner membrane protein variant of YjeH as described herein compared to unmodified proteins.

Brief description of graphic materials

FIG. 1 is a schematic representation of plasmid pDCM2.

The best way to carry out the invention

The present invention will be described in detail. However, each description and embodiment of the invention disclosed in the description of the present invention can be applied to the various descriptions and embodiments presented herein. In other words, all combinations of the various components disclosed in the specification of the present invention are included within the scope of the present invention. Moreover, the scope of the description of the present invention should not be limited to the following descriptions.

In one aspect of the present invention, to achieve the above-described objects, a variant of the inner membrane protein YjeH is provided, comprising i) replacing the amino acid corresponding to position 92 with asparagine, ii) replacing the amino acid corresponding to position 351 with leucine, or iii) replacing both positions i) and ii), in the amino acid sequence of SEQ ID NO: 1, and having 95% or more but less than 100% homology with SEQ ID NO: 1.

A variant of the inner membrane protein YjeH may have the ability to export O-acetylhomoserine or homoserine, i.e. O-acetylhomoserine or homoserine export activity.

As used herein, the term “homoserine” is an α-amino acid having a hydroxyl group on the side chain. It is known that homoserine, as an intermediate in the biosynthesis of threonine and methionine in microorganisms and plants, is formed from aspartic acid 4-semialdehyde and has the chemical formula C ₄ H ₉ NO ₃ . Homoserine can be converted to O-acetylcysteine by homoserine acetyltransferase in the presence of acetyl coenzyme A (acetyl-CoA).

As used herein, the term “O-acetylhomoserine” refers to the acetyl derivative of L-homoserine, which is a specific intermediate in the methionine biosynthesis pathway in microorganisms. It is known that O-acetylhomoserine is formed by the interaction between homoserine and acetyl-CoA, catalyzed by homoserine acetyltransferase. It has _the chemical _{formula C6H11NO4} . O-Acetylhomoserine may also be called O-acetyl-L-homoserine.

Specifically, in a protein variant as described herein, i) the amino acid corresponding to position 92 may be replaced by another amino acid, ii) the amino acid corresponding to position 351 may be replaced by another amino acid, or iii) both amino acids, corresponding to position 92 and position 351 may be replaced by other amino acids in the amino acid sequence of SEQ ID NO: 1, but without limitation. The amino acid substitution may be i) a substitution of the amino acid corresponding to position 92 with asparagine, or ii) a substitution of the amino acid corresponding to position 351 with leucine, and more specifically, the protein variant may be a variant of the inner membrane protein YjeH, in which i) the amino acid corresponding to position 92 is replaced by asparagine, ii) the amino acid corresponding to position 351 is replaced by leucine, or iii) the amino acid corresponding to position 92 is replaced by asparagine and the amino acid corresponding to position 351 is replaced by leucine, in the amino acid sequence of SEQ ID NO: 1, but without limitation.

As used herein, the term “inner membrane protein” refers to a protein that is a member of the Amino Acid Efflux (AAE) family of proteins, a member of the superfamily of amino acid polyamine organocation (APC) transport proteins that mediate O-acetylhomoserine export. and/or homoserine. This protein is predicted to contain 12 transmembrane α-helices, 10 of which form the inverted repeat fold that is characteristic of the APC superfamily. The inner membrane protein may be, for example, a protein comprising the amino acid sequence of SEQ ID NO: 1. A protein comprising the amino acid sequence of SEQ ID NO: 1 may be used interchangeably with a protein having the amino acid sequence of SEQ ID NO: 1, or a protein consisting from the amino acid sequence SEQ ID NO: 1. In the specification of the present invention, inner membrane protein may be used interchangeably with O-acetylhomoserine or homoserine exporting protein, protein having the ability to export O-acetylhomoserine or homoserine, protein having O-acetylhomoserine or homoserine-exporting activity, YjeH or YjeH protein.

As used herein, SEQ ID NO: 1 may refer to the amino acid sequence of YjeH protein, which is an inner membrane protein. In particular, SEQ ID NO: 1 may be the amino acid sequence of an inner membrane protein encoded by the yjeH gene and having O-acetylhomoserine or homoserine exporting activity. The amino acid sequence of SEQ ID NO: 1 can be obtained from the known GenBank database of the National Center for Biotechnology Information (NCBI). For example, the amino acid sequence may be derived from Escherichia coli (E. coli), but is not limited to, and any sequence having activity identical to that of the amino acid sequence is included without limitation. In addition, although an inner membrane protein in the specification of the present invention may be defined as a protein comprising the amino acid sequence of SEQ ID NO: 1, this inner membrane protein does not exclude mutation, which may occur naturally or by adding a minor sequence upstream or downstream of the amino acid sequence SEQ ID NO: 1, or a silent mutation thereof. If a protein has an activity identical or equivalent to that of a protein containing the amino acid sequence of SEQ ID NO: 1, it clearly belongs to the inner membrane protein described in the present invention. For example, the inner membrane protein of the present invention may be a protein consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.7% homology or identity with it. It will also be apparent that any protein having an amino acid sequence including deletion, modification, substitution or addition of portions of the amino acid sequence is within the scope of the present invention if the amino acid sequence retains the above-described homology or identity and effects equivalent to those of the protein.

Thus, although the description of the present invention uses the expressions “a protein or polypeptide having the amino acid sequence represented by the specified sequence of SEQ ID NO” and “a protein or polypeptide comprising the amino acid sequence represented by the specified sequence of SEQ ID NO”, it is obvious that any protein having an amino acid sequence including deletion, modification, substitution or addition of portions of the amino acid sequence may also be used in the description of the present invention if the protein has an activity identical or equivalent to that of a polypeptide consisting of a given amino acid sequence. For example, it is apparent that “a protein comprising the amino acid sequence of SEQ ID NO: 1” or “a polypeptide comprising the amino acid sequence of SEQ ID NO: 1” refers to “a protein consisting of the amino acid sequence of SEQ ID NO: 1” or “a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1" if the former has the same or equivalent activity as the latter.

As used herein, the term “variant” refers to a protein produced by conservative substitution and/or modification of at least one amino acid different from an amino acid of the specified sequence while maintaining its functions or properties. A variant may have an amino acid sequence that differs from the identified sequence due to substitution, deletion, or addition of multiple amino acids. Such variants can typically be identified by modifying at least one amino acid of the above protein amino acid sequence and evaluating the properties of the modified protein. Thus, the activity of the variant may be enhanced, unchanged, or reduced relative to the native protein. In addition, some variants may include variants from which at least one region, such as an N-terminal leader sequence or transmembrane domain, is deleted. Other variants may include variants in which a region is removed from the N- and/or C-terminus of the mature protein. The term “variant” may also be used interchangeably with other terms such as modification, modified protein, modified polypeptide, mutant, mutein and divergent, as well as any terms used to indicate variation, which may also be used without limitation. In light of the purposes of the description of the present invention, the ability of the variant may be enhanced relative to the native or unmodified protein, but without limitation.

As used herein, the term “conservative substitution” refers to the replacement of one amino acid with another amino acid having a similar structural and/or chemical property. For example, a variant may include at least one conservative substitution while maintaining at least one biological activity. Such amino acid substitution may generally occur on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residue. For example, amino acids with an electrical charge and side chains include positively charged (basic) amino acids such as arginine, lysine and histidine, and negatively charged (acidic) amino acids such as glutamic acid and aspartic acid; and uncharged amino acids with side chains include non-polar amino acids such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline, and polar or hydrophilic amino acids such as serine, threonine, cysteine, tyrosine, asparagine and glutamine . Among the nonpolar amino acids, aromatic amino acids include phenylalanine and tryptophan.

Variations may also include deletion or addition of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus of the protein that co-translationally or post-translationally directs protein transfer. The polypeptide may also be conjugated to another sequence or linker to identify, purify, or synthesize the polypeptide.

In the description of the present invention, “replaced with another amino acid” is not particularly limited as long as the amino acid after the replacement is different from the amino acid before the replacement.

In the description of the present invention, “replaced with another amino acid” is not particularly limited as long as the amino acid after the replacement is different from the amino acid before the replacement. In particular, the amino acid may be replaced by any of lysine, histidine, glutamic acid, aspartic acid, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine and glutamine, in difference from the previous amino acid sequence.

Specifically, in the amino acid sequence of SEQ ID NO: 1, phenylalanine, which is the amino acid corresponding to position 92, may be replaced by asparagine, or phenylalanine, which is the amino acid corresponding to position 351, may be replaced by leucine. At the same time, in the description of the present invention, it is obvious that unless the expression “replaced by another amino acid” is used, the expression “replaced by a certain amino acid” means that the amino acid after the replacement is different from the amino acid before the replacement.

The substituted amino acid residue may include not only a naturally occurring amino acid, but also a non-naturally occurring amino acid. The unnatural amino acid may be, for example, a D-amino acid, a (homo)-amino acid, a (beta-homo)-amino acid, an N-methyl amino acid, an α-methyl amino acid, and a rare amino acid (for example, citrulline or naphthylalanine), but without restrictions. At the same time, in the description of the present invention, it is obvious that unless the expression “replaced by another amino acid” is used, the expression “replaced by a certain amino acid” means that the amino acid after the replacement is different from the amino acid before the replacement.

The variant may be one in which at least one of the amino acids corresponding to position 92 and position 351 is replaced by an amino acid different from the amino acid before the substitution in the amino acid sequence SEQ ID NO: 1, or is replaced by an uncharged side chain acid , different from the one that was before the replacement, but without restrictions.

In particular, the variant may be a protein variant in which i) the amino acid corresponding to position 92 may be replaced by another amino acid, ii) the amino acid corresponding to position 351 may be replaced by another amino acid, or iii) both amino acids, corresponding to position 92 and position 351 may be replaced by other amino acids in the amino acid sequence of SEQ ID NO: 1, but without limitation. The amino acid substitution may be i) substitution of the 92nd amino acid with asparagine, or ii) substitution of the 351st amino acid with leucine. More specifically, the variant may be a variant of the inner membrane protein YjeH in which i) the 92nd amino acid is replaced by asparagine, ii) the 351st amino acid is replaced by leucine, or iii) the 92nd amino acid is replaced by asparagine, and the 351st amino acid is replaced by leucine in amino acid sequence SEQ ID NO: 1, but without limitation.

As used herein, the term “corresponds to (something)” refers to an amino acid residue at a specified position in a protein or polypeptide, or an amino acid residue that is similar, identical or homologous to a residue specified in a protein or polypeptide. As used herein, the term “corresponding region” generally refers to a region similar to it in a related or reference protein.

In the description of the present invention, a specific numbering may be used for the position of the amino acid residue of the protein used in the description of the present invention. For example, a position corresponding to an amino acid residue of a protein of the present invention may be renumbered by aligning the amino acid sequence of the protein being compared to the amino acid sequence of the protein of the present invention.

The YjeH inner membrane protein variant proposed herein may refer to a variant having enhanced O-acetylhomoserine or homoserine export ability compared to the unmodified protein as a result of an amino acid substitution at a specific position in the above described inner membrane protein having O export ability. -acetylhomoserine or homoserine.

A variant of the inner membrane protein YjeH comprising i) a substitution of the amino acid corresponding to position 92 with asparagine, ii) a substitution of the amino acid corresponding to position 351 with leucine, or a substitution at both positions i) and ii) in the amino acid sequence SEQ ID NO: 1 may include any of the amino acid sequences of SEQ ID NOs: 35 and 36, in particular consisting of any of the amino acid sequences of SEQ ID NOs: 35 and 36, more particularly consisting of any of the amino acid sequences of SEQ ID NOs: 35 and 36, but without limitation .

In addition, the variant may include the amino acid sequences of SEQ ID NOs: 35 and 36, or an amino acid sequence wherein at least any one of the 92nd and 351st amino acids is fixed in the amino acid sequences of SEQ ID NOs: 35 and 36, and having at least 80% homology to, but not limited to, any of the amino acid sequences of SEQ ID NOs: 35 and 36. In particular, a variant as described herein may include a polypeptide having 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with any of the amino acid sequences of SEQ ID NOs: 35 and 36. Also It is apparent that any protein having an amino acid sequence involving deletion, modification, substitution or addition in a portion of the amino acid sequence other than the 92nd position and the 351st position is within the scope of the present invention if the amino acid sequence retains the above-described homology or identity and effects equivalent to those of that protein.

As used herein, the term “homology” or “identity” refers to the degree of correspondence between two given amino acid or base sequences and may be expressed as a percentage. The terms homology and identity can often be used interchangeably.

The homology or identity of conserved polynucleotides or polypeptides can be determined using a standard sequence matching algorithm, together with which can be used the default gap penalties set by the program. Substantially homologous or identical sequences may hybridize to each other over at least about 50%, 60%, 70%, 80%, or 90% of the full-length sequence, or throughout the entire full-length sequence under moderate to high stringency conditions. Hybrid polynucleotides may also include polynucleotides that include a degenerate codon in place of a codon.

Homology, similarity or sequence identity between two specific polypeptides or polynucleotides can be determined using any known computer algorithm, such as the FASTA program, using default parameters as introduced, for example, by Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85: 2444. Alternatively, one can use the Needleman-Wunsch algorithm (1970, J. Mol. Biol. 48: 443-453) run in the Needleman program from The European Molecular Biology Open Software Suite (EMBOSS) (Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) for this definition (including the GCG software package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S.F. et al., J MOLEC BIOL 215: 403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and CARILLO et al. (1988) SIAM J Applied Math 48: 1073) For example, homology, similarity, or identity can be determined using the BLAST program from the National Center for Biotechnology Information database, or ClustalW.

Homology, similarity or identity between polynucleotides or a polypeptide can be determined, for example, by comparing sequence information using a GAP computer program, such as the program introduced by Needleman et al. (1970), J Mol Biol. 48: 443, for example, as described in Smith and Waterman, Adv. Appl. Math (1981) 2: 482. Briefly, the GAP program defines similarity as the number of matched characters (ie, nucleotides or amino acids) that are similar divided by the total number of characters in the shorter of the two sequences. Default parameters for the GAP program may include: (1) a binary comparison matrix (containing the values 1 for identity and 0 for non-identity), a weighted comparison matrix Gribskov et al. (1986) Nucl. Acids Res. 14: 6745, described in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or EDNAFULL substitution matrix (EMBOSS NCBI NUC version 4.4)); (2) a 3.0 penalty for each gap and an additional 0.10 penalty for each symbol in each gap (or a 10 penalty for opening a gap and a 0.5 penalty for extending a gap); and (3) no penalty for end gaps.

Homology, similarity or identity between two specific polynucleotides or polypeptides can also be identified by comparing their sequences by Southern hybridization under specified stringency conditions, and these specified stringency conditions are within the scope of the art and can be determined by a method well known to one of ordinary skill in the art. (e.g. J. Sambrook et al., Molecular Cloning, A Laboratory Manual, ^2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

As used herein, the term "inner membrane protein variant" or "inner membrane protein variant YjeH" may have the ability to export O-acetylhomoserine or homoserine and may be used interchangeably with a modified polypeptide having the ability to produce O-acetylhomoserine or homoserine, O-acetylhomoserine - or a homoserine-producing modified polypeptide, a modified polypeptide having O-acetylhomoserine or homoserine-producing ability, a modified polypeptide having O-acetylhomoserine or homoserine export activity, a variant with O-acetylhomoserine or homoserine export activity, O-acetylhomoserine or homoserine -exporting variant, modified homoserine exporting protein, modified O-acetylhomoserine exporting protein, modified YjeH, YjeH variant, YjeH variant, YjeH mutant, etc.

In addition, the protein may be derived from strains belonging to the genus Escherichia, genus Erwinia, genus Serratia, genus Providencia, genus Corynebacterium, genus Pseudomonas, genus Leptospira, genus Salmonella, genus Brevibacterium, genus Hypomononas, genus Chromobacterium and genus Norcardia, or from yeasts and fungi, in particular having origin from strains belonging to the genus Escherichia, the genus Corynebacterium and the genus Leptospira, and from yeasts, and more particularly having origin from strains belonging to the genus Escherichia, for example Escherichia coli (E. coli), but without restrictions.

The inner membrane YjeH protein variant may include a mutation at position 92 and/or position 351 in the amino acid sequence of SEQ ID NO: 1, and any variant involving the addition of an amino acid to or deletion from the amino acid sequence of SEQ ID NO: 1 may include within the scope of the description of the present invention, if the amino acid corresponding to the 92nd position and/or the 351st position from the N-terminus of SEQ ID NO: 1 is replaced. The YjeH inner membrane protein variant may include an amino acid sequence comprising the substitution of the 92nd amino acid and/or the 351st amino acid with a different amino acid in the amino acid sequence of SEQ ID NO: 1 and may have enhanced activity compared to the inner membrane protein comprising the amino acid sequence of SEQ ID NO: 1 or not including mutation. The YjeH inner membrane protein variant may have homology or identity of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or more and less than 100 % with SEQ ID NO: 1.

A mutation at amino acid 92 or amino acid 351 may be a substitution of the 92nd amino acid with asparagine or a substitution of the 351st amino acid with leucine.

In particular, the YjeH inner membrane protein variant may include i) a substitution of the 92nd amino acid with asparagine, ii) a substitution of the 351st amino acid with leucine, or a substitution of both amino acids i) and ii) in the amino acid sequence of SEQ ID NO: 1 and may have enhanced activity compared to a protein comprising the amino acid sequence of SEQ ID NO: 1 or an inner membrane protein derived from a wild-type microorganism without modification.

In another aspect of the present invention, there is provided a polynucleotide encoding a variant of the inner membrane protein YjeH.

The inner membrane protein YjeH and its variant are as described above.

As used herein, the term "polynucleotide" refers to a strand of DNA or RNA having a specified minimum or greater length, in the form of a polymer of nucleotides in which the nucleotide monomers are linked together by covalent bonds to form a long chain; more specifically, the polynucleotide may be a polynucleotide fragment encoding a variant.

The polynucleotide may include any nucleotide sequence encoding a variant of the inner membrane protein YjeH as described herein, without limitation. In the present invention, the gene encoding the amino acid sequence of the inner membrane protein YjeH is the yjeH gene, which may be derived from Escherichia coli (E. coli) without limitation.

In particular, the polynucleotide described in the present invention may include various modifications made to the coding region, provided that the amino acid sequence expressed from the coding region as a result of codon degeneracy or taking into account codon preferences in the living organism in which the protein is expressed is not changed . In particular, any polynucleotide sequence encoding a variant of the inner membrane protein YjeH in which the 92nd or 351st amino acid is replaced by another amino acid in the amino acid sequence of SEQ ID NO: 1 may be included, without limitation.

In addition, any sequence encoding an inner membrane protein may be included in which the 92nd or 351st amino acid of amino acid sequence SEQ ID NO: 1 is replaced by another amino acid by hybridization with a probe that can be derived from known gene sequences, e.g. sequences complementary to the full-length nucleotide sequence or part thereof, under strict conditions, without restrictions. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are disclosed in detail in known documents (eg, J. Sambrook et al.). For example, these conditions may include conditions for performing hybridization between genes having high homology or identity, for example, homology or identity of 40% or more, particularly 90% or more, more particularly 95% or more, even more particularly 97% or more, and most specifically 99% or more, without performing hybridization between genes having lower homology than the homology or identity values mentioned above, or performing hybridization once, in particular two or three times, under conventional washing conditions for Southern hybridization at concentration salt and a temperature of 60°C, 1×sodium citrate and chloride (SSC) solution and 0.1% sodium dodecyl sulfate (SDS), in particular 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68 °C, 0.1×SSC and 0.1% SDS.

Hybridization requires that the two polynucleotides have complementary sequences, although the bases may not be the same, depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing with each other. For example, as far as DNA is concerned, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, not only a substantially similar nucleic acid, but also an isolated nucleic acid fragment, but complementary to the full-length sequence, may be included in the description of the present invention.

In particular, polynucleotides having homology or identity can be detected using the above-described hybridization conditions and using a hybridization process at a Tm value of 55°C. The Tm value may also be, but is not limited to, 60°C, 63°C, or 65°C, and may be adjusted accordingly by those skilled in the art according to intended purposes.

The appropriate degree of stringency for polynucleotide hybridization may depend on the lengths and degree of complementarity of the polynucleotides, as well as factors well known to those skilled in the art (Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

In another aspect of the present invention, there is provided a vector comprising a polynucleotide encoding a variant of the inner membrane protein YjeH.

The inner membrane protein YjeH, its variant and polynucleotide are as described above.

A vector as described herein may be a DNA construct containing a polynucleotide encoding a target polypeptide and operably linked to a suitable expression regulatory region (expression regulatory sequence) so as to enable expression of the target polypeptide in a suitable host cell. The expression control sequence may include a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable ribosome binding site on the mRNA, and a sequence for regulating termination of transcription and translation. When a suitable host cell is transformed with a vector, the vector can replicate or function independently of the host cell genome or can integrate into the human genome.

As used herein, the term “expression” includes any process associated with the production of a polypeptide, such as transcription, post-transcriptional modification, translation, post-translational modification and secretion, etc., without limitation.

As used herein, the term “expression vector” refers to a linear or circular nucleic acid molecule comprising a coding sequence and an operably linked regulatory sequence for its expression.

As used herein, the term "operably linked" refers to the arrangement of a regulatory sequence at an appropriate position to regulate the expression of a coding sequence. Thus, “operably linked” includes the binding between a regulatory region of a functional domain having a known or desired activity, such as a promoter, stop codon, signal sequence, or enhancer, and a target sequence (gene or polypeptide) regulating the expression, secretion, or function of the target sequence according to known or required activity.

The vector used in describing the present invention is not particularly limited, and any known vector can be used. Examples of known vectors may include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A and Charon21A, etc. can be used as a phage or cosmid vector, and vectors based on pBR, on pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based and pET-based. In particular, the vectors pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118 and pCC1BAC can be used. More specifically, the vector used in the description of the present invention may be pDCM2 (FIG. 1, SEQ ID NO: 37), designed to insert and replace a gene on the Corynebacterium chromosome, but is not limited to, and any expression vector may be used.

For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a cell chromosome insertion vector. The insertion of a polynucleotide into a chromosome can be accomplished by any method known in the art, such as homologous recombination, without limitation. The polynucleotide may further include a selectable marker to confirm insertion into the chromosome. A selectable marker is used to select cells transformed with a vector, that is, to confirm insertion of a desired nucleic acid molecule, and examples of a selectable marker may include markers conferring selective phenotypes such as drug resistance, nutrient requirement, resistance to cytotoxic agents, or surface polypeptide expression . In an environment treated with a selection agent, only cells expressing the selectable marker are able to survive or exhibit different phenotypes, and thus transformed cells can be selected.

In another aspect of the present invention, there is provided a microorganism comprising at least one of: a variant of the inner membrane protein YjeH, comprising i) replacing the amino acid corresponding to position 92 with asparagine, ii) replacing the amino acid corresponding to position 351 with leucine, or iii) a substitution at both positions i) and ii) in the amino acid sequence of SEQ ID NO: 1, and having 95% or more but less than 100% homology with SEQ ID NO: 1; a polynucleotide encoding this variant; and a vector encoding this polynucleotide.

SEQ ID NO: 1, inner membrane protein YjeH, a variant thereof, a polynucleotide encoding the variant, and a vector including the polynucleotide are as described above.

As used herein, the term “microorganism comprising a variant of the inner membrane protein YjeH” refers to a microorganism with an enhanced ability to export O-acetylhomoserine or homoserine obtained by introducing a variant of the inner membrane protein YjeH into a microorganism having a weak ability to export O-acetylhomoserine or homoserine. Specifically, the microorganism is a microorganism expressing a variant of the inner membrane protein YjeH comprising a modification of at least one amino acid in the amino acid sequence of SEQ ID NO: 1, and the amino acid modification may include replacing the 92nd or 351st amino acid from the N-terminus with another amino acid.

In light of the purposes of describing the present invention, the extracellular export of O-acetylhomoserine or homoserine of a microorganism comprising at least any one of the inner membrane protein variant YjeH; a polynucleotide encoding this variant; and the vector including the polynucleotide is increased, and thus the microorganism is characterized in that its ability to produce O-acetylhomoserine or homoserine is enhanced. Although wild-type or unmodified microorganisms cannot export or can produce trace amounts of O-acetylhomoserine or homoserine, the ability to produce O-acetylhomoserine or homoserine can be enhanced as described herein by increasing the exportable amounts of O-acetylhomoserine or homoserine by administering at least any one of the inner membrane protein YjeH variants; a polynucleotide encoding this variant; and a vector including this polynucleotide.

As used herein, the term “unmodified microorganism” does not exclude strains that include a mutation that may occur naturally in microorganisms, and refers to a wild-type strain or microorganism that does not include a variant of the inner membrane protein YjeH. "Unmodified microorganism" may be used interchangeably with "strain before modification", "microorganism before modification", "unmutated strain", "unmutated microorganism", "unmodified strain" or "comparative microorganism".

As used herein, the term “protein to be expressed/is expressed” means a state in which the target protein is introduced into a microorganism or modified for expression in a microorganism. If the target protein is present in the microorganism, the activity of this protein can be enhanced compared to the activity of the endogenous protein or the protein before modification. In light of the subject matter of the present invention, the “target protein” may be a variant of the inner membrane protein YjeH described above.

In particular, the term "introducing a protein" refers to providing the activity of a particular protein in a microorganism that does not have that protein, or enhancing the activity of the protein compared to the protein's own activity or its activity before modification. For example, protein introduction may refer to the introduction into a chromosome of a polynucleotide encoding a particular protein, or to the introduction into a microorganism of a vector comprising a polynucleotide encoding that particular protein, thereby expressing the activity of that protein. In addition, “enhanced activity” may mean that the activity of a particular microorganism protein is enhanced relative to its own activity or activity before modification. The term "intrinsic activity" may refer to the activity of a particular protein possessed by the parent strain prior to transformation when the microorganism is transformed with a natural or artificial natural variant.

In particular, the enhancement of activity as described in the present invention can be achieved by at least one method selected from the group consisting of: a method of increasing the copy number of a gene encoding a protein variant; a method for introducing a mutation into the expression regulatory sequence of a gene encoding a protein variant; a method of replacing a gene expression regulatory sequence with a sequence encoding a protein variant and having stronger activity; a method of replacing a gene encoding a wild-type inner membrane protein on a chromosome with a gene encoding a variant protein; a method for additionally introducing a mutation into a gene encoding a protein variant to enhance the activity of the protein variant; and a method of administering the protein variant, without limitation.

In the above description, the increase in the number of copies of a gene, although there is no specific limitation, can be performed in a state of functional association with the vector or in a state integrated into the chromosome of the host cell. In particular, increasing the copy number of a gene can be accomplished by introducing a vector to which a polynucleotide encoding a protein variant as described herein is operably linked and which can replicate and function in a host cell independently of the host cell. Alternatively, a vector is introduced into the cell to which the polynucleotide is operably linked and is capable of inserting the polynucleotide into the chromosome of the host cell. The insertion of a polynucleotide into a chromosome can be accomplished by any method known in the art, such as homologous recombination.

Modification of an expression control sequence to increase the expression level of a polynucleotide can be accomplished by, but without particular limitation, inducing a mutation on the sequence by deletion, insertion, non-conservative substitution, conservative substitution, or any combination thereof to further enhance the activity of the expression control sequence or substitution with a sequence having stronger activity. The expression control sequence may include a promoter, an operator sequence, a sequence encoding a suitable ribosome binding site, and a sequence for regulating transcription and translation termination.

A promoter stronger than its own promoter may be linked upstream of a polynucleotide expression unit without limitation. Examples of a stronger promoter known in the art may include the CJ1-CJ7 promoters (US Pat. No. 7,662,943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7, SPL13 promoter (sm3) (US 10584338 B2), O2 promoter (US 10273491 B2), tkt promoter and uscA promoter, without limitation.

In addition, modification of a polynucleotide sequence on a chromosome can be accomplished by inducing a mutation in an expression regulatory sequence by deletion, insertion, non-conservative substitution, conservative substitution, or any combination thereof to further enhance the activity of the polynucleotide sequence, or by replacing the nucleotide sequence with a modified nucleotide sequence, having stronger activity, but without restrictions.

The introduction of protein and enhancement of activity may increase the activity or concentration of the protein compared to the activity or concentration of a wild-type strain or an unmodified strain of the microorganism, without limitation.

In the present invention, the microorganism comprising the YjeH variant of the inner membrane protein may be a recombinant microorganism obtained by transformation using a vector comprising a polynucleotide encoding the YjeH variant of the inner membrane protein, but is not limited to.

As used herein, the term “transformation” refers to the process of introducing into a host cell a vector comprising a polynucleotide encoding a target protein such that the protein encoded by the polynucleotide is expressed in the host cell. The transformed polynucleotide can be either in a form integrated into the chromosome of the host cell or in a form located outside the chromosome, and in either case the protein is expressed in the host cell. In addition, the polynucleotide includes DNA and/or RNA encoding the target protein. The polynucleotide can be introduced into a host cell provided that the polynucleotide is introduced into the host cell and the protein is expressed therein. For example, a polynucleotide can be introduced into a host cell in the form of an expression cassette, which is a gene construct that includes all the essential elements required for self-expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal that are operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into a host cell in its original form and operably linked to a sequence required for expression in the host cell, without limitation.

The types of microorganism having the ability to produce homoserine are not particularly limited as long as the microorganism is capable of producing O-acetylhomoserine or homoserine, and in particular the microorganism can be any microorganism belonging to the Corynebacterium genus, Escherichia genus, Enterobacter genus, Erwinia genus, Serratia genus , the genus Providencia and the genus Brevibacterium, more specifically a microorganism belonging to the genus Corynebacterium.

More specifically, the microorganism belonging to the genus Corynebacterium may be Corynebacterium glutamicum, Corynebacterium ammoniagenes, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, and any microorganisms belonging to the genus Corynebacterium can also be used without limitation.

In the present invention, the parent strain of the microorganism may be a microorganism in which a gene weakening the O-acetylhomoserine or homoserine biosynthesis pathway is further inactivated, or the O-acetylhomoserine or homoserine biosynthesis pathway is enhanced to enhance the production of O-acetylhomoserine or homoserine, without limitation.

In particular, in order to inactivate a gene that weakens the O-acetylhomoserine or homoserine biosynthetic pathway, the expression of the metB gene (SEQ ID NO: 17), encoding cystathionine hexyl synthase involved in the O-acetylhomoserine degradation pathway, can be attenuated or inactivated, for example. or the metY gene (SEQ ID NO: 22), encoding O-acetylhomoserine (thiol)-lyase involved in the O-acetylhomoserine degradation pathway.

To enhance the O-acetylhomoserine or homoserine biosynthetic pathway, for example, a genetic mutation can be introduced into the lysC gene (SEQ ID NO: 27), encoding aspartokinase, or the expression of the metX gene (SEQ ID NO: 32), encoding O-acetylhomoserine transferase, can be amplified.

However, this embodiment is not limited to this, and the ability to produce O-acetylhomoserine or homoserine can be enhanced by any known methods for regulating gene expression.

As used herein, the term "enhancement" is a concept that includes an increase in activity relative to one's own activity.

Enhancement or enhancement of gene activity can be achieved by using various methods well known in the art. Examples of these methods may include at least one selected from the group consisting of: a method for increasing the copy number of a gene; a method for introducing a mutation into a gene expression regulatory sequence; a method of replacing a gene expression regulatory sequence with a sequence having stronger activity; a method for additionally introducing a mutation into a gene in order to enhance the activity of the gene; a method of introducing a foreign gene into a microorganism; and any combination thereof, without restrictions.

As used herein, the term “inactivation” of a protein means that the activity of the protein is reduced relative to its own activity, or that the protein has no activity.

Protein inactivation can be achieved by using various methods well known in the art. Examples of methods may include: a method of deleting part or all of a gene encoding a protein on a chromosome, comprising removing the activity of the protein; a method of replacing a gene encoding a protein on a chromosome with a mutant gene to reduce enzymatic activity; a method for inducing a mutation in the expression regulatory sequence of a gene encoding a protein on a chromosome; a method of replacing an expression regulatory sequence of a gene encoding a protein with a sequence having weaker or no activity (for example, a method of replacing a gene promoter with a promoter weaker than an endogenous promoter); a method of deleting a part or full-length gene encoding a protein on a chromosome; a method of introducing an antisense nucleotide (eg, antisense RNA) that inhibits translation from mRNA to protein by complementary binding to a gene transcript on the chromosome; a method for making it impossible to attach a ribosome due to the formation of a secondary structure by artificially attaching a complementary sequence to the Shine-Dalgarno sequence (SD) above the SD sequence of the gene encoding the protein; and a reverse transcription engineering (RTE) method that attaches a reverse transcription promoter to the 3' end of an open reading frame (ORF) of a corresponding sequence, and a combination thereof, but without particular limitation.

In another aspect of the description of the present invention, a method of obtaining the target product is provided, comprising cultivating a microorganism in a culture medium, where the microorganism includes at least one of: a variant of the inner membrane protein YjeH, comprising i) replacing the amino acid corresponding to the 92nd position with asparagine, ii) substitution of the amino acid corresponding to position 351 with leucine, or iii) substitution at both positions i) and ii) in the amino acid sequence of SEQ ID NO: 1, and having 95% or more but less than 100% homology with SEQ ID NO: 1; a polynucleotide encoding this variant; and a vector encoding this polynucleotide.

SEQ ID NO: 1, inner membrane protein YjeH, a variant thereof, a polynucleotide encoding the variant, a vector including the polynucleotide, and a microorganism including them are as described above.

The desired product may be O-acetylhomoserine or homoserine, but is not limited to.

As used herein, the term "cultivation" refers to growing a microorganism in an appropriately regulated environment. The culture process described in the present invention can be performed in accordance with suitable media and culture conditions known in the art. The cultivation process can be easily adjusted for use by one skilled in the art according to the strain being selected. The cultivation of the microorganism can be performed in a batch process, a continuous process, a fed-batch process, etc. no limits.

As used herein, the term “culture medium” refers to a substance in which the nutritional components required for the cultivation of a microorganism are mixed as the main ingredients and supplemented with nutritional components and growth factors, as well as water, which are necessary for survival and growth. In particular, although the culture medium and other cultivation conditions for cultivating the microorganism of the present invention are not particularly limited, if the culture medium is widely used in cultivating microorganisms; the microorganism of the present invention can be cultivated in a conventional medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and/or vitamins, under aerobic conditions under control of temperature, pH, etc.

In the present invention, carbohydrates such as glucose, fructose, sucrose and maltose can be used as carbon sources; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid and citric acid; and amino acids such as glutamic acid, methionine and lysine. In addition, natural organic nutritional components such as starch hydrolysates, molasses, raw molasses, rice bran, cassava, cane bagasse and corn syrup can be used, and in particular carbohydrates such as glucose and pre-sterile processed molasses (t i.e. molasses converted to reduce sugar content), and suitable amounts of any other carbon sources may be used without restriction. These carbon sources can be used individually or as a combination of at least two of them.

As nitrogen sources, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used; and organic nitrogen sources such as amino acids such as glutamic acid, methionine and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn syrup, casein hydrolysate, fish or its degradation products, and defatted soybean meal or products of its degradation. These nitrogen sources can be used individually or as a combination of at least two of them.

As sources of phosphorus, you can use monopotassium phosphate, dipotassium phosphate or their corresponding sodium salts. As inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, calcium carbonate and the like can be used. Additionally, amino acids, vitamins and/or a suitable precursor may also be included. The nutrient medium and precursors can be added to the culture medium in a batch or continuous process without limitation.

In the present invention, during the process of cultivating a microorganism, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture to adjust the pH of the culture medium. During the culture process, an antifoam agent such as a polyglycol fatty acid ester may also be added to suppress foam formation. In addition, oxygen or an oxygen-containing gas may be introduced into the culture medium to maintain the culture medium in an aerobic state, and nitrogen, hydrogen, or carbon dioxide gases may be introduced into the cultures to maintain anaerobic and microaerobic conditions without introducing any other gases, but the embodiment is not limited to this.

The temperature of the culture medium can be maintained at 25°C - 50°C, more particularly at 30°C - 37°C without limitation. Cultivation can be continued until the required amount of product is obtained, in particular for 10 hours to 100 hours, without limitation.

The target product obtained as a result of cultivation can be exported to the culture medium or remain in the cells.

The method may further include isolating the target product from the culture medium or microorganism.

Isolation of the target product obtained in the cultivation step of the present invention can be accomplished by collecting the target product from the culture using any known method selected in accordance with the cultivation method. For example, centrifugation, filtration, anion exchange chromatography, crystallization and high performance liquid chromatography (HPLC) can be used, and the target amino acid can be isolated from the culture medium or microorganism using any suitable method in the art without limitation. The culture may also be called culture fluid.

The isolation step may also include a purification process, which can be performed using a suitable method well known in the art. Thus, the isolated target product can be purified from the fermentation broth of the microorganism containing the target product (Introduction to Biotechnology and Genetic Engineering, A.J. Nair., 2008).

In another aspect of the present invention, there is provided a method for producing methionine, comprising: producing O-acetylhomoserine by cultivating a microorganism in a culture medium; and converting O-acetylhomoserine to methionine by reacting O-acetylhomoserine with a sulfide, wherein the microorganism comprises at least one of: a variant of the inner membrane protein YjeH, comprising i) replacing the amino acid corresponding to position 92 with asparagine, ii) replacing the amino acid corresponding to the 351st position, leucine, or iii) a substitution at both positions i) and ii) in the amino acid sequence of SEQ ID NO: 1, and having 95% or more but less than 100% homology with SEQ ID NO: 1; a polynucleotide encoding this variant; and a vector encoding this polynucleotide.

SEQ ID NO: 1, inner membrane protein YjeH, a variant thereof, a polynucleotide encoding the variant, a vector including the polynucleotide, a microorganism including them, and O-acetylhomoserine are as described above.

In the present invention, O-acetyl homoserine as a methionine precursor can be converted to methionine by reaction with sulfide.

Sulfide reaction refers to the process of forming L-methionine from O-succinylhomoserine using any known method. For example, L-methionine can be prepared by reacting methyl mercaptan as a sulfide with O-acetylhomoserine. In addition, a catalyst or enzyme can be added or the reaction carried out in a microorganism containing the enzyme to increase the reaction rate and yield.

"Sulfide" may be, for example, methyl mercaptan, and methyl mercaptan may be any methyl mercaptan derivatives capable of providing sulfur atoms, such as dimethyl sulfide (DMS), disclosed in international publication WO 2010/098629), as well as sodium methyl mercaptan (CH ₃ S -Na) in the liquid phase and methyl mercaptan (CH ₃ SH) in the gaseous or liquid state.

As the enzyme used in the methionine conversion reaction, cystathionine-γ-synthase, O-acetylhomoserine sulfhydrylase or O-succinylhomoserine sulfhydrylase, which are known in the art, can be used.

The methionine conversion reaction process can be expressed by the following reaction scheme:

O-acetyl-L-homoserine+CH ₃ SH↔L-methionine+acetate

In this reaction, the CH ₃ S residue of methyl mercaptan is replaced by an O-acetylhomoserine acetate residue to produce L-methionine.

This reaction can be carried out at a temperature of from 20°C to 45°C, while maintaining the pH of the reaction mixture in the range of 6 to 7, and pyridoxal 5'-phosphate can be added as a coenzyme.

The conversion of the L-methionine precursor to L-methionine in the presence of methyl mercaptan is an enzymatic reaction and is disclosed in International Publication No. WO 2008/013432. International Publication No. WO 2008/013432 provides a detailed description of the properties and preparation of methionine converting enzymes having the appropriate activity to convert the L-methionine precursor to L-methionine in the presence of methyl mercaptan. Such a suitable methionine converting enzyme can be produced biotechnologically using gene expression.

The sequence of the gene encoding the enzyme having methionine-converting activity can be obtained, but without limitation, from the NCBI database (USA) and the DNA data bank (KEGG, Japan). For biological conversion, the gene can be cloned from the resulting gene sequence and then introduced into an expression vector. In particular, the gene can be expressed in active form from the vector pCL-CJI (CJ, Korea), which is an expression vector for E. coli in recombinant strains, and the expressed protein can be obtained from an enzyme solution in which sonicated cells are lysed. Both the strain expressing the methionine converting enzyme and the expressed methionine converting enzyme can be directly used in the methionine conversion reaction.

To initiate the reaction, the methionine-converting enzyme expressed from the gene or from the strain of microorganism expressing it can be mixed directly, partially or completely, with the fermentation supernatant or with the fermentation broth in which L-methionine accumulates. Thus, the reaction can be initiated by adding a methionine-converting enzyme or a strain of microorganism expressing it to the fermentation solution in which O-acetylhomoserine accumulates and adding methyl mercaptan thereto.

In particular, O-acetylhomoserine, which accumulates in the fermentation solution, can be converted to L-methionine by cystathionine-γ-synthase, O-acetylhomoserine sulfhydrylase or O-succinylhomoserine sulfhydrylase.

The conversion reaction to methionine can be confirmed using DTNB (5,5-dithiobis(2-nitrobenzoic acid), Sigma, USA) and the reaction product can be analyzed by HPLC. The conversion reaction to methionine can further produce by-products such as acetic acid without a separate production process, through the reaction between methyl mercaptan and O-acetylhomoserine.

The enzymatic reaction between the L-methionine precursor and methyl mercaptan can be represented by the following reaction scheme:

The method for producing methionine can be easily determined by one of ordinary skill in the art, provided the optimal culture conditions and enzyme activation conditions are known. The specific conditions and media for cultivation are as defined above.

In another aspect of the present invention, there is provided a method for producing glufosinate, comprising producing O-acetylhomoserine or homoserine by cultivating a microorganism in a culture medium and converting O-acetylhomoserine or homoserine to glufosinate, wherein the microorganism comprises at least one of: a variant of the inner membrane protein YjeH, comprising i) replacing the amino acid corresponding to position 92 with asparagine, ii) replacing the amino acid corresponding to position 351 with leucine, or iii) replacing at both positions i) and ii), in the amino acid sequence SEQ ID NO: 1, and having homology of 95% or more but less than 100% with SEQ ID NO: 1; a polynucleotide encoding this variant; and a vector encoding this polynucleotide.

SEQ ID NO: 1, inner membrane protein YjeH, a variant thereof, a polynucleotide encoding the variant, a vector including the polynucleotide, the microorganism including them, and homoserine are as described above.

As used herein, the term “glufosinate” refers to a wide range of natural herbicides produced by several species of soil bacteria Streptomyces and is also known as “phosphinothricin”.

In the present invention, O-acetylhomoserine or homoserine can be converted to glufosinate through a variety of steps, including halogenation.

The conversion reaction to glufosinate is disclosed, for example, in CN 108516991 A. In particular, the conversion reaction to glucosinate may include: (1) the step of performing azeotropic dehydration of homoserine (Formula II) using an organic solvent in the presence of an acid catalyst; and the step of adding a halogenating agent to the azeotropically dehydrated homoserine and performing halogenation to obtain L-3,6-bis(2-haloethyl)-2,5-dionepiperazine (Formula III); (2) the step of adding diester or methyl phosphite diester of methyl phosphinic acid to L-3,6-bis(2-haloethyl)-2,5-dionepiperazine and performing an Abbov reaction using a high boiling point solvent in the presence of a catalyst to obtain a compound formula IV; and (3) the step of performing a hydrolysis reaction by dissolving a compound of formula IV in an acid and heating the solution, removing the solvent after completion of the reaction, adding an alcohol thereto, and performing a conversion reaction by adding an alkylene oxide thereto to produce glufosinate (formula I).

The conversion reaction to glufosinate using homoserine as a precursor can be represented using the following scheme:

The method for producing glufosinate can be carried out using homoserine obtained by biological fermentation as a precursor as described in the present invention. Glufosinate can be prepared by preparing L-3,6-bis(2-haloethyl)-2,5-dionepiperazine by reacting with methylphosphonium by azeotropic dehydration and halogenation, performing the Abbow reaction, and then performing the hydrolysis reaction as described above.

In another aspect of the present invention, a composition for producing homoserine is provided, comprising a microorganism or a culture of a microorganism in a culture medium, wherein the microorganism comprises at least one of: a variant of the inner membrane protein YjeH, comprising i) replacing the amino acid corresponding to position 92 with asparagine, ii) substitution of the amino acid corresponding to position 351 with leucine, or iii) substitution at both positions i) and ii), in the amino acid sequence of SEQ ID NO: 1, and having 95% or more but less than 100% homology with SEQ ID NO: 1; a polynucleotide encoding this variant; and a vector encoding this polynucleotide.

In another aspect of the present invention, a composition for producing O-acetylhomoserine is provided, comprising a microorganism or a culture of a microorganism in a culture medium, wherein the microorganism comprises at least one of: a variant of the inner membrane protein YjeH, comprising i) an amino acid substitution corresponding to the 92nd position, asparagine, ii) substitution of the amino acid corresponding to position 351 with leucine, or iii) substitution at both positions i) and ii), in the amino acid sequence of SEQ ID NO: 1, and having 95% or more but less than 100% homology with SEQ ID NO: 1; a polynucleotide encoding this variant; and a vector encoding this polynucleotide.

SEQ ID NO: 1, inner membrane protein YjeH, a variant thereof, a polynucleotide encoding the variant, a vector including the polynucleotide including the microorganism thereof, homoserine and O-acetylhomoserine are as described above.

The composition for producing O-acetylhomoserine or homoserine may refer to the composition used for producing O-acetylhomoserine or homoserine by the inner membrane protein YjeH variant described in the present invention. This composition may include a variant of the inner membrane protein YjeH or a component capable of performing the function of the inner membrane protein YjeH without limitation. The inner membrane protein YjeH variant may be in the form of an insert into a vector capable of expressing a functionally related gene in a host cell.

The composition may further include a cryoprotectant or excipient. The cryoprotectant or excipient may be a natural or non-naturally occurring substance, but is not limited to. In another example, the cryoprotectant or excipient may be a substance that the microorganism does not naturally come into contact with and that is not naturally included in the microorganism, but is not limited to.

Another aspect of the present invention provides the use of a variant of the inner membrane protein YjeH to produce O-acetylhomoserine or homoserine.

Another aspect of the present invention provides the use of a microorganism to produce O-acetylhomoserine or homoserine, wherein the microorganism comprises at least one of: a variant of the inner membrane protein YjeH, comprising i) replacing the amino acid corresponding to position 92 with asparagine, ii) replacing the amino acid corresponding to position 351, leucine, or iii) a substitution at both positions i) and ii) in the amino acid sequence of SEQ ID NO: 1, and having 95% or more but less than 100% homology with SEQ ID NO: 1; a polynucleotide encoding this variant; and a vector encoding this polynucleotide.

Homoserine, O-acetylhomoserine, inner membrane protein YjeH, a variant thereof, a polynucleotide encoding the variant, a vector including the polynucleotide, and a microorganism including them are as described above.

Method for carrying out the invention

The present invention will now be described in more detail with reference to the following examples. However, the following examples are presented solely to illustrate the description of the present invention, and the scope of the description of the present invention is not limited to them.

Example 1: Plasmid Construction

The plasmid (pDCM2, FIG. 1, SEQ ID NO: 37) was designed to insert into and replace a gene on the Corynebacterium chromosome and was synthesized using the gene synthesis service of Bionics Co., Ltd. The plasmid was designed to contain a restriction enzyme efficient for cloning, with reference to an article relating to the sacB system, well known in the art (Gene, 145 (1994) 69-73). The synthesized plasmid pDCM2 has the following characteristics.

1) Due to the fact that the origin of replication is active only in E. coli, the plasmid independently replicates only in E. coli and cannot independently replicate in Corynebacterium.

2) The plasmid contains the kanamycin resistance gene as a selective marker.

3) The plasmid contains the levansucrase gene (sacB) as a secondary positive selection marker.

4) The final strain obtained does not retain genetic information originating from the pDCM2 plasmid.

Example 2: Construction of strains with an introduced variant of an exogenous membrane protein (YjeH) and assessment of the ability to produce O-acetylhomoserine and homoserine

2-1. Construction of strains with introduced exogenous membrane protein (YjeH) and YjeH variant

In order to evaluate the effectiveness of YjeH, which was introduced into Corynebacterium glutamicum ATCC13032 as an exogenous membrane protein and an O-acetylhomoserine exporting protein, a chromosomal insertion vector was obtained that included the yjeH gene (SEQ ID NO: 2) encoding YjeH originating from E. coli

Specifically, in order to construct a transposase deletion vector, a pair of primers (SEQ ID NO: 4 and 5) were designed to amplify the 5' upstream region and a pair of primers (SEQ ID NO: 6 and 7) to amplify the 3' downstream region of the gene encoding the transposase (SEQ ID NO: 3, Gene No. NCg12335). Primers SEQ ID NO: 5 and 6 were designed to overlap each other. The restriction enzyme sequence SmaI was localized in this region. The primer sequences are presented in Table 1 below.

PCR was performed using wild type (WT) chromosome ATCC13032 as template and primers SEQ ID NO: 4 and 5 and SEQ ID NO: 6 and 7. PCR was performed under the following conditions. After denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds were repeated, followed by polymerization at 72°C. C for 7 minutes. As a result, DNA fragments were obtained: 851 bp. 5' of the overlying region and 847 bp. 3' downstream of the deletion region of the NCg12335 gene.

PCR was performed using two amplified DNA fragments as templates and primers SEQ ID NO: 4 and 7. PCR was performed under the following conditions. After denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 90 seconds were repeated, followed by polymerization at 72°C. C for 7 minutes. As a result, a DNA fragment of 1648 bp was amplified, including a region capable of deleting the gene encoding transposase (SEQ ID NO: 3, gene no. NCg12335).

The resulting PCR products were cloned by fusion using the SmaI restriction enzyme-treated pDCM2 vector and the In-Fusion® HD Cloning Kit (Clontech). E. coli DH5α was transformed with the cloned vector, and the transformed E. coli were smeared onto solid Luria-Bertani (LB) medium containing kanamycin (25 mg/L). Colonies transformed with the plasmid with the insertion of the target gene were selected by PCR and the plasmid was obtained using the plasmid extraction method. Finally, the recombinant vector pDCM2-ΔNCg12335 was constructed, including the cloned NCg12335 deletion cassette.

In order to evaluate the effectiveness of the O-acetylcholine export protein, a chromosomal insertion vector containing the gene (SEQ ID NO: 2) encoding YjeH, which is derived from E. coli, was developed. For this purpose, a vector expressing the yjeH gene was constructed using the CJ7 promoter (SEQ ID NO: 8, US 7662943 B2). A pair of primers (SEQ ID NOs: 9 and 10) to amplify the CJ7 promoter region and a pair of primers (SEQ ID NOs: 11 and 12) to amplify the yjeH region of E. coli were designed. The primer sequences are presented in Table 2 below.

PCR was performed using pECCG117-PCJ7-gfp (US 7662943 B2) as a template and primers SEQ ID NO: 9 and 10, and PCR was also performed using the E. coli wild type chromosome as a template and primers SEQ ID NO: 11 and 12 respectively. PCR was performed under the following conditions. After denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 90 seconds were repeated, followed by polymerizations at 72°C. C for 7 minutes. As a result, a DNA fragment of 360 bp was obtained. promoter region of CJ7 and a DNA fragment of 1297 bp. yjeH gene of E. coli.

PCR was performed using two amplified DNA fragments as templates and primers SEQ ID NO: 9 and 12. PCR was performed under the following conditions. After denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 90 seconds were repeated, followed by polymerization at 72°C. C for 7 minutes. As a result, a 1614 bp DNA fragment was amplified, including the region in which the CJ7 promoter and gene inserts were located. yjeH.

The gene deletion DNA fragment obtained by PCR was cloned into the pDCM2-ΔNCg12335 vector treated with the restriction enzyme SmaI using the In-Fusion® HD Cloning Kit (Clontech). The recombinant vector pDCM2-ΔNCg12335::PCJ7-yjeH (eco, WT) was finally constructed.

Specifically, phenylalanine (Phe), which is the 92nd amino acid of the amino acid sequence of YjeH, was replaced by asparagine (Asn) (F92N) using plasmid pDCM2-ΔNCg12335::PCJ7-yjeH (eco, WT) as template and primers SEQ ID NOs: 13 and 14. The constructed plasmid including the gene encoding the YjeH variant (F92N) was named pDCM2-ΔNCg12335::PCJ7-yjeH (eco, F92N).

Phenylalanine (Phe), which is the 351st amino acid of the YjeH amino acid sequence, was replaced with leucine (F351L) using plasmid pDCM2-ΔNCg12335::PCJ7-yjeH (eco, WT) as template and primers SEQ ID NO: 15 and 16 The constructed plasmid including the gene encoding the YjeH variant (F351L) was named pDCM2-ΔNCg12335::PCJ7-yjeH (eco, F351L). The primer sequences are presented in Table 3 below.

Strain ATCC13032 was transformed with each of the constructs pDCM2-ΔNCg12335, pDCM2-ΔNCg12335::PCJ7-yjeH (eco, WT), pDCM2-ΔNCg12335::PCJ7-yjeH (eco, F92N) and pDCM2-ΔNCg12335::PCJ7-yjeH (eco, F351L ) using the electric pulse method and subjected to a secondary crossing over process to obtain ATCC13032 ΔNCg12335, ATCC13032 ΔNCg12335::PCJ7-yjeH (eco, WT), ATCC13032 ΔNCg12335::PCJ7-yjeH (eco, F92N) and ATCC13032 ΔNCg12335::PCJ7-yjeH (eco , F351L), in which the NCg12335 gene was deleted from the chromosome. The insertion of the inactivated NCg12335 gene and the newly introduced E. coli yjeH gene was identified by performing PCR using primers SEQ ID NOs: 4 and 7 and comparing the results with those for ATCC13032, in which the NCg12335 gene was not inactivated.

2-2. Assessment of the ability to produce O-acetylhomoserine

To compare the O-acetylhomoserine (O-AH)-producing capacity of ATCC13032ΔNCg12335, ATCC13032ΔNCg12335::PCJ7-yjeH (eco, WT), ATCC13032ΔNCg12335::PCJ7-yjeH (eco, F92N) and ATCC13032Δ NCg12335::PCJ7-yjeH (eco, F351L), which were constructed in Example 2-1, and the wild-type strain ATCC13032, O-acetylhomoserine contained in the cultures was analyzed by the following method.

One microbiological inoculation loop for each strain was inoculated into a 250 ml corner flask containing 25 ml of the medium described below and then cultured with shaking at 33°C for 20 hours at 200 rpm. O-acetylhomoserine concentrations were analyzed using HPLC, and the analyzed concentrations are shown in Table 4.

Medium for the production of O-acetylhomoserine (pH 7.2)

30 g glucose, 2 g KH ₂ PO ₄ , 3 g urea, 40 g (NH ₄ ) ₂ SO ₄ , 2.5 g peptone, 5 g (10 ml) corn syrup (CSL, Sigma), 0.5 g MgSO ₄ ⋅7H ₂ O and 20 g CaCO ₃ (calculated per 1 liter of distilled water)

As a result, it was confirmed that when ATCC13032 was cultured as a control, O-acetyl-L-homoserine accumulated at a concentration of 0.3 g/L, and, as shown in Table 4, deletion of the NCg12335 gene (transposase) did not affect the production of O-acetyl -L-homoserine. In particular, it was confirmed that when the wild type yjeH gene was expressed, O-acetyl-L-homoserine accumulated at a concentration of 0.7 g/l, and when yjeH gene variants were expressed, O-acetyl-L-homoserine accumulated at concentrations of 1.2 g /li 1.0 g/l.

Thus, based on the results presented above, it was confirmed that the wild type and yjeH variant genes as described in the present invention lead to a significant increase in the production of the amino acid of interest as a result of the export of O-acetylhomoserine from ATCC13032.

Strains ATCC13032 ΔNCg12335::PCJ7-yjeH (eco, F92N) and ATCC13032 ΔNCg12335::PCJ7-yjeH (eco, F351L) were named Corynebacterium glutamicum CM04-0651 and Corynebacterium glutamicum CM04-0652, respectively, and deposited in Cor. Yeisk Center for Microbial Cultures in accordance with the Budapest Treaty December 4, 2019 under accession numbers KССМ12633Р and KССМ12634Р respectively.

2-3. Assessment of the ability to produce homoserine

To compare the homoserine-producing capacity of ATCC13032ΔNCg12335, ATCC13032ΔNCg12335::PCJ7-yjeH (eco, WT), ATCC13032ΔNCg12335::PCJ7-yjeH (eco, F92N) and ATCC13032ΔNCg12335::PC J7-yjeH (eco, F351L), which were constructed in Example 2-1, and the wild-type strain ATCC13032 was analyzed for homoserine contained in the cultures by the following method.

One microbiological inoculation loop for each strain was inoculated into a 250 ml corner flask containing 25 ml of the medium described below and then cultured with shaking at 33°C for 20 hours at 200 rpm. Homoserine concentrations were analyzed using HPLC, and the analyzed concentrations are shown in Table 5.

Medium for homoserine production (pH 7.2)

30 g glucose, 2 g KH ₂ PO ₄ , 3 g urea, 40 g (NH ₄ ) ₂ SO ₄ , 2.5 g peptone, 5 g (10 ml) corn syrup (CSL, Sigma), 0.5 g MgSO ₄ ⋅7H ₂ O and 20 g CaCO ₃ (calculated per 1 liter of distilled water)

As a result, it was confirmed that when ATCC13032 was cultured as a control, homoserine did not accumulate (concentration 0.0 g/L), and, as shown in Table 5, deletion of the NCg12335 (transposase) gene did not affect homoserine production. In particular, it was confirmed that when the wild-type yjeH gene was expressed, homoserine did not accumulate (concentration of 0.0 g/L), and when the yjeH gene variants were expressed, homoserine accumulated at concentrations of 0.2 g/L and 0.2 g/L.

Thus, based on the results presented above, it was confirmed that the tepa option. yjeH as described in the present invention leads to a significant increase in the production of the amino acid of interest as a result of the export of homoserine from ATCC13032.

Example 3: Construction of a strain with enhanced ability to produce O-acetylhomoserine and evaluation of the ability to produce O-acetylhomoserine and homoserine

3-1. Inactivation of cystathionine gamma (γ) synthase (MetB)

Using PCR using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template, the metB gene encoding cystathionine-γ-synthase involved in the O-acetylhomoserine degradation pathway was obtained. Information on the nucleotide sequence of the metB gene (NCBI No. Ncg12360, SEQ ID NO: 17) was obtained from GenBank of the National Institutes of Health (USA), and on its basis primers (SEQ ID NO: 18 and 19) including the N-terminus of the gene were synthesized metB and linker, and primers (SEQ ID NOs: 20 and 21) including its C-terminus and linker. The primer sequences are presented in Table 6 below.

PCR was performed using ATCC13032 chromosomal DNA as a template and primers SEQ ID NOs: 18 and 19 and SEQ ID NOs: 20 and 21. PfuUltra™ High Confidence DNA Polymerase (Stratagene) was used as the polymerase and PCR was performed by repeating 30 cycles of denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds and curing at 72°C for 1 minute. As a result, we obtained: a 558 bp amplified gene containing the N-terminus of the metB gene and a linker, and a 527 bp amplified gene containing the C-terminus of the metB gene and a linker, respectively.

PCR was performed using the two amplified genes obtained as described above as templates by repeating 10 cycles of denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds and polymerization at 72°C for 1 minute , and then adding primers SEQ ID NO: 18 and 21, followed by polymerization 20 times. As a result, an inactivation cassette of 1064 bp was obtained, including the N-terminus-linker-C-terminus of the metB gene. The resulting PCR products were treated with the restriction enzymes XbaI and SalI and ligated with the pDCM2 vector treated with the restriction enzymes XbaI and SaiI for cloning to obtain the final construct of the recombinant vector pDCM2-ΔmetB with the cloned metB inactivation cassette.

ATCC13032 was transformed with the constructed vector pDCM2-ΔmetB using the electropulse method and subjected to a secondary crossover process to obtain ATCC13032ΔmetB, in which the metB gene was inactivated in the chromosome. The inactivated metB gene was ultimately confirmed by comparison with ATCC13032, in which the metB gene was not inactivated, after performing PCR using primers SEQ ID NO: 18 and 21.

3-2. Inactivation of O-acetylhomoserine(tnol)-lyase (MetY)

Using PCR using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template, the metY gene encoding O-acetylhomoserine(thiol)-lyase involved in the O-acetylhomoserine degradation pathway was obtained. Information on the nucleotide sequence of the metY gene (NCBI No. Ncg10625, SEQ ID NO: 22) was obtained from GenBank of the National Institutes of Health (USA), and on its basis primers (SEQ ID NO: 23 and 24) were synthesized, including the N-terminus of the gene metY and linker, and primers (SEQ ID NOs: 25 and 26) including its C-terminus and linker. The primer sequences are presented in Table 7 below.

PCR was performed using ATCC13032 chromosomal DNA as a template and primers SEQ ID NOs: 23 and 24 and SEQ ID NOs: 25 and 26. PfuUltra™ High Confidence DNA Polymerase (Stratagene) was used as the polymerase and PCR was performed by repeating 30 cycles of denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds and curing at 72°C for 1 minute. As a result, we obtained: a 548 bp amplified gene containing the N-terminus of the metY gene and a linker, and a 550 bp amplified gene containing the C-terminus of the metY gene and a linker, respectively. PCR was performed using the two amplified genes obtained as described above as templates by repeating 10 cycles of denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds and polymerization at 72°C for 1 minute , and then adding primers SEQ ID NO: 23 and 26, followed by polymerization 20 times. As a result, an inactivation cassette of 1077 bp was obtained, including the N-terminal linker and the C-terminal end of the metY gene. The resulting PCR products were treated with the restriction enzymes XbaI and SalI and ligated with the pDCM2 vector treated with the restriction enzymes XbaI and SalI for cloning to obtain the final construct of the recombinant vector pDCM2-ΔmetY with the cloned metY inactivation cassette.

Strain ATCC13032ΔmetB was transformed with the constructed vector pDCM2-ΔmetY using the electropulse method and subjected to a secondary crossover process to obtain ATCC13032ΔmetBΔmetY, in which the metY gene is inactivated in the chromosome. The inactivated metY gene was ultimately confirmed by comparison with ATCC13032, in which the metY gene was not inactivated, after performing PCR using primers SEQ ID NOs: 23 and 26.

3-3. Introduction of aspartokinase variant (LysC)

In order to maximize the production of O-acetylhomoserine, a mutation (L377K) was introduced into the lysC gene (SEQ ID NO: 27) obtained from Corynebacterium glutamicum ATCC13032, encoding aspartokinase, to enhance the expression of the lysC gene and weaken the inhibition by L-lysine and L-threonine by the reverse mechanism communications (US 10662450 B2). In order to obtain a vector including the lysC gene variant, a pair of primers (SEQ ID NO: 28 and 29) was designed to amplify the 5' upstream region and a pair of primers (SEQ ID NO: 30 and 31) to amplify the 3' downstream region of position of the mutation. The insertion of XbaI and SalI restriction enzyme regions was introduced at both ends of primers SEQ ID NOs: 28 and 31, and primers SEQ ID NOs: 29 and 30 were designed to overlap each other and the nucleotide change was localized to this region. The primer sequences are as shown in Table 8 below.

PCR was performed using Corynebacterium glutamicum ATCC13032 as template and primers SEQ ID NO: 28 and 29 and SEQ ID NO: 30 and 31. PCR was performed under the following conditions. After denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds were repeated, followed by polymerization at 72°C. C for 7 minutes. As a result, a DNA fragment of 512 bp was obtained. 5' upstream region and 522 bp DNA fragment. 3' downstream of the lysC gene mutation.

PCR was performed using two amplified DNA fragments as templates and primers SEQ ID NO: 28 and 31. PCR was performed under the following conditions. After denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 60 seconds were repeated, followed by polymerization at 72°C. C for 7 minutes. As a result, a 1011 bp DNA fragment was amplified, including a variant of the lysC gene (L377K), encoding a variant of aspartokinase, in which leucine at position 377 was replaced by lysine.

The lysC fragment (L377K), obtained by PCR, was treated with restriction enzymes XbaI and SalI at both ends and ligated with the vector pDCM2, treated with restriction enzymes XbaI and SalI, after which it was cloned and finally obtained the recombinant vector pDCM2-lysC (L377K) with cloned lysC replacement cassette (L377K).

Strain ATCC13032 ΔmetB ΔmetY was transformed with the resulting vector pDCM2-lysC (L377K) by the electric pulse method and subjected to the process of secondary crossing over to obtain Corynebacterium glutamicum ATCC13032 ΔmetB ΔmetY lysC (L377K) with an introduced variant of the lysC gene on the chromosome. The gene variant was conclusively identified by PCR performed using primers SEQ ID NOs: 28 and 31, and the sequence was then compared to the wild-type lysC gene sequence by sequencing.

3-4. Construction of a plasmid with the introduction of O-acetylhomoserine transferase (MetX)

In order to amplify the gene encoding O-acetylhomoserine transferase (MetX), the nucleotide sequence information of the metX gene (NCBI No. NCg10624, SEQ ID NO: 32) was obtained from the GenBank of the National Institutes of Health (USA), the restriction enzyme BamHI was inserted into both ends of each of primers (SEQ ID NOs: 33 and 34) based on this sequence to amplify the region from the promoter (localized approximately 300 bp upstream of the start codon) to the terminator (localized approximately 100 bp downstream of the stop codon). codon PCR was performed under the following conditions: After denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 90 seconds were repeated. , and then polymerization was performed at 72°C for 7 minutes. As a result, a DNA fragment of 1546 bp of the coding region of the metX gene was obtained. The vector pECCG117 (KR 10-0057684 B1) and the metX DNA fragment were treated with the restriction enzyme BamHI, ligated using DNA ligase and cloned to obtain a plasmid named pECCG117-metX WT. The primer sequences are as shown in Table 9 below.

3-5. Construction of an O-acetylhomoserine-producing strain and assessment of the ability to produce O-acetylhomoserine

After obtaining the pECCG117-metX WT vector in example 3-4, it was introduced into Corynebacterium glutamicum ATCC13032 ΔmetB ΔmetY lysC (L377K) using the electric pulse method, the strain was sown by the smear method on a selective medium containing kanamycin (25 mg/l), and cultivated until transformants.

To compare the O-acetylhomoserine-producing ability between strains, the strains were cultured as described below and the O-acetylhomoserine contained in the cultures was analyzed.

One microbiological inoculation loop for each strain was inoculated into a 250 ml corner flask containing 25 ml of the medium described below, and then cultured with shaking at 37°C for 20 hours at 200 rpm. O-acetylhomoserine concentrations were analyzed using HPLC, and the analyzed concentrations are shown in Table 10.

Medium for the production of O-acetylhomoserine (pH 7.2)

30 g glucose, 2 g KH ₂ PO ₄ , 3 g urea, 40 g (NH ₄ ) ₂ SO ₄ , 2.5 g peptone, 5 g (10 ml) corn syrup (CSL, Sigma), 0.5 g MgSO ₄ ⋅7H ₂ O, 400 mg of methionine and 20 g of CaCO ₃ (calculated per 1 liter of distilled water)

As a result, it was confirmed that when ATCC13032 ΔmetB ΔmetY lysC (L377K) was cultivated as a control, O-acetyl-L-homoserine accumulated at a concentration of 0.7 g/l, and when the strain ATCC13032 ΔmetB ΔmetY lysC (L377K)/pECCG117-metX was cultivated WT O-acetyl-L-homoserine accumulated at a concentration of 1.3 g/L, as shown in Table 10.

Thus, the increased production of O-acetyl-L-homoserine in ATCC13032 ΔmetB ΔmetY lysC (L377K)/pECCG117-metX WT strain was confirmed based on the results presented above.

3-6. Assessment of the ability to produce homoserine

In Corynebacterium glutamicum ATCC13032 ΔmetB ΔmetY lysC (L377K), constructed in example 3-3, the pECCG117-metX WT vector, constructed in example 3-4, was introduced using the electric pulse method, sown using the smear method on a selective medium containing (25 mg/l) kanamycin and cultured until transformants were obtained.

To compare the homoserine-producing ability between strains, the strains were cultured as described below and the homoserine contained in the cultures was analyzed.

One microbiological inoculation loop for each strain was inoculated into a 250 ml corner flask containing 25 ml of the medium described below, and then cultured with shaking at 37°C for 20 hours at 200 rpm. Homoserine concentrations were analyzed using HPLC, and the analyzed concentrations are shown in Table 11.

Medium for homoserine production (pH 7.2)

30 g glucose, 2 g KH ₂ PO ₄ , 3 g urea, 40 g (NH ₄ ) ₂ SO ₄ , 2.5 g peptone, 5 g (10 ml) corn syrup (CSL, Sigma), 0.5 g MgSO ₄ ⋅7H ₂ O, 400 mg of methionine and 20 g of CaCO ₃ (calculated per 1 liter of distilled water)

As a result, it was confirmed that when cultivating ATCC13032 ΔmetB ΔmetY lysC (L377K) as a control, homoserine accumulated at a concentration of 0.2 g/l, and when cultivating the ATCC13032 ΔmetB ΔmetY lysC (L377K)/pECCG117-metX WT strain, homoserine accumulated at a concentration of 0 .2 g/l, as shown in Table 11.

Thus, based on these results, it was confirmed that the ATCC13032 ΔmetB ΔmetY lysC (L377K)/pECCG117-metX WT strain had the same level of homoserine production.

Example 4: Construction of strains with an introduced variant of an exogenous membrane protein (YjeH) having an enhanced ability to produce O-acetylhomoserine, and evaluation of the ability to produce O-acetylhomoserine

4-1. Introduction of an exogenous protein variant (YjeH)

The Corynebacterium glutamicum strain ATCC13032 ΔmetB ΔmetY lysC (L377K), constructed in example 3-3, was transformed with the vectors pDCM2-ΔNCg12335, pDCM2-ΔNCg12335::PCJ7-yjeH (eco, WT), pDCM2-ΔNCg12335::PCJ7 -yjeH (eco, F92N ) and pDCM2-ΔNCg12335::PCJ7-yjeH (eco, F351L), previously constructed in Example 2-1, and subjected to a secondary crossover process to obtain ATCC13032 ΔmetB ΔmetY lysC (L377K) ΔNCg12335, ATCC13032 ΔmetB Δmet Y lysC (L377K) ΔNCg12335:: PCJ7-yjeH (eco, WT), ATCC13032 ΔmetB ΔmetY lysC (L377K) ΔNCg12335::PCJ7-yjeH (eco, F92N) and ATCC13032 ΔmetB ΔmetY lysC (L377K) ΔNCg12335::PCJ7-yjeH (eco , F351L), from whose chromosomes the NCg12335 (transposase) gene was deleted. The integration of the inactivated NCg12335 gene and the newly introduced E. coli yjeH gene was identified by performing PCR using primers SEQ ID NOs: 4 and 7 and comparing the results with those for ATCC13032, in which the NCg12335 gene was not inactivated.

4-2. Construction of an O-acetylhomoserine-producing strain and assessment of the ability to produce O-acetylhomoserine

After obtaining the pECCG117-metX WT vector in example 3-4, it was introduced into ATCC13032 ΔmetB ΔmetY lysC (L377K) ΔNCg12335, ATCC13032 ΔmetB ΔmetY lysC (L377K) ΔNCg12335::PCJ7-yjeH (eco, WT), ATCC 13032 ΔmetB ΔmetY lysC (L377K) ΔNCg12335::PCJ7-yjeH (eco, F92N) and ATCC13032 ΔmetB ΔmetY lysC (L377K) ΔNCg12335::PCJ7-yjeH (eco, F351L), obtained in example 4-1, using the electric pulse method, the strains were smeared onto a selective medium, containing (25 mg/l) kanamycin and cultivated until transformants were obtained.

To compare the O-acetylhomoserine production ability between strains, the strains were cultured as described below and the O-acetylhomoserine contained in the cultures was analyzed.

One microbiological loop for inoculation of each strain was inoculated into a flask with corner partitions with a capacity of 250 ml containing 25 ml of medium for the production of O-acetylhomoserine (pH 7.2) of example 3-5, and then cultured with shaking at 33°C for 20 hours at 200 rpm. O-acetylhomoserine concentrations were analyzed using HPLC, and the analyzed concentrations are shown in Table 12.

As a result, it was confirmed that when Corynebacterium glutamicum ATCC13032 ΔmetB ΔmetY lysC (L377K) was cultivated as a control, O-acetyl-L-homoserine accumulated at a concentration of 1.3 g/L, and, as shown in Table 12, deletion of the NCg12335 (transposase) gene ) did not affect the production of O-acetyl-L-homoserine. In particular, it was confirmed that when the wild-type yjeH gene was expressed, O-acetyl-L-homoserine accumulated at a concentration of 2.1 g/L, and when the tepa variants were expressed. yjeH O-acetyl-L-homoserine accumulated at concentrations of 2.7 g/L and 2.3 g/L.

Thus, based on the results presented above, it was confirmed that the wild type and yjeH variant genes as described in the present invention lead to a significant increase in the production of the amino acid of interest as a result of the export of O-acetyl-L-homoserine from Corynebacterium glutamicum ATCC13032.

4-3. Assessment of the ability to produce homoserine

After obtaining the pECCG117-metX WT vector in example 3-4, it was introduced into ATCC13032 ΔmetB ΔmetY lysC (L377K) ΔNCg12335, ATCC13032 ΔmetB ΔmetY lysC (L377K) ΔNCg12335::PCJ7-yjeH (eco, WT), ATCC 13032 ΔmetB ΔmetY lysC (L377K) ΔNCg12335::PCJ7-yjeH (eco, F92N) and ATCC13032 ΔmetB ΔmetY lysC (L377K) ΔNCg12335::PCJ7-yjeH (eco, F351L), obtained in example 4-1, using the electric pulse method, the strains were smeared onto a selective medium, containing (25 mg/l) kanamycin and cultivated until transformants were obtained.

To compare the homoserine-producing ability between strains, the strains were cultured as described below and the homoserine contained in the cultures was analyzed.

One microbiological inoculation loop for each strain was inoculated into a 250 ml corner flask containing 25 ml of the medium described below and then cultured with shaking at 33°C for 20 hours at 200 rpm. Homoserine concentrations were analyzed using HPLC, and the analyzed concentrations are shown in Table 13.

Medium for homoserine production (pH 7.2)

30 g glucose, 2 g KH ₂ PO ₄ , 3 g urea, 40 g (NH ₄ ) ₂ SO ₄ , 2.5 g peptone, 5 g (10 ml) corn syrup (CSL, Sigma), 0.5 g MgSO ₄ ⋅7H ₂ O, 400 mg of methionine and 20 g of CaCO ₃ (calculated per 1 liter of distilled water)

As a result, it was confirmed that when Corynebacterium glutamicum ATCC13032 ΔmetB ΔmetY lysC (L377K) was cultivated as a control, homoserine accumulated at a concentration of 0.2 g/L, and, as shown in Table 13, deletion of the NCg12335 (transposase) gene did not affect the production of homoserine . In particular, it was confirmed that upon expression of the wild-type yjeH gene, homoserine accumulated at a concentration of 0.2 g/L, and upon expression of variants of the yjeH gene, homoserine accumulated at concentrations of 1.2 g/L and 0.7 g/L.

Thus, based on the results presented above, it was confirmed that the wild type and yjeH variant genes as described in the present invention lead to a significant increase in the production of the amino acid of interest as a result of homoserine export from Corynebacterium glutamicum ATCC13032.

Example 5: Production of methionine (MET) using the conversion reaction of O-acetylhomoserine produced by the strain

After obtaining the pECCG117-metX WT vector in example 3-4, it was introduced into ATCC13032 ΔmetB ΔmetY lysC (L377K) ΔNCg12335, ATCC13032 ΔmetB ΔmetY lysC (L377K) ΔNCg12335::PCJ7-yjeH (eco, WT), ATCC 13032 ΔmetB ΔmetY lysC (L377K) ΔNCg12335::PCJ7-yjeH (eco, F92N) and ATCC13032 ΔmetB ΔmetY lysC (L377K) ΔNCg12335::PCJ7-yjeH (eco, F351L), obtained in example 4-1, using the electric pulse method, the strains were smeared onto a selective medium, containing (25 mg/l) kanamycin and cultivated until transformants were obtained.

In accordance with the method disclosed in a previously filed patent document (WO 2008/013432), the metZ gene encoding O-succinylhomoserine sulfhydrylase, originating from Pseudomonas aeruginosa and Pseudomonas putida, which is to be used to convert O-acetylhomoserine to methionine, was also cloned. PCR was performed using the chromosome of each strain as a template and primers to obtain the metZ gene from Pseudomonas aeruginosa and Pseudomonas putida as described below: 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 2 minutes to obtain DNA fragments. The resulting DNA fragments were digested with NdeI/PacI and cloned into the pCL-CJ1 vector (CJ, Korea), digested with the same enzymes. E. coli W3110 cells were transformed with the cloned vector and cultured in a Petri dish with LB medium containing 50 μg/L spectinomycin, and then colonies were selected. Selected colonies were inoculated with 3 ml of LB medium containing 50 μg/L spectinomycin and cultured overnight at 37°C. Cells were isolated from culture, washed with 0.1 M potassium phosphate buffer (pH 7.5), suspended in 200 μl potassium phosphate buffer, and lysed by sonication 5 times at 30-second intervals. The cell lysate was centrifuged at 12,000 rpm for 10 min, and the resulting supernatant was quantified for total protein levels using Bio-Rad Protein Quantification Solution (Bio-Rad, USA). Protein expression was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The supernatant obtained from the cell extract was used for the enzymatic conversion reaction.

The metZ gene, encoding O-succinylhomoserine sulfhydrylase, originating from Hyphomonas neptunium, was also cloned. PCR was performed using the Hyphomonas neptunium chromosome as template and primer in the same manner as described above to obtain DNA fragments. The resulting DNA fragments were digested with NdeI/AvrII and cloned into the pCL-CJ1 vector (CJ, Korea), digested with the same enzymes. The supernatant obtained using the cloned vector was used in the same manner as described above and used in the enzymatic conversion reaction.

Each of the strains W3110 transformed with the Pseudomonas-derived metZ expression vector and transformed with the Hyphomonas-derived metZ expression vector was inoculated into a plate of LB culture medium containing spectinomycin as an antibiotic and cultured overnight at 30°C. up to 40°C. A single colony was then inoculated with 40 ml of LB medium containing spectinomycin and cultured for 5 hours at 30°C to 40°C. Strains expressing metZ of Pseudomonas origin and metZ of Hypomononas origin were cultured in a 1 L fermentation vessel at 600-900 rpm and a temperature of 30°C to 40°C for 15-30 hours to obtain fermentation broths. The nutrient medium used for cultivation is described below. The cells of the resulting fermentation broth were lysed by sonication to obtain enzyme solutions.

Wednesday 2XYT

10 g yeast extract, 16 g tryptophan, 40 g glucose and 50 g spectinomycin (based on 1 liter of distilled water)

The conversion reaction to methionine was performed by adding a solution of the enzyme O-succinylhomoserine sulfhydrylase derived from Pseudomonas and Hyphomonas to the culture of the transformant producing O-acetylhomoserine. After adding 0.1 L of enzyme solution to 2.0 L of O-acetylhomoserine culture from which cells were not removed, 0.3 L of 15% sodium methyl mercaptan solution was added to initiate the reaction. After 2 hours, the fermentation broth was removed, the cells were removed from it, and the concentrations of the resulting methionine were identified by HPLC. The analyzed concentrations are presented in Table 14 below.

The above description of the present invention is presented for purposes of illustration, and it will be understood by those skilled in the art that various changes and modifications can be made without changing the technical concept and essential features of the present invention. Thus, it is clear that the embodiments described above are illustrative in all aspects and are not limiting of the present invention. Thus, the scope of this specification is determined not by the detailed description, but by the claims and their equivalents, as well as all variations within the scope of the claims, and their equivalents should be construed as included within the scope of the specification.

--->

LIST OF SEQUENCES

<110>CJ CheilJedang Corporation

<120> Modified inner membrane protein and methods of production

target product with its use

<130> OPA20205

<150> KR 10-2019-0172055

<151> 2019-12-20

<160> 37

<170> KoPatentIn 3.0

<210> 1

<211> 418

<212>PRT

<213> Unknown

<220>

<223> Escherichia coli YjeH

<400> 1

Met Ser Gly Leu Lys Gln Glu Leu Gly Leu Ala Gln Gly Ile Gly Leu

1 5 10 15

Leu Ser Thr Ser Leu Leu Gly Thr Gly Val Phe Ala Val Pro Ala Leu

20 25 30

Ala Ala Leu Val Ala Gly Asn Asn Ser Leu Trp Ala Trp Pro Val Leu

35 40 45

Ile Ile Leu Val Phe Pro Ile Ala Ile Val Phe Ala Ile Leu Gly Arg

50 55 60

His Tyr Pro Ser Ala Gly Gly Val Ala His Phe Val Gly Met Ala Phe

65 70 75 80

Gly Ser Arg Leu Glu Arg Val Thr Gly Trp Leu Phe Leu Ser Val Ile

85 90 95

Pro Val Gly Leu Pro Ala Ala Leu Gln Ile Ala Ala Gly Phe Gly Gln

100 105 110

Ala Met Phe Gly Trp His Ser Trp Gln Leu Leu Leu Ala Glu Leu Gly

115 120 125

Thr Leu Ala Leu Val Trp Tyr Ile Gly Thr Arg Gly Ala Ser Ser Ser

130 135 140

Ala Asn Leu Gln Thr Val Ile Ala Gly Leu Ile Val Ala Leu Ile Val

145 150 155 160

Ala Ile Trp Trp Ala Gly Asp Ile Lys Pro Ala Asn Ile Pro Phe Pro

165 170 175

Ala Pro Gly Asn Ile Glu Leu Thr Gly Leu Phe Ala Ala Leu Ser Val

180 185 190

Met Phe Trp Cys Phe Val Gly Leu Glu Ala Phe Ala His Leu Ala Ser

195 200 205

Glu Phe Lys Asn Pro Glu Arg Asp Phe Pro Arg Ala Leu Met Ile Gly

210 215 220

Leu Leu Leu Ala Gly Leu Val Tyr Trp Gly Cys Thr Val Val Val Leu

225 230 235 240

His Phe Asp Ala Tyr Gly Glu Lys Met Ala Ala Ala Ala Ser Leu Pro

245 250 255

Lys Ile Val Val Gln Leu Phe Gly Val Gly Ala Leu Trp Ile Ala Cys

260 265 270

Val Ile Gly Tyr Leu Ala Cys Phe Ala Ser Leu Asn Ile Tyr Ile Gln

275 280 285

Ser Phe Ala Arg Leu Val Trp Ser Gln Ala Gln His Asn Pro Asp His

290 295 300

Tyr Leu Ala Arg Leu Ser Ser Arg His Ile Pro Asn Asn Ala Leu Asn

305 310 315 320

Ala Val Leu Gly Cys Cys Val Val Ser Thr Leu Val Ile His Ala Leu

325 330 335

Glu Ile Asn Leu Asp Ala Leu Ile Ile Tyr Ala Asn Gly Ile Phe Ile

340 345 350

Met Ile Tyr Leu Leu Cys Met Leu Ala Gly Cys Lys Leu Leu Gln Gly

355 360 365

Arg Tyr Arg Leu Leu Ala Val Val Gly Gly Leu Leu Cys Val Leu Leu

370 375 380

Leu Ala Met Val Gly Trp Lys Ser Leu Tyr Ala Leu Ile Met Leu Ala

385 390 395 400

Gly Leu Trp Leu Leu Leu Pro Lys Arg Lys Thr Pro Glu Asn Gly Ile

405 410 415

Thr Thr

<210> 2

<211> 1257

<212> DNA

<213> Unknown

<220>

<223> Escherichia coli yjeH

<400> 2

atgagtggac tcaaacaaga actggggctg gccccagggca ttggcctgct atcgacgtca 60

ttattaggca ctggcgtgtt tgccgttcct gcgttagctg cgctggtagc gggcaataac 120

agcctgtggg cgtggcccgt tttgattatc ttagtgttcc cgattgcgat tgtgtttgcg 180

attctgggtc gccactatcc cagcgcaggc ggcgtcgcgc acttcgtcgg tatggcgttt 240

ggttcgcggc ttgagcgagt caccggctgg ctgtttttat cggtcattcc cgtgggtttg 300

cctgccgcac tacaaattgc cgccgggttc ggccaggcga tgtttggctg gcatagctgg 360

caactgttgt tggcagaact cggtacgctg gcgctggtgt ggtatatcgg tactcgcggt 420

gccagttcca gtgctaatct acaaaccgtt attgccggac ttatcgtcgc gctgattgtc 480

gctatctggt gggcgggcga tatcaaacct gcgaatatcc cctttccggc acctggtaat 540

atcgaactta ccgggttatt tgctgcgtta tcagtgatgt tctggtgttt tgtcggtctg 600

gaggcatttg cccatctcgc ctcggaattt aaaaatccag agcgtgattt tcctcgtgct 660

ttgatgattg gtctgctgct ggcaggatta gtctactggg gctgtacggt agtcgtctta 720

cacttcgacg cctatggtga aaaaatggcg gcggcagcat cgcttccaaa aattgtagtg 780

cagttgttcg gtgtaggagc gttatggatt gcctgcgtga ttggctatct ggcctgcttt 840

gccagtctca acatttatat acagagcttc gcccgcctgg tctggtcgca ggcgcaacat 900

aatcctgacc actacctggc acgcctctct tctcgccata tcccgaataa tgccctcaat 960

gcggtgctcg gctgctgtgt ggtgagcact ttggtgattc atgctttaga gatcaatctg 1020

gacgctctta ttatttatgc caatggcatc tttattatga tttatctgtt atgcatgctg 1080

gcaggctgta aattattgca aggacgttat cgactactgg cggtggttgg cgggctgtta 1140

tgcgttctgt tactggcaat ggtcggctgg aaaagtctct atgcgctgat catgctggcg 1200

gggttatggc tgttgctgcc aaaacgaaaa acgccggaaa atggcataac cacataa 1257

<210> 3

<211> 267

<212> DNA

<213> Artificial sequence

<220>

<223> Tn2335

<400> 3

gtggcctaca ccttcgacca cgtcgtcgcc tggcgctggt gcaccaaaga agacgcctac 60

aactacaccc acctcttcga tcaactccaa ccacccttaa tcgtgaccac cgacggacaa 120

aaaaggcgca ctcaaagcca tcaccacgac ctggccgaca acgaaaatcc aacgctgcct 180

cgtccacgtc aaacgcaacg tccaaaaaca cgtcacccta agacccgtgc tgagctcgcc 240

gaaaagcact ccggggtctc tccttga 267

<210> 4

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Tn_5 F

<400> 4

tgaattcgag ctcggtaccc caccgacgcg catctgcct 39

<210> 5

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> Tn_5 R

<400> 5

ggtgtggtga ctttcagcag ttcccggggg gggaggaggca tgtggtgttg 50

<210> 6

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> Tn_3 F

<400> 6

caacaccaca tgcctcctcc cccccgggaa ctgctgaaag tcaccacacc 50

<210> 7

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Tn_3 R

<400> 7

gtcgactcta gaggatcccc ctcccaaacc attgaggaat gg 42

<210> 8

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223>CJ7

<400> 8

agaaacatcc cagcgctact aatagggagc gttgaccttc cttccacgga ccggtaatcg 60

gagtgcctaa aaccgcatgc ggcttaggct ccaagatagg ttctgcgcgg ccgggtaatg 120

catcttcttt agcaacaagt tgaggggtag gtgcaaataa gaacgacata gaaatcgtct 180

cctttctgtt tttaatcaac atacaccacc acctaaaaat tccccgacca gcaagttcac 240

agtattcggg cacaatatcg ttgccaaaat attgtttcgg aatatcatgg gatacgtacc 300

caacgaaagg aaacactc 318

<210> 9

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> CJ7_yjeH F

<400> 9

acaccacatg cctcctcccc agaaacatcc cagcgctac 39

<210> 10

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> CJ7_yjeH R

<400> 10

agttcttgtt tgagtccact catagtgttt cctttcgttg ggt 43

<210> 11

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> yjeH F

<400> 11

acccaacgaa aggaaacact atgagtggac tcaaacaaga actg 44

<210> 12

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> yjeH R

<400> 12

gactttcagc agttcccggg ttatgtggtt atgccatttt ccgg 44

<210> 13

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223>F92N F

<400> 13

cggctggctg aatttatcgg tca 23

<210> 14

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223>F92N R

<400> 14

tgaccgataa attcagccag ccg 23

<210> 15

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223>F351L F

<400> 15

caatggcatc cttattatga ttt 23

<210> 16

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223>F351L R

<400> 16

aaatcataat aaggatgcca ttg 23

<210> 17

<211> 1161

<212> DNA

<213> Unknown

<220>

<223> Corynebacterium glutamicum metB

<400> 17

ttgtcttttg acccaaacac cccaggtttc tccactgcat cgattcacgc tgggtatgag 60

cgacgact actacggttc gattaacacc ccaatctatg cctccaccac cttcgcgcag 120

aacgctccaa acgaactgcg caaaggctac gagtacaccc gtgtgggcaa ccccaccatc 180

gtggcattag agcagaccgt cgcagcactc gaaggcgcaa agtatggccg cgcattctcc 240

tccggcatgg ctgcaaccga catcctgttc cgcatcatcc tcaagccggg cgatcacatc 300

gtcctcggca acgatgctta cggcggaacc taccgcctga tcgacaccgt attcaccgca 360

tggggcgtcg aatacaccgt tgttgatacc tccgtcgtgg aagaggtcaa ggcagcgatc 420

aaggacaaca ccaagctgat ctgggtggaa accccaacca acccagcact tggcatcacc 480

gacatcgaag cagtagcaaa gctcaccgaa ggcaccaacg ccaagctggt tgttgacaac 540

accttcgcat ccccatacct gcagcagcca ctaaaactcg gcgcacacgc agtcctgcac 600

tccaccacca agtacatcgg aggacactcc gacgttgttg gcggccttgt ggttaccaac 660

gaccaggaaa tggacgaaga actgctgttc atgcagggcg gcatcggacc gatcccatca 720

gttttcgatg catacctgac cgcccgtggc ctcaagaccc ttgcagtgcg catggatcgc 780

cactgcgaca acgcagaaaa gatcgcggaa ttcctggact cccgcccaga ggtctccacc 840

gtgctctacc caggtctgaa gaaccaccca ggccacgaag tcgcagcgaa gcagatgaag 900

cgcttcggcg gcatgatctc cgtccgtttc gcaggcggcg aagaagcagc taagaagttc 960

tgtacctcca ccaaactgat ctgtctggcc gagtccctcg gtggcgtgga atccctcctg 1020

gagcacccag caaccatgac ccaccagtca gctgccggct ctcagctcga ggttccccgc 1080

gacctcgtgc gcatctccat tggtattgaa gacattgaag acctgctcgc agatgtcgag 1140

caggccctca ataaccttta g 1161

<210> 18

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> metB_N_del F

<400> 18

tctagacgcc cgcatactgg cttc 24

<210> 19

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> metB_N_del R

<400> 19

cccatccact aaacttaaac agatgtgatc gcccggc 37

<210> 20

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> metB_C_del F

<400> 20

tgtttaagtt tagtggatgg ggaagaacca cccaggcc 38

<210> 21

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> metB_C_del R

<400> 21

gtcgaccaat cgtccagagg gcg 23

<210> 22

<211> 1314

<212> DNA

<213> Artificial sequence

<220>

<223> Corynebacterium glutamicum metY

<400> 22

atgccaaagt acgacaattc caatgctgac cagtggggct ttgaaacccg ctccattcac 60

gcaggccagt cagtagacgc acagaccagc gcacgaaacc ttccgatcta ccaatccacc 120

gctttcgtgt tcgactccgc tgagcacgcc aagcagcgtt tcgcacttga ggatctaggc 180

cctgtttact cccgcctcac caacccaacc gttgaggctt tggaaaaccg catcgcttcc 240

ctcgaaggtg gcgtccacgc tgtagcgttc tcctccggac aggccgcaac caccaacgcc 300

attttgaacc tggcaggagc gggcgaccac atcgtcacct ccccacgcct ctacggtggc 360

accgagactc tattccttat cactcttaac cgcctgggta tcgatgtttc cttcgtggaa 420

aaccccgacg accctgagtc ctggcaggca gccgttcagc caaacaccaa agcattcttc 480

ggcgagactt tcgccaaccc acaggcagac gtcctggata ttcctgcggt ggctgaagtt 540

gcgcaccgca acagcgttcc actgatcatc gacaacacca tcgctaccgc agcgctcgtg 600

cgcccgctcg agctcggcgc agacgttgtc gtcgcttccc tcaccaagtt ctacaccggc 660

aacggctccg gactgggcgg cgtgcttatc gacggcggaa agttcgattg gactgtcgaa 720

aaggatggaa agccagtatt cccctacttc gtcactccag atgctgctta ccacggattg 780

aagtacgcag accttggtgc accagccttc ggcctcaagg ttcgcgttgg ccttctacgc 840

gacaccggct ccaccctctc cgcattcaac gcatgggctg cagtccaggg catcgacacc 900

ctttccctgc gcctggagcg ccacaacgaa aacgccatca aggttgcaga attcctcaac 960

aaccacgaga aggtggaaaa ggttaacttc gcaggcctga aggattcccc ttggtacgca 1020

accaaggaaa agcttggcct gaagtacacc ggctccgttc tcaccttcga gatcaagggc 1080

ggcaaggatg aggcttgggc atttatcgac gccctgaagc tacactccaa ccttgcaaac 1140

atcggcgatg ttcgctccct cgttgttcac ccagcaacca ccacccattc acagtccgac 1200

gaagctggcc tggcacgcgc gggcgttacc cagtccaccg tccgcctgtc cgttggcatc 1260

gagaaccattg atgatatcat cgctgacctc gaaggcggct ttgctgcaat ctag 1314

<210> 23

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> metY_N_del F

<400> 23

tctagaccat cctgcaccat ttag 24

<210> 24

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> metY_N_del R

<400> 24

cccatccact aaacttaaac acgctcctgc caggttc 37

<210> 25

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> metY_C_del F

<400> 25

tgtttaagtt tagtggatgg gcttggtacg caaccaagg 39

<210> 26

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> metY_C_del R

<400> 26

gtcgacgatt gctccggctt cgg 23

<210> 27

<211> 1266

<212> DNA

<213> Unknown

<220>

<223> Corynebacterium glutamicum lysC

<400> 27

gtggccctgg tcgtacagaa atatggcggt tcctcgcttg agagtgcgga acgcattaga 60

aacgtcgctg aacggatcgt tgccaccaag aaggctggaa atgatgtcgt ggttgtctgc 120

tccgcaatgg gagacaccac ggatgaactt ctagaacttg cagcggcagt gaatcccgtt 180

ccgccagctc gtgaaatgga tatgctcctg actgctggtg agcgtatttc taacgctctc 240

gtcgccatgg ctattgagtc ccttggcgca gaagcccaat ctttcacggg ctctcaggct 300

ggtgtgctca ccaccgagcg ccacggaaac gcacgcattg ttgatgtcac tccaggtcgt 360

gtgcgtgaag cactcgatga gggcaagatc tgcattgttg ctggtttcca gggtgttaat 420

aaagaaaccc gcgatgtcac cacgttgggt cgtggtggtt ctgacaccac tgcagttgcg 480

ttggcagctg ctttgaacgc tgatgtgtgt gagatttact cggacgttga cggtgtgtat 540

accgctgacc cgcgcatcgt tcctaatgca cagaagctgg aaaagctcag cttcgaagaa 600

atgctggaac ttgctgctgt tggctccaag attttggtgc tgcgcagtgt tgaatacgct 660

cgtgcattca atgtgccact tcgcgtacgc tcgtcttata gtaatgatcc cggcactttg 720

attgccggct ctatggagga tattcctgtg gaagaagcag tccttaccgg tgtcgcaacc 780

gacaagtccg aagccaaagt aaccgttctg ggtatttccg ataagccagg cgaggctgcg 840

aaggttttcc gtgcgttggc tgatgcagaa atcaacattg acatggttct gcagaacgtc 900

tcttctgtag aagacggcac caccgacatc accttcacct gccctcgttc cgacggccgc 960

cgcgcgatgg agatcttgaa gaagcttcag gttcagggca actggaccaa tgtgctttac 1020

gacgaccagg tcggcaaagt ctccctcgtg ggtgctggca tgaagtctca cccaggtgtt 1080

accgcagagt tcatggaagc tctgcgcgat gtcaacgtga acatcgaatt gatttccacc 1140

tctgagattc gtatttccgt gctgatccgt gaagatgatc tggatgctgc tgcacgtgca 1200

ttgcatgagc agttccagct gggcggcgaa gacgaagccg tcgtttatgc aggcaccgga 1260

cgctaa 1266

<210> 28

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> lysC_L377K_5 F

<400> 28

tcctctagag ctgcgcagtg ttgaatacg 29

<210> 29

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> lysC_L377K_5 R

<400> 29

aggtggaaat cttttcgatg ttc 23

<210> 30

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> lysC_L377K_3 F

<400> 30

gaacatcgaa aagatttcca cct 23

<210> 31

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> lysC_L377K_3 R

<400> 31

gactctagag ttcacctcag agacgatta 29

<210> 32

<211> 1134

<212> DNA

<213> Artificial sequence

<220>

<223> Corynebacterium glutamicum metX

<400> 32

atgcccaccc tcgcgccttc aggtcaactt gaaatccaag cgatcggtga tgtctccacc 60

gaagccggag caatcattac aaacgctgaa atcgcctatc accgctgggg tgaataccgc 120

gtagataaag aaggacgcag caatgtcgtt ctcatcgaac acgccctcac tggagattcc 180

aacgcagccg attggtgggc tgacttgctc ggtcccggca aagccatcaa cactgatatt 240

tactgcgtga tctgtaccaa cgtcatcggt ggttgcaacg gttccaccgg acctggctcc 300

atgcatccag atggaaattt ctggggtaat cgcttccccg ccacgtccat tcgtgatcag 360

gtaaacgccg aaaaacaatt cctcgacgca ctcggcatca ccacggtcgc cgcagtactt 420

ggtggttcca tgggtggtgc ccgcacccta gagtgggccg caatgtaccc agaaactgtt 480

ggcgcagctg ctgttcttgc agtttctgca cgcgccagcg cctggcaaat cggcattcaa 540

tccgcccaaa ttaaggcgat tgaaaacgac caccactggc acgaaggcaa ctactacgaa 600

tccggctgca acccagccac cggactcggc gccgcccgac gcatcgccca cctcacctac 660

cgtggcgaac tagaaatcga cgaacgcttc ggcaccaaag cccaaaagaa cgaaaaccca 720

ctcggtccct accgcaagcc cgaccagcgc ttcgccgtgg aatcctactt ggactaccaa 780

gcagacaagc tagtacagcg tttcgacgcc ggctcctacg tcttgctcac cgacgccctc 840

aaccgccacg acattggtcg cgaccgcgga ggcctcaaca aggcactcga atccatcaaa 900

gttccagtcc ttgtcgcagg cgtagatacc gatattttgt acccctacca ccagcaagaa 960

cacctctcca gaaacctggg aaatctactg gcaatggcaa aaatcgtatc ccctgtcggc 1020

cacgatgctt tcctcaccga aagccgccaa atggatcgca tcgtgaggaa cttcttcagc 1080

ctcatctccc cagacgaaga caacccttcg acctacatcg agttctacat ctaa 1134

<210> 33

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> metX F

<400> 33

ggatcccctc gttgttcacc cagcaacc 28

<210> 34

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> metX R

<400> 34

ggatcccaaa gtcacaacta cttatgttag 30

<210> 35

<211> 418

<212>PRT

<213> Artificial sequence

<220>

<223> YjeH F92N

<400> 35

Met Ser Gly Leu Lys Gln Glu Leu Gly Leu Ala Gln Gly Ile Gly Leu

1 5 10 15

Leu Ser Thr Ser Leu Leu Gly Thr Gly Val Phe Ala Val Pro Ala Leu

20 25 30

Ala Ala Leu Val Ala Gly Asn Asn Ser Leu Trp Ala Trp Pro Val Leu

35 40 45

Ile Ile Leu Val Phe Pro Ile Ala Ile Val Phe Ala Ile Leu Gly Arg

50 55 60

His Tyr Pro Ser Ala Gly Gly Val Ala His Phe Val Gly Met Ala Phe

65 70 75 80

Gly Ser Arg Leu Glu Arg Val Thr Gly Trp Leu Asn Leu Ser Val Ile

85 90 95

Pro Val Gly Leu Pro Ala Ala Leu Gln Ile Ala Ala Gly Phe Gly Gln

100 105 110

Ala Met Phe Gly Trp His Ser Trp Gln Leu Leu Leu Ala Glu Leu Gly

115 120 125

Thr Leu Ala Leu Val Trp Tyr Ile Gly Thr Arg Gly Ala Ser Ser Ser

130 135 140

Ala Asn Leu Gln Thr Val Ile Ala Gly Leu Ile Val Ala Leu Ile Val

145 150 155 160

Ala Ile Trp Trp Ala Gly Asp Ile Lys Pro Ala Asn Ile Pro Phe Pro

165 170 175

Ala Pro Gly Asn Ile Glu Leu Thr Gly Leu Phe Ala Ala Leu Ser Val

180 185 190

Met Phe Trp Cys Phe Val Gly Leu Glu Ala Phe Ala His Leu Ala Ser

195 200 205

Glu Phe Lys Asn Pro Glu Arg Asp Phe Pro Arg Ala Leu Met Ile Gly

210 215 220

Leu Leu Leu Ala Gly Leu Val Tyr Trp Gly Cys Thr Val Val Val Leu

225 230 235 240

His Phe Asp Ala Tyr Gly Glu Lys Met Ala Ala Ala Ala Ser Leu Pro

245 250 255

Lys Ile Val Val Gln Leu Phe Gly Val Gly Ala Leu Trp Ile Ala Cys

260 265 270

Val Ile Gly Tyr Leu Ala Cys Phe Ala Ser Leu Asn Ile Tyr Ile Gln

275 280 285

Ser Phe Ala Arg Leu Val Trp Ser Gln Ala Gln His Asn Pro Asp His

290 295 300

Tyr Leu Ala Arg Leu Ser Ser Arg His Ile Pro Asn Asn Ala Leu Asn

305 310 315 320

Ala Val Leu Gly Cys Cys Val Val Ser Thr Leu Val Ile His Ala Leu

325 330 335

Glu Ile Asn Leu Asp Ala Leu Ile Ile Tyr Ala Asn Gly Ile Phe Ile

340 345 350

Met Ile Tyr Leu Leu Cys Met Leu Ala Gly Cys Lys Leu Leu Gln Gly

355 360 365

Arg Tyr Arg Leu Leu Ala Val Val Gly Gly Leu Leu Cys Val Leu Leu

370 375 380

Leu Ala Met Val Gly Trp Lys Ser Leu Tyr Ala Leu Ile Met Leu Ala

385 390 395 400

Gly Leu Trp Leu Leu Leu Pro Lys Arg Lys Thr Pro Glu Asn Gly Ile

405 410 415

Thr Thr

<210> 36

<211> 418

<212>PRT

<213> Artificial sequence

<220>

<223> YjeH F351L

<400> 36

Met Ser Gly Leu Lys Gln Glu Leu Gly Leu Ala Gln Gly Ile Gly Leu

1 5 10 15

Leu Ser Thr Ser Leu Leu Gly Thr Gly Val Phe Ala Val Pro Ala Leu

20 25 30

Ala Ala Leu Val Ala Gly Asn Asn Ser Leu Trp Ala Trp Pro Val Leu

35 40 45

Ile Ile Leu Val Phe Pro Ile Ala Ile Val Phe Ala Ile Leu Gly Arg

50 55 60

His Tyr Pro Ser Ala Gly Gly Val Ala His Phe Val Gly Met Ala Phe

65 70 75 80

Gly Ser Arg Leu Glu Arg Val Thr Gly Trp Leu Phe Leu Ser Val Ile

85 90 95

Pro Val Gly Leu Pro Ala Ala Leu Gln Ile Ala Ala Gly Phe Gly Gln

100 105 110

Ala Met Phe Gly Trp His Ser Trp Gln Leu Leu Leu Ala Glu Leu Gly

115 120 125

Thr Leu Ala Leu Val Trp Tyr Ile Gly Thr Arg Gly Ala Ser Ser Ser

130 135 140

Ala Asn Leu Gln Thr Val Ile Ala Gly Leu Ile Val Ala Leu Ile Val

145 150 155 160

Ala Ile Trp Trp Ala Gly Asp Ile Lys Pro Ala Asn Ile Pro Phe Pro

165 170 175

Ala Pro Gly Asn Ile Glu Leu Thr Gly Leu Phe Ala Ala Leu Ser Val

180 185 190

Met Phe Trp Cys Phe Val Gly Leu Glu Ala Phe Ala His Leu Ala Ser

195 200 205

Glu Phe Lys Asn Pro Glu Arg Asp Phe Pro Arg Ala Leu Met Ile Gly

210 215 220

Leu Leu Leu Ala Gly Leu Val Tyr Trp Gly Cys Thr Val Val Val Leu

225 230 235 240

His Phe Asp Ala Tyr Gly Glu Lys Met Ala Ala Ala Ala Ser Leu Pro

245 250 255

Lys Ile Val Val Gln Leu Phe Gly Val Gly Ala Leu Trp Ile Ala Cys

260 265 270

Val Ile Gly Tyr Leu Ala Cys Phe Ala Ser Leu Asn Ile Tyr Ile Gln

275 280 285

Ser Phe Ala Arg Leu Val Trp Ser Gln Ala Gln His Asn Pro Asp His

290 295 300

Tyr Leu Ala Arg Leu Ser Ser Arg His Ile Pro Asn Asn Ala Leu Asn

305 310 315 320

Ala Val Leu Gly Cys Cys Val Val Ser Thr Leu Val Ile His Ala Leu

325 330 335

Glu Ile Asn Leu Asp Ala Leu Ile Ile Tyr Ala Asn Gly Ile Leu Ile

340 345 350

Met Ile Tyr Leu Leu Cys Met Leu Ala Gly Cys Lys Leu Leu Gln Gly

355 360 365

Arg Tyr Arg Leu Leu Ala Val Val Gly Gly Leu Leu Cys Val Leu Leu

370 375 380

Leu Ala Met Val Gly Trp Lys Ser Leu Tyr Ala Leu Ile Met Leu Ala

385 390 395 400

Gly Leu Trp Leu Leu Leu Pro Lys Arg Lys Thr Pro Glu Asn Gly Ile

405 410 415

Thr Thr

<210> 37

<211> 5803

<212> DNA

<213> Artificial sequence

<220>

<223> pDCM2

<400> 37

gttcgcttgc tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag 60

ctacctgctt tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat 120

tcatccgggg tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc 180

agcccttgcg ccctgagtgc ttgcggcagc gtgaagctag cttttatcgc cattcgccat 240

tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc 300

tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt 360

cacgacgttg taaaacgacg gccagtgaat tcgagctcgg tacccgggga tcctctagag 420

tcgacctgca ggcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 480

tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 540

ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 600

tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 660

ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 720

ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 780

gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 840

gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 900

cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 960

ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 1020

tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg 1080

gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 1140

tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 1200

ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 1260

ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 1320

ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 1380

accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 1440

tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 1500

cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttgggg 1560

tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc ctgatagaaa 1620

cagaagccac tggagcacct caaaaacacc atcatacact aaatcagtaa gttggcagca 1680

tcacccgacg cactttgcgc cgaataaata cctgtgacgg aagatcactt cgcagaataa 1740

ataaatcctg gtgtccctgt tgataccggg aagccctggg ccaacttttg gcgaaaatga 1800

gacgttgatc ggcacgtaag aggttccaac tttcaccata atgaaataag atcactaccg 1860

ggcgtatttt ttgagttatc gagatttca ggagctgata gaaacagaag ccactggagc 1920

acctcaaaaa caccatcata cactaaatca gtaagttggc agcatcaccc gacgcacttt 1980

gcgccgaata aatacctgtg acggaagatc acttcgcaga ataaataaat cctggtgtcc 2040

ctgttgatac cgggaagccc tgggccaact tttggcgaaa atgagacgtt gatcggcacg 2100

taagaggttc caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt 2160

tatcgagatt ttcaggagct ctttggcatc gtctctcgcc tgtcccctca gttcagtaat 2220

ttcctgcatt tgcctgtttc cagtcggtag atattccaca aaacagcagg gaagcagcgc 2280

ttttccgctg cataaccctg cttcggggtc attatagcga ttttttcggt atatccatcc 2340

tttttcgcac gatatacagg attttgccaa agggttcgtg tagactttcc ttggtgtatc 2400

caacggcgtc agccgggcag gataggtgaa gtaggcccac ccgcgagcgg gtgttccttc 2460

ttcactgtcc cttattcgca cctggcggtg ctcaacggga atcctgctct gcgaggctgg 2520

ccggctaccg ccggcgtaac agatgagggc aagcggatgg ctgatgaaac caagccaacc 2580

aggaagggca gcccacctat caaggtgtac tgccttccag acgaacgaag agcgattgag 2640

gaaaaggcgg cggcggccgg catgagcctg tcggcctacc tgctggccgt cggccagggc 2700

tacaaaatca cgggcgtcgt ggactatgag cacgtccgcg agggcgtccc ggaaaacgat 2760

tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt 2820

gggcgtcgct tggtcggtca tttcgaaaaa ggttaggaat acggttagcc atttgcctgc 2880

ttttatatag ttcantatgg gattcacctt tatgttgata agaaataaaa gaaaatgcca 2940

ataggatatc ggcattttct tttgcgtttt tatttgttaa ctgttaattg tccttgttca 3000

aggatgctgt ctttgacaac agatgttttc ttgcctttga tgttcagcag gaagctcggc 3060

gcaaacgttg attgtttgtc tgcgtagaat cctctgtttg tcatatagct tgtaatcacg 3120

acattgtttc ctttcgcttg aggtacagcg aagtgtgagt aagtaaaggt tacatcgtta 3180

ggcggatcaa gatccatttt taacacaagg ccagttttgt tcagcggctt gtatgggcca 3240

gttaaagaat tagaaacata accaagcatg taaatatcgt tagacgtaat gccgtcaatc 3300

gtcatttttg atccgcggga gtcagtgaac aggtaccatt tgccgttcat tttaaagacg 3360

ttcgcgcgtt caatttcatc tgttactgtg ttagatgcaa tcagcggttt catcactttt 3420

ttcagtgtgt aatcatcgtt tagctcaatc ataccgagag cgccgtttgc taactcagcc 3480

gtgcgttttt tatcgctttg cagaagtttt tgactttctt gacggaagaa tgatgtgctt 3540

ttgccatagt atgctttgtt aaataaagat tcttcgcctt ggtagccatc ttcagttcca 3600

gtgtttgctt caaatactaa gtatttgtgg cctttatctt ctacgtagtg aggatctctc 3660

agcgtatggt tgtcgcctga gctgtagttg ccttcatcga tgaactgctg tacattttga 3720

tacgtttttc cgtcaccgtc aaagattgat ttataatcct ctacaccgtt gatgttcaaa 3780

gagctgtctg atgctgatac gttaacttgt gcagttgtca gtgtttgttt gccgtaatgt 3840

ttaccggaga aatcagtgta gaataaacgg atttttccgt cagatgtaaa tgtggctgaa 3900

cctgaccatt cttgtgtttg gtcttttagg atagaatcat ttgcatcgaa tttgtcgctg 3960

tctttaaaga cgcggccagc gtttttccag ctgtcaatag aagtttcgcc gactttttga 4020

tagaacatgt aaatcgatgt gtcatccgca tttttaggat ctccggctaa tgcaaagacg 4080

atgtggtagc cgtgatagtt tgcgacagtg ccgtcagcgt tttgtaatgg ccagctgtcc 4140

caaacgtcca ggccttttgc agaagata tttttaattg tggacgaatc aaattcagaa 4200

acttgatatt tttcattttt ttgctgttca gggatttgca gcatatcatg gcgtgtaata 4260

tgggaaatgc cgtatgtttc cttatatggc ttttggttcg tttctttcgc aaacgcttga 4320

gttgcgcctc ctgccagcag tgcggtagta aaggttaata ctgttgcttg ttttgcaaac 4380

tttttgatgt tcatcgttca tgtctccttt tttatgtact gtgttagcgg tctgcttctt 4440

ccagccctcc tgtttgaaga tggcaagtta gttacgcaca ataaaaaaag acctaaaata 4500

tgtaaggggt gacgccaaag tatacacttt gccctttaca cattttaggt cttgcctgct 4560

ttatcagtaa caaacccgcg cgatttactt ttcgacctca ttctattaga ctctcgtttg 4620

gattgcaact ggtctatttt cctcttttgt ttgatagaaa atcataaaag gatttgcaga 4680

ctacgggcct aaagaactaa aaaatctatc tgtttctttt cattctctgt attttttata 4740

gtttctgttg catgggcata aagttgcctt tttaatcaca attcagaaaa tatcataata 4800

tctcatttca ctaaataata gtgaacggca ggtatatgtg atgggttaaa aaggatcacc 4860

cccagtccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc 4920

gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc 4980

agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc 5040

acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc 5100

gccatgggtc acgacgagat cctcgccgtc gggcatccgc gccttgagcc tggcgaacag 5160

ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc 5220

ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt 5280

agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc 5340

aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc 5400

ccttcccgct tcagtgacaa cgtcgagaca gctgcgcaag gaacgcccgt cgtggccagc 5460

cacgatagcc gcgctgcctc gtcttggagt tcattcaggg caccggacag gtcggtcttg 5520

acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg 5580

attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct 5640

gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcagat 5700

cttgatcccc tgcgccatca gatccttggc ggcaagaaag ccatccagtt tactttgcag 5760

ggcttcccaa ccttaccaga gggcgcccca gctggcaatt ccg 5803

<---

Claims (17)

1. A protein having the activity of the inner membrane YjeH protein, comprising i) replacement of the amino acid corresponding to ^position 92 with asparagine, ii) replacement of the amino acid corresponding to position ³⁵¹ with leucine, or iii) replacement at both positions i) and ii) in the amino acid sequence of SEQ ID NO: 1, and having 95% or more but less than 100% homology with SEQ ID NO: 1. 2. Protein according to claim 1, having the ability to export O-acetylhomoserine or homoserine. 3. Polynucleotide encoding the protein according to claim 1. 4. An expression vector containing a polynucleotide according to claim 3. 5. A microorganism for producing O-acetylhomoserine, homoserine, methionine or glufosinate, containing at least any one of: a protein comprising i) replacement of the amino acid corresponding to ^position 92 with asparagine, ii) replacement of the amino acid corresponding to position ³⁵¹ with leucine, or iii) a substitution at both positions i) and ii) in the amino acid sequence of SEQ ID NO: 1, and having 95% or more but less than 100% homology with SEQ ID NO: 1; and a polynucleotide encoding this protein. 6. The microorganism according to claim 5, which has an enhanced ability to produce O-acetylhomoserine or homoserine compared to an unmodified microorganism. 7. The microorganism according to claim 5, which belongs to the genus Corynebacterium. 8. A method for obtaining the target product, including cultivating a microorganism in a culture medium, where the microorganism contains at least any one of: a protein comprising i) replacement of the amino acid corresponding to the ^92nd position with asparagine, ii) replacement of the amino acid corresponding to ^{the 351st} position with leucine , or iii) a substitution at both positions i) and ii) in the amino acid sequence of SEQ ID NO: 1, and having 95% or more but less than 100% homology with SEQ ID NO: 1; and a polynucleotide encoding the protein, wherein the target product is O-acetyl homoserine or homoserine. 9. The method according to claim 8, further comprising isolating the target product from the culture medium or microorganism. 10. A method for producing methionine, including: obtaining O-acetylhomoserine by cultivating a microorganism in a culture medium wherein the microorganism contains at least any one of: a protein comprising i) replacing the amino acid corresponding to ^position 92 with asparagine, ii) replacing the amino acid corresponding to position ³⁵¹ with leucine, or iii ) a substitution at both positions i) and ii) in the amino acid sequence of SEQ ID NO: 1, and having 95% or more but less than 100% homology with SEQ ID NO: 1; and a polynucleotide encoding this variant; and the conversion of O-acetylhomoserine to methionine by reacting O-acetylhomoserine with sulfide. 11. A method for producing glufosinate, including: obtaining O-acetylhomoserine or homoserine by cultivating the microorganism in a culture medium, where the microorganism contains at least any one of: a protein comprising i) replacement of the amino acid corresponding to position 92 ^with asparagine, ii) replacement of the amino acid corresponding to position ³⁵¹ with leucine, or iii) a substitution at both positions i) and ii) in the amino acid sequence of SEQ ID NO: 1, and having 95% or more but less than 100% homology with SEQ ID NO: 1; and a polynucleotide encoding this protein; and the conversion of O-acetylhomoserine or homoserine to glufosinate. 12. Composition for producing homoserine, containing a microorganism according to claim 5 or a culture of a microorganism and an excipient. 13. Composition for producing O-acetylhomoserine, containing a microorganism according to claim 5 or a microorganism culture and an excipient. 14. Use of the protein according to claim 1 to obtain O-acetylhomoserine or homoserine. 15. Use of a microorganism according to claim 5 to obtain O-acetylhomoserine or homoserine.