Recombinant microorganism in which the activity of manganese uptake protein is regulated, and a method for producing O-phosphoserine, cysteine, and their derivatives using the same

Abstract

The present application relates to a recombinant microorganism in which the activity of a protein having manganese uptake activity is regulated, and a method for producing O-phosphoserine, cysteine, and derivatives thereof using the same.

Description

[Technical Field]

[0001] The present application relates to a recombinant microorganism in which the activity of a protein having manganese uptake activity is regulated, and a method for producing O-phosphoserine, cysteine, and derivatives thereof using the same. [Background technology]

[0002] L-cysteine ​​is an important amino acid in sulfur metabolism in all living organisms, and is used not only in the synthesis of endogenous proteins such as hair keratin, glutathione, biotin, methionine, and other sulfur-containing metabolites, but also as a precursor for coenzyme A biosynthesis.

[0003] Known methods for producing L-cysteine ​​using microorganisms include: 1) a method of biologically converting D,L-ATC (D,L-2-amino-2-thiazoline-4-carboxylate) using microorganisms; 2) a direct fermentation method for producing L-cysteine ​​using Escherichia coli (Patent Document 1, Non-Patent Document 1); and 3) a method of fermentatively producing O-phosphoserine (hereinafter referred to as "OPS") using microorganisms, followed by a reaction with sulfide under the catalytic action of O-phosphoserine sulfhydrylase (hereinafter referred to as "OPSS") to convert it to L-cysteine ​​(Patent Document 2).

[0004] Here, in order to produce cysteine ​​at a high yield by the above method 3), it was necessary to produce the precursor OPS in excess. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] European Patent No. 0885962 [Patent Document 2] U.S. Patent No. 8,557,549 [Patent Document 3] US Patent Application Publication No. 2012 / 0190081 [Patent Document 4] US Patent Application Publication No. 2020 / 0048619 [Patent Document 5] U.S. Patent No. 7,662,943 [Patent Document 6] U.S. Patent No. 10,584,338 [Patent Document 7] U.S. Patent No. 10,273,491 [Patent Document 8] Korean Patent No. 1381048 [Patent Document 9] Korean Patent Publication No. 2012-0041115 [Patent Document 10] Korean Patent No. 1208267 [Patent Document 11] International Publication No. 2016 / 024771 [Non-patent literature]

[0006] [Non-Patent Document 1] Wada M and Takagi H, Appl.Microbiol. Biochem., 73:48-54, 2006 [Non-patent document 2] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-patent document 3] Rice et al., 2000, Trends Genet. 16:276-277 [Non-patent document 4] Needleman and Wunsch, 1970, J.Mol. Biol. 48: 443-453 [Non-patent document 5] Devereux, J., et al, Nucleic Acids Research 12: 387 (1984) [Non-patent document 6] Atschul, [S.] [F.,] [ET AL, JMOLEC BIOL 215]: 403 (1990)

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[0007] An objective of the present application is to provide a recombinant microorganism in which the activity of a protein having manganese uptake activity is regulated, and a method for producing O-phosphoserine, cysteine, and derivatives thereof using the same. [Means for solving the problem]

[0008] An object of the present application is to provide an O-phosphoserine-producing recombinant microorganism in which the activity of the MntH protein is enhanced compared to the endogenous activity.

[0009] Another object of the present application is to provide a method for producing O-phosphoserine using the O-phosphoserine-producing recombinant microorganism of the present application.

[0010] Another object of the present application is to provide a method for producing cysteine ​​or a derivative thereof using the O-phosphoserine-producing recombinant microorganism of the present application. [Effects of the Invention]

[0011] When O-phosphoserine is produced using an O-phosphoserine-producing recombinant microorganism in which the activity of the MntH protein of the present application is enhanced compared to the endogenous activity, O-phosphoserine can be produced at a higher yield than when a conventional unmodified strain is used. DETAILED DESCRIPTION OF THE INVENTION

[0012] These will be described in detail below. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. Furthermore, this application is not limited to the specific descriptions below. Furthermore, many papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety, thereby more clearly explaining the state of the art to which this application pertains and the contents of this application.

[0013] One aspect of the present application provides an O-phosphoserine-producing recombinant microorganism in which the activity of the MntH protein is enhanced compared to the endogenous activity.

[0014] In the present application, "O-phosphoserine (hereinafter referred to as "OPS")" refers to a phosphoric acid ester of serine and is a component of various proteins. OPS is a precursor of L-cysteine ​​and is converted to cysteine ​​by reacting with sulfide under the catalytic action of O-phosphoserine sulfhydrylase (OPS sulfhydrylase, OPSS), but is not limited thereto (Patent Document 3).

[0015] In the present application, the term "MntH protein" refers to a protein that is classified into the NRAMP family and has divalent ion transporter activity, such as Mn 2+ In this application, "Mn 2+ Uptake of NRAMP (natural resistance-associated macrophage protein) transporter (Mn 2+ "NRAMP uptake transporter, MntH)" refers to Mn 2+ / Fe 2+ :H + It refers to a protein with symporter activity, and has the activity of taking up manganese (Mn) into cells.

[0016] The amino acid sequence of the MntH protein can be obtained from a publicly known database such as NCBI's Genebank.

[0017] By way of example, the MntH protein of the present application may be derived from a microorganism, specifically, but not limited to, a microorganism of the genus Escherichia.

[0018] As another example, the amino acid sequence of the MntH protein of the present application is WP000186369.1 or EEW8215783.1 derived from Escherichia coli, but it goes without saying that proteins having MntH protein activity of various origins are included.

[0019] In the present application, the MntH protein may have the amino acid sequence of SEQ ID NO: 1, may contain the amino acid sequence, may consist of the amino acid sequence, or may essentially consist of the amino acid sequence.

[0020] In the present application, the MntH protein may comprise an amino acid sequence that is at least 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, or 99.9% or more homologous or identical to the amino acid sequence of SEQ ID NO: 1. It goes without saying that the present application also encompasses proteins with amino acid sequences in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, so long as the amino acid sequence has such homology or identity and exhibits efficacy equivalent to that of a protein comprising the amino acid sequence of SEQ ID NO: 1. For example, the MntH protein may be composed of 412 to 428 amino acids including the amino acid sequence of SEQ ID NO: 1.

[0021] For example, the amino acid sequence may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally that do not change the function of the protein of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0022] The term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0023] In this application, "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0024] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, optionally with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize to all or part of the sequence under moderately or highly stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides containing common codons or codons that take into account codon degeneracy in the polynucleotide.

[0025] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms, such as the "FASTA" program, with default parameters, as described in, for example, Non-Patent Document 2. Alternatively, the Needleman-Wunsch algorithm (Non-Patent Document 4) can be used, as implemented in the Needle program (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 3) (version 5.0.0 or later) of the EMBOSS package (which includes the GCG program package (Non-Patent Document 5), BLASTP, BLASTN, and FASTA (Non-Patent Documents 6, 7, and 8)). For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0026] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 4, as disclosed in, for example, Non-Patent Document 9. Briefly, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include: (1) a unitary matrix (identity takes a value of 1 and non-identity takes a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBINUC4.4) substitution matrix) as disclosed in Non-Patent Document 10; (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 and a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0027] The MntH protein of the present application may be encoded by the mntH gene.

[0028] By way of example, but not limitation, the mntH gene is a polynucleotide encoding EEW8215783.1 from Escherichia coli, another example is a polynucleotide encoding WP_000186369.1 from Escherichia coli, and yet another example is a sequence contained in CP116188.1 from Escherichia coli, but needless to say, the mntH gene of various origins encodes a protein having MntH protein activity.

[0029] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and refers to a DNA or RNA chain longer than a certain length, and more specifically refers to a polynucleotide fragment that encodes the protein.

[0030] The polynucleotide encoding the MntH protein of the present application may comprise a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1. As an example of the present application, the polynucleotide of the present application may have the nucleotide sequence of SEQ ID NO: 2 or may comprise said nucleotide sequence. Furthermore, the polynucleotide of the present application may consist of the nucleotide sequence of SEQ ID NO: 2 or may essentially consist of said nucleotide sequence. Specifically, the mntH gene may be encoded by a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 2.

[0031] The polynucleotide of the present application can be modified in various ways in the coding region without changing the amino acid sequence of the MntH protein, taking into account codon degeneracy or codons preferred in the organism in which the MntH protein of the present application is to be expressed. Specifically, the polynucleotide of the present application has, comprises, consists of, or consists essentially of a nucleotide sequence that has at least 70%, 75%, 76%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity to the sequence of SEQ ID NO: 2, but is not limited to these.

[0032] Furthermore, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 12 and 13). For example, conditions include those under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, but polynucleotides with lower homology or identity do not hybridize with each other; or conditions for washing once, specifically two to three times, at a salt concentration and temperature equivalent to those used in conventional Southern hybridization, namely, 60°C, 1×SSC, 0.1% SDS, specifically, 60°C, 0.1×SSC, 0.1% SDS, more specifically, 68°C, 0.1×SSC, 0.1% SDS.

[0033] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0034] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0035] The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables known in the art (eg, Non-Patent Document 12).

[0036] In this application, the term "vector" refers to a DNA product comprising a base sequence of a polynucleotide encoding a target protein operably linked to a suitable regulatory sequence so as to enable the target protein to be expressed in a suitable host. The regulatory sequence includes a promoter that initiates transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that regulates the termination of transcription and translation. When transformed into a suitable host, the vector can replicate or function independently of the host genome, or can be integrated into the genome itself.

[0037] The vector used in the present application is not particularly limited as long as it is replicable in host cells, and any vector known in the art can be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pSKH130 (Patent Document 4), pSK, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0038] The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination.

[0039] In this application, "transformation" refers to the introduction of a recombinant vector containing a polynucleotide encoding a target protein into a host cell, thereby expressing the protein encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide that is expressed in the host cell, regardless of whether it is located intrachromosomally or extrachromosomally. The transformation method may be any method for introducing nucleic acid into a cell, and as known in the art, can be performed using a standard technique appropriate for the host cell. Examples include, but are not limited to, electroporation, calcium phosphate (CaPO) precipitation, calcium chloride (CaCl) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0040] Furthermore, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence or expression regulatory region that initiates and mediates transcription of the polynucleotide encoding the target protein of the present application. Operable linking can be achieved using recombinant DNA techniques known in the art, and site-specific DNA cleavage and ligation can be achieved using cleavage and ligation enzymes known in the art, but is not limited to these.

[0041] The microorganisms of the present application are not particularly limited in type, and may be either prokaryotic or eukaryotic cells, as long as they are capable of producing OPS. Examples include strains of microorganisms belonging to the genera Escherichia, Erwinia, Seratia, Providencia, Corynebacterium, and Brevibacterium. Specific examples include, but are not limited to, Escherichia microorganisms, and more specifically, Escherichia coli (E. coli). For example, Escherichia microorganisms can produce OPS and L-serine using SerA, SerC, and SerB, enzymes in the L-serine biosynthetic pathway (Non-Patent Documents 14, 15, and 16).

[0042] In the present application, the term "microorganism that produces O-phosphoserine (OPS)" refers to a microorganism that naturally has the ability to produce OPS, or a microorganism in which OPS-producing ability has been imparted to a parent strain that does not have the ability to produce OPS. Specifically, the microorganism may be a microorganism that has been genetically modified, either naturally or artificially, to enhance MntH protein activity and thereby produces OPS. For the purposes of the present application, the OPS-producing microorganism may be any microorganism that produces O-phosphoserine by enhancing MntH protein activity using the method disclosed in the present application. In the present application, the term "microorganism that produces O-phosphoserine (OPS)" is used interchangeably with "O-phosphoserine (OPS)-producing microorganism" and "microorganism having the ability to produce O-phosphoserine (OPS)."

[0043] In one embodiment, the OPS-producing microorganism of the present application is a genetically modified or recombinant microorganism in which the activity of the MntH protein is enhanced, thereby improving the ability to produce the desired OPS, but is not limited thereto. The recombinant microorganism may be a microorganism in which the ability to produce O-phosphoserine is enhanced beyond its endogenous ability to produce O-phosphoserine.

[0044] The microorganism of the present application may have an MntH protein activity that is improved compared to the endogenous activity. Specifically, the microorganism of the present application is a microorganism in which the MntH protein or the mntH gene encoding it has been enhanced, or a microorganism that has been genetically modified (e.g., a recombinant microorganism) so that the MntH protein or the mntH gene encoding it has been enhanced, but is not limited thereto.

[0045] In the present application, "enhancement" of a polypeptide (including, for example, proteins identified by the name of each enzyme) means improving the activity of the polypeptide compared to its endogenous activity. The term "enhancement" is used interchangeably with "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and enhancement all encompass the development of an activity not originally present, as well as an improvement in activity compared to endogenous activity or activity prior to modification. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or unmodified microorganism prior to the transformation, when a trait is altered through genetic mutation due to natural or artificial factors. This term is used interchangeably with "activity prior to transformation." "Enhancement," "up-regulation," "overexpression," or "improvement" of a polypeptide compared to its endogenous activity means an improvement in the activity and / or concentration (expression level) of a specific polypeptide inherently possessed by a parent strain or unmodified microorganism prior to the transformation.

[0046] The enhancement may be achieved by introducing a foreign polypeptide, or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide has been enhanced can be confirmed by an increase in the level of activity of the polypeptide, the expression level, or the amount of a product produced from the polypeptide.

[0047] For the purposes of this application, the microorganisms of the present application have improved OPS-producing ability due to enhanced MntH protein activity. Unmodified microorganisms without enhanced MntH protein, which are used to compare whether the OPS-producing ability or MntH protein is improved, include, but are not limited to, the OPS-producing strain CA07-0012 (KCCM11121P, Patent Document 2), which lacks endogenous phosphoserine phosphatase (SerB) and has reduced OPS-degrading ability, the CA07-0012 strain with enhanced YhhS expression that has OPS-producing ability (CA07-4821 of the present application), and the CA07-4821 strain with further enhanced NADH:quinone oxidoreductase activity (CA07-4828 of the present application).

[0048] Various methods well known in the art can be applied to enhance the polypeptide activity, and any method can be used as long as it can enhance the activity of the target polypeptide compared to the unmodified microorganism. Specifically, the method can be, but is not limited to, conventional methods in molecular biology that use genetic engineering and / or protein engineering well known to those skilled in the art (e.g., Non-Patent Documents 17 and 18).

[0049] Specifically, the activity of a polypeptide of the present application can be enhanced by 1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide, 2) modifying an expression regulatory sequence on a chromosome encoding the polypeptide, 3) modifying a nucleotide sequence encoding the start codon or 5'UTR region of a gene transcript encoding the polypeptide, 4) modifying the amino acid sequence of the polypeptide so as to enhance the polypeptide activity, 5) modifying the polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide activity (for example, modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide activity), 6) introducing a foreign polypeptide that exhibits the activity of the polypeptide or a foreign polynucleotide encoding it, 7) optimizing the codons of a polynucleotide encoding the polypeptide, 8) analyzing the tertiary structure of the polypeptide and selecting and modifying or chemically modifying exposed portions, 9) adjusting the cellular localization of the polypeptide, or 10) a combination of two or more selected from 1) to 9) above, but is not particularly limited thereto.

[0050] More specifically, 1) increasing the intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a host cell a vector to which a polynucleotide encoding the polypeptide is operably linked, the vector replicating and functioning independently of the host. Alternatively, the increase may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into a chromosome in the host cell. The introduction into a chromosome can be achieved by, but is not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell.

[0051] The 2) replacement of the expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide with a sequence with stronger activity can be carried out, for example, by generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by replacing the expression regulatory region with a sequence with higher activity, so as to further enhance the activity of the expression regulatory region. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, and the like. Specifically, the above method is carried out by inserting a strong promoter after the original promoter, but is not limited to this.

[0052] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (Patent Document 5), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (Patent Document 6), the O2 promoter (Patent Document 7), the tkt promoter, the yccA promoter, the rmf promoter, and the serC promoter.

[0053] The nucleotide sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide (3) can be modified, for example, by substituting it with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon, but is not limited to this.

[0054] The modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above can be carried out by, but is not limited to, generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to enhance the activity of the polypeptide, or by substituting an amino acid sequence or polynucleotide sequence improved to have higher activity or an amino acid sequence or polynucleotide sequence improved to improve activity. Specifically, the substitution can be carried out by, but is not limited to, inserting a polynucleotide into a chromosome by homologous recombination.

[0055] The vector used here may further contain a selection marker to confirm whether or not it has been inserted into a chromosome. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether or not the target nucleic acid molecule has been inserted. Examples of the selection marker include, but are not limited to, markers that confer a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, and expression of a surface polypeptide. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit a different phenotype, allowing the selection of transformed cells.

[0056] 6) Introduction of a foreign polynucleotide that exhibits the activity of a polypeptide may be carried out by introducing into a host cell a foreign polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide. The foreign polynucleotide may be of any origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The introduction can be carried out by a known transformation method appropriately selected by those skilled in the art, and the introduced polynucleotide is expressed in the host cell as described above, thereby producing the polypeptide and improving its activity.

[0057] 7) Optimizing the codons of a polynucleotide encoding a polypeptide may be carried out by optimizing the codons of an endogenous polynucleotide so as to increase transcription or translation within a host cell, or by optimizing the codons of an exogenous polynucleotide so as to achieve optimized transcription and translation within a host cell.

[0058] 8) Analyzing the tertiary structure of a polypeptide and selecting and altering or chemically modifying exposed portions may be carried out, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins based on the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and altering or modifying exposed portions to be altered or chemically modified.

[0059] 9) The intracellular location of the polypeptide may be controlled by targeting the polypeptide to a specific intracellular organelle or specific intracellular space, for example, but not limited to, targeting the polypeptide to the periplasm or cytoplasm by adding or removing a leader sequence that functions to target the polypeptide.

[0060] In one example, the activity of the MntH protein may be enhanced by modifying the expression regulatory region of the gene on the chromosome that encodes the polypeptide of 2) above.

[0061] In any of the above-described embodiments, the activity of the MntH protein of the present application may be enhanced by increasing the expression of a gene encoding the MntH protein. In any of the above-described embodiments, the activity of the MntH protein may be enhanced by including a gene expression regulatory sequence with enhanced activity upstream of the gene encoding it. Specifically, the upstream of the gene encoding the MntH protein may be upstream of the mntH gene. In one embodiment, the activity of the MntH protein may be enhanced by modifying the expression regulatory sequence of the mntH gene to enhance expression of the gene sequence. Specifically, the modification of the expression regulatory sequence is achieved by inserting an additional gene expression regulatory sequence with enhanced activity between the endogenous promoter of the mntH gene and the mntH gene. For example, but not limited to, the gene expression regulatory sequence may be a promoter.

[0062] Such enhancement of polypeptide activity can be achieved by, but is not limited to, improving the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in a wild-type or unmodified microbial strain, or by increasing the amount of product produced from the polypeptide.

[0063] In this application, the terms "pre-modification strain" and "pre-modification microorganism" do not exclude strains containing naturally occurring mutations in microorganisms, but refer to the native strain itself or a strain before its traits are changed due to genetic mutation caused by natural or artificial factors. In this application, the trait change may be enhanced activity of the MntH protein. The terms "pre-modification strain" and "pre-modification microorganism" are also used interchangeably with "non-mutated strain," "non-modified strain," "non-mutated microorganism," "non-modified microorganism," or "reference microorganism."

[0064] In the microorganisms of the present application, partial or complete modification of a polynucleotide can be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal introduction into the microorganism, or genome editing using engineered nucleases (e.g., CRISPR-Cas9), and / or (b) light and / or chemical treatment, such as ultraviolet light or radiation. Methods for partially or completely modifying the gene include methods using DNA recombination techniques. For example, partial or complete deletion of a gene can be achieved by injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism, causing homologous recombination. The introduced nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

[0065] The microorganism of the present application may be a microorganism with improved OPS production ability.

[0066] For the purposes of this application, a recombinant microorganism of this application refers to a naturally occurring wild-type microorganism or a microorganism that produces OPS containing an MntH protein or a polynucleotide encoding it, in which the MntH protein or the polynucleotide encoding it has been enhanced, resulting in improved OPS production ability compared to the naturally occurring wild-type microorganism or a microorganism that produces OPS containing an MntH protein or a polynucleotide encoding it, but is not limited thereto. For example, the naturally occurring wild-type microorganism or a microorganism that produces OPS containing an MntH protein or a polynucleotide encoding it are subject strains for comparison to determine whether or not the OPS production ability or MntH protein activity has been improved, as described above, but is not limited thereto.

[0067] For example, the recombinant strain with improved OPS production ability has an OPS production ability that is improved by at least about 1%, specifically at least about 1.7%, at least 2%, at least about 2.3%, at least about 3%, at least about 4%, at least about 5%, at least about 5.3%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10% (there is no particular upper limit, for example, at most about 200%, at most about 150%, at most about 100%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, or at most about 15%) compared to the OPS production ability of the parent strain or unmodified microorganism before mutation. However, any recombinant strain with improved OPS production ability may be used as long as it has an increase in the + value compared to the production ability of the parent strain or unmodified microorganism before mutation. In other examples, the microorganism with improved production ability has an OPS production ability that is about 1.01-fold or more, about 1.017-fold or more, about 1.02-fold or more, about 1.023-fold or more, about 1.03-fold or more, about 1.04-fold or more, about 1.05-fold or more, about 1.053-fold or more, about 1.06-fold or more, about 1.07-fold or more, about 1.08-fold or more, about 1.09-fold or more, or about 1.10-fold or more (there is no particular upper limit, and for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, or about 2-fold or less) improved compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto.

[0068] Such production ability can be evaluated by measuring the amount of target product produced after culturing in a medium. The evaluation can be performed by measuring the amount of target product produced using a suitable method known in the art. For example, the amount of target product produced can be measured using a suitable method known in the art, such as HPLC (High Performance Liquid Chromatography), GC (Gas Chromatography), GC / MS (Gas Chromatography-Mass Spectrometry), LC / MS (Liquid Chromatography-Mass Spectrometry), GPC (Gel Permeation Chromatography), or a combination thereof.

[0069] The microorganisms of the present application may be modified to enhance their ability to produce OPS and / or their ability to export OPS to the outside of the cell, or may be modified to enhance their ability to degrade OPS and / or their ability to take up OPS.

[0070] Examples of modifications that enhance OPS production ability and / or extracellular export ability, or modifications that enhance OPS degradation ability and / or uptake ability include, but are not limited to, enhanced activity of NADH:quinone oxidoreductase, weakened activity of phosphoserine phosphatase (SerB), enhanced activity of phosphoserine export protein (YhhS), or combinations of these modifications.

[0071] In the present application, "attenuation" of a polypeptide refers to a decrease in activity compared to the endogenous activity or the absence of activity. The term "attenuation" is interchangeable with other terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0072] The reduction includes at least one of the following: a decrease or elimination of the activity of the polypeptide itself compared to the activity of the polypeptide originally possessed by the microorganism due to, for example, a mutation in the polynucleotide encoding the polypeptide; a decrease in the overall level and / or concentration (expression level) of polypeptide activity in the cell compared to that of a native strain due to, for example, inhibition of gene expression of the encoding polynucleotide or inhibition of translation into the polypeptide; a complete absence of expression of the polynucleotide; and a lack of polypeptide activity even if the polynucleotide is expressed. The term "endogenous activity" refers to the activity of a specific polypeptide originally possessed by a parent strain, wild-type, or unmodified microorganism before the trait change, when the trait is changed due to genetic mutation caused by natural or artificial factors. This term is used interchangeably with "activity before modification." A polypeptide activity that is "weakened," "inactivated," "absent," "reduced," "down-regulated," "decreased," or "attenuated" compared to the endogenous activity refers to a decrease in the activity of a specific polypeptide originally possessed by a parent strain or unmodified microorganism before the trait change.

[0073] Such attenuation of polypeptide activity may be achieved by applying various methods well known in the art, including, but not limited to, those described in Non-Patent Documents 18 and 19.

[0074] Specifically, the activity of a polypeptide of the present application can be attenuated by 1) deleting all or part of a gene encoding the polypeptide, 2) modifying an expression regulatory region (or expression regulatory sequence) so as to reduce the expression of a gene encoding the polypeptide, 3) modifying the amino acid sequence constituting the polypeptide so as to delete or attenuate the activity of the polypeptide (for example, by deleting / substituting / adding one or more amino acids in the amino acid sequence), or 4) modifying the gene sequence encoding the polypeptide so as to delete or attenuate the activity of the polypeptide (for example, by modifying the nucleic acid base sequence of the polypeptide gene so as to encode a polypeptide modified so as to delete or attenuate the activity of the polypeptide). 5) modifying the base sequence encoding the start codon, Shine-Dalgarno sequence, or 5'UTR region of the gene transcript encoding the polypeptide; 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding the polypeptide; 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide so that a secondary structure that prevents ribosome attachment is formed; 8) adding a promoter to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide so that it is reverse transcribed (reverse transcription engineering, RTE); 9) adjusting the cellular localization of the polypeptide; or 10) a combination of two or more selected from 1) to 9) above, but this is not particularly limited thereto.

[0075] For example, 1) deleting a part or all of the gene encoding the polypeptide may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, or by substituting a polynucleotide with a partial deletion of nucleotides or a marker gene.

[0076] The modification of the expression regulatory region (or expression regulatory sequence) may be carried out by generating a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting a sequence having a lower activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0077] The modification of the amino acid sequence or polynucleotide sequence in 3) and 4) above can be carried out by, but is not limited to, generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to attenuate the activity of the polypeptide, or by substituting an amino acid sequence or polynucleotide sequence that has been improved to have lower activity or to eliminate activity. For example, gene expression can be inhibited or attenuated by, but is not limited to, introducing a mutation into a polynucleotide sequence to form a stop codon.

[0078] The base sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide (5) can be modified, for example, by substituting it with a base sequence encoding another start codon that has a lower polypeptide expression rate than the endogenous start codon, but is not limited to this.

[0079] 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcription product encoding the polypeptide may be carried out, for example, as described in Non-Patent Document 20.

[0080] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide so that a secondary structure that prevents ribosome attachment is formed may be achieved by disabling or slowing down mRNA translation.

[0081] Furthermore, the above 8) adding a promoter to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide so as to reverse transcribe it (reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the gene transcript encoding the polypeptide to attenuate its activity.

[0082] 9) The intracellular location of the polypeptide may be controlled by targeting the polypeptide to a specific intracellular organelle or specific intracellular space, for example, but not limited to, targeting the polypeptide to the periplasm or cytoplasm by adding or removing a leader sequence that functions to target the polypeptide.

[0083] Such attenuation of polypeptide activity can be achieved by, but is not limited to, attenuating the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in a wild-type or unmodified microbial strain, or by increasing the amount of the product produced from the polypeptide.

[0084] In one embodiment, the recombinant microorganism of the present application may be a microorganism in which at least one activity selected from NADH:quinone oxidoreductase and O-phosphoserine export protein is further enhanced compared to the endogenous activity.

[0085] In any of the above-mentioned embodiments, the recombinant microorganism of the present application may be a microorganism in which the activity of NADH:quinone oxidoreductase is enhanced.

[0086] In this application, "NADH:quinone oxidoreductase (NADH:quinone oxidoreductase, hereinafter referred to as "Nuo")" refers to an enzyme that oxidizes NADH in the electron transport system of a microorganism and reduces quinone in the intracellular membrane. This enzyme protein is also called NADH dehydrogenase-1 (NDH-1). A gene encoding this protein is, for example, but not limited to, the nuoABCEFGHIJKLMN gene cluster. The nuoABCEFGHIJKLMN gene cluster constitutes the nuo operon, and its expression can be regulated by a promoter upstream of the operon and a polynucleotide in a ribosome binding site. In this application, the term "nuoABCEFGHIJKLMN gene" is used interchangeably with "gene encoding NADH:quinone oxidoreductase," "nuoABCEFGHIJKLMN gene," "nuo operon," and "nuo gene."

[0087] Nuo is a complex of 13 subunit proteins (NuoA, NuoB, NuoC, NuoE, NuoF, NuoG, NuoH, NuoI, NuoJ, NuoK, NuoL, NuoM, and NuoN). Translation of each subunit protein uses two operons, nuoABCEFGHIJKL and nuoMN, as templates. The structure of the nuo operon can be confirmed in EcoCyc (Non-Patent Document 21) (Accession Number: EG12082). The nuo operon is known to contain structural genes and expression regulatory regions. The "expression regulatory region" of the nuo operon refers to a site located upstream of the structural genes that make up the nuo operon and capable of regulating the expression of the structural genes. The expression regulatory region of the nuo operon may include a promoter (nuoA promoter and / or nuoM promoter) excluding the structural gene and an operator, and specifically may include a promoter.

[0088] The operon is as described above.

[0089] In one embodiment, the activity of the NADH:quinone oxidoreductase may be enhanced by mutation so that the activity of the NADH:quinone oxidoreductase is enhanced compared to the endogenous activity, or by enhancing or introducing the nuo operon.

[0090] The nuo operon of the present application reduces NADH accumulation due to the enhancement of the OPS biosynthetic pathway, and therefore, a microorganism mutated to enhance the activity of the NADH:quinone oxidoreductase or a microorganism into which the nuo operon has been introduced is characterized by increased OPS production and is useful for OPS production.

[0091] The nuo operon may comprise a base sequence encoding an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity to at least one of the amino acid sequences represented by SEQ ID NOs: 45 to 57. Specifically, the nuo operon may comprise a structural gene sequence encoding an amino acid sequence represented by SEQ ID NOs: 45 to 57, or an amino acid sequence having homology or identity thereto and corresponding function, and an expression regulatory region that regulates the expression of the structural gene sequence. The sequences of SEQ ID NOs: 45 to 57 can be confirmed in the publicly known database NCBI Genbank.

[0092] Specifically, the nuo operon may be SEQ ID NO: 3, and / or a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to SEQ ID NO: 3. Needless to say, the present application also includes nucleotide sequences in which a portion of the sequence has been deleted, modified, substituted, or added, as long as the nucleotide sequence has such homology or identity and has a function corresponding to the nuo operon.

[0093] In any of the above-mentioned embodiments, the recombinant microorganism of the present application may be a microorganism in which SerB activity is attenuated.

[0094] Since "SerB" in the present application has the activity of converting OPS to L-serine, microorganisms mutated to reduce the SerB activity are characterized by accumulating OPS and are useful for producing OPS. The SerB in the present application may be, but is not limited to, a protein having the amino acid sequence set forth in SEQ ID NO: 4, a protein containing the amino acid sequence, a protein consisting of the amino acid sequence, or a protein substantially consisting of the amino acid sequence. Furthermore, the SerB in the present application may have or contain an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 4, as long as it exhibits SerB activity. Furthermore, the SerB in the present application may be, but is not limited to, an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 4, or a protein that is substantially consisting of the amino acid sequence. Furthermore, the polynucleotide encoding SerB may have a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 4, or may include said nucleotide sequence. Furthermore, the polynucleotide encoding SerB may consist of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 4, or may essentially consist of said nucleotide sequence. The polynucleotide encoding SerB of the present application may undergo various modifications in the coding region, taking into account codon degeneracy or codons preferred in the organism in which the SerB protein is to be expressed, as long as the amino acid sequence of the SerB protein is not changed. The polynucleotide encoding SerB of the present application may have a nucleotide sequence that is at least 70%, 80%, 90%, 95%, or 99% homologous or identical to, but less than 100%, the nucleotide sequence of SEQ ID NO: 5, or may include said nucleotide sequence.Furthermore, the polynucleotide encoding SerB of the present application may consist of a base sequence that has at least 70%, 80%, 90%, 95%, or 99% or more, but less than 100%, homology or identity to the base sequence of SEQ ID NO: 5, or may be substantially composed of the base sequence, but is not limited to these.

[0095] In any of the above-described embodiments, the recombinant microorganism of the present application may be a microorganism in which the activity of an O-phosphoserine export protein is enhanced, for example, a microorganism in which the activity of YhhS is enhanced.

[0096] Because the "YhhS" of the present application has the activity of excreting OPS, microorganisms mutated to enhance the YhhS activity are characterized by their ability to excrete OPS and are useful for producing OPS. The YhhS of the present application may be, but is not limited to, a protein having the amino acid sequence set forth in SEQ ID NO: 6, a protein containing the amino acid sequence, a protein consisting of the amino acid sequence, or a protein essentially consisting of the amino acid sequence. Furthermore, the YhhS of the present application may have or contain an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 6, as long as it exhibits O-phosphoserine excretion activity. Furthermore, the YhhS of the present application may be, but is not limited to, an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 6, or a protein essentially consisting of the amino acid sequence. Furthermore, the polynucleotide encoding YhhS may have or contain a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 6. Furthermore, the polynucleotide encoding YhhS may consist of or essentially consist of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 6. The polynucleotide encoding YhhS of the present application can undergo various modifications in the coding region, taking into account codon degeneracy or preferred codons in the organism in which the YhhS protein is to be expressed, as long as the amino acid sequence of the YhhS protein is not altered. The polynucleotide encoding YhhS of the present application may have or contain a nucleotide sequence that is at least 70%, 80%, 90%, 95%, or 99% homologous or identical to, but less than 100%, the nucleotide sequence of SEQ ID NO: 7.Furthermore, the polynucleotide encoding YhhS of the present application may consist of a base sequence that is at least 70%, 80%, 90%, 95%, or 99% homologous or identical to, but less than 100% identical to, the base sequence of SEQ ID NO: 7, or may essentially consist of the base sequence, but is not limited to these.

[0097] In one embodiment, microorganisms modified to enhance OPS production and / or extracellular export ability or to enhance OPS degradation and / or uptake ability include, but are not limited to, CA07-0012 (KCCM11121P; Patent Document 3), CA07-4821, or CA07-4828. In addition to the above, other OPS-producing microorganisms disclosed in Patent Documents 3 and 8 are incorporated herein by reference, but are not limited thereto.

[0098] Another aspect of the present application provides a method for producing O-phosphoserine, comprising the step of culturing in a medium an O-phosphoserine-producing recombinant microorganism in which the activity of the MntH protein is enhanced compared to the endogenous activity.

[0099] The MntH protein, endogenous activity, enhancement, O-phosphoserine, and microorganisms are as described above.

[0100] The term "culturing" in the present application means growing the microorganism under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using a suitable medium and culture conditions known in the art. Those skilled in the art can easily adjust such a culturing process depending on the selected strain. Specifically, the culturing may be batch, continuous, or fed-batch culture, but is not limited thereto.

[0101] The culture medium for culturing the microorganism may further contain glycine or serine. Glycine is supplied in the form of purified glycine, a yeast extract containing glycine, or tryptone, and its concentration in the culture medium is usually 0.1 to 10 g / L, specifically 0.5 to 3 g / L. Serine is supplied in the form of purified serine, a yeast extract containing serine, or tryptone, and its concentration in the culture medium is usually 0.1 to 5 g / L, specifically 0.1 to 1 g / L.

[0102] Carbon sources contained in the medium include, but are not limited to, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose, fats and oils such as soybean oil, sunflower oil, castor oil, and coconut oil, fatty acids such as palmitic acid, stearic acid, and linoleic acid, alcohols such as glycerin and ethanol, and organic acids such as acetic acid. These substances can be used alone or in combination.

[0103] Nitrogen sources contained in the medium include, but are not limited to, organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal, and urea, and inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. These nitrogen sources can be used alone or in combination.

[0104] Phosphorus sources contained in the medium include, but are not limited to, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding sodium-containing salts.

[0105] The medium may also contain metal salts such as magnesium sulfate and iron sulfate, as well as amino acids, vitamins, suitable precursors, etc. These media or precursors can be added to the culture in a batch or continuous manner, but are not limited thereto.

[0106] The pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in a suitable manner during cultivation. Furthermore, foam formation can be suppressed during cultivation using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, oxygen or an oxygen-containing gas may be injected into the culture to maintain an aerobic state, while anaerobic and microaerobic states can be maintained without gas injection, or by injecting nitrogen, hydrogen, or carbon dioxide gas. The temperature of the culture is usually 25°C to 40°C, specifically 30°C to 35°C. The cultivation period of the culture is continued until the desired amount of useful substance is produced, specifically 10 to 100 hours. However, the cultivation period is not limited to these.

[0107] The OPS production method of the present application may further include a step of preparing the microorganism of the present application, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example, before the culturing step.

[0108] The OPS production method of the present application may further include a step of recovering OPS from the culture medium (the medium in which the culture was carried out) or the cultured microorganism. The recovery step may be further included after the culture step.

[0109] The recovery may involve collecting the target OPS using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof can be used. The target OPS can be recovered from the medium or the microorganism using a suitable method known in the art.

[0110] The OPS production method of the present application may further include a purification step. The purification can be carried out by any suitable method known in the art. For example, when the OPS production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out continuously or discontinuously in any order, or may be carried out simultaneously or integrated into a single step, but are not limited to these. Therefore, as described above, the recovered OPS may be in a purified form or may be a microbial fermentation broth containing OPS. Furthermore, OPS can be efficiently recovered by adding a suitable method known in the art before or after the culturing step or before or after the recovery step.

[0111] In yet another aspect, the present application provides a method for producing cysteine ​​or a derivative thereof, the method comprising: a) culturing in a medium an O-phosphoserine-producing recombinant microorganism in which the activity of the MntH protein is enhanced compared to the endogenous activity, to produce O-phosphoserine or a medium containing O-phosphoserine; and b) reacting the O-phosphoserine produced in step a) or the medium containing O-phosphoserine with sulfide in the presence of O-phosphoserine sulfhydrylase (OPSS) or a microorganism containing the same.

[0112] The steps a) and b) are not necessarily limited to being performed consecutively or in order, and there may be no time interval between the steps, and the steps may be performed simultaneously, or may be performed with an interval of several seconds, several minutes, several hours, or several days.

[0113] The MntH protein, endogenous activity, enhancement, O-phosphoserine (OPS), and microorganisms are as described above.

[0114] In this application, the term "derivative" refers to a similar compound obtained by chemically changing a part of a compound, and generally refers to a compound in which a hydrogen atom or a specific atomic group in the compound is replaced with another atom or atomic group.

[0115] In the present application, the term "cysteine ​​derivative" refers to a compound in which a hydrogen atom or a specific atomic group of cysteine ​​is substituted with another atom or atomic group. Examples include forms in which another atom or atomic group is bound to the nitrogen atom of the amino group (-NH2) or the sulfur atom of the thiol group (-SH) of cysteine, such as N-acetylcysteine ​​(NAC), S-carboxymethylcysteine ​​(SCMC), BOC-CYS(ME)-OH, (R)-S-(2-Amino-2-carboxyethyl)-L-homocysteine, (R)-2-Amino-3-sulfopropionic acid, D-2-Amino-4-(ethylthio)butyric acid, 3-sulfino-L-alanine, Fmoc-Cys(Boc-methyl)-OH, seleno-L-cystine, S-(2-Thiazolyl)-L-cysteine, S-(2-Thienyl)-L-cysteine, and S-(4-Tolyl)-L-cysteine, but are not limited to these.

[0116] If cysteine ​​is produced by the method of the present application, it may be easily converted into various cysteine ​​derivatives by methods well known in the art.

[0117] In the present application, the method for producing a cysteine ​​derivative may further comprise the step of converting the cysteine ​​produced in step b) into a cysteine ​​derivative.

[0118] Specifically, in the present application, a method for producing a cysteine ​​derivative may include the steps of producing cysteine ​​by the method of the present application described above, and converting the produced cysteine ​​into a cysteine ​​derivative as described above.

[0119] The step of converting the cysteine ​​produced as described above into a cysteine ​​derivative can be carried out by a method known in the art, for example, by a method known in the art, such as reacting cysteine ​​with an acetylation agent to synthesize N-acetylcysteine ​​(NAC), or reacting cysteine ​​with haloacetic acid under basic conditions to synthesize S-carboxymethylcysteine ​​(SCMC), but is not limited to these.

[0120] The cysteine ​​derivatives are mainly used as pharmaceutical raw materials in antitussives, cough suppressants, and therapeutic agents for bronchitis, bronchial asthma, sore throat, etc., but are not limited thereto.

[0121] In this application, "O-phosphoserinesulfhydrylase (OPSS)" refers to an enzyme that catalyzes the reaction of converting OPS to cysteine ​​by donating a thiol group (SH group) to OPS. This enzyme has been found in Aeropyrum pernix, Mycobacterium tuberculosis, Mycobacterium smegmatis, and Trichomonas vaginalis (Non-Patent Documents 22, 23). Furthermore, the OPSS includes not only wild-type OPSS protein, but also mutant proteins in which a portion of the polynucleotide sequence encoding the OPSS has been deleted, substituted, or added, and which exhibit biological activity equivalent to or greater than that of the wild-type OPSS protein, including all of the OPSS proteins and their mutant proteins disclosed in Patent Documents 9 and 10.

[0122] The sulfide may be supplied not only in the form of a solid commonly used in the art, but also in the form of a liquid or gas depending on differences in pH, pressure, and solubility. Any sulfide that can be converted to a thiol group (SH group) in the form of a sulfide (S-) or thiosulfate (S-) may be used. Specific examples include, but are not limited to, NaS, NaSH, (NH)S, HS, and NaSO, which donate thiol groups to OPS. The reaction involves donating one thiol group to one OPS functional group to produce one cysteine ​​or cysteine ​​derivative. The amount of sulfide added in the reaction is 0.1 to 3 times, specifically 1 to 2 times, the molar concentration of OPS, but is not limited thereto.

[0123] The present invention may further include a step of recovering the cysteine ​​produced in the reaction step, in which the target cysteine ​​can be separated, purified, and recovered from the reaction solution using a suitable reaction known in the art.

[0124] Yet another aspect of the present application provides a composition for producing O-phosphoserine, cysteine, or a cysteine ​​derivative, comprising an O-phosphoserine-producing recombinant microorganism in which the activity of the MntH protein is enhanced compared to the endogenous activity, a medium in which the same is cultured, or a combination thereof.

[0125] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing O-phosphoserine, cysteine, or cysteine ​​derivatives, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0126] Yet another aspect of the present application provides a method for producing a recombinant microorganism that produces O-phosphoserine, the method comprising the step of enhancing the activity of the MntH protein above its endogenous activity.

[0127] Yet another aspect of the present application provides use of an O-phosphoserine-producing recombinant microorganism in which the activity of the MntH protein is enhanced compared to the endogenous activity, for producing O-phosphoserine, cysteine, or a cysteine ​​derivative.

[0128] The MntH protein, intrinsic activity, enhancement, O-phosphoserine (OPS), cysteine, cysteine ​​derivatives, and microorganisms are as described above. [Example]

[0129] The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the present application is not limited thereto. Note that technical matters not described in this specification are well understood and easily implemented by skilled artisans in the technical field of the present application or a similar technical field. [Example 1]

[0130] Construction of an O-phosphoserine (OPS)-producing strain with enhanced YhhS expression 1-1. Construction of a vector for enhancing YhhS expression Using the chromosomal DNA of wild-type Escherichia coli ATCC27325 as a template, PCR was performed to obtain a gene fragment upstream of the wild-type promoter of the yhhS gene (SEQ ID NO: 7), which undergoes homologous recombination, using the primer pair of SEQ ID NOs: 10 and 11, and to obtain a gene fragment downstream of the wild-type promoter of the yhhS gene using the primer pair of SEQ ID NOs: 14 and 15. PCR was also performed to obtain the Ptrc promoter (SEQ ID NO: 59) using pCL_Ptrc-gfp (Patent Document 11) as a template and the primer pair of SEQ ID NOs: 12 and 13.

[0131] To perform PCR, Solg was used as the polymerase. TMPfu-X DNA polymerase was used, and the PCR amplification conditions were as follows: denaturation at 95°C for 2 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 60 seconds, followed by polymerization at 72°C for 5 minutes.

[0132] The upstream and downstream fragments of the yhhS promoter and the Ptrc promoter fragment obtained above were cloned into the chromosomal transformation vector pSKH130 (SEQ ID NO: 58, Patent Document 4) digested with EcoRV restriction enzyme using an in-fusion cloning kit (Clontech Laboratories, Inc.) to obtain a recombinant plasmid designated pSKH_Ptrc-yhhS.

[0133] The primer sequences used here are shown in Table 1.

[0134] [Table 1]

[0135] 1-2. Construction of YhhS-enhanced strain The pSKH_Ptrc-yhhS prepared in Example 1-1 was introduced into the OPS-producing strain CA07-0012 (KCCM11121P, Patent Document 2), which is a wild-type Escherichia coli K-12 W3110 strain lacking endogenous phosphoserine phosphatase (SerB) and thus exhibiting weakened OPS degradation ability. By enhancing expression of YhhS (SEQ ID NO: 6), a protein with OPS export ability, the OPS export ability was further improved.

[0136] The CA07-0012 strain was transformed with pSKH_Ptrc-yhhS prepared in Example 1-1 by electroporation (Non-Patent Document 24), followed by a first crossover using R6K and kanamycin to obtain the desired strain. A second crossover was then performed in sucrose-containing medium to obtain a strain in which the kanamycin resistance gene had been deleted and the Ptrc promoter sequence had been inserted at the end of the wild-type promoter sequence of the yhhS gene. The insertion of the Ptrc promoter sequence was confirmed by PCR amplification using the primer pair of SEQ ID NOs: 16 and 17, which amplify the exogenous regions upstream and downstream of the homologous recombination, respectively, and genome sequencing. The resulting strain was designated CA07-4821 (CA07-0012ΔPn_yhhS::Ptrc_yhhS).

[0137] The primer sequences used here are shown in Table 2.

[0138] [Table 2] [Example 2]

[0139] Construction of an OPS-producing strain carrying the nuo operon 2-1. Construction of a vector for introducing the nuo operon Using the chromosomal DNA of wild-type E. coli ATCC27325 as a template, PCR was performed to obtain a gene fragment of the upstream region of the wild-type promoter of the nuo operon (SEQ ID NO: 3) using the primer pair of SEQ ID NO: 18 and SEQ ID NO: 19, and to obtain a gene fragment of the downstream region of the wild-type promoter of the nuo operon using the primer pair of SEQ ID NO: 20 and SEQ ID NO: 21. PCR was also performed to obtain the promoter region of the rmf gene (SEQ ID NO: 8) using the chromosomal DNA of wild-type E. coli ATCC27325 as a template and the primer pair of SEQ ID NO: 22 and SEQ ID NO: 23.

[0140] To perform PCR, Solg was used as the polymerase. TMPfu-X DNA polymerase was used, and the PCR amplification conditions were as follows: denaturation at 95°C for 2 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 60 seconds, followed by polymerization at 72°C for 5 minutes.

[0141] The upstream and downstream fragments of the nuo promoter and the rmf promoter fragment obtained above were cloned into the chromosomal transformation vector pSKH130 cleaved with EcoRV restriction enzyme using an infusion cloning kit to obtain a recombinant plasmid designated pSKH_Prmf-nuoA.

[0142] The primer sequences used here are shown in Table 3.

[0143] [Table 3]

[0144] 2-2. Construction of nuo operon-introduced strain The OPS-producing strain CA07-4821, which lacks endogenous phosphoserine phosphatase and thus has reduced OPS degradation ability, was transformed by electroporation with pSKH_Prmf-nuoA prepared in Example 2-1. The desired strain was then isolated using R6K and kanamycin in a first crossover step. A second crossover step was then performed in sucrose-containing medium to obtain a strain in which the kanamycin resistance gene was deleted and the promoter sequence of the rmf gene was inserted at the end of the wild-type promoter sequence of the nuo operon. The insertion of the rmf promoter sequence was confirmed by PCR amplification using the primer pair of SEQ ID NOs: 24 and 25, which amplify the exogenous regions upstream and downstream of the homologous recombination, respectively, and genome sequencing. The resulting strain was designated CA07-4828 (CA07-4821ΔPn_nuoA::Prmf_nuoA).

[0145] The primer sequences used here are shown in Table 4.

[0146] [Table 4] [Example 3]

[0147] Generation of strains with enhanced or deleted MntH expression 3-1. Construction of a vector for enhancing MntH expression Previous research has shown that the average transcription levels of the mntH, serC, and rmf genes in OPS-producing host strains were 698, 6215, and 32205, respectively, as shown in Table 5. Compared to the average transcription level of the mntH gene, the average transcription level of the rmf gene was 46-fold higher, and the average transcription level of the serC gene was 8.9-fold higher. This confirms that the rmf and serC gene promoters are relatively stronger promoters than the mntH gene promoter.

[0148] [Table 5]

[0149] Therefore, the rmf promoter and serC promoter (SEQ ID NO: 9), which have been confirmed to have stronger activity, were further inserted at the end of the promoter of the mntH gene (SEQ ID NO: 2) in the OPS-producing microorganism to create a strain with enhanced MntH expression.

[0150] Using the chromosomal DNA of wild-type E. coli ATCC27325 as a template, PCR was performed to obtain a gene fragment in the upstream region of the wild-type promoter of the mntH gene using the primer pair of SEQ ID NOs: 26 and 27, and to obtain a gene fragment in the downstream region of the wild-type promoter of the mntH gene using the primer pair of SEQ ID NOs: 28 and 29. PCR was also performed to obtain the promoter region of the rmf gene using the chromosomal DNA of wild-type E. coli ATCC27325 as a template and the primer pair of SEQ ID NOs: 30 and 31.

[0151] To perform PCR, Solg was used as the polymerase.TM Pfu-X DNA polymerase was used, and the PCR amplification conditions were as follows: denaturation at 95°C for 2 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 60 seconds, followed by polymerization at 72°C for 5 minutes.

[0152] The upstream and downstream fragments of the mntH promoter and the rmf promoter fragment obtained above were cloned into the chromosomal transformation vector pSKH130 cleaved with EcoRV restriction enzyme using an infusion cloning kit to obtain a recombinant plasmid designated pSKH130_Prmf-mntH.

[0153] Using the chromosomal DNA of wild-type E. coli ATCC27325 as a template, PCR was performed to obtain a gene fragment in the upstream region of the wild-type promoter of the mntH gene using the primer pair of SEQ ID NOs: 32 and 33, and to obtain a gene fragment in the downstream region of the wild-type promoter of the mntH gene using the primer pair of SEQ ID NOs: 34 and 35. PCR was also performed to obtain the promoter region of the serC gene using the chromosomal DNA of wild-type E. coli ATCC27325 as a template and the primer pair of SEQ ID NOs: 36 and 37.

[0154] To perform PCR, Solg was used as the polymerase. TM PCR was performed using Pfu-X DNA polymerase under the following PCR conditions: denaturation at 95°C for 2 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 60 seconds, followed by polymerization at 72°C for 5 minutes.

[0155] The upstream and downstream fragments of the mntH promoter and the serC promoter fragment obtained above were cloned into the chromosomal transformation vector pSKH130 cleaved with EcoRV restriction enzyme using an infusion cloning kit to obtain a recombinant plasmid designated pSKH130_PserC-mntH.

[0156] 3-2. Construction of a vector for MntH deficiency Using the chromosomal DNA of wild-type E. coli ATCC27325 as a template, PCR was performed using the primer pair of sequence numbers 38 and 39 to obtain a gene fragment in the upstream region of the wild-type promoter of the mntH gene, and the primer pair of sequence numbers 40 and 41 to obtain a gene fragment in the downstream region of the wild-type promoter of the mntH gene.

[0157] PCR was performed in the same manner as in Example 3-1 to obtain upstream and downstream fragments of the mntH promoter, which were then cloned in the same manner as in Example 3-1 to obtain a recombinant plasmid, which was named pSKH_ΔmntH.

[0158] The primer sequences used in Examples 3-1 and 3-2 are shown in Table 6.

[0159] [Table 6]

[0160] 3-3. Construction of strains with enhanced MntH expression The CA07-4821 strain, an OPS-producing strain with enhanced YhhS expression (Example 1-2), was transformed by electroporation with the pSKH_PserC-mntH gene prepared in Example 3-1. A secondary crossover process was then performed to obtain a strain in which the promoter sequence of the serC gene was inserted at the end of the wild-type promoter sequence of the mntH gene. The insertion of the serC promoter sequence was confirmed by PCR amplification using the primer pair of SEQ ID NOs: 42 and 43, which amplify the exogenous regions upstream and downstream of the homologous recombination, respectively, and genome sequencing. The resulting strain was designated CA07-4895 (CA07-4821ΔPn_mntH::PserC_mntH).

[0161] The CA07-4828 strain, an OPS-producing strain incorporating the nuo operon of Example 2-2, was transformed by electroporation with pSKH_Prmf-mntH and pSKH_PserC-mntH prepared in Example 3-1. Subsequently, a secondary crossover process was performed to obtain strains in which the promoter sequences of the serC and rmf genes, respectively, were inserted at the termini of the native promoter sequences of the mntH gene. The insertion of the serC or rmf promoter sequences was confirmed by PCR amplification using the primer pair of SEQ ID NOs: 42 and 43, which amplify the exogenous regions upstream and downstream of the homologous recombination, respectively, and genome sequencing. The resulting strains were designated CA07-4898 (CA07-4828ΔPn_mntH::PserC_mntH) and CA07-4899 (CA07-4828ΔPn_mntH::Prmf_mntH), respectively.

[0162] 3-4. Construction of MntH-deficient strain The CA07-4828 strain, an OPS-producing strain incorporating the nuo operon of Example 2-2, was transformed by electroporation with the pSKH_ΔmntH prepared in Example 3-2, followed by a secondary crossover process to obtain a strain lacking the mntH gene. The deletion of the mntH nucleotide sequence was confirmed by PCR amplification using the primer pair of SEQ ID NOs: 42 and 44, which amplify the exogenous regions upstream and downstream of the homologous recombination, respectively, and genome sequencing. The resulting strain was designated CA07-4897 (CA07-4828ΔmntH).

[0163] The primer sequences used in Examples 3-3 and 3-4 are shown in Table 7.

[0164] [Table 7] [Example 4]

[0165] Evaluation of strain OPS production ability Flask fermentation titer evaluation was performed to measure the OPS production ability of the MntH expression-enhanced strains CA07-4895, CA07-4898, and CA07-4899 of Example 3-3, the MntH-deficient strain CA07-4897 of Example 3-4, the parent strain CA07-0012 of Example 1-2 as a control strain, the CA07-4821 of Example 1-2, and the CA07-4828 of Example 2-2.

[0166] Each strain was smeared on LB solid medium and then cultured overnight in an incubator at 33°C. The strain cultured overnight on LB solid medium was inoculated into 25 mL of the next titer medium, which was then cultured in an incubator at 33°C and 200 rpm for 48 hours. After the culture was completed, the OPS concentration was measured using HPLC. The results are shown in Table 8. <Titer medium> Glucose 40g / L, KH2PO4 6g / L, (NH4)2SO4 17g / L, MgSO4·7H2O 1g / L, MnSO4·4H2O 5mg / L, FeSO4·7H2O 10mg / L, L-glycine 1.5g / L, yeast extract 2.5g / L, CaCO3 30g / L, pH 6.8

[0167] [Table 8]

[0168] As shown in Table 8, the MntH-enhanced strain CA07-4895, which was derived from the YhhS-enhanced strain CA07-4821 as a parent strain, had a 9.4% improvement in OPS production ability compared to the parent strain.

[0169] The CA07-4828 strain, in which the YhhS and nuo operons were simultaneously enhanced, showed a 7.9% improvement in OPS production compared to the CA07-4821 strain in which the YhhS was enhanced.

[0170] Furthermore, the MntH-enhanced strains CA07-4898 and CA07-4899, derived from the nuo operon-enhanced parent strain CA07-4828, showed 2.3% and 5.3% higher OPS production, respectively, than the parent strain. The MntH-deficient strain CA07-4897 showed a 10.3% decrease in OPS production compared to the parent strain.

[0171] Therefore, it was confirmed that strengthening MntH improves OPS production.

[0172] From the above description, those skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing the technical idea or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present application should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalent concepts.

Claims

1. The activity of the MntH protein is enhanced compared to the endogenous activity. O-phosphoserine-producing recombinant microorganisms.

2. The recombinant microorganism has an ability to produce O-phosphoserine that is enhanced compared to its endogenous ability to produce O-phosphoserine. The microorganism described in claim 1.

3. The MntH protein comprises the amino acid sequence of SEQ ID NO:

1. The microorganism described in claim 1.

4. The polynucleotide encoding the MntH protein comprises the base sequence of SEQ ID NO:

2. The microorganism described in claim 1.

5. The recombinant microorganism further has at least one activity selected from NADH:quinone oxidoreductase and O-phosphoserine export protein enhanced compared to its endogenous activity. The microorganism described in claim 1.

6. The recombinant microorganism is of the genus Escherichia. The microorganism described in claim 1.

7. Cultivating in a medium an O-phosphoserine-producing recombinant microorganism in which the activity of the MntH protein is enhanced compared to the endogenous activity; A method for producing O-phosphoserine.

8. The method further comprises recovering O-phosphoserine from the culture medium or the microorganism. The method for producing O-phosphoserine according to claim 7.

9. The MntH protein comprises the amino acid sequence of SEQ ID NO:

1. The method for producing O-phosphoserine according to claim 7.

10. The polynucleotide encoding the MntH protein comprises the base sequence of SEQ ID NO:

2. The method for producing O-phosphoserine according to claim 7.

11. a) culturing an O-phosphoserine-producing recombinant microorganism in which the activity of the MntH protein is enhanced compared to the endogenous activity in a medium to produce O-phosphoserine or a medium containing O-phosphoserine; b) reacting the O-phosphoserine produced in step a) or a medium containing the same with sulfide in the presence of O-phosphoserinesulfhydrylase (OPSS) or a microorganism containing the same; A method for producing cysteine ​​or a derivative thereof.

12. The sulfide is Na ２ S, NaSH, (NH ４ ) ２ S, H ２ S and Na ２ S ２ O ３ At least one selected from the group consisting of: The method for producing cysteine ​​or a derivative thereof according to claim 11.

13. The MntH protein comprises the amino acid sequence of SEQ ID NO:

1. The method for producing cysteine ​​or a derivative thereof according to claim 11.

14. The polynucleotide encoding the MntH protein comprises the base sequence of SEQ ID NO:

2. The method for producing cysteine ​​or a derivative thereof according to claim 11.

15. Use of an O-phosphoserine-producing recombinant microorganism in which the activity of the MntH protein is enhanced compared to the endogenous activity for producing O-phosphoserine, cysteine ​​or a cysteine ​​derivative.

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