Specification
Title of the invention: L-methionine-producing microorganism into which protein encoded by exogenous metZ gene is introduced and L-methionine production method using the same
technical field
[One]
The present application relates to an L-methionine-producing microorganism into which a protein encoded by a foreign metZ gene is introduced, and a method for producing L-methionine using the same.
[2]
background
[3]
L-methionine (L-methionine) is one of the essential amino acids in the body, and is used as a raw material for pharmaceuticals, such as feed, infusions, and synthetic raw materials for pharmaceuticals, and as a food additive. Methionine is an important amino acid involved in the methyl group transfer reaction in vivo, and serves to provide sulfur.
[4]
The chemical synthesis of methionine mainly involves hydrolysis of 5-(β-methylmercaptoethyl)-hydantoin to form a mixture of L- and D-forms. Methods for producing methionine are being used. However, in the chemical synthesis, L-form and D-form are produced in a mixed form.
[5]
Meanwhile, L-methionine can be produced using a biological method. More specifically, one of the methods for the production of L-methionine by microorganisms is direct sulfhydration using O-acyl homoserine (O-acetyl homoserine or O-succinyl homoserine) and hydrogen sulfide as substrates. to produce methionine. For example, it is known that an enzyme encoded by a metY gene in a coryneform microorganism performs a direct sulfhydrylation function. Another method for microorganisms to produce L-methionine is to produce methionine by transsulfuration using O-acyl homoserine (O-acetyl homoserine or O-succinyl homoserine) and cysteine ​​as substrates. For example, it is known that an enzyme encoded by the metB gene in a coryneform microorganism performs a transsulfuration function. However, the enzyme encoded by metB produces a lot of byproducts and the metY gene has disadvantages that feedback is inhibited, making it difficult to industrially apply it to mass production of L-methionine (Kromer JO et al., J Bacteriol 188(2):609-618, 2006, Yeom HJ et al., J Microbiol Biotechnol 14(2): 373-378, 2004 et al.).
[6]
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[7]
The present inventors completed the present application by confirming that the microorganism introduced with the protein encoded by the metZ gene produces L-methionine in high yield after earnest efforts to discover a protein that can replace the protein.
[8]
means of solving the problem
[9]
An object of the present application is to provide an L-methionine-producing microorganism into which a protein encoded by a foreign metZ gene is introduced.
[10]
Another object of the present application is to provide a method for producing L-methionine comprising culturing the microorganism in a medium containing thiosulfate.
[11]
Another object of the present application is to provide a composition for producing L-methionine comprising the microorganism and thiosulfate.
[12]
Effects of the Invention
[13]
The microorganism into which the protein activity encoded by metZ of the present application has been introduced produces fewer by-products compared to metY and has a high yield, and unlike metB, which is feedback-inhibited by methionine, does not receive feedback inhibition, so L-methionine in high yield can be produced, so it can be usefully used in the industrial production of L-methionine.
[14]
Brief description of the drawing
[15]
1 is a schematic diagram of a pDCM2 plasmid.
[16]
Best mode for carrying out the invention
[17]
This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the specific descriptions described below.
[18]
In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present application described herein. Also, such equivalents are intended to be covered by this application.
[19]
[20]
One aspect of the present application provides an L-methionine-producing microorganism into which a protein encoded by a foreign metZ gene is introduced.
[21]
Another aspect of the present application provides a method for producing L-methionine, comprising culturing the L-methionine-producing microorganism in a medium containing thiosulfate.
[22]
The term 'metZ gene' in the present application is a gene encoding an enzyme involved in sulfhydration of acylhomoserine as a substrate.
[23]
In the present application, "acylhomoserine" refers to a compound in which an acyl group is bonded to homoserine, and includes both succinylhomoserine and acetylhomoserine. For example, the acylhomoserine may be O-succinylhomoserine or O-acetylhomoserine, but is not limited thereto.
[24]
In the present application, the enzyme encoded by the metZ gene is succinylhomoserine sulfhydrylase, acetylhomoserine sulfhydrylase, or O-succinylhomoserine as a substrate for sulfhydration It may be a gene known to encode an enzyme involved in, but is not limited thereto.
[25]
In the present application, the term "sulfhydration" is used interchangeably with the term "sulfhydrylation", and refers to a reaction in which a sulfhydryl (-SH) functional group is provided to a specific molecule. The term may mean a reaction in the methionine synthesis process for the purpose of the present application, but is not limited thereto. The enzyme involved in the "sulfhydration" may be referred to as "sulfhydrylase", but is not limited thereto.
[26]
[27]
Conventionally, in the methionine fermentation reaction, the enzyme expressed by the metZ gene was used in vitro for the following reactions:
[28]
CH 3SH + O-acetyl-L-homoserine => acetate + methionine
[29]
CH 3SH + O-succinyl-L-homoserine => succinate + methionine
[30]
[31]
That is, the first step of preparing a methionine precursor using a microorganism; and a two-step reaction of performing an in vitro enzymatic reaction by adding methylmercaptan and a methionine converting enzyme to a fermentation broth containing a methionine precursor, wherein the enzyme expressed by the metZ gene is methionine in vitro. used as a converting enzyme (see US 2010-0184164 A1)
[32]
On the other hand, methionine fermentation in microorganisms of the genus Corynebacterium uses two types of sulfhydrylation steps. (Hwang BJ et al., J Bacteriol 184(5):1277-1286, 2002) One uses an enzyme encoded by the gene called metB to convert O-acetyl homoserine (AH) to cystathionine ( cystationine), in which case cysteine ​​is used as a sulfur source. That is, the reaction of converting acylhomoserine and cysteine ​​into cystathionine as reactants is called "transsulfuration", and the enzyme involved in this is also called "transsulfurase". The other is to convert O-acetyl homoserine to homocysteine ​​using an enzyme encoded by the gene called metY. In this case, an inorganic sulfur compound such as hydrogen sulfide is used as a sulfur source. do. Unlike the above-described transsulfuration, the reaction of converting acylhomoserine and hydrogen sulfide to homocysteine ​​as a reactant does not generate cystathionine, an intermediate product, in the process of generating homocysteine, a precursor of methionine, so it is a direct This is called direct sulfhydrylation.
[33]
That is, the sulfhydration pathway may refer to a reaction pathway for conversion to another material through the reaction of acylhomoserine and a sulfur source, and can be largely divided into transsulfuration and direct sulfhydration.
[34]
However, both enzymes involved in the sulfhydration in Corynebacterium strains have disadvantages. For example, the protein encoded by the metB gene uses acetylhomoserine and homocysteine ​​in addition to cystathionine to produce a byproduct called homolanthionine (Kromer JO et al., J Bacteriol 188(2): 609-618, 2006) Also, it is known that the metY gene is highly subjected to feedback inhibition by methionine (Yeom HJ et al., J Microbiol Biotechnol 14(2): 373-378, 2004).
[35]
[36]
[37]
The present application is characterized in that the introduction of the exogenous metZ gene into a Corynebacterium strain and the introduction of the metZ gene can be usefully used for methionine fermentation to biologically produce methionine only in a single-step reaction.
[38]
In the methionine synthesis pathway involving the protein encoded by the metZ gene of the present application, the amount of by-products may be reduced. The by-product may be homolanthionine. The reduction in the amount of by-products may mean, but is not limited to, compared to wild-type microorganisms or reduced compared to the amount of by-products produced in a synthetic pathway involving a protein encoded by metB.
[39]
Accordingly, the microorganism into which the exogenous metZ gene has been introduced and the methionine production method comprising culturing the microorganism of the present application include the methionine-producing microorganism into which the foreign metZ gene is not introduced and the methionine production method using the sameIt may be that the amount of by-products produced is reduced compared to that. The protein encoded by the metZ gene of the present application may be one that does not receive feedback inhibition by methionine.
[40]
The protein encoded by the metZ gene of the present application is O-acylhomoserine sulfhydrylase, and not only can hydrogen sulfide be used as a sulfur source, but also O-acylhomoserine transsulfure As an acylhomoserine transsulfurase, it may be one that can use cysteine ​​as a sulfur source. More specifically, the protein is O-acetylhomoserine sulfhydrylase, O-acetylhomoserine transsulfurase, O-succinylhomoserine sulfhydrylase or O-succinylhomoserine transsulfuraseyl can Therefore, in the present application, the protein encoded by the metZ gene may be a protein having O-acylhomoserine sulfhydrylase activity, and specifically, O-acetylhomoserine sulfhydrylase, O-acetylhomoserine transsul It may be a protein having the activity of at least one of purase, O-succinylhomoserine sulfhydrylase, and O-succinylhomoserine transsulfurase.
[41]
[42]
For example, the exogenous metZ gene of the present application may be a gene derived from a different origin from the L-methionine-producing microorganism into which the gene is introduced, or may be different from a gene inherently present in the L-methionine-producing microorganism into which the gene is introduced. have. Specifically, the gene may be a gene named metZ derived from Chromobacterium violaceum, Hyphomonas neptunium or Rhodobacter sphaeroides, but is not limited thereto. And, for the purpose of the present application, the gene is not limited as long as it can enhance L-methionine production ability, and any one may be included. The sequence of the metZ gene can be obtained from GenBank of NCBI, which is a known database, and various methods well known in the art can be applied to obtain the corresponding sequence.
[43]
In the present application, the protein encoded by the exogenous metZ is any one or more amino acid sequence (polypeptide sequence), but is not limited thereto. For example, the protein comprises a polypeptide sequence of any one of SEQ ID NOs: 60, 61 and 62 and 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 97.7%, 97.8% , 98%, 98.5%, 98.7%, 98.8%, 99%, 99.5%, 99.7%, 99.8% or more may include a polypeptide sequence having less than 100% homology or identity, for example, SEQ ID NOs: 66 to It may include, but is not limited to, a sequence selected from the polypeptide sequence of any one of 71 and a polypeptide sequence having 90% or more homology or identity thereto.
[44]
The metZ gene of the present application may include a polynucleotide sequence having 90% or more homology or identity to any one or more polynucleotide sequences selected from SEQ ID NOs: 63, 64 and 65. For example, the gene comprises at least one polynucleotide sequence selected from SEQ ID NOs: 63, 64 and 65 and 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 97.7% , 97.8%, 98%, 98.5%, 98.7%, 98.8%, 99%, 99.5%, 99.7%, 99.8% or more polynucleotide sequences having less than 100% homology or identity.
[45]
As used herein, the term "polynucleotide" refers to a DNA strand of a certain length or more as a polymer of nucleotides in which nucleotide monomers are connected in a long chain form by covalent bonds.
[46]
In the present application, the metZ gene, a protein encoded by any one or more polynucleotide sequences selected from SEQ ID NOs: 63, 64 and 65, or a protein having an amino acid sequence of any one or more of SEQ ID NOs: 60, 61 and 62 Effect corresponding to In the case of including a polynucleotide encoding a protein having
[47]
For example, the metZ gene may encode an amino acid sequence in which a part of the amino acid sequence of any one of SEQ ID NOs: 60, 61 and 62, for example, 1 to 20 amino acids is substituted. In another embodiment, the metZ gene has 20, 19, 18, 17, 16, 15, 14, 13, 12 or 11 amino acid sequences added before and after the amino acid sequence. It may be a sequence encoding a given sequence. In another embodiment, the metZ gene may be a sequence encoding an amino acid sequence including all of the aforementioned substitutions and additions, but is not limited thereto.
[48]
In addition, probes that can be prepared from known gene sequences, for example, polynucleotides that hybridize under stringent conditions with a sequence complementary to all or part of the polynucleotide sequence may be included without limitation.
[49]
That is, even if described in the present application as a polynucleotide comprising a nucleotide sequence of a specific SEQ ID NO: a polynucleotide comprising a nucleotide sequence of a specific SEQ ID NO: or a polynucleotide having a nucleotide sequence of a specific SEQ ID NO: polynucleotide of the SEQ ID NO: In the case of having the same or corresponding activity as the polypeptide encoded by the nucleotide sequence consisting of The inclusion is self-evident. For example, the amino acid sequence N-terminus and/or C-terminal addition of a sequence that does not alter the function of the protein, a naturally occurring mutation, a silent mutation or a conservative substitution thereof. .
[50]
The term “conservative substitution” means substituting an amino acid for another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues.
[51]
In the present application, the terms "homology" and "identity" refer to a degree related to two given nucleotide sequences and may be expressed as a percentage.
[52]
The terms homology and identity can often be used interchangeably.
[53]
The sequence homology or identity of a conserved polynucleotide is determined by standard alignment algorithms, and the default gap penalty established by the program used may be used together. Substantially, homologous or identical sequences generally have moderate or high stringency conditions along at least about 50%, 60%, 70%, 80% or 90% of the entire or full-length sequence. It can hybridize under stringent conditions. It is self-evident that hybridization also includes polynucleotides containing degenerate codons instead of common codons in polynucleotides.
[54]
Whether any two polynucleotide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, using a known computer algorithm such as the "FASTA" program. or, as performed in the Needleman program (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) of the EMBOSS package, The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine. (GCG program 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 ETA/.] (1988) SIAM J Applied Math 48: 1073) For example, BLAST of the National Center for Biotechnology Information Database, or ClustalW, can be used to determine homology, similarity or identity.
[55]
Homology, similarity or identity of polynucleotides is described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. 48: 443 by comparing the sequence information using a GAP computer program. In summary, the GAP program is defined as the total number of symbols in the shorter of two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids). Default parameters for GAP programs are (1) binary comparison matrix (identityvalues ​​of 1 for hazard and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.
[56]
In addition, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be confirmed by comparing the sequences by Southern hybridization experiments under defined stringent conditions, and the defined appropriate hybridization conditions are within the scope of the art. , methods well known to those skilled 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).
[57]
In addition, in the polynucleotide of the present application, various modifications are made to the coding region within a range that does not change the polypeptide sequence due to codon degeneracy or in consideration of codons preferred in the organism to express the polynucleotide. can be done In addition, by hybridizing under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the nucleotide sequence, L is not a sequence naturally present in the microorganism to be introduced. - Any polynucleotide sequence capable of increasing the ability to produce methionine may be included without limitation. The "stringent condition" means a condition that enables specific hybridization between polynucleotides. Such conditions have been specifically described in several literatures (eg, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989), and those in the art It is well known. For example, genes with high homology or identity, 40% or more, specifically 70% or more, 80% or more, 85% or more, 90% or more, more specifically 95% or more, More specifically, the conditions under which genes having homology or identity of 97% or more, particularly specifically 99% or more, hybridize with each other and genes with lower homology or identity do not hybridize, or normal Southern hybridization (southern hybridization). hybridization) at a salt concentration and temperature corresponding to 60°C, 1XSSC, 0.1% SDS, specifically 60°C, 0.1XSSC, 0.1% SDS, more specifically 68°C, 0.1XSSC, 0.1% SDS, Conditions for washing once, specifically 2 to 3 times, can be enumerated.
[58]
Hybridization requires that two polynucleotides have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present application may also include substantially similar polynucleotide sequences as well as isolated polynucleotide fragments complementary to the overall sequence.
[59]
Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the above-described conditions. In addition, the Tm value may be 60 °C, 63 °C, or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.
[60]
The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementarity, and the parameters are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).
[61]
[62]
The term "introduction of protein" of the present application means that the microorganism exhibits the activity of a specific protein that it did not originally have, or exhibits improved activity compared to the intrinsic activity or activity before modification of the protein. For example, a specific protein is introduced, a polynucleotide encoding a specific protein is introduced into a chromosome in a microorganism, or a vector including a polynucleotide encoding a specific protein is introduced into a microorganism to exhibit its activity. In the present application, protein introduction can also be expressed as enhancement of protein activity in microorganisms without specific protein activity.
[63]
Introduction of the protein may be performed by introducing a foreign polynucleotide encoding a protein exhibiting the same/similar activity as the protein, or a codon-optimized mutant polynucleotide thereof into a host cell. The foreign polynucleotide may be used without limitation in origin or sequence as long as it exhibits the same/similar activity as the protein. In addition, the introduced foreign polynucleotide may be introduced into the host cell by optimizing its codon so that the optimized transcription and translation are performed in the host cell. The introduction can be performed by appropriately selecting a known transformation method by those skilled in the art, and the introduced polynucleotide is expressed in a host cell to generate a protein and increase its activity.
[64]
Enhancing the activity of the introduced protein,
[65]
1) an increase in the intracellular copy number of a gene or polynucleotide encoding the protein;
[66]
2) a method of replacing the gene expression control region on the chromosome encoding the protein with a sequence with strong activity;
[67]
3) a method of modifying the nucleotide sequence of the start codon or 5'-UTR region of the protein,
[68]
4) a method of modifying a polynucleotide sequence on a chromosome to increase the activity of the protein, or
[69]
5) It may be performed by a combination of the above methods, but is not limited thereto.
[70]
[71]
As used herein, the term “vector” refers to a DNA preparation containing a target polynucleotide sequence operably linked to a suitable regulatory sequence so as to introduce a target gene in a suitable host. Such regulatory sequences may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself. For example, a target polynucleotide in a chromosome may be replaced with a mutated polynucleotide through a vector for intracellular chromosome insertion. The insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto.
[72]
The vector of the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, and pBluescriptII-based plasmid vectors may be used as plasmid vectors. , pGEM-based, pTZ-based, pCL-based, pET-based and the like can be used. Specifically, pDZ, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.
[73]
[74]
As used herein, the term “transformation” refers to introducing a vector including a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome, as long as they can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding a target protein. As long as the polynucleotide can be introduced and expressed into a host cell, it may be introduced in any form. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression. The expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.
[75]
Also, the term "operably linked" in the present application refers to theIt means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding an enemy protein and the gene sequence are functionally linked.
[76]
The method for transforming the vector of the present application includes any method of introducing a nucleic acid into a cell, and may be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO 4) precipitation, calcium chloride (CaCl 2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and Lithium acetate-DMSO method and the like, but is not limited thereto.
[77]
[78]
The microorganisms of the present application include wild-type microorganisms or microorganisms in which genetic modification has occurred naturally or artificially, and microorganisms into which the foreign metZ gene as described in the present application is introduced or included may be included without limitation.
[79]
The microorganism, the foreign metZ gene of the present application; proteins encoded thereby; and any one or more of vectors including the metZ gene, may be a microorganism producing L-methionine.
[80]
[81]
The term "L-methionine-producing microorganism" in the present application includes both wild-type microorganisms and microorganisms in which genetic modification has occurred naturally or artificially. As a result of which a specific mechanism is weakened or enhanced, it may be a microorganism containing genetic modification for the desired production of L-methionine.
[82]
The L-methionine-producing microorganism may be a microorganism having improved L-methionine-producing ability than the parent strain or unmodified microorganism, including the protein encoded by the foreign metZ gene of the present application.
[83]
As used herein, the term "strain before modification" or "microbe before modification" does not exclude strains containing mutations that may occur naturally in microorganisms, and is either a wild-type strain or a natural-type strain itself, or caused by natural or artificial factors. It may refer to a strain before the trait is changed due to genetic mutation. The "pre-modified strain" or "pre-modified microorganism" may be used interchangeably with "unmodified strain", "unmodified strain", "unmodified microorganism", "unmodified microorganism" or "reference microorganism". Alternatively, the expression level of the gene involved in the L-methionine biosynthesis pathway may be unregulated, or it may be a microorganism into which the metZ gene, which does not exist intrinsically, is not introduced.
[84]
The L-methionine-producing microorganism of the present application may be a microorganism in which the activity of a part of the protein in the L-methionine biosynthesis pathway is enhanced, or the activity of a part of the protein in the L-methionine degradation pathway is weakened, so that the L-methionine-producing ability is enhanced.
[85]
Specifically, examples of proteins or genes capable of regulating expression to enhance L-methionine biosynthetic pathway or attenuate/inactivate degradation pathways are as follows. Proteins, representative genes encoding proteins, and representative EC numbers were described in order. Proteins were written in capital letters, and genes were written in italics. For example, Rdl2p, GlpE, PspE, YgaP, ThiI, YbbB, SseA, YnjE, YceA, YibN, NCgl0671, NCgl1369, NCgl2616, NCgl0053, NCgl0054, NCGl2678, NCgl2890 thiosulphate sulfur transferase; sulfite reductase, cysI; thiosulphate/sulphate transport system, cysPUWA (EC 3.6.3.25); 3'-phosphoadenosine 5'-phosphosulphate reductase (3'-phosphoadenosine 5'-phosphosulphate reductase), cysH (EC 1.8.4.8); sulphite reductase, cysJI (EC 1.8.1.2); cysteine ​​synthase, cysK (EC 2.5.1.47); cysteine ​​synthase B, cysM (EC 2.5.1.47); serine acetyltransferase, cysE (EC 2.3.1.30); glycine cleavage system, gcvTHP-lpd (EC 2.1.2.10, EC 1.4.4.2, EC 1.8.1.4); lipoyl synthase, lipA (EC 2.8.1.8); lipoyl protein ligase, lipB (EC 2.3.1.181); phosphoglycerate dehydrogenase, serA (EC 1.1.1.95); 3-phosphoserine phosphatase, serB (EC 3.1.3.3); 3-phosphoserine/phosphohydroxythreonine aminotransferase, serC (EC 2.6.1.52); serine hydroxymethyltransferase, glyA (EC 2.1.2.1); aspartokinase I (EC 2.7.2.4); homoserine dehydrogenase I, thrA (EC 1.1.1.3); aspartate kinase, lysC (EC 2.7.2.4); homoserine dehydrogenase, hom (EC 1.1.1.3); homoserine O-acetyltransferase, metX (EC 2.3.1.31); homoserine O-succinyltransferase, metA (EC 2.3.1.46); cystathionine gamma-synthase, metB (EC 2.5.1.48); β-C-S-lyase (β-C-S-lyase), aecD (EC 4.4.1.8, beta-lyase); cystathionine beta-lyase, metC (EC 4.4.1.8); B12-independent homocysteine ​​S-methyltransferase, metE (EC 2.1.1.14); methionine synthase, metH (EC 2.1.1.13); methylenetetrahydrofolate reductase, metF (EC 1.5.1.20); L-methionine exotransporter BrnFE; valine exotransporters YgaZH (B2682, B2683), ygaZH (b2682. b2683); exotransporter YjeH, b4141; pyridine nucleotide transhydrogenase PntAB, pntAB (EC 1.6.1.2); And phosphoenolpyruvate carboxylase (phosphoenolpyruvate carboxylase), Pyc (EC 4.1.1.31) at least one protein selected from or enhancing the activity of some proteins constituting the system or a polynucleotide encoding the same is overexpressed to L-amino acid It can enhance biosynthetic pathways or weaken degradation pathways. or, glucose 6-phosphate isomerase, pgi (EC 5.3.1.9); homoserine kinase, thrB (EC 2.7.1.39); S-adenosylmethionine synthetase, metK (EC 2.5.1.6); dihydrodipicolinate synthetase, dapA (EC 4.2.1.52); phosphoenolpyruvate carboxykinase, pck (EC 4.1.1.49);, formyltetrahydrofolate hydrolase, purU (EC 3.5.1.10); pyruvate kinase I, pykF (EC 2.7.1.40); pyruvate kinase II, pykA (EC 2.7.1.40); cystathionine γ-lyase, cg3086 (EC 4.4.1.1); cystathionine β-synthetase, cg2344 (EC 4.2.1.22); regulatory proteins Cg3031, cg3031; methionine and cysteine ​​biosynthesis repressor protein McbR, mcbR; L-methionine synthesis transcriptional regulator (Met transcriptional repressor protein), metJ; L-methionine transporters MetQNI, metQ, metN, metI; N-acyltransferase, yncA; sRNA fnrS; And L-methionine transporter, the activity of one or more proteins selected from the group consisting of metP may be inactivated or attenuated, or the expression of a gene encoding the protein is suppressed or removed.
[86]
In one embodiment, the L-methionine-producing microorganism of the present application, in addition to the introduction of metZ, weakened or inactive cystathionine gamma synthase activity; attenuation or inactivation of O-acetylhomoserine sulfidylase; attenuation or inactivation of inhibitors of methionine-cysteine ​​biosynthesis; enhancing the activity of methionine synthase; and one or more genetic modifications selected from the group consisting of enhancing the activity of sulfite reductase. Alternatively, the genetic modification may include deletion/inhibition of expression of the metB gene; deletion of the metY gene; deletion/expression inhibition of the mcbR gene; And one or more mutations selected from the group consisting of enhanced metH expression and enhanced cysI gene expression may be additionally introduced, for example, the metB gene is SEQ ID NO: 25, the metY gene is SEQ ID NO: 26, and the mcbR gene is SEQ ID NO: 1, metH gene is SEQ ID NO: 39, cysI gene is poly of SEQ ID NO: 40nucleotide sequence and at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 polynucleotide sequences having %, 98% or 99% homology or identity. The above description of homology or identity is the same for the metB, metY, mcbR, metH and cysI genes.
[87]
However, the above genes are one example and are not limited thereto, and may be microorganisms that enhance protein activity of various known L-methionine biosynthetic pathways or inactivate/ weaken protein activity of degradation pathways.
[88]
As used herein, the term “enhancement” of a polypeptide or protein activity means that the activity of the polypeptide or protein is increased compared to the intrinsic activity. The reinforcement may be used interchangeably with terms such as up-regulation, overexpression, and increase. Here, the increase may include both exhibiting an activity that it did not originally have, or exhibiting an improved activity compared to intrinsic activity or activity before modification. The "intrinsic activity" refers to the activity of a specific polypeptide or protein originally possessed by the parent strain or unmodified microorganism before the transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before modification". "Enhancement" or "increase" in the activity of a polypeptide or protein compared to the intrinsic activity means that it is improved compared to the activity of a specific polypeptide or protein originally possessed by the parent strain or unmodified microorganism before transformation. The "increase in activity" " can be achieved by introducing an exogenous polypeptide or protein or enhancing the activity of the endogenous polypeptide or protein, but specifically, it may be achieved through enhancing the activity of the endogenous polypeptide or protein. Whether or not the activity of the polypeptide or protein is enhanced can be confirmed from the increase in the activity level, expression level, or amount of a product excreted from the corresponding polypeptide or protein.
[89]
The enhancement of the activity of the polypeptide or protein can be applied by various methods well known in the art, and may not be limited as long as the activity of the target polypeptide or protein can be enhanced compared to the microorganism before modification. The method is not limited thereto, but may use genetic engineering and/or protein engineering well known to those skilled in the art, which are routine methods of molecular biology (Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology). 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012 et al.).
[90]
The method for enhancing polypeptide or protein activity using genetic engineering, for example,
[91]
1) an increase in the intracellular copy number of a gene or polynucleotide encoding the polypeptide or protein,
[92]
2) a method of replacing the gene expression control region on the chromosome encoding the polypeptide or protein with a sequence with strong activity;
[93]
3) a method of modifying the base sequence of the start codon or 5'-UTR region of the polypeptide or protein,
[94]
4) a method of modifying a polynucleotide sequence on a chromosome to increase the polypeptide or protein activity,
[95]
5) introduction of a foreign polynucleotide exhibiting the activity of the polypeptide or protein or a polynucleotide with a codon-optimized variant of the polynucleotide, or
[96]
6) It may be performed by a combination of the above methods, but is not limited thereto.
[97]
The method for enhancing polypeptide or protein activity using the protein engineering may be carried out by, for example, a method of selecting an exposed site by analyzing the tertiary structure of the polypeptide or protein and modifying or chemically modifying it, but is limited thereto. doesn't happen
[98]
1) The increase in the intracellular copy number of the gene or polynucleotide encoding the polypeptide or protein is performed by any method known in the art, for example, the gene or polynucleotide encoding the polypeptide or protein is operably linked, This can be accomplished by introducing a vector capable of replicating and functioning independently of the host into a host cell. Alternatively, a vector capable of inserting the gene or polynucleotide into a chromosome in the host cell, to which the gene is operably linked, may be introduced into the host cell, but is not limited thereto. The vector is the same as described above.

Claims
[Claim 1]
A method for producing L-methionine, comprising culturing a microorganism into which a protein encoded by a foreign metZ gene has been introduced in a medium containing thiosulfate.
[Claim 2]
The method of claim 1, wherein the protein has O-acylhomoserine transsulfurase activity.
[Claim 3]
The method of claim 1, wherein the protein is derived from Chromobacterium violaceum, Hyphomonas neptunium, or Rhodobacter sphaeroides. .
[Claim 4]
The L- Method for preparing methionine.
[Claim 5]
According to claim 1, wherein the microorganism, cystathionine gamma synthase activity attenuated or inactive; attenuation or inactivation of O-acetylhomoserine sulfidylase; attenuation or inactivation of inhibitors of methionine-cysteine ​​biosynthesis; enhancing the activity of methionine synthase; and one or more genetic modifications selected from the group consisting of enhancing the activity of sulfite reductase, L-methionine production method.
[Claim 6]
According to claim 1, wherein the microorganism is a microorganism of the genus Corynebacterium, L-methionine production method.
[Claim 7]
The method of claim 6, wherein the microorganism is Corynebacterium glutamicum.
[Claim 8]
The method of claim 1, wherein the production method comprises recovering L-methionine from the microorganism or medium.
[Claim 9]
The method according to claim 1, wherein the method is to reduce the amount of homolanthionine produced, L-methionine production method.
[Claim 10]
An L-methionine-producing microorganism into which a protein encoded by a foreign metZ gene has been introduced.
[Claim 11]
The microorganism according to claim 10, wherein the protein has O-acylhomoserine transsulfurase activity.
[Claim 12]
11. The method of claim 10, wherein the protein is Chromobacterium violaceum (Chromobacterium violaceum), Hyphomonas neptunium (Hyphomonas neptunium) or Rhodobacter spheroids (Rhodobacter sphaeroides) It is derived from, L- methionine production microorganisms that do.
[Claim 13]
The L- Microorganisms that produce methionine.
[Claim 14]
11. The method of claim 10, wherein the microorganism, cystathionine gamma synthase activity attenuated or inactive; attenuation or inactivation of O-acetylhomoserine sulfidylase; attenuation or inactivation of inhibitors of methionine-cysteine ​​biosynthesis; enhancing the activity of methionine synthase; and L-methionine-producing microorganisms comprising one or more genetic modifications selected from the group consisting of enhancing the activity of sulfite reductase.
[Claim 15]
The microorganism according to claim 10, wherein the microorganism is a microorganism of the genus Corynebacterium, L-methionine-producing microorganism.
[Claim 16]
The microorganism according to claim 10, wherein the microorganism is Corynebacterium glutamicum.
[Claim 17]
11. The method of claim 10, wherein the microorganism is a reduced amount of homolanthionine production, L- methionine-producing microorganism.
[Claim 18]
A composition for producing L-methionine, comprising the microorganism of any one of claims 10 to 17 and thiosulfate.
[Claim 19]
Use of a microorganism into which a protein encoded by an exogenous metZ gene has been introduced for the production of L-methionine.