Recombinant microorganism with regulated expression of alkylhydroperoxide reductase and 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 alkyl hydroperoxide reductase 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 expression of alkyl hydroperoxide reductase 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 in which D,L-ATC (D,L-2-amino-2-thiazoline-4-carboxylate) is biologically converted using microorganisms; 2) a direct fermentation method in which L-cysteine ​​is produced using Escherichia coli (European Patent EP 0885962B; Wada M and Takagi H, Appl. Microbiol. Biochem., 73:48-54, 2006); and 3) a method in which O-phosphoserine (hereinafter referred to as "OPS") is produced by fermentation 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 ​​(US Pat. No. 8,557,549 B2).

[0004] In order to produce cysteine ​​in high yield using the method 3), it is necessary to overproduce the precursor OPS. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] European Patent EP0885962B [Patent Document 2] US 8557549 B2 [Patent Document 3] US 2012-0190081 A1 [Patent Document 4] US Published Patent US 2020-0048619 [Patent Document 5] US Patent US 8557549 B2 [Patent Document 6] US Patent US 7662943 B2 [Patent Document 7] US Patent US 10584338 B2 [Patent Document 8] US Patent US 10273491 B2 [Patent Document 9] Korean Patent No. 1381048 [Patent Document 10] Korean Patent Publication No. 2012-0041115 [Patent Document 11] Korean Patent No. 1208267 [Patent Document 12] International Patent Publication WO 2016-024771 A1 [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

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[0007] An object of the present application is to provide a recombinant microorganism in which expression of alkylhydroperoxide reductase is regulated, and a method for producing O-phosphoserine, cysteine, and derivatives thereof using the same. [Means for solving the problem]

[0008] One object of the present application is to provide a recombinant microorganism that produces O-phosphoserine, in which the activity of the Ahp 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 aspect 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 Ahp protein of the present application is enhanced compared to the endogenous activity, O-phosphoserine can be produced at a higher yield than when using existing non-transformed strains. DETAILED DESCRIPTION OF THE INVENTION

[0012] This will be explained in more detail as follows: Meanwhile, each description and embodiment disclosed in this application also applies to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the specific descriptions set forth below are not deemed to limit the category of this application. Furthermore, numerous 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 to more clearly explain the state of the art to which this application pertains and the contents of this application.

[0013] One aspect of the present application provides a recombinant microorganism that produces O-phosphoserine, in which the activity of the Ahp protein is enhanced compared to the endogenous activity.

[0014] In this application, the term "O-phosphoserine (hereinafter "OPS")" refers to a phosphoric acid ester of serine and is a component of various proteins. OPS is a precursor of L-cysteine ​​and can be converted to cysteine ​​by reacting with sulfide under the catalytic action of O-phosphoserine sulfhydrylase (OPS sulfhydrylase, OPSS), but is not limited thereto (US 2012-0190081 A1).

[0015] In this application, the term "alkyl hydroperoxide reductase (Ahp)" refers to an enzyme that converts NADH to NAD + The alkyl hydroperoxide reductase may be referred to as an Ahp protein or Ahp.

[0016] The Ahp protein may consist of AhpC (alkyl hydroperoxide reductase subunit C) and AhpF (alkyl hydroperoxide reductase subunit F). The amino acid sequence of AhpC or AhpF can be obtained from a publicly known database such as NCBI Genebank.

[0017] As an example, the AhpC or AhpF 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 AhpC in the present application may be UMQ15446.1 derived from Escherichia coli, and the amino acid sequence of AhpF may be WP_000979839.1 derived from Escherichia coli. However, it is clear that the present application encompasses proteins having AhpC or AhpF activity from various sources. For example, AhpC may be UMQ15446.1, EFF4225430.1, or WP_000052796.1, and AhpF may be NCBI Accession No. ANK05951.1, EFS6382721.1, or HCD8516410.1.

[0019] In the present application, AhpC may have, comprise, consist of, or essentially consist of the amino acid sequence set forth in SEQ ID NO: 1. In the present application, AhpF may have, comprise, consist of, or essentially consist of the amino acid sequence set forth in SEQ ID NO: 3.

[0020] In the present application, the amino acid sequence of the AhpC may comprise an amino acid sequence having 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 homology or identity to the amino acid sequence of SEQ ID NO: 1. In the present application, the amino acid sequence of the AhpF may comprise an amino acid sequence having 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 homology or identity to the amino acid sequence of SEQ ID NO: 3. It is also clear that the scope of the present application also includes proteins having an amino acid sequence 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 corresponding to the protein comprising the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 3. As an example, AhpC may be composed of 187 to 193 amino acids including the amino acid sequence of SEQ ID NO: 1. As an example, AhpF may be composed of 521 to 531 amino acids including the amino acid sequence of SEQ ID NO: 3.

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

[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 the amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0023] As used herein, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid or nucleotide sequences, which can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0024] Homology or identity of conserved polynucleotide or polypeptide sequences can be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences are generally capable of hybridizing to all or a portion of the sequence under moderately or highly stringent conditions. Hybridization obviously includes hybridization to polynucleotides containing common codons in polynucleotides or codons that take codon degeneracy into account.

[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 using default parameters, e.g., as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later), as implemented in the 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 ET AL] (1988) SIAM J Applied Math 48:1073. 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 of Needleman et al. (1970), J Mol Biol. 48:443, as known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines a match as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a unitary matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (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.

[0027] AhpC and AhpF of the present application may be encoded by the ahpCF operon genes.

[0028] In this application, the term "operon" refers to a functional unit of DNA containing a group of genes whose expression is regulated by a single expression control sequence, specifically a single promoter. The mRNA transcribed by an operon may be polycistronic, in which a single mRNA molecule encodes one or more proteins, or monocistronic, in which a single mRNA molecule encodes one protein.

[0029] The term "ahpCF operon genes" may be used interchangeably with "ahpCF operon," "ahpCF genes," and "ahp genes."

[0030] The ahpCF operon genes may include the ahpC gene and the ahpF gene.

[0031] As an example, the ahpC gene may be a polynucleotide encoding UMQ15446.1 or a portion of CP101971.1 derived from Escherichia coli, and the ahpF gene may be a polynucleotide encoding WP_000979839.1 derived from Escherichia coli. However, without being limited thereto, it is clear that the invention includes ahpC and / or ahpF genes of various origins that encode proteins having AhpC and / or AhpF activity.

[0032] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, such as a DNA or RNA chain of a certain length or more, and more specifically, a polynucleotide fragment that encodes the protein.

[0033] A polynucleotide encoding the Ahp protein of the present application may comprise a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 3. As an example of the present application, a polynucleotide of the present application may have or comprise the nucleotide sequence of SEQ ID NO: 2 and / or SEQ ID NO: 4. Alternatively, a polynucleotide of the present application may consist of or consist essentially of the nucleotide sequence of SEQ ID NO: 2 and / or SEQ ID NO: 4. Specifically, the Ahp protein may be encoded by a polynucleotide described by the nucleotide sequence of SEQ ID NO: 2 and / or SEQ ID NO: 4.

[0034] The polynucleotide of the present application may have various modifications in the coding region thereof without altering the amino acid sequence of the Ahp protein, taking into account codon degeneracy or preferred codons in the organism in which the Ahp protein of the present application is to be expressed. Specifically, the polynucleotide of the present application may have, comprise, or consist essentially of a nucleotide sequence that is 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 homologous or identical to the sequence of SEQ ID NO: 2 and / or SEQ ID NO: 4, but is not limited thereto.

[0035] Furthermore, the polynucleotides of the present application may include, without limitation, any sequences that can hybridize under stringent conditions to probes prepared from known gene sequences, for example, sequences complementary to all or part of the polynucleotide sequences 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 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, pp. 9.50-9.51, 11.7-11.8). For example, conditions include conditions 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 under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization, such as 60°C, 1×SSC, and 0.1% SDS, specifically 60°C, 0.1×SSC, and 0.1% SDS, more specifically 68°C, 0.1×SSC, and 0.1% SDS.

[0036] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can also include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0037] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the above-mentioned hybridization conditions, including a hybridization step 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.

[0038] The appropriate stringency for hybridizing such polynucleotides depends on the length and degree of complementation of the polynucleotides, variables well known in the art (eg, J. Sambrook et al., supra).

[0039] As described above, an operon is a group of genes whose expression is regulated by a single promoter, and the expression of ahpC and ahpF in the ahpCF operon may be regulated by the ahpC promoter. Therefore, in one embodiment of the present application, the ahpCF operon promoter (ahp operon promoter) may be the ahpC promoter.

[0040] As used herein, the term "vector" refers to a DNA construct containing a polynucleotide sequence encoding a protein of interest in a suitable host, operably linked to regulatory sequences suitable for expressing the protein of interest. The expression regulatory sequences may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and sequences regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector can replicate or function independently of the host genome, or it can be integrated into the genome itself.

[0041] The vectors used in the present application are not particularly limited as long as they are 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, vectors such as pSKH130 (U.S. Patent Publication US 2020-0048619), pSK, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC can be used.

[0042] The insertion of the polynucleotide into the chromosome may be performed by any method known in the art, including, but not limited to, homologous recombination.

[0043] As used herein, the term "transformation" refers to the introduction of a recombinant vector containing a polynucleotide encoding a protein of interest into a host cell, thereby enabling the expression of the protein encoded by the polynucleotide in the host cell. The transformed polynucleotide may be located either intrachromosomally or extrachromosomally, as long as it can be expressed in the host cell. The transformation method may include any method for introducing nucleic acid into a cell, and may be performed using standard techniques known in the art depending on 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.

[0044] Furthermore, the term "operably linked" as used herein 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. The operable linkage can be produced using recombinant DNA techniques known in the art, and site-specific DNA cleavage and ligation can be produced using cleavage and ligation enzymes known in the art, but is not limited thereto.

[0045] The microorganism of the present application is not particularly limited in type as long as it is capable of producing OPS, and may be either a prokaryotic or eukaryotic cell, specifically a prokaryotic cell. Examples include microbial strains belonging to the genera Escherichia, Erwinia, Seratia, Providencia, Corynebacterium, and Brevibacterium, specifically, a microorganism of the genus Escherichia, more specifically, Escherichia coli (E. coli), but are not limited thereto. For example, microorganisms of the genus Escherichia can produce OPS and L-serine through the enzymes SerA, SerC, and SerB in the L-serine biosynthetic pathway (Ahmed Zahoor, Computational and structural biotechnology journal, vol. 3, 2012 October; Wendisch VF et al., Curr Opin Microbiol. 2006 Jun;9(3):268-74; Peters-Wendisch P et al., Appl Environ Microbiol. 2005 Nov;71(11):7 139-44.).

[0046] In the present application, the term "microorganism capable of producing O-phosphoserine (OPS)" refers to a microorganism that naturally has the ability to produce OPS or a microorganism in which the ability to produce OPS has been imparted to a parent strain that does not have the ability to produce OPS. Specifically, the microorganism may be an OPS-producing microorganism in which Ahp protein activity has been enhanced by natural or artificial genetic modification. For the purposes of the present application, the OPS-producing microorganism may be any microorganism in which Ahp protein activity has been enhanced by the method disclosed in the present application and which is capable of producing O-phosphoserine. In the present application, the term "microorganism capable of producing O-phosphoserine (OPS)" may be used interchangeably with "microorganism capable of producing O-phosphoserine (OPS)" or "microorganism capable of producing O-phosphoserine (OPS)."

[0047] In one embodiment, the OPS-producing microorganism of the present application may be, but is not limited to, a genetically modified or recombinant microorganism in which the activity of the Ahp protein is enhanced and the ability to produce the desired OPS is increased. The recombinant microorganism may be a microorganism in which the ability to produce O-phosphoserine is enhanced compared to its endogenous ability to produce O-phosphoserine.

[0048] The microorganism of the present application may have an increased activity of the Ahp protein compared to the endogenous activity. Specifically, the microorganism of the present application may be, but is not limited to, a microorganism in which the Ahp protein or the ahpCF operon genes encoding it have been enhanced; or a microorganism (e.g., a recombinant microorganism) that has been genetically modified to enhance the Ahp protein or the ahpCF operon genes encoding it.

[0049] As used herein, the term "enhancement" of a polypeptide (including, for example, the proteins specified in the names of each enzyme) means that the activity of the polypeptide is increased compared to its endogenous activity. The term "enhancement" may be used interchangeably with terms such as "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and increase can all encompass the display of an activity not inherently possessed, or the display of an activity improved compared to the endogenous activity or the activity before modification. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or an unaltered microorganism before the trait has been altered due to genetic mutation caused by natural or artificial factors. This term may be used interchangeably with "activity before modification." The term "enhancement," "up-regulation," "overexpression," or "increase" of a polypeptide compared to its endogenous activity means that the activity and / or concentration (expression level) of a specific polypeptide is improved compared to the activity and / or concentration (expression level) of a specific polypeptide inherently possessed by a parent strain or an unaltered microorganism before the trait has been altered.

[0050] The enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. The enhancement of the activity of the polypeptide can be confirmed by an increase in the activity, expression level, or amount of a product secreted from the polypeptide.

[0051] For the purposes of this application, the microorganism of the present application has improved OPS-producing ability due to enhanced Ahp protein activity. Non-transformed microorganisms without enhanced Ahp protein, which are used as control strains for comparing the presence or absence of increases in OPS-producing ability or Ahp protein, may include, but are not limited to, the OPS-producing strain CA07-0012 (KCCM 11121P, U.S. Patent No. US 8557549 B2), which lacks endogenous phosphoserine phosphatase (SerB) and has reduced OPS-degrading ability, and a strain of the CA07-0012 strain with enhanced YhhS expression, which has the ability to excrete OPS (CA07-4821 of the present application).

[0052] The activity of the polypeptide can be enhanced by various methods well known in the art, and is not limited thereto, as long as the activity of the polypeptide of interest can be enhanced compared to that of the microorganism before transformation. Specifically, the enhancement may be achieved by using genetic engineering and / or protein engineering, which are routine methods in molecular biology and well known to those skilled in the art, but is not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2, pp. 1-16; Sambrook et al., Molecular Cloning 2012, etc.).

[0053] Specifically, the activity of the polypeptide of the present application is enhanced by: 1) an increase in the intracellular copy number of the polynucleotide encoding the polypeptide; 2) modification of the gene expression regulatory sequence on the chromosome encoding the polypeptide; 3) a modification of the nucleotide sequence encoding the initiation codon or 5'UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the polypeptide activity is enhanced; 5) modifying a polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide's activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide's activity); 6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same; 7) codon optimization of the polynucleotide encoding the polypeptide; 8) Analyzing the tertiary structure of the polypeptide and selectively deforming or chemically modifying exposed sites; 9) Regulation of the cellular localization of a polypeptide; or 10) It may be a combination of two or more selected from the above 1) to 9), but is not particularly limited thereto.

[0054] More specifically, The 1) increase in the intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a host cell a vector that is operably linked to a polynucleotide encoding the polypeptide and that can replicate and function 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 that can insert the polynucleotide into a chromosome in the host cell.

[0055] 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 may be, for example, by introducing a mutation in the sequence through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or by replacing the sequence with a sequence with stronger activity. The expression regulatory region may include, 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. Specifically, the method may involve, but is not limited to, inserting a strong promoter after the original promoter.

[0056] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (U.S. Patent No. 7,662,943 B2), 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 (U.S. Patent No. 10,584,338 B2), the O2 promoter (U.S. Patent No. 10,273,491 B2), the tkt promoter, the yccA promoter, the rmf promoter, and the serC promoter.

[0057] The modification of the nucleotide sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide (3) may be, for example, but is not limited to, substituting a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon.

[0058] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above may be, but is not limited to, the generation of mutations in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to enhance the activity of the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity or an amino acid sequence or polynucleotide sequence improved to increase activity.Specifically, the replacement can be performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited to these.

[0059] The vector used in this case may further include a selection marker to confirm whether or not the gene 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 gene to be introduced has been inserted. A marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein, may be used, but is not limited to these. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0060] The introduction of an exogenous polynucleotide that exhibits the activity of a polypeptide (6) may be the introduction of an exogenous polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide into a host cell. The exogenous polynucleotide is not limited in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be any known transformation method appropriately selected by those skilled in the art. The introduced polynucleotide is expressed in a host cell to produce a polypeptide, and its activity can be increased.

[0061] The codon optimization of the polynucleotide encoding the polypeptide (7) may be an optimization of the codons of an endogenous polynucleotide to increase transcription and translation in a host cell, or an optimization of the codons of an exogenous polynucleotide to optimize transcription and translation in a host cell.

[0062] 8) Analyzing the tertiary structure of a polypeptide and selecting and modifying or chemically modifying exposed sites may involve, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and modifying exposed sites to be modified or chemically modified.

[0063] 9) The intracellular location control of the polypeptide may be to target the polypeptide to a specific organelle or a specific intracellular space within the cell, for example, but not limited to, targeting the polypeptide to the periplasm or cytoplasm through the addition or removal of a leader sequence that functions to target the polypeptide.

[0064] In one embodiment, the activity of the protein can be enhanced by modifying the expression regulatory region of the gene on the chromosome encoding the polypeptide of 2).

[0065] In one embodiment of the above-mentioned embodiments, the enhanced activity of the Ahp protein of the present application may be increased expression of a gene encoding the Ahp protein. In one embodiment of the above-mentioned embodiments, the enhanced activity of the Ahp protein may be achieved by including a gene expression regulatory sequence with enhanced activity upstream of the gene encoding the Ahp protein. Specifically, the upstream of the gene encoding the Ahp protein may be upstream of the ahpC gene. In one embodiment, the enhanced activity of the Ahp protein may be achieved by modifying the expression regulatory sequence of the ahpC gene to enhance expression of the gene sequence. Specifically, the modification of the expression regulatory sequence may be achieved by inserting a gene expression regulatory sequence with enhanced activity between the endogenous promoter of the ahpC gene and ahp operon genes. For example, the gene expression regulatory sequence may be, but is not limited to, a promoter.

[0066] In one embodiment among the above-mentioned embodiments, the enhancement of the activity of the Ahp protein of the present application may be enhancement of the activity of one or more proteins selected from AhpC and AhpF.

[0067] Such enhanced polypeptide activity may be, but is not limited to, an increase in the activity or expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in a wild-type or untransformed microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0068] In this application, the terms "pre-transformed strain" or "pre-transformed microorganism" do not exclude strains containing mutations that may occur naturally in microorganisms, but refer to a naturally occurring strain itself or a strain before its traits are changed due to genetic mutations caused by natural or artificial factors. In this application, the trait change may be an enhancement of the activity of an Ahp protein. The terms "pre-transformed strain" or "pre-transformed microorganism" may be used interchangeably with "non-mutated strain," "non-transformed strain," "non-mutated microorganism," "non-transformed microorganism," or "reference microorganism."

[0069] Modification of a portion or all of a polynucleotide in the microorganism of the present application can be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal insertion in the microorganism or genome editing using engineered nucleases (e.g., CRISPR-Cas9) and / or (b) treatment with light and / or chemicals, such as ultraviolet light and radiation. Methods for modifying a portion or all of the gene include DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene can be injected into the microorganism to cause homologous recombination, thereby deleting a portion or all of the gene. The injected nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

[0070] The microorganism of the present application may be a microorganism with improved OPS production ability.

[0071] For purposes of this application, the recombinant microorganism of this application may be, but is not limited to, a naturally occurring wild-type microorganism, or a microorganism that produces OPS containing an Ahp protein or a polynucleotide encoding it, in which the Ahp protein or a polynucleotide encoding it has been enhanced to have increased OPS production ability compared to the naturally occurring wild-type microorganism or a microorganism that produces OPS containing an Ahp protein or a polynucleotide encoding it. For example, the naturally occurring wild-type microorganism or a microorganism that produces OPS containing an Ahp protein or a polynucleotide encoding it may be, but is not limited to, a subject strain for comparing the presence or absence of increased OPS production ability or Ahp protein activity as described above.

[0072] For example, the recombinant strain with increased OPS production ability may have an increase in OPS production ability of at least about 1%, specifically at least about 2%, at least about 3%, at least about 3.2%, at least about 4%, at least about 4.4%, at least about 5%, 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, and the increase may be, 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, but is not limited thereto as long as there is an increase in OPS production ability compared to the parent strain or unmodified microorganism before mutation. In other examples, the microorganism with increased production ability may have an OPS production ability that is increased by about 1.01-fold or more, about 1.02-fold or more, about 1.03-fold or more, about 1.032-fold or more, about 1.04-fold or more, about 1.044-fold or more, about 1.05-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 the increase may be, for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, or about 2-fold or less) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto.

[0073] Such productivity can be evaluated by measuring the amount of the target product produced after culturing in a medium. The evaluation can be performed by measuring the amount of the target product produced using an appropriate method known in the art. For example, high-performance liquid chromatography (HPLC), gas chromatography (GC), gas chromatography-mass spectrometry (GC / MS), liquid chromatography-mass spectrometry (LC / MS), gel permeation chromatography (GPC), or a combination of these methods can be used. The amount of the target product produced can be measured using an appropriate method known in the art.

[0074] The microorganisms of the present application may further include modifications that enhance the ability to produce and / or excrete OPS from the cell; or that enhance the ability to degrade and / or influx OPS.

[0075] Examples of modifications that enhance the ability to produce and / or export OPS to the outside of the cell; or that enhance the ability to degrade and / or import OPS include, but are not limited to, weakening the activity of phosphoserine phosphatase (SerB); enhancing the activity of phosphoserine export protein (YhhS); or a combination of these modifications.

[0076] In the present application, the term "attenuation" of the activity of a polypeptide is a concept that encompasses a decrease in activity or no activity compared to the endogenous activity. The term "attenuation" may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0077] The term "attenuation" can also include cases where the activity of a polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by a microorganism due to, for example, a mutation in the polynucleotide encoding the polypeptide; cases where the overall polypeptide activity and / or concentration (expression level) in cells is lower than that of a wild-type strain due to, for example, inhibition of gene expression of the encoding polynucleotide or inhibition of translation into the polypeptide; cases where the polynucleotide is not expressed at all; and / or cases where the polypeptide activity is absent despite the expression of the polynucleotide. The term "endogenous activity" refers to the activity of a specific polypeptide originally possessed by a parent strain, wild-type, or untransformed microorganism before transformation, in cases where a trait has been altered due to genetic mutation caused by natural or artificial factors. This term may be used interchangeably with "activity before transformation." A polypeptide activity that is "attenuated, inactivated, deficient, reduced, down-regulated, decreased, or attenuated" compared to its endogenous activity means that the activity of the specific polypeptide is reduced compared to that originally possessed by a parent strain or untransformed microorganism before transformation.

[0078] The activity of such a polypeptide can be attenuated by any method known in the art, but is not limited to this and can be achieved by applying various methods well known in the art (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

[0079] Specifically, the activity of the polypeptide of the present application can be attenuated by: 1) Deletion of all or part of a gene encoding a polypeptide; 2) modification of the expression control region (or expression control sequence) so that expression of the gene encoding the polypeptide is reduced; 3) modification of the amino acid sequence constituting the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence) so as to eliminate or attenuate the activity of the polypeptide; 4) modification of the gene sequence encoding the polypeptide so that the activity of the polypeptide is eliminated or attenuated (e.g., deletion / substitution / addition of one or more nucleic acid bases in the nucleic acid base sequence of the polypeptide gene so as to encode a polypeptide that has been altered so that the activity of the polypeptide is eliminated or attenuated); 5) A modification of the nucleotide sequence encoding the initiation codon, Shine-Dalgarno sequence, or 5'UTR region of a gene transcript encoding a polypeptide; 6) introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide in order to form a secondary structure to which ribosomes cannot attach; 8) Addition of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE); 9) Regulation of the cellular localization of a polypeptide; or 10) It may be a combination of two or more selected from the above 1) to 9), but is not particularly limited thereto.

[0080] for example, The deletion of a part or all of the gene encoding the polypeptide (1) may be removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacement with a polynucleotide lacking some nucleotides, or replacement with a marker gene.

[0081] The modification of the expression regulatory region (or expression regulatory sequence) in 2) above may be the generation of a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacement with a sequence having a weaker 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.

[0082] The modifications of the amino acid sequence or polynucleotide sequence in 3) and 4) above may be, but are not limited to, mutations in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to attenuate the activity of the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence that has been improved to have weaker activity or no activity. For example, but not limited to, introducing a mutation into a polynucleotide sequence to form a stop codon can inhibit or attenuate gene expression.

[0083] The modification of the nucleotide sequence encoding the initiation codon or 5'UTR region of the gene transcript encoding the polypeptide (5) may be, for example, a substitution with a nucleotide sequence encoding another initiation codon that has a lower polypeptide expression rate than the endogenous initiation codon, but is not limited thereto.

[0084] 6) The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide can be carried out by referring to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0085] 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure that prevents ribosome attachment may disable or slow down mRNA translation.

[0086] Furthermore, 8) the addition of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE) may create an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide, thereby weakening the activity.

[0087] 9) The intracellular localization of the polypeptide may be targeted to a specific organelle or specific intracellular space within the cell, for example, but not limited to, targeting the polypeptide to the periplasm or cytoplasm through the addition or removal of a leader sequence that functions to target the polypeptide.

[0088] Such attenuation of polypeptide activity may be, but is not limited to, attenuation of the activity or expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in a wild-type or untransformed microbial strain, or an increase in the amount of product produced from the polypeptide.

[0089] In any one of the above-described embodiments, the recombinant microorganism of the present application may be a microorganism in which SerB activity is attenuated.

[0090] Because "SerB" in the present application has the activity of converting OPS to L-serine, microorganisms mutated to attenuate the SerB activity are characterized by OPS accumulation and are useful for OPS production. The SerB in the present application may be, but is not limited to, a protein having or containing the amino acid sequence set forth in SEQ ID NO: 5, or a protein consisting of or essentially consisting of the amino acid sequence set forth in SEQ ID NO: 5. 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: 6, so long as it exhibits SerB activity. The SerB in the present application may also consist of, or essentially consist of, 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: 5, but is not limited to this. Furthermore, the polynucleotide encoding SerB may have or include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 5. The polynucleotide encoding SerB may consist of, or essentially consist of, a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 5. The polynucleotide encoding SerB of the present application may have various modifications in the coding region within a range that does not change the amino acid sequence of the SerB protein, taking into account codon degeneracy or codons preferred in the organism in which the SerB protein is to be expressed. The polynucleotide encoding SerB of the present application may have or include 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: 6. In addition, the polynucleotide encoding SerB of the present application may consist of or essentially 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: 6, but is not limited thereto.

[0091] In any one of the above-described embodiments, the recombinant microorganism of the present application may be a microorganism having enhanced activity of an O-phosphoserine export protein, for example, a microorganism having enhanced YhhS activity.

[0092] Because the "YhhS" of the present application has the activity of excreting OPS, microorganisms mutated to enhance the YhhS activity have the characteristic of excreting OPS and are useful for producing OPS. The YhhS of the present application may be, but is not limited to, a protein having or comprising the amino acid sequence set forth in SEQ ID NO: 7, or a protein consisting of or essentially consisting of the amino acid sequence set forth in SEQ ID NO: 7. Furthermore, the YhhS of the present application may have or comprise 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: 8, so long as it exhibits O-phosphoserine excretion activity. The YhhS of the present application may also consist of, or essentially consist of, 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: 7, but is not limited to this. The polynucleotide encoding YhhS may have or comprise a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 7. 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: 7. The polynucleotide encoding YhhS of the present application may have 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, without changing the amino acid sequence of the YhhS protein. The polynucleotide encoding YhhS of the present application may have or comprise a nucleotide sequence that is at least 70%, 80%, 90%, 95%, or 99% homologous or identical to the nucleotide sequence of SEQ ID NO: 8, but less than 100% identical. Furthermore, the polynucleotide encoding YhhS of the present application may consist of, or essentially 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: 8, but is not limited thereto.

[0093] In one embodiment, a microorganism containing a mutation that enhances OPS production and / or extracellular export ability or enhances OPS degradation and / or influx ability may be, but is not limited to, CA07-0012 (KCCM 11121P; US2012-0190081A) or CA07-4821. In addition to the above, the disclosures of Korean Patent No. 1381048 or U.S. Patent Publication No. 2012-0190081, etc., regarding such OPS-producing microorganisms, may be used as reference material in this application, but are not limited thereto.

[0094] Another aspect of the present application provides a method for producing O-phosphoserine, comprising culturing in a medium an O-phosphoserine-producing recombinant microorganism in which the activity of an Ahp protein is enhanced relative to the endogenous activity.

[0095] The Ahp protein, intrinsic activity, enhancement, O-phosphoserine, and microorganisms are as described above.

[0096] In the present application, the term "culturing" refers to growing the microorganism under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using an appropriate medium and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected microorganism. Specifically, the culturing may be, but is not limited to, a batch, continuous, or fed-batch culture.

[0097] The culture medium for culturing the microorganism may further contain glycine or serine. Glycine may be provided in the form of purified glycine, a yeast extract containing glycine, or tryptone, and the concentration of glycine in the culture medium may be typically 0.1 to 10 g / L, specifically 0.5 to 3 g / L. Serine may be provided in the form of purified serine, a yeast extract containing serine, or tryptone, and the concentration of glycine in the culture medium may be typically 0.1 to 5 g / L, specifically 0.1 to 1 g / L.

[0098] Carbon sources contained in the medium may include, but are not limited to, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats 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 glycerol and ethanol; and organic acids such as acetic acid, which may be used individually or as a mixture.

[0099] Nitrogen sources contained in the medium may include, but are not limited to, organic nitrogen sources such as peptone, yeast extract, broth, malt extract, corn steep liquor, and soybean meal, and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate, which may be used alone or in combination.

[0100] The phosphorus source contained in the medium may include, but is not limited to, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salts.

[0101] The medium may also contain metal salts such as magnesium sulfate or iron sulfate, as well as amino acids, vitamins, and appropriate precursors. These mediums or precursors may be added to the culture in a batch or continuous manner, but are not limited thereto.

[0102] During cultivation, chemicals such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. Furthermore, during cultivation, antifoaming agents such as fatty acid polyglycol esters can be used to suppress the generation of bubbles. Furthermore, oxygen or an oxygen-containing gas can be injected into the culture to maintain an aerobic state, or nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection to maintain anaerobic and microaerobic states. The temperature of the culture can usually be 25°C to 40°C, specifically 30°C to 35°C. The cultivation period of the culture can be continued until the desired amount of useful substance is produced, specifically 10 to 100 hours. However, the cultivation period is not limited to these examples.

[0103] The OPS production method of the present application can further include, for example, before the culturing step, 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).

[0104] 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 performed) or the cultured microorganism. The recovery step may be further included after the culturing step.

[0105] The recovery may involve collecting the target OPS using a suitable method known in the art based on the microbial culture method of the present application, such as a batch, continuous, or fed-batch culture method. For example, the target OPS can be recovered from the medium or the microorganism using a suitable method known in the art, such as centrifugation, filtration, treatment with a crystallized 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 of these methods.

[0106] The OPS production method of the present application may further include a purification step. The purification can be carried out using a 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 can be carried out continuously or discontinuously, in any order, simultaneously, or integrated into a single step, but are not limited thereto. Therefore, the recovered OPS may be in a purified form or in the form of 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.

[0107] In another aspect, the present application provides a method for producing cysteine ​​or a derivative thereof, comprising the steps of: (a) culturing an O-phosphoserine-producing recombinant microorganism, in a medium, in which Ahp protein activity is enhanced compared to 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.

[0108] The steps a) and b) are not necessarily limited to being performed consecutively or sequentially, and these steps may be performed without a time interval between them, simultaneously, or with an interval of several seconds, minutes, hours, or days.

[0109] The Ahp protein, intrinsic activity, enhancement, O-phosphoserine (OPS), and microorganisms are as described above.

[0110] 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.

[0111] In this application, the term "cysteine ​​derivative" refers to a compound in which a hydrogen atom or a specific atomic group of cysteine ​​is replaced by another atom or atomic group. Examples of such cysteine ​​include those in which another atom or atomic group is attached to the nitrogen atom of the amine group (-NH2) or the sulfur atom of the thiol group (-SH) of cysteine. Examples include, but are not limited to, NAC (N-acetylcysteine), SCMC (S-carboxymethylcysteine), 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.

[0112] As long as cysteine ​​can be produced by the method of the present application, it can be easily converted into various cysteine ​​derivatives by methods well known in the art.

[0113] In the present application, the method for producing a cysteine ​​derivative may further comprise a step of converting the cysteine ​​produced in step b) into a cysteine ​​derivative.

[0114] Specifically, in the present application, the 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.

[0115] The produced cysteine ​​may be converted into a cysteine ​​derivative by a method well known in the art, for example, by reacting cysteine ​​with an acetylation agent to synthesize NAC (N-acetylcysteine) or by reacting cysteine ​​with haloacetic acid under basic conditions to synthesize SCMC (S-carboxymethylcysteine), but is not limited thereto.

[0116] The cysteine ​​derivatives may be used primarily as pharmaceutical raw materials, such as antitussives, cough relievers, and therapeutic agents for bronchitis, bronchial asthma, sore throat, etc., but are not limited thereto.

[0117] As used herein, the term "O-phosphoserine sulfhydrylase (OPSS)" refers to an enzyme that catalyzes the reaction of OPS by donating a thiol group (SH group) to convert the OPS to cysteine. This enzyme was first identified in Aeropyrum pernix, Mycobacterium tuberculosis, Mycobacterium smegmatis, and Trichomonas vaginalis (Mino K and Ishikawa K, FEBS Letters, 551:133-138, 2003; Bums KE et al., J. Am. Chem. Soc, 127:11602-11603, 2005). In addition, the OPSS includes not only wild-type OPSS protein, but also mutant proteins in which a portion of the polynucleotide sequence encoding the OPSS is deleted, substituted, or added, and which exhibit biological activity equivalent to or greater than that of the wild-type OPSS protein, and may include all of the OPSS proteins and their mutant proteins disclosed in Korean Patent Publication No. 2012-0041115 and Korean Patent Registration No. 1208267.

[0118] The sulfide is not only provided in the solid form commonly used in the art, but also in the liquid or gas form depending on the pH, pressure, and solubility. 2- ), thiosulfate (thiosulfate, S203 2-Any sulfide that can be converted into a thiol group (SH group) in the form of, for example, thiol group (SH group) can be used without limitation. Specifically, Na2S, NaSH, (NH4)2S, H2S, or Na2S2O3, which provide a thiol group to OPS, may be used, but is not limited to these. The reaction is a reaction in which one thiol group is provided to one OPS reactive group to produce one cysteine ​​or cysteine ​​derivative, and the amount of sulfide added during the reaction may be 0.1 to 3 times the molar concentration of OPS, specifically, 1 to 2 times, but is not limited to these.

[0119] The present invention may further include a step of recovering the cysteine ​​produced through the reaction step, in which the target cysteine ​​can be separated, purified, and collected from the reaction solution using a suitable reaction known in the art.

[0120] 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 an Ahp protein is enhanced compared to the endogenous activity; a medium in which the same is cultured; or a combination thereof.

[0121] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing O-phosphoserine, cysteine, or cysteine ​​derivatives, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonic agents.

[0122] Another embodiment of the present application provides a method for producing a recombinant microorganism that produces O-phosphoserine, comprising the step of enhancing the activity of the Ahp protein compared to the endogenous activity.

[0123] Another aspect of the present application provides use of an O-phosphoserine-producing recombinant microorganism in which the activity of the Ahp protein is enhanced compared to the endogenous activity, for producing O-phosphoserine, cysteine, or a cysteine ​​derivative.

[0124] The Ahp protein, intrinsic activity, enhancement, O-phosphoserine (OPS), cysteine, cysteine ​​derivatives, and microorganisms of the above-mentioned aspects are as described above. [Example]

[0125] The present application will be described in more detail below with reference to experimental examples. However, the following examples are merely preferred embodiments for illustrating the present application, and are not intended to limit the scope of the present application. Meanwhile, technical matters not described in this specification can be fully understood and easily implemented by those of ordinary skill in the technical field of the present application or a similar technical field.

[0126] Example 1: Construction of an O-phosphoserine (OPS)-producing strain with enhanced YhhS expression 1-1. Construction of vector for enhanced YhhS expression Using wild-type Escherichia coli ATCC27325 chromosomal DNA as a template, PCR was performed to obtain a gene fragment upstream of the wild-type promoter of the yhhS gene (SEQ ID NO: 8), where homologous recombination occurs on the chromosome, using the primer pair of SEQ ID NOs: 11 and 12. PCR was also performed to obtain a gene fragment downstream of the wild-type promoter of the yhhS gene using the primer pair of SEQ ID NOs: 15 and 16. PCR was also performed to obtain the Ptrc promoter (SEQ ID NO: 37) using the primer pair of SEQ ID NOs: 13 and 14, using pCL_Ptrc-gfp (International Patent Publication WO 2016-024771 A1) as a template.

[0127] To perform PCR, the polymerase Solg TM PCR amplification was performed using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 2 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes.

[0128] The upstream and downstream fragments of the yhhS promoter and the Ptrc promoter fragment obtained in the above process were cloned together with the chromosomal transformation vector pSKH130 (SEQ ID NO: 36, U.S. Patent Publication US 2020-0048619) cleaved with EcoRV restriction enzyme using an in-fusion cloning kit (Clontech Laboratories, Inc.) to obtain a recombinant plasmid designated pSKH_Ptrc-yhhS.

[0129] The primer sequences used above are as shown in Table 1 below.

[0130] [Table 1]

[0131] 1-2. Construction of strains with enhanced YhhS expression The OPS-producing strain CA07-0012 (KCCM 11121P, U.S. Patent No. US 8557549 B2), which is a wild-type E. coli K-12 W3110 strain lacking endogenous phosphoserine phosphatase (SerB) and thus exhibiting reduced OPS-degrading ability, was introduced with pSKH_Ptrc-yhhS prepared in Example 1-1 above to enhance expression of YhhS (SEQ ID NO: 7), a protein with OPS-exporting ability, thereby further improving OPS-exporting ability.

[0132] The CA07-0012 strain was transformed with pSKH_Ptrc-yhhS prepared in Example 1-1 by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), and the desired strain was isolated using R6K and kanamycin in a primary crossover step. A secondary crossover step was then performed in sucrose medium to delete the kanamycin resistance gene and obtain a strain in which the Ptrc promoter sequence was 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 NO: 17 and SEQ ID NO: 18, which amplify the upstream and downstream regions of the homologous recombination region, respectively, and genome sequencing. The resulting strain was designated CA07-4821 (CA07-0012ΔPn_yhhS::Ptrc_yhhS).

[0133] The primer sequences used above are as shown in Table 2 below.

[0134] [Table 2]

[0135] Example 2: Construction of strains with enhanced or attenuated Ahp expression 2-1. Construction of vector for enhanced Ahp expression In previous studies, the average transcription levels of the ahpC, rhtB, serC, and rmf genes in an OPS-producing host strain were determined to be 11123, 1391, 24861, and 32205, respectively, as shown in Table 3 below. Compared to the average transcription level of the ahpC gene, the average transcription level of the rmf gene was 2.9-fold higher, and the average transcription level of the serC gene was 2.2-fold higher. In contrast, the average transcription level of the rhtB gene was found to be 0.1-fold higher than the average transcription level of the ahpC gene. This confirmed that the rmf and serC gene promoters are relatively stronger promoters than the Ahp operon promoter, while the rhtB gene promoter is a relatively weaker promoter.

[0136] [Table 3]

[0137] In addition, the rmf promoter and serC promoter (SEQ ID NO: 10), which have been confirmed to have stronger activity, were further inserted at the end of the ahpCF operon promoter in the OPS-producing microorganism to create a strain with enhanced Ahp expression.

[0138] 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 ahpC gene (SEQ ID NO: 2) using the primer pair of SEQ ID NO: 19 and SEQ ID NO: 20, and to obtain a gene fragment of the downstream region of the wild-type promoter of the ahpC gene using the primer pair of SEQ ID NO: 21 and SEQ ID NO: 22. 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 using the primer pair of SEQ ID NO: 23 and SEQ ID NO: 24.

[0139] To perform PCR, the polymerase Solg TM PCR amplification was performed using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 2 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes.

[0140] The upstream and downstream fragments of the ahpC promoter and the rmf promoter fragment obtained in the above process were cloned together with the chromosomal transformation vector pSKH130 cleaved with EcoRV restriction enzyme using an infusion cloning kit to obtain a recombinant plasmid, which was named pSKH130_Prmf-ahp.

[0141] 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 ahpC gene using the primer pair of SEQ ID NOs: 19 and 25, and to obtain a gene fragment of the downstream region of the wild-type promoter of the ahpC gene using the primer pair of SEQ ID NOs: 22 and 26. 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: 27 and 28.

[0142] To perform PCR, the polymerase Solg TM PCR was performed using Pfu-X DNA polymerase. The PCR was performed under the following conditions: denaturation at 95°C for 2 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes.

[0143] The upstream and downstream fragments of the ahpC promoter and the serC promoter fragment obtained in the above process were cloned together with the chromosomal transformation vector pSKH130 cleaved with EcoRV restriction enzyme using an infusion cloning kit to obtain a recombinant plasmid named pSKH130_PserC-ahp.

[0144] 2-2. Construction of vector for attenuating Ahp expression A strain with enhanced Ahp expression was constructed by further inserting the rhtB promoter (SEQ ID NO: 35), which has weaker activity, at the end of the ahpCF operon promoter in the OPS-producing microorganism.

[0145] 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 ahpC gene using the primer pair of SEQ ID NOs: 19 and 29, and to obtain a gene fragment of the downstream region of the wild-type promoter of the ahpC gene using the primer pair of SEQ ID NOs: 22 and 20. PCR was also performed to obtain the promoter region of the rhtB gene using the chromosomal DNA of wild-type E. coli ATCC27325 as a template and the primer pair of SEQ ID NOs: 31 and 32.

[0146] To perform PCR, the polymerase Solg TM PCR amplification was performed using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 2 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes.

[0147] The upstream and downstream fragments of the ahpC promoter and the rhtB promoter fragment obtained in the above process were cloned together with the chromosomal transformation vector pSKH130 cleaved with EcoRV restriction enzyme using an infusion cloning kit to obtain a recombinant plasmid named pSKH130_PrhtB-ahp.

[0148] The primer sequences used in Examples 2-1 and 2-2 are shown in Table 4 below.

[0149] [Table 4]

[0150] 2-3. Construction of strains with enhanced Ahp expression The CA07-4821 strain, an OPS-producing strain with enhanced YhhS expression in Example 1-2, was transformed with pSKH_Prmf-ahp and pSKH_PserC-ahp constructed in Example 2-1 by electroporation, respectively, and then a secondary crossover process was performed to obtain strains in which the promoter sequence of the serC or rmf gene, respectively, was inserted at the end of the wild-type promoter sequence of the ahp gene. The insertion of the serC or rmf promoter sequence was confirmed through PCR amplification and genome sequencing using primer pairs of SEQ ID NOs: 33 and 34, which can amplify the exogenous regions of the upstream and downstream regions of the homologous recombination, respectively. The strains obtained above were designated CA07-4888 (CA07-4821ΔPn_ahpC-ahpF::PserC-ahpC-ahpF) and CA07-4887 (CA07-4821ΔPn_ahpC-ahpF::Prmf_ahpC-ahpF), respectively.

[0151] 2-4. Construction of an attenuated Ahp expression strain The CA07-4821 strain, an OPS-producing strain with enhanced YhhS expression (Example 1-2), was transformed with pSKH130_PrhtB-ahp (Example 2-2) by electroporation, followed by a secondary crossover to obtain a strain in which the promoter sequence of the rhtB gene was inserted at the end of the native promoter sequence of the ahp gene. The insertion of the rhtB promoter sequence was confirmed through PCR amplification and genome sequencing using a primer pair (SEQ ID NO: 33 and SEQ ID NO: 34) capable of amplifying the upstream and downstream regions of the homologous recombination, respectively. The resulting strain was designated CA07-4889 (CA07-4821ΔPn_ahpC-ahpF::PrhtB_ahpC-ahpF).

[0152] The primer sequences used in Examples 2-3 and 2-4 are shown in Table 5 below.

[0153] [Table 5]

[0154] Example 4: Evaluation of strains' OPS production ability Flask fermentation titers were evaluated to measure the OPS production ability of the CA07-4888 and CA07-4887 strains, which are strains with enhanced Ahp expression in Example 2-3, and the CA07-4889 strain, which is a strain with attenuated Ahp expression in Example 2-4, as well as the parent strain CA07-0012 in Example 1-2 and the CA07-4821 strain in Example 1-2, which served as control strains.

[0155] Each strain was smeared on LB solid medium and cultured overnight in an incubator at 33°C. The strains cultured overnight on LB solid medium were inoculated into 25 mL of 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 and is shown in Table 6 below.

[0156] <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

[0157] [Table 6]

[0158] As shown in Table 6, the Ahp-enhanced CA07-4887 and CA07-4888 strains, which were derived from the YhhS-enhanced CA07-4821 strain as a parent strain, had OPS production abilities increased by 4.4% and 3.2%, respectively, compared to the parent strain. The Ahp-weakened CA07-4889 strain showed the lowest OPS production ability, with an increase of 1.5% compared to the parent strain.

[0159] This confirmed that strengthening Ahp increases OPS production capacity.

[0160] From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including within the meaning and scope of the claims below, and any modifications or variations derived from the equivalent concepts thereof, rather than the above detailed description.

Claims

1. A recombinant microorganism producing O-phosphoserine, in which the activity of Ahp protein is enhanced compared to the endogenous activity.

2. The microorganism according to claim 1, wherein the recombinant microorganism has enhanced O-phosphoserine-producing ability compared to endogenous O-phosphoserine-producing ability.

3. The microorganism according to claim 1, wherein the activity of one or more selected from the group consisting of AhpC (Alkyl hydroperoxide reductase subunit C) and AhpF (Alkyl hydroperoxide reductase subunit F) is enhanced.

4. The microorganism according to claim 1 , wherein the Ahp protein is encoded by ahpCF operon genes.

5. The microorganism according to claim 3, wherein the AhpC is composed of the amino acid sequence of SEQ ID NO:

1.

6. The microorganism according to claim 3 , wherein the AhpF is composed of the amino acid sequence of SEQ ID NO:

3.

7. The microorganism according to claim 1, wherein the recombinant microorganism further has enhanced activity of an O-phosphoserine excretion protein compared to its endogenous activity.

8. 2. The microorganism of claim 1, wherein the recombinant microorganism is of the genus Escherichia.

9. A method for producing O-phosphoserine, comprising culturing in a medium an O-phosphoserine-producing recombinant microorganism in which the activity of Ahp protein is enhanced compared to the endogenous activity.

10. 10. The method for producing O-phosphoserine according to claim 9, further comprising recovering O-phosphoserine from the culture medium or the microorganism.

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

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

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

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