Recombinant microorganism having modulated manganese import protein activity and method for producing o-phosphoserine, cysteine or cysteine derivative using same

Abstract

The present disclosure relates to a recombinant microorganism in which the activity of a protein having manganese import activity is regulated, and a method for producing O-phosphoserine, cysteine or a cysteine derivative using the microorganism.

Description

Technical Field

[0001] This disclosure relates to recombinant microorganisms having regulated manganese input protein activity, and methods for using them to produce O-phosphoserine, cysteine, and cysteine ​​derivatives. Background Technology

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

[0003] Methods for producing L-cysteine ​​using microorganisms known in the art include: 1) methods for bioconverting D,L-2-aminothiazoline-4-carboxylic acid (D,L-ATC) to L-cysteine ​​using microorganisms; 2) methods for producing L-cysteine ​​by direct fermentation using Escherichia coli (EP0885962B; Wada M and Takagi H, Appl. Microbiol. Biochem., 73:48-54, 2006); and 3) methods for producing O-phosphoserine (hereinafter “OPS”) by microbial fermentation, followed by conversion of O-phosphoserine to L-cysteine ​​by reacting O-phosphoserine with sulfides under the catalysis of O-phosphoserine hydrogen sulfide hydrolase (hereinafter “OPSS”) (US 8557549 B2), etc.

[0004] In particular, in order to produce cysteine ​​in high yield via method 3), the precursor OPS should be produced in large quantities. Summary of the Invention

[0005] [Technical Issues]

[0006] The technical problem of this disclosure is to provide a recombinant microorganism in which the activity of a protein with manganese input activity is regulated, and a method for producing O-phosphoserine, cysteine ​​and cysteine ​​derivatives using the microorganism.

[0007] [Technical Solution]

[0008] One object of this disclosure is to provide a recombinant microorganism that produces O-phosphoserine, wherein the activity of the MntH protein is enhanced compared to its endogenous activity.

[0009] Another object of this disclosure is to provide a method for producing O-phosphoserine using the recombinant microorganisms that produce O-phosphoserine of this disclosure.

[0010] Another object of this disclosure is to provide a method for producing cysteine ​​or cysteine ​​derivatives using recombinant microorganisms that produce O-phosphoserine as disclosed herein.

[0011] [Beneficial Effects]

[0012] When O-phosphoserine is produced using the recombinant microorganisms of this disclosure that produce O-phosphoserine, wherein the activity of the MntH protein is enhanced compared to endogenous activity, O-phosphoserine can be produced in high yield compared to using existing unmodified strains. Detailed Implementation

[0013] This disclosure will now be described in detail. Furthermore, each description and embodiment disclosed herein can be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed herein fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited by the specific descriptions below. In addition, numerous papers and patent documents are cited in this specification. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety and will more clearly describe the level of the technical field to which this disclosure pertains and the content of this disclosure.

[0014] One aspect of this disclosure provides a recombinant microorganism that produces O-phosphoserine, wherein the activity of the MntH protein is enhanced compared to its endogenous activity.

[0015] As used herein, the term “O-phosphoserine (hereinafter “OPS”)” refers to the phosphate ester of serine, which is a component of many proteins. OPS is a precursor of L-cysteine ​​and can be converted to cysteine ​​by reaction with sulfides under the catalysis of OPS hydrogen sulfide hydrolase (hereinafter “OPSS”), but is not limited thereto (US 2012-0190081 A1).

[0016] As used herein, the term "MntH protein" refers to a protein with divalent ion transporter activity, classified as belonging to the NRAMP family; in one instance, it could be Mn 2+ Uptake-type NRAMP. As used herein, the term "Mn" refers to... 2+ "Uptake-type NRAMP (natural resistance-associated macrophage protein) transporter (MntH)" refers to a transporter containing Mn... 2+ / Fe 2+ H + It is a protein with cotransporter activity and the ability to introduce manganese (Mn) into cells.

[0017] The amino acid sequence of the MntH protein can be obtained from known databases such as NCBI GenBank.

[0018] In one instance, the MntH protein of this disclosure may be derived from a microorganism. The microorganism may specifically be derived from, but is not limited to, microorganisms of the genus *Escherichia*.

[0019] In another instance, the amino acid sequence of the MntH protein disclosed herein may be WP000186369.1 or EEW8215783.1 derived from Escherichia coli (E. coli), but obviously it may include proteins with MntH protein activity from various sources.

[0020] In this disclosure, the MntH protein may have or include the amino acid sequence of SEQ ID NO: 1, or be composed of the amino acid sequence of SEQ ID NO: 1, or may be substantially composed of the above-described amino acid sequence.

[0021] In this disclosure, the MntH protein may include an amino acid sequence having at least 70% or higher, 75% or higher, 76% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.5% or higher, 99.7% or higher, or 99.9% or higher homology or identity with the amino acid sequence of SEQ ID NO: 1. Furthermore, it is apparent that any protein whose amino acid sequence is partially deleted, modified, substituted, conservatively substituted, or added, provided that the amino acid sequence has such homology or identity and exhibits the corresponding efficacy to a protein including the amino acid sequence of SEQ ID NO: 1, may also fall within the scope of this disclosure. In one example, the MntH protein may consist of 412 to 428 amino acids comprising the amino acid sequence shown in SEQ ID NO: 1.

[0022] For example, it could be a sequence addition that does not alter the protein function of this disclosure, a naturally occurring mutation, a silent mutation therein, or a conserved substitution within the N-terminus, C-terminus, and / or amino acid sequence.

[0023] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitutions typically occur based on the similarity of residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity. Generally, conserved substitutions have little or no effect on the activity of a protein or peptide.

[0024] As used herein, the terms “homology” or “identity” refer to the degree of correlation between two given amino acid sequences or nucleotide sequences, expressed as a percentage. The terms homology and identity are generally used interchangeably.

[0025] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined using standard alignment algorithms and can be used in conjunction with default gap penalties established by the program used. Essentially, homologous or identical sequences are generally expected to hybridize to all or part of the sequence under moderately or highly stringent conditions. Obviously, hybridization with polynucleotides containing universal or degenerate codons is also included.

[0026] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined by known computer algorithms, such as the “FASTA” program, using default parameters as described 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), which uses the Needleman program (version 5.0.0 or higher) of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (GCG package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF 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 AppliedMath 48: 1073) Execution. For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0027] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by aligning sequence information using, for example, a GAP computer program (such as Needleman et al., (1970), J Mol Biol. 48:443), as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In short, the GAP program defines homology, similarity, or identity as a value obtained by dividing the number of similarly aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter sequence of the two sequences. The default parameters of the GAP program may include: (1) a binary alignment moment (with a value of 1 for agreement and a value of 0 for disagreement) and a weighted alignment matrix (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) permutation matrix as disclosed in Atlas of Protein Sequence and Structure edited by Schwartz and Dayhoff, National Biomedical Research Foundation, pp. 353–358 (1979)); (2) a penalty of 3.0 for each vacancy and an additional penalty of 0.10 for each symbol in each vacancy (or a penalty of 10 for vacancy opening and 0.5 for vacancy extension); and (3) no penalty for terminal vacancy.

[0028] The MntH protein disclosed herein can be encoded by the mntH gene.

[0029] In one instance, the mntH gene may be a polynucleotide encoding EEW8215783.1 derived from *E. coli*, but is not limited thereto. In another instance, the mntH gene may be a polynucleotide encoding WP_000186369.1 derived from *E. coli*, and in yet another instance, it may be a sequence contained in CP116188.1 derived from *E. coli*, but is not limited thereto, and obviously the gene may include mntH genes encoding proteins with MntH protein activity from various sources.

[0030] As used herein, the term "polynucleotide" is a polymer of nucleotides composed of nucleotide monomers linked together by covalent bonds to form a long chain, which is a DNA or RNA chain of at least a certain length. More specifically, it can refer to a polynucleotide segment that encodes a protein.

[0031] The polynucleotide encoding the MntH protein of this disclosure may include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1. In one example of this disclosure, the polynucleotide may have or include the nucleotide sequence of SEQ ID NO: 2. Furthermore, the polynucleotide of this disclosure may consist of or substantially consist of the nucleotide sequence of SEQ ID NO: 2. Specifically, mntH may be encoded by the polynucleotide shown in the nucleotide sequence of SEQ ID NO: 2.

[0032] Due to codon degeneracy or considering preferred codons in organisms expressing the MntH protein, the polynucleotides of this disclosure can be modified in various ways in the coding region without altering the amino acid sequence of the MntH protein. Specifically, the polynucleotides of this disclosure may have or include nucleotide sequences having 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 homology or identity with the sequence of SEQ ID NO: 2, or may consist of or substantially consist of nucleotide sequences having 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 homology or identity with the sequence of SEQ ID NO: 2, but are not limited thereto.

[0033] Furthermore, the polynucleotides disclosed herein may include probes that can be prepared from known gene sequences, such as any polynucleotide sequence that can hybridize with all or part of the complementary sequence of the polynucleotide sequence disclosed herein under stringent conditions, without limitation. “Stringent conditions” refers to conditions that allow specific hybridization between polynucleotides. These conditions are described in detail in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold SpringHarbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50–9.51, 11.7–11.8). For example, stringent conditions may include polynucleotides with high homology or identity, i.e., polynucleotides with 70% or higher, 75% or higher, 76% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology or identity hybridizing with each other, while polynucleotides with lower homology or identity do not hybridize with each other; or may include ordinary washing conditions for Southern hybridization, i.e., washing once, especially twice or three times, at a salt concentration and temperature corresponding to 60°C, 1×SSC, 0.1% SDS, particularly 60°C, 0.1×SSC, 0.1% SDS, and even more particularly 68°C, 0.1×SSC, 0.1% SDS.

[0034] Hybridization requires two nucleic acids to have complementary sequences, although mismatches between bases are possible depending on the strictness of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this disclosure may also include isolated nucleic acid fragments complementary to the whole sequence as well as nucleic acid sequences substantially similar to them.

[0035] Specifically, polynucleotides homologous to or identical with the polynucleotides disclosed herein can be detected using hybridization conditions, which include a hybridization step at a Tm value of 55°C under the conditions described above. Furthermore, the Tm value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art for their purposes.

[0036] The appropriate stringency of polynucleotide hybridization depends on the length and complementarity of the polynucleotides, variables that are well known in the field (e.g., Sambrook et al., ibid.).

[0037] As used herein, the term "vector" refers to a DNA construct containing a nucleotide sequence encoding a target protein, said nucleotide sequence being operatively linked to a suitable expression regulatory sequence to enable expression of the target protein in a suitable host cell. The expression regulatory sequence may include a promoter capable of initiating transcription, any operon sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences for regulating transcription and translation termination. Once transformed into a suitable host cell, the vector may replicate or function independently of the host genome, or it may integrate into the host genome.

[0038] There are no particular limitations on the vectors used in this disclosure, as long as they can replicate in host cells, and any vector known in the art can be used. Examples of commonly used vectors can include natural or recombinant plasmids, colloids, viruses, and bacteriophages. For example, as phage vectors or colloid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used; as plasmid vectors, plasmid vectors based on pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used. Specifically, vectors such as pSKH130 (US Patent Application Publication No. 2020-0048619), pSK, pDZ, pDC, pDCM2, pDC24, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC can be used.

[0039] Polynucleotides can be inserted into chromosomes by any method known in the art (e.g., homologous recombination, but not limited thereto).

[0040] As used herein, the term "transformation" refers to the introduction of a recombinant vector containing a polynucleotide encoding a target protein into a host cell, enabling the expression of the protein encoded by the polynucleotide within the host cell. It is not important whether the transformed polynucleotide is integrated into and located within the host cell's chromosome or outside of it, as long as the transformed polynucleotide can be expressed in the host cell; both are acceptable. Methods for transforming vectors include any method of introducing nucleic acids into cells and can be performed using appropriate standard techniques known in the art, depending on the host cell. For example, transformation can be performed via electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) technology, DEAE-dextran technology, cationic liposome technology, lithium acetate-DMSO technology, etc., but the method is not limited to these.

[0041] Furthermore, as used herein, the term "operably ligated" refers to the functional ligation of a polynucleotide sequence to a promoter sequence or expression regulatory region that initiates and mediates the transcription of a polynucleotide encoding the target protein disclosed herein. Operable ligations can be prepared using gene recombination techniques known in the art, and site-specific DNA cutting and ligation can be prepared using, but are not limited to, cutting and ligases known in the art.

[0042] The microorganisms disclosed herein are not limited by type, as long as they can produce OPS, and can be any prokaryotic or eukaryotic microorganism, specifically, a prokaryotic microorganism. In one instance, it may include strains of microorganisms belonging to the genera *Escherichia*, *Erwinia*, *Serratia*, *Providencia*, *Corynebacterium*, and *Brevibacterium*, specifically, microorganisms belonging to the genus *Escherichia*, more specifically, *Escherichia coli*, but not limited thereto. For example, in the case of microorganisms belonging to the genus Escherichia, OPS and L-serine can be produced by SerA, SerC and SerB, which are enzymes in the L-serine biosynthesis 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;7 1( ll):7 139-44.).

[0043] As used herein, the term "O-phosphoserine (OPS)-producing microorganism" refers to a microorganism with the natural ability to produce O-phosphoserine, or a microorganism in which the ability to produce O-phosphoserine has been conferred upon a parent strain that does not possess the ability to produce O-phosphoserine. Specifically, the microorganism can be an OPS-producing microorganism with enhanced MntH protein activity due to natural or artificial genetic modification. For the purposes of this disclosure, an OPS-producing microorganism can be any microorganism capable of producing O-phosphoserine by enhancing the activity of the MntH protein through the methods disclosed herein. As used herein, "O-phosphoserine (OPS)-producing microorganism" may be used interchangeably with "O-phosphoserine (OPS)-producing microorganism" or "microorganism with the ability to produce O-phosphoserine."

[0044] In one embodiment, the OPS-producing microorganism of this disclosure may be a genetically modified microorganism or a recombinant microorganism, wherein the activity of the MntH protein is enhanced, thereby increasing the desired OPS-producing capacity, but is not limited thereto. The recombinant microorganism may be a microorganism whose ability to produce O-phosphoserine is enhanced compared to endogenous O-phosphoserine production.

[0045] The microorganisms disclosed herein may have increased MntH protein activity compared to endogenous activity. Specifically, the microorganisms disclosed herein may be microorganisms in which the MntH protein or the mntH gene encoding it is enhanced; or microorganisms that have been genetically modified to enhance the MntH protein or the mntH gene encoding it (e.g., recombinant microorganisms), but are not limited thereto.

[0046] As used herein, “enhancement” of the term polypeptide (e.g., protein specified by the name of each enzyme) refers to an increase in the activity of the polypeptide compared to its endogenous activity. Enhancement can be used interchangeably with terms such as activation, upregulation, overexpression, and increase. Specifically, activation, enhancement, upregulation, overexpression, and increase can include both cases of exhibiting activity not initially present, or activity enhanced compared to endogenous or unmodified activity. “Endogenous activity” refers to the activity of a specific polypeptide originally possessed by the pre-transformation parental strain or unmodified microorganism when the trait is altered through genetic modification caused by natural or artificial factors, and can be used interchangeably with “unmodified activity.” “Enhancement,” “upregulation,” “overexpression,” or “increase” of polypeptide activity compared to its endogenous activity refers to an increase in the activity and / or concentration (expression level) of the specific polypeptide compared to the activity and / or concentration (expression level) originally possessed by the pre-transformation parental strain or unmodified microorganism.

[0047] This enhancement can be achieved by introducing exogenous peptides or by increasing the activity and / or concentration (expression level) of endogenous peptides. Enhanced peptide activity can be confirmed by increases in peptide activity levels, expression levels, or the amount of peptide secreted products.

[0048] For the purposes of this disclosure, the microorganisms of this disclosure possess enhanced MntH protein activity, thereby enhancing their ability to produce OPS. The unmodified microorganisms without enhanced MntH protein (which are target strains for comparing OPS production capacity or increased MntH protein) may be CA07-0012 (KCCM 11121P, US 8557549 B2), an OPS-producing strain (in which endogenous phosphatase (SerB) is absent and thus its ability to decompose OPS is weakened), a strain in which the expression of YhhS with OPS exporting capacity is enhanced in CA07-0012 (CA07-4821 of this disclosure), and a strain in which the activity of NADH:quinone oxidoreductase is further enhanced in CA07-4821 (CA07-4828 of this disclosure), but are not limited thereto.

[0049] Enhancement of peptide activity can be achieved through a variety of methods well known in the art, and such methods are not limited, as long as they enhance the activity of the target peptide compared to the unmodified microorganism. Specifically, genetic engineering and / or protein engineering, which are well known to those skilled in the art and are common methods in molecular biology, can be used, but the method is not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16; Sambrook et al., Molecular Cloning 2012, etc.).

[0050] Specifically, the enhancement of the peptide activity disclosed herein can be achieved in the following ways:

[0051] 1) Increase the intracellular copy number of polynucleotides encoding polypeptides;

[0052] 2) Modify the expression regulatory sequences of genes encoding polypeptides on chromosomes;

[0053] 3) Modify the nucleotide sequence of the start codon or 5'-UTR of a gene transcript encoding a polypeptide;

[0054] 4) Modifying the amino acid sequence of peptides to enhance their activity;

[0055] 5) Modifying the polynucleotide sequence encoding the polypeptide to enhance its activity (e.g., modifying the polynucleotide sequence of a polypeptide gene to encode a polypeptide that has been modified to enhance its activity).

[0056] 6) Import exogenous peptides that exhibit peptide activity or exogenous polynucleotides encoding such peptides;

[0057] 7) Codon optimization for polynucleotides encoding polypeptides;

[0058] 8) Analyze the tertiary structure of the peptide to select and modify exposed sites, or chemically modify them;

[0059] 9) Regulating the cellular localization of peptides; or

[0060] 10) Selected from two or more of the above 1) to 9), but not limited thereto.

[0061] More specifically,

[0062] 1) Increasing the intracellular copy number of a polynucleotide encoding a polypeptide can be achieved by introducing a vector into a host cell, the vector being operatively linked to the polynucleotide encoding the polypeptide and capable of replication and functioning regardless of the host cell. Alternatively, this method can be achieved by introducing one or two copies of the polynucleotide encoding the polypeptide into the chromosome of the host cell. Chromosomal insertion can be performed by introducing a vector capable of inserting the polynucleotide into the host cell chromosome, but is not limited thereto.

[0063] 2) Replacing the expression regulatory region (or expression regulatory sequence) of a gene encoding a polypeptide on a chromosome with a highly active sequence can be achieved, for example, by inducing sequence modifications through deletion, insertion, non-conserved or conserved substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or by replacing the sequence with a more active one. The expression regulatory region may include, but is not limited to, promoters, operon sequences, sequences encoding ribosome binding sites, and sequences regulating transcription and translation termination. This method may specifically include, but is not limited to, inserting a strong promoter downstream of the original promoter.

[0064] Examples of known strong promoters include CJ1 to CJ7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, λ phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13(sm3) promoter (US 10584338 B2), O2 promoter (US 10273491 B2), tkt promoter, yccA promoter, rmf promoter, serC promoter, etc., but strong promoters are not limited to these.

[0065] 3) The method of modifying the nucleotide sequence of the start codon or 5'-UTR of the gene transcript (which encodes a polypeptide) can be achieved, for example, by replacing the nucleotide sequence with a nucleotide sequence that encodes another polypeptide with a higher expression rate than the endogenous start codon, but is not limited thereto.

[0066] Methods 4) and 5) for modifying amino acid or polynucleotide sequences can be achieved by inducing sequence modifications to enhance peptide activity through deletions, insertions, non-conserved or conserved substitutions, or combinations thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or by replacing the sequence with a modified amino acid or polynucleotide sequence that has stronger activity, or a modified amino acid or polynucleotide sequence that enhances activity, but are not limited thereto. Specifically, substitution can be achieved by inserting polynucleotides into chromosomes through homologous recombination, but is not limited thereto.

[0067] The vectors used in this article may also include selection markers to confirm insertion into the chromosome. Selection markers are used to select cells transformed with the vector (i.e., to confirm the insertion of the gene to be introduced), and may include markers that provide selectable phenotypes (e.g., drug resistance, auxotrophic phenotypes, resistance to cytotoxic agents, or expression of surface proteins), but are not limited to these. Furthermore, in an environment treated with a selection agent, only cells expressing the selection marker can survive or exhibit different phenotypes, thus allowing selection of transformed cells.

[0068] 6) The method of introducing exogenous polynucleotides exhibiting peptide activity can be achieved by introducing exogenous polynucleotides encoding peptides exhibiting the same or similar activity as the peptide into host cells. Exogenous polynucleotides can be used without restriction, regardless of their origin or sequence, as long as they exhibit the same or similar activity as the peptide. Those skilled in the art can perform the introduction by appropriately selecting transformation methods known in the art, and the expression of the introduced polynucleotide in the host cells can produce the peptide, thereby increasing its activity.

[0069] 7) Codon optimization of polynucleotides encoding polypeptides can be achieved by optimizing the codons of endogenous polynucleotides to increase transcription or translation in host cells, or by optimizing the codons of exogenous polynucleotides to enable optimized transcription and translation in host cells.

[0070] 8) Methods for analyzing the tertiary structure of a polypeptide to select and modify exposed sites, or to chemically modify it, can be achieved by, for example, comparing the sequence information of the polypeptide to be analyzed with a database (which stores the sequence information of known proteins) to determine template protein candidates based on sequence similarity, and thus confirming the structure based on this information, thereby selecting and transforming or modifying the exposed sites to be modified or chemically modified.

[0071] 9) Methods for regulating the cellular localization of peptides can be achieved by targeting the peptide to specific organelles or specific spaces within the cell. For example, this can be achieved by adding or removing a leader sequence that functions in the target peptide, thus targeting the periplasm or cytoplasm, but is not limited to this.

[0072] In one implementation, the enhancement of MntH protein activity can be achieved by modifying the expression regulatory region of the chromosome gene encoding the polypeptide described in 2) above.

[0073] In any of the above embodiments, the enhancement of MntH protein activity disclosed herein may be an increase in the expression of the gene encoding the MntH protein. In any of the above embodiments, the enhancement of MntH protein activity may include a gene expression regulatory sequence with enhancing activity upstream of the gene encoding the protein. Specifically, the upstream of the gene encoding the MntH protein may be upstream of the mntH gene. In one embodiment, the enhancement of MntH protein activity may be achieved by modifying the expression regulatory sequence of the mntH gene to enhance gene sequence expression. Specifically, the modification of the expression regulatory sequence may involve the additional insertion of a gene expression regulatory sequence with enhancing activity between the endogenous promoter of the mntH gene and the mntH gene itself; for example, the gene expression regulatory sequence may be a promoter, but is not limited thereto.

[0074] This enhancement of peptide activity may mean, but is not limited to, an increase in the activity or concentration of the peptide relative to the peptide expressed in the wild-type strain or the pre-modified microbial strain, or an increase in the amount of product produced by the peptide.

[0075] As used herein, the terms "pre-modification strain" or "pre-modification microorganism" do not exclude strains containing mutations that may occur naturally in microorganisms, and can refer to the natural strain itself, or to a strain whose traits have been altered due to genetic modifications caused by natural or artificial factors. In this disclosure, trait modification can be an enhancement of MntH protein activity. "Pre-modification strain" or "pre-modification microorganism" may be used interchangeably with "unmutated strain," "unmodified strain," "unmutated microorganism," "unmodified microorganism," or "reference microorganism."

[0076] The modification of some or all polynucleotides in microorganisms disclosed herein can be achieved, but is not limited to, by (a) homologous recombination involving chromosomal insertion in the microorganism using a vector or genome editing using engineered nucleases (e.g., CRISPR-Cas9), and / or (b) induction by light (e.g., ultraviolet light and radiation) and / or chemical treatment. Methods for modifying some or all genes can include, but are not limited to, methods using DNA recombination technology. For example, some or all genes can be deleted by injecting a nucleotide sequence or a vector containing a nucleotide sequence homologous to a target gene into the microorganism to induce homologous recombination. The injected nucleotide sequence or vector can include, but is not limited to, dominant selection markers.

[0077] The microorganisms disclosed herein may be microorganisms with enhanced OPS production capacity.

[0078] For the purposes of this disclosure, the recombinant microorganisms of this disclosure may be microorganisms that have increased OPS production capacity by enhancing the MntH protein or the polynucleotide encoding it in natural wild-type microorganisms; or OPS-producing microorganisms that contain the MntH protein or the polynucleotide encoding it compared to natural wild-type microorganisms; or OPS-producing microorganisms that contain the MntH protein or the polynucleotide encoding it, but are not limited thereto. For example, natural wild-type microorganisms containing the MntH protein or the polynucleotide encoding it, or OPS-producing microorganisms, may be target strains used to compare increased OPS production capacity or increased MntH protein activity, but are not limited thereto.

[0079] In one instance, a recombinant strain with increased OPS production capacity compared to the parental strain before modification or the unmodified microorganism may have an increased OPS production capacity of about 1% or more, specifically about 1.7% or more, 2% or more, about 2.3% or more, about 3% or more, about 4% or more, about 5% or more, about 5.3% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, or about 10% or more (with no particular upper limit, e.g., about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 15% or less), but is not limited thereto, as long as it has an increased positive value compared to the production capacity of the parental strain before modification or the unmodified microorganism. In another instance, a microorganism with increased OPS production capacity compared to the parental strain before modification or the unmodified microorganism may have an increased OPS production capacity of about 1.01 times or more, about 1.017 times or more, about 1.02 times or more, about 1.023 times or more, about 1.03 times or more, about 1.04 times or more, about 1.05 times or more, about 1.053 times or more, about 1.06 times or more, about 1.07 times or more, about 1.08 times or more, about 1.09 times or more, or about 1.10 times or more (with no particular upper limit, e.g., about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less), but is not limited thereto.

[0080] Production capacity can be assessed by measuring the yield of the desired product obtained from cultivation in a culture medium. This assessment can be performed using suitable methods known in the art to measure the yield of the desired product. For example, 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 combinations thereof can be used, and the yield of the desired product can be measured using suitable methods known in the art.

[0081] The microorganisms disclosed herein may further enhance the ability to produce OPS and / or export OPS from cells; or may include modifications that enhance the ability to decompose and / or import OPS.

[0082] Examples of modifications that enhance the ability to produce and / or export OPS from cells; or enhance the ability to break down and / or import OPS may include enhancing the activity of NADH:quinone oxidoreductase; weakening the activity of phosphoserine phosphatase (SerB); enhancing the activity of phosphoserine export protein (YhhS); or combinations of these modifications, but are not limited thereto.

[0083] As used herein, the term “attenuation” of peptide activity is a broad concept, encompassing reduced or absent activity compared to its intrinsic activity. Attenuation can be used interchangeably with terms such as inactivation, lack, downregulation, reduction, decrease, and weakening.

[0084] The attenuation may also include situations where the polypeptide activity itself is reduced or eliminated compared to the polypeptide activity originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide; situations where the overall level of intracellular polypeptide activity and / or concentration (expression level) is reduced compared to the natural strain due to inhibition of gene expression encoding the polypeptide polynucleotide, or inhibition of translation into the polypeptide, etc.; situations where the polynucleotide is not expressed at all; and / or situations where polypeptide activity is not observed even if the polynucleotide is expressed. As used herein, the term "endogenous activity" refers to the activity of a specific polypeptide originally possessed by the pre-transformation parental strain, wild-type, or unmodified microorganism when the trait is altered by genetic modifications caused by natural or artificial factors, and may be used interchangeably with "pre-modification activity". The expression "attenuated, inactivated, lacking, reduced, downregulated, decreased, or weakened" of polypeptide activity compared to its endogenous activity means that the polypeptide activity is reduced compared to the activity of a specific polypeptide originally possessed by the pre-transformation parental strain or unmodified microorganism.

[0085] The attenuation of peptide activity can be achieved by any method known in the art, but is not limited to this method, and can be achieved by applying a variety of methods well known in the art (e.g., Nakashima N et al., Bacterialcellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014; 15(2): 2773–2793; Sambrook et al., Molecular Cloning 2012, etc.).

[0086] Specifically, the reduction of peptide activity disclosed herein can be achieved in the following ways:

[0087] 1) Missing part or all of the gene encoding the polypeptide;

[0088] 2) Modify expression regulatory regions (expression regulatory sequences) to reduce the expression of genes encoding polypeptides;

[0089] 3) Modify the amino acid sequence that makes up the polypeptide so that the polypeptide activity is removed or weakened (e.g., by deleting / replacing / adding one or more amino acids in the amino acid sequence).

[0090] 4) Modify the gene sequence encoding the polypeptide to remove or reduce the polypeptide activity (e.g., delete / replace / add one or more nucleotides in the nucleotide sequence of the polypeptide gene to encode a polypeptide that has been modified to remove or reduce the polypeptide activity).

[0091] 5) Modify the nucleotide sequence of the coding start codon, Shine-Dalgarno (SD) sequence, or 5'-UTR of a gene transcript that encodes a polypeptide;

[0092] 6) Introduce antisense oligonucleotides (e.g., antisense RNA) that bind complementary to the transcript of the gene encoding the polypeptide.

[0093] 7) Add a sequence complementary to the Shine-Dalgarno (SD) sequence to the front of the SD sequence of the gene encoding the polypeptide to form a secondary structure, thereby inhibiting ribosome attachment;

[0094] 8) Reverse transcription engineering (RTE), which involves adding a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of a gene sequence encoding a polypeptide;

[0095] 9) Regulating the cellular localization of peptides; or

[0096] 10) A combination of two or more of the methods 1) to 9) above, but not specifically limited thereto.

[0097] For example,

[0098] 1) The method of deleting part or all of the gene encoding the polypeptide can be achieved by deleting all polynucleotides encoding the endogenous target polypeptide within the chromosome, or by replacing the polynucleotides with polynucleotides that have partially deleted nucleotides, or by replacing the polynucleotides with marker genes.

[0099] 2) Modification of the expression regulatory region (expression regulatory sequence) can be achieved through deletion, insertion, non-conservative substitution, or conservative substitution, or a combination thereof; or by replacing the sequence with a less active sequence. The expression regulatory region may include, but is not limited to, promoters, operon sequences, sequences encoding ribosome binding sites, and sequences regulating transcription and translation termination.

[0100] Methods 3) and 4) for modifying amino acid sequences or polynucleotide sequences can be achieved by inducing sequence modifications, such as deletions, insertions, non-conserved or conserved substitutions, or combinations thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to reduce the activity of the polypeptide, or by replacing the sequence with a modified amino acid sequence or polynucleotide sequence that has weaker activity, or a modified amino acid sequence or polynucleotide sequence that is inactive, but are not limited thereto. For example, gene expression can be suppressed or reduced by introducing mutations into the polynucleotide sequence to form a stop codon, but are not limited thereto.

[0101] 5) Modifying the nucleotide sequence of the start codon or 5'-UTR of a gene transcript (which encodes a polypeptide) can be achieved, for example, by replacing the nucleotide sequence with a nucleotide sequence encoding another polypeptide with a lower expression rate than the endogenous start codon, but is not limited thereto.

[0102] 6) The method of introducing antisense oligonucleotides (e.g., antisense RNA) that are complementary to the transcript of the gene encoding the polypeptide can be found in the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0103] 7) Adding a sequence complementary to the Shine-Dalgarno (SD) sequence to the front of the SD sequence of a gene encoding a polypeptide to form a secondary structure, thereby inhibiting ribosome attachment, can be achieved by inhibiting mRNA translation or slowing down its rate.

[0104] In addition, 8) reverse transcription engineering (RTE), which adds a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide, can be achieved by forming an antisense nucleotide complementary to the gene transcript encoding the polypeptide to reduce activity.

[0105] 9) Methods for regulating the cellular localization of peptides can be achieved by targeting the peptide to specific organelles or specific spaces within the cell. For example, this can be achieved by adding or removing a leader sequence that functions in the targeting peptide, thus targeting the periplasm or cytoplasm, but is not limited to this.

[0106] This reduction in peptide activity may mean, but is not limited to, a decrease in the activity or concentration of the peptide relative to the peptide expressed in the wild-type strain or the pre-modified microbial strain, or a decrease in the amount of product produced by the peptide.

[0107] In one embodiment, the recombinant microorganism of this disclosure may be a microorganism whose activity is further enhanced compared to endogenous activity, selected from one or more of NADH:quinone oxidoreductase and O-phosphoserine export protein.

[0108] In any of the above embodiments, the recombinant microorganisms disclosed herein may be microorganisms in which the activity of NADH:quinone oxidoreductase is enhanced.

[0109] As used herein, the term "NADH:quinone oxidoreductase (hereinafter referred to as "Nuo")" is an enzyme that reduces quinone in the cell membrane by oxidizing NADH in the microbial electron transport chain. This enzyme protein may also be named NADH dehydrogenase-1 (NDH-1). The gene encoding this protein may be, for example, the nuoABCEFGHIJKLMN gene cluster, but is not limited thereto. The nuoABCEFGHIJKLMN gene cluster constitutes the nuo operon, the expression of which is regulated by a promoter preceding the operon and by polynucleotides at the ribosome binding site. In this disclosure, "nuoABCEFGHIJKLMN gene" may be used interchangeably with "gene encoding NADH:quinone oxidoreductase," "nuoABCEFGHIJKLMN gene," "nuo operon," and "nuo gene."

[0110] Nuo is a complex of 13 subunit proteins (NuoA, NuoB, NuoC, NuoE, NuoF, NuoG, NuoH, NuoI, NuoJ, NuoK, NuoL, NuoM, NuoN). Two types of operons, nuoABCEFGHIJKL and nuoMN, serve as templates in the translation of each subunit protein. The structure of the nuo operon can be found in EcoCyc (www.biocyc.org) (accession number: EG12082). The nuo operon is known to include a structural gene and an expression regulatory region. The "expression regulatory region" of the nuo operon refers to the region located upstream of the structural gene constituting the nuo operon, and therefore can regulate the expression of the structural gene. The expression regulatory region of the nuo operon may include promoters (nuoA promoter and / or nuoM promoter) in addition to the structural gene and the operon, and may specifically include promoters.

[0111] The operator is as described above.

[0112] In one implementation, the enhancement of NADH:quinone oxidoreductase activity can be achieved by modification that enhances the activity of NADH:quinone oxidoreductase compared to endogenous activity, or by strengthening or introducing the nuo operon.

[0113] Since the nuo operon described in this disclosure can reduce the accumulation of NADH by enhancing the OPS biosynthetic pathway, microorganisms modified to enhance the activity of NADH:quinone oxidoreductase, or microorganisms introduced with the nuo operon, may possess the characteristic of increasing OPS production and thus can be used to produce OPS.

[0114] The nuo operon may include a nucleotide sequence encoding an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher homology or identity with the amino acid sequences shown in SEQ ID NO:45-57. Specifically, the nuo operon may include a structural gene sequence encoding the amino acid sequence of SEQ ID NO:45-57 or an amino acid sequence homologous to or identical with it and exhibiting a corresponding function; and an expression regulatory region regulating the expression of said structural gene sequence. The sequence of SEQ ID NO:45-57 can be confirmed in the known database NCBI's GenBank.

[0115] Specifically, the nuo operon can be the nucleotide sequence of SEQ ID NO: 3 and / or a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher homology or identity with SEQ ID NO: 3. Furthermore, it is apparent that any nucleotide sequence in which a portion of the sequence is deleted, modified, substituted, or added may also fall within the scope of this disclosure, provided that the nucleotide sequence has such homology or identity and exhibits a function corresponding to that of the nuo operon.

[0116] In any of the above embodiments, the recombinant microorganisms disclosed herein may be microorganisms in which SerB activity is weakened.

[0117] The "SerB" of this disclosure has the activity of converting OPS to L-serine, therefore microorganisms modified to attenuate SerB activity have the property of accumulating OPS therein, which can be used for OPS production. The SerB of this disclosure may be a protein having or including the amino acid sequence shown in SEQ ID NO: 4, or may be a protein composed of or substantially composed of the amino acid sequence shown in SEQ ID NO: 4, but is not limited thereto. Furthermore, the SerB of this disclosure may have or include an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% or higher homology or identity with the amino acid sequence shown in SEQ ID NO: 4, as long as it exhibits SerB activity. Furthermore, the SerB of this disclosure may be composed of or substantially composed of an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% or higher homology or identity with the amino acid sequence shown in SEQ ID NO: 4, but is not limited thereto. Furthermore, the polynucleotide encoding SerB may have or include a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4. Furthermore, the polynucleotide encoding SerB may consist of or substantially consist of a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4. Due to codon degeneracy or considering preferred codons in the organism to which the SerB protein is to be expressed, the polynucleotide encoding SerB of this disclosure may be modified in various ways in the coding region without altering the amino acid sequence of the SerB protein. The polynucleotide encoding SerB of this disclosure may have or include a nucleotide sequence having at least 70%, 80%, 90%, 95%, or 99% or higher and less than 100% homology or identity with the nucleotide sequence of SEQ ID NO: 5, but is not limited thereto.

[0118] In any of the above embodiments, the recombinant microorganism of this disclosure may be a microorganism in which the activity of the O-phosphoserine export protein is enhanced. For example, it may be a microorganism with enhanced YhhS activity.

[0119] The "YhhS" disclosed herein possesses OPS-exporting activity; therefore, microorganisms modified to enhance YhhS activity possess the characteristic of OPS exporting and can be used for OPS production. The YhhS of this disclosure may be a protein having or including the amino acid sequence shown in SEQ ID NO: 6, or may be a protein composed of or substantially composed of the amino acid sequence shown in SEQ ID NO: 6, but is not limited thereto. Furthermore, the YhhS of this disclosure may have or include amino acid sequences having at least 70%, 80%, 90%, 95%, or 99% or higher homology or identity with the amino acid sequence shown in SEQ ID NO: 6, provided it exhibits YhhS activity. Furthermore, the YhhS of this disclosure may be composed of or substantially composed of amino acid sequences having at least 70%, 80%, 90%, 95%, or 99% or higher homology or identity with the amino acid sequence shown in SEQ ID NO: 6, but is not limited thereto. Furthermore, the polynucleotide encoding YhhS may have or include a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 6. Furthermore, the polynucleotide encoding YhhS may consist of or substantially consist of a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 6. Due to codon degeneracy or considering preferred codons in the organism to which the YhhS protein is to be expressed, the polynucleotide encoding YhhS of this disclosure may be modified in various ways in the coding region without altering the amino acid sequence of the YhhS protein. The polynucleotide encoding YhhS of this disclosure may have or include a nucleotide sequence having at least 70%, 80%, 90%, 95%, or 99% or higher and less than 100% homology or identity with the nucleotide sequence of SEQ ID NO: 7, but is not limited thereto.

[0120] In one embodiment, the microorganism includes modifications that enhance the ability to produce OPS and / or export OPS from the cell; or modifications that enhance the ability to decompose and / or import OPS, such as CA07-0012 (KCCM 11121 p; US2012-0190081A), CA07-4821, or CA07-4828, but are not limited thereto. Regarding the content of the OPS-producing microorganism, in addition to those described above, disclosures in Korean Patent Publication No. 1381048 or US Application Publication No. 2012-0190081, etc., may be used as references in this disclosure, but are not limited thereto.

[0121] Another aspect of this disclosure provides a method for producing O-phosphoserine, comprising culturing a recombinant microorganism producing O-phosphoserine in a culture medium, wherein the activity of the MntH protein in the recombinant microorganism is enhanced compared to endogenous activity.

[0122] MntH protein, endogenous activity, enhancement, O-phosphoserine, and microorganisms are as described above.

[0123] As used herein, the term "culture" refers to the growth of microorganisms under appropriately controlled environmental conditions. The culture process of this disclosure can be carried out in suitable culture media and culture conditions known in the art. Those skilled in the art can readily adapt this culture process for use depending on the strain to be selected. Specifically, the culture can be a batch culture, a continuous culture, or a fed-batch culture, but is not limited thereto.

[0124] When culturing microorganisms, the culture medium may also contain glycine or serine. Glycine can be provided in the form of purified glycine, glycine-containing yeast extract, or tryptone. The concentration of glycine in the culture medium is typically from 0.1 g / L to 10 g / L, particularly from 0.5 g / L to 3 g / L. Similarly, serine can be provided in the form of purified serine, serine-containing yeast extract, or tryptone. The concentration of serine in the culture medium is typically from 0.1 g / L to 5 g / L, particularly from 0.1 g / L to 1 g / L.

[0125] Examples of carbon sources in culture media can include 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. These carbon sources can be used alone or in combination, but are not limited to these.

[0126] Examples of nitrogen sources in culture media can include organic nitrogen sources such as peptone, yeast extract, meat 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. These nitrogen sources can be used alone or in combination, but are not limited thereto.

[0127] Examples of phosphorus sources in the culture medium may include, but are not limited to, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and their corresponding sodium salts.

[0128] In addition, the culture medium may include metal salts, such as magnesium sulfate or ferric sulfate, and may also contain amino acids, vitamins, and appropriate precursors. These media or precursors may be added to the culture in batch or continuous culture, but are not limited thereto.

[0129] The pH of the culture can be adjusted by adding compounds (such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid) in an appropriate manner during the culture process. Furthermore, the use of antifoaming agents (such as polyethylene glycol esters of fatty acids) during the culture process can prevent bubble formation. Additionally, oxygen or oxygen-containing gas can be injected into the culture to maintain aerobic conditions; or nitrogen, hydrogen, or carbon dioxide can be injected to maintain anaerobic or micro-aerobic conditions, or no gas injection may be necessary. The culture temperature can be in the range of 25°C to 40°C, specifically 30°C to 35°C. The culture can continue until the production of useful substances can be obtained, specifically 10 hours to 100 hours, but is not limited to these illustrative examples.

[0130] For example, prior to the cultivation step, the method for producing OPS of this disclosure may also include the steps of preparing the microorganisms of this disclosure, preparing the culture medium for culturing the microorganisms, or a combination thereof (in any order, regardless of the order).

[0131] The method for producing OPS disclosed herein may further include a step of recovering OPS from a culture medium (on which the culture grows) or from cultured microorganisms. A recovery step may also be included after the culturing step.

[0132] In the recovery step, the desired OPS can be collected using the methods for culturing microorganisms disclosed herein, for example, using suitable methods known in the art, depending on the batch culture, continuous culture, or fed-batch culture method. For example, methods such as centrifugation, filtration, treatment with a protein crystallizing precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatography methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or combinations thereof can be used, and the desired OPS can be recovered from the culture medium or microorganisms using suitable methods known in the art.

[0133] Furthermore, the method for producing OPS disclosed herein may also include a purification step, which may be performed using suitable methods known in the art. In one example, when the method for producing OPS disclosed herein includes a recovery step and a purification step, the recovery step and the purification step may be performed continuously or intermittently, simultaneously, or integrated into a single step, but the method is not limited thereto. Therefore, the recovered OPS may be in a purified state or a microbial fermentation broth containing OPS. Moreover, OPS recovery can be effectively performed by adding suitable methods known in the art before and after the cultivation step and before and after the recovery step.

[0134] Another aspect of this disclosure provides a method for producing cysteine ​​or a derivative thereof, comprising:

[0135] a) Culturing recombinant microorganisms producing O-phosphoserine in a medium for producing O-phosphoserine or a medium containing O-phosphoserine, wherein the activity of the MntH protein is enhanced compared to endogenous activity in the recombinant microorganisms; and

[0136] b) In the presence of O-phosphoserine hydrogen sulfide hydrolase (OPSS) or microorganisms containing the enzyme, react the O-phosphoserine produced in step a) or the culture medium containing it with the sulfide.

[0137] Steps a) and b) are not necessarily restricted by order, i.e., they can be performed continuously or sequentially, and there is no time interval between these steps. These steps can be performed simultaneously or at intervals of seconds, minutes, hours, or days.

[0138] MntH protein, endogenous activity, enhancement, O-phosphoserine (OPS), and microorganisms are as described above.

[0139] As used herein, the term "derivative" refers to a similar compound obtained by chemically modifying a portion of any compound. The term generally refers to a compound in which one hydrogen atom or a particular group of atoms is replaced by another atom or group of atoms.

[0140] As used herein, the term "cysteine ​​derivative" refers to a compound in which a hydrogen atom or a specific group of atoms in cysteine ​​is replaced by another atom or group of atoms. For example, cysteine ​​derivatives can have a form in which the nitrogen atom of the amino group (-NH2) or the sulfur atom of the thiol group (-SH) in cysteine ​​has another atom or group attached to it. Examples of cysteine ​​derivatives may include, but are not limited to, 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-sulfinyl-L-alanine, Fmoc-Cys(Boc-methyl)-OH, seleno-L-cysteine, S-(2-thiazolyl)-L-cysteine, S-(2-thienyl)-L-cysteine, S-(4-tolyl)-L-cysteine, etc.

[0141] As long as cysteine ​​is produced according to the method of this disclosure, it can be readily converted into various cysteine ​​derivatives by methods known in the art.

[0142] In this disclosure, the method for producing cysteine ​​derivatives may further include converting the cysteine ​​produced in step b) into cysteine ​​derivatives.

[0143] Specifically, in this disclosure, a method for producing cysteine ​​derivatives may include the following steps: producing cysteine ​​according to the method of this disclosure described above; and converting the produced cysteine ​​into cysteine ​​derivatives.

[0144] The conversion of produced cysteine ​​into cysteine ​​derivatives can be carried out by methods known in the art. For example, according to methods known in the art, cysteine ​​can be synthesized into N-acetylcysteine ​​(NAC) by reacting with an acetylation agent, or into S-carboxymethylcysteine ​​(SCMC) by reacting with haloacetic acid under alkaline conditions, but examples are not limited thereto.

[0145] These cysteine ​​derivatives are primarily used as pharmaceutical materials for antitussives, cough suppressants, and treatments for bronchitis, bronchial asthma, pharyngitis, etc., but are not limited to these applications.

[0146] As used herein, the term “O-phosphoserine hydrogen sulfide hydrolase (OPSS)” refers to an enzyme that catalyzes the reaction of converting OPS to cysteine ​​by donating a sulfhydryl group (SH group) to OPS. This enzyme may have been first discovered in thermophilic archaea (Aeropyrumpernix), Mycobacterium tuberculosis, Mycobacterium smegmatics, and Trichomonas vaginalis (Mino K and Ishikawa K, FEBS Letters, 551: 133-138, 2003; Burns KE et al., J. Am. Chem. Soc., 127: 11602-11603, 2005). In addition, OPSS may include not only wild-type OPSS protein, but also variant proteins in which a portion of the sequence encoding OPSS is deleted, substituted, or added, and the activity of the variant protein is equal to or higher than that of the wild-type OPSS protein. Furthermore, it may include all OPSS proteins and their variant proteins disclosed in Korean Application Publication No. 2012-0041115 and Korean Patent Publication No. 1208267.

[0147] Sulfides can be any sulfide, provided not only in the solid form commonly used in the art, but also in liquid or gaseous forms due to differences in pH, pressure, and solubility, and thus can be converted into sulfides (S). 2− ), thiosulfate (S2O3) 2−The sulfide can be any form of thiol (SH) group, without limitation. Specifically, the sulfide may include Na2S, NaSH, (NH4)2S, H2S, or Na2S2O3, which can provide a thiol group to the OPS, but is not limited thereto. In the reaction, a single thiol group is provided to a single reactive OPS group to produce a single cysteine ​​or a derivative thereof. In this document, the sulfide may be added in an amount of 0.1 to 3 molar equivalents, specifically 1 to 2 molar equivalents, based on the molar concentration of the OPS, but is not limited thereto.

[0148] Furthermore, methods for producing cysteine ​​derivatives may also include a step of recovering cysteine ​​produced by the reaction steps. In this document, the desired cysteine ​​can be collected by separating and purifying cysteine ​​from the reaction solution using suitable reactions known in the art.

[0149] Another aspect of this disclosure provides compositions for producing O-phosphoserine, cysteine, or cysteine ​​derivatives, comprising: a recombinant microorganism producing O-phosphoserine, wherein the activity of the MntH protein is enhanced compared to endogenous activity; a culture medium for culturing the microorganism; or a combination thereof.

[0150] The compositions disclosed herein may also include any suitable excipient commonly used in the production of O-phosphoserine, cysteine, or cysteine ​​derivatives, and such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0151] Another aspect of this disclosure provides a method for producing recombinant microorganisms that produce O-phosphoserine, the method comprising enhancing the activity of MntH protein compared to endogenous activity.

[0152] Another aspect of this disclosure provides the use of recombinant microorganisms producing O-phosphoserine in the production of O-phosphoserine, cysteine, or cysteine ​​derivatives, wherein the activity of the MntH protein is enhanced compared to endogenous activity in said recombinant microorganisms.

[0153] The MntH protein, endogenous activity, enhancement, O-phosphoserine (OPS), cysteine, cysteine ​​derivatives, and microorganisms in this regard are as described above.

[0154] [Methods of Implementing the Invention]

[0155] The present disclosure will be described in detail below by way of embodiments. However, these embodiments are merely preferred embodiments given for illustrative purposes, and therefore the scope of the present disclosure is not limited to these embodiments. Furthermore, those skilled in the art within the scope of this disclosure or similar technical fields will fully understand and readily implement technical features not described herein.

[0156] Example 1: Preparation of a strain of YhhS with enhanced expression of O-phosphoserine (OPS) production

[0157] 1-1. Preparation of vectors for enhancing YhhS expression

[0158] Using wild-type Escherichia coli ATCC27325 chromosomal DNA as a template, PCR was performed using primer pairs SEQ ID NO: 10 and 11 to obtain the gene fragment upstream of the wild-type promoter of the yhhS gene (SEQ ID NO: 7) (in which homologous recombination occurred on the chromosome), and primer pairs SEQ ID NO: 14 and 15 to obtain the gene fragment downstream of the wild-type promoter of the yhhS gene. Furthermore, using pCL_Ptrc-gfp (WO 2016-024771 A1) as a template, PCR was performed using primer pairs SEQ ID NO: 12 and 13 to obtain the Ptrc promoter (SEQ ID N: 59).

[0159] Use Solg TM PCR was performed using Pfu-X DNA polymerase under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by denaturation at 95°C for 30 seconds and 30 cycles, annealing at 60°C for 30 seconds, polymerization at 72°C for 60 seconds, and then polymerization at 72°C for 5 minutes.

[0160] The upstream and downstream fragments of the yhhS promoter, as well as the Ptrc promoter fragment obtained by the above method, were cloned using the pSKH130 vector (SEQ ID NO: 58, US Application Publication No. 2020-0048619). These fragments were used for chromosome transformation after digestion with the restriction enzyme EcoRV. The recombinant plasmid was obtained using an in-fusion cloning kit (Clontech Laboratories, Inc.) and named pSKH_Ptrc-yhhS.

[0161] The primer sequences used above are shown in Table 1.

[0162] [Table 1]

[0163]

[0164] 1-2. Preparation of strains with enhanced YhhS expression

[0165] The pSKH_Ptrc-yhhS prepared in Example 1-1 was introduced into CA07-0012 (KCCM 11121P, US Patent Publication No. US 8557549 B2). CA07-0012 is an OPS-producing strain in which the OPS degradation ability is weakened by the deletion of endogenous phosphatase (SerB) in wild-type Escherichia coli K-12W3110, thereby enhancing the expression of YhhS (SEQ ID NO: 6). YhhS is a protein with OPS exporting ability, thereby further enhancing the OPS exporting ability.

[0166] The pSKH_Ptrc-yhhS prepared in Example 1-1 was transformed into strain CA07-0012 by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), followed by a first crossover using R6K and kanamycin to obtain the desired strain. Subsequently, the strain underwent a second crossover in a sucrose-containing medium to obtain a strain in which the kanamycin resistance gene was deleted and the Ptrc promoter nucleotide sequence was inserted at the end of the wild-type promoter sequence of the yhhS gene. The insertion of the Ptrc promoter nucleotide sequence was confirmed by genome sequencing and PCR amplification using primer pairs SEQ ID NO: 16 and 17, which amplified the outer regions of the upstream and downstream regions of homologous recombination, respectively. The resulting strain was named CA07-4821 (CA07-0012ΔPn_yhhS::Ptrc_yhhS).

[0167] The primer sequences used above are shown in Table 2.

[0168] [Table 2]

[0169]

[0170] Example 2: Preparation of OPS-producing strains with the introduction of the nuo operon

[0171] 2-1. Preparation of vectors for introducing the nuo operon

[0172] Using wild-type Escherichia coli ATCC27325 chromosomal DNA as a template, PCR was performed using primer pairs SEQ ID NO: 18 and 19 to obtain the gene fragment upstream of the wild-type promoter of the nuo operon (SEQ ID NO: 3), and primer pairs SEQ ID NO: 20 and 21 to obtain the gene fragment downstream of the wild-type promoter of the nuo operon. Furthermore, using wild-type Escherichia coli ATCC27325 chromosomal DNA as a template, PCR was performed using primer pairs SEQ ID NO: 22 and 23 to obtain the promoter region of the rmf gene (SEQ ID NO: 8).

[0173] Use Solg TM PCR was performed using Pfu-X DNA polymerase under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by denaturation at 95°C for 30 seconds and 30 cycles, annealing at 60°C for 30 seconds, polymerization at 72°C for 60 seconds, and then polymerization at 72°C for 5 minutes.

[0174] The upstream and downstream fragments of the nuo promoter, as well as the rmf promoter fragment obtained by the above method, were cloned using pSKH130 for chromosome transformation after being cut with the restriction enzyme EcoRV. The recombinant plasmid was obtained using an in-fusion cloning kit and named pSKH_Prmf-nuoA.

[0175] The primer sequences used above are shown in Table 3.

[0176] [Table 3]

[0177]

[0178] 2-2. Preparation of strains incorporating the nuo operon

[0179] pSKH_Prmf-nuoA prepared in Example 2-1 was transformed into strain CA07-4821, an OPS-producing strain whose OPS degradation ability was weakened by the absence of endogenous phosphatase SerB. The desired strain was obtained using R6K and kanamycin in the first exchange. Subsequently, the strain underwent a second exchange in a sucrose-containing medium to obtain a strain in which the kanamycin resistance gene was deleted and the promoter nucleotide sequence of the rmf gene was inserted at the end of the wild-type promoter nucleotide sequence of the nuo gene. The insertion of the rmf promoter nucleotide sequence was confirmed by genome sequencing and PCR amplification using primer pairs SEQ ID NO: 24 and 25, which amplified the outer regions of the upstream and downstream regions of homologous recombination, respectively. The resulting strain was named CA07-4828 (CA07-4821ΔPn_nuoA::Prmf_nuoA).

[0180] The primer sequences used above are shown in Table 4 below.

[0181] [Table 4]

[0182]

[0183] Example 3: Preparation of strains with enhanced or absent MntH expression

[0184] 3-1. Preparation of vectors for enhancing YhhS expression

[0185] As a result of previous studies, the average transcription levels of the mntH, serC, and rmf genes in OPS-producing host strains were found to be 698, 6215, and 32205, respectively, as shown in Table 5 below. This confirms that the average transcription level of the rmf gene is 46-fold higher than that of the mntH gene, and the average transcription level of the serC gene is 8.9-fold higher. Therefore, this confirms that the rmf and serC gene promoters are relatively stronger promoters compared to the mntH promoter.

[0186] [Table 5]

[0187]

[0188] Therefore, strains with enhanced MntH expression were prepared by further inserting the rmf promoter and serC promoter (SEQ ID NO: 9) (which showed stronger activity) at the promoter end of the mntH gene (SEQ ID NO: 2) in OPS-producing microorganisms.

[0189] PCR was performed using wild-type *E. coli* ATCC27325 chromosomal DNA as a template. Primers SEQ ID NO: 26 and 27 were used to obtain the gene fragment upstream of the wild-type promoter of the *mntH* gene, and primers SEQ ID NO: 28 and 29 were used to obtain the gene fragment downstream of the wild-type promoter of the *mntH* gene. Furthermore, PCR was performed using wild-type *E. coli* ATCC27325 chromosomal DNA as a template, and primers SEQ ID NO: 30 and 31 were used to obtain the promoter region of the *rmf* gene.

[0190] Use Solg TM PCR was performed using Pfu-X DNA polymerase under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by denaturation at 95°C for 30 seconds and 30 cycles, annealing at 60°C for 30 seconds, polymerization at 72°C for 60 seconds, and then polymerization at 72°C for 5 minutes.

[0191] The upstream and downstream fragments of the mntH promoter, as well as the rmf promoter fragment obtained by the above method, were cloned using pSKH130 for chromosome transformation after being cut with the restriction enzyme EcoRV. The recombinant plasmid was obtained using an in-fusion cloning kit and named pSKH130_Prmf-mntH.

[0192] PCR was performed using wild-type *E. coli* ATCC27325 chromosomal DNA as a template. Primers SEQ ID NO: 32 and 33 were used to obtain the gene fragment upstream of the wild-type promoter of the *mntH* gene, and primers SEQ ID NO: 34 and 35 were used to obtain the gene fragment downstream of the wild-type promoter of the *mntH* gene. Furthermore, PCR was performed using wild-type *E. coli* ATCC27325 chromosomal DNA as a template, and primers SEQ ID NO: 36 and 37 were used to obtain the promoter region of the *serC* gene.

[0193] Use Solg TM PCR was performed using Pfu-X DNA polymerase under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by denaturation at 95°C for 30 seconds and 30 cycles, annealing at 60°C for 30 seconds, polymerization at 72°C for 60 seconds, and then polymerization at 72°C for 5 minutes.

[0194] The upstream and downstream fragments of the mntH promoter, along with the serC promoter fragment obtained by the above method, were cloned using pSKH130. These fragments were then used for chromosome transformation after being cut with the restriction enzyme EcoRV. The recombinant plasmid was obtained using an in-fusion kit and named pSKH130_PserC-mntH.

[0195] 3-2. Preparation of vectors for MntH deletion

[0196] Using wild-type Escherichia coli ATCC27325 chromosomal DNA as a template, PCR was performed. The gene fragment upstream of the wild-type promoter of the mntH gene was obtained using primer pairs SEQ ID NO: 38 and 39, and the gene fragment downstream of the wild-type promoter of the mntH gene was obtained using primer pairs SEQ ID NO: 40 and 41.

[0197] PCR was performed in the same manner as in Example 3-1 to obtain upstream and downstream fragments of the mntH promoter, and these fragments were cloned in the same manner as in Example 3-1 to obtain a recombinant plasmid. The plasmid thus obtained was named pSKH_ΔmntH.

[0198] The primer sequences used in Examples 3-1 and 3-2 are shown in Table 6 below.

[0199] [Table 6]

[0200]

[0201] 3-3. Preparation of strains with enhanced MntH expression

[0202] The pSKH_PserC-mntH prepared in Example 3-1 was transformed into the CA07-4821 strain of Example 1-2, a YhhS-enhanced OPS-producing strain, by electroporation. A second exchange was then performed to obtain a strain in which the promoter nucleotide sequence of the serC gene was inserted at the end of the wild-type promoter nucleotide sequence of the mntH gene. The insertion of the serC promoter nucleotide sequence was confirmed by genome sequencing and PCR amplification using primer pairs SEQ ID NO: 42 and 43, which amplified the upstream and downstream regions of the homologous recombination, respectively. The resulting strain was named CA07-4895 (CA07-4821ΔPn_mntH::PserC_mntH).

[0203] The pSKH_Prmf-mntH and pSKH_PserC-mntH prepared in Example 3-1 were transformed into strain CA07-4828 of Example 2-2, an OPS-producing strain with the nuo operon introduced, by electroporation. A second exchange was then performed to obtain strains in which the promoter nucleotide sequence of the serC or rmf gene was inserted at the end of the wild-type promoter nucleotide sequence of the mntH gene, respectively. The insertion of the serC or rmf promoter nucleotide sequence was confirmed by genome sequencing and PCR amplification using primer pairs SEQ ID NO: 42 and 43, which could amplify the outer regions of the upstream and downstream regions of homologous recombination, respectively. The resulting strains were named CA07-4898 (CA07-4828ΔPn_mntH::PserC_mntH) and CA07-4899 (CA07-4828ΔPn_mntH::Prmf_mntH), respectively.

[0204] 3-4. Preparation of MntH-deleted strains

[0205] The pSKH_ΔmntH prepared in Example 3-2 was transformed into the CA07-4828 strain of Example 2-2 by electroporation. The CA07-4828 strain is an OPS-producing strain with an introduced nuo operon. A second crossover was then performed to obtain a strain lacking the mntH gene. The deletion of the mntH nucleotide sequence was confirmed by genome sequencing and PCR amplification using primer pairs SEQ ID NO: 42 and 44, which amplified the upstream and downstream regions of homologous recombination, respectively. The resulting strain was named CA07-4897 (CA07-4828ΔmntH).

[0206] The primer sequences used in Examples 3-3 and 3-4 are shown in Table 7 below.

[0207] [Table 7]

[0208]

[0209] Example 4: Evaluation of the strain's OPS production capacity

[0210] To evaluate the potency of shake-flask fermentation, the following strains were tested: CA07-4895, CA07-4898 and CA07-4899 (both strains with enhanced MntH expression) from Example 3-3, CA07-4897 (a strain lacking MntH) from Example 3-4, CA07-0012 (the parental strain) from Example 1-2, CA07-4821 from Example 1-2, and CA07-4828 (the control strains) from Example 2-2.

[0211] Each strain was plated on solid LB medium and incubated overnight at 33°C. The strains incubated overnight on solid LB medium were then inoculated into 25 mL of the following potency medium and incubated at 33°C for 48 hours at 200 rpm. After incubation, the OPS concentration was measured using HPLC, and the results are shown in Table 8 below.

[0212] <Potency Culture Medium>

[0213] Glucose 40 g / L, KH₂PO₄ 6 g / L, (NH₄)₂SO₄ 17 g / L, MgSO₄·7H₂O 1 g / L, MnSO₄·4H₂O 5 mg / L, FeSO₄·7H₂O 10 mg / L, L-glycine 1.5 g / L, yeast extract 2.5 g / L, CaCO₃ 30 g / L, pH 6.8

[0214] [Table 8]

[0215]

[0216] As shown in Table 8, the MntH-enhanced CA07-4895 strain (which used the YhhS-enhanced CA07-4821 strain as the parent strain) showed a 9.4% increase in OPS production capacity compared to the parent strain.

[0217] The CA07-4828 strain, enhanced by both the YhhS and nuo operons, showed a 7.9% increase in OPS production compared to the YhhS-enhanced CA07-4821 strain.

[0218] Furthermore, the MntH-enhanced CA07-4898 and CA07-4899 strains (which used the nuo operon-enhanced CA07-4828 as a parent strain) showed increased OPS production capacity of 2.3% and 5.3%, respectively, compared to the parent strain. The MntH-deficient CA07-4897 strain showed a 10.3% decrease in OPS production capacity compared to the parent strain.

[0219] Therefore, it has been confirmed that enhancing MntH increases the ability to produce OPS.

[0220] Based on the foregoing, those skilled in the art will understand that this disclosure can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of this disclosure. Rather, this disclosure is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of this disclosure as defined by the appended claims.

Claims

1. A recombinant microorganism producing O-phosphoserine, wherein the activity of the MntH protein is enhanced compared to its endogenous activity.

2. The microorganism according to claim 1, wherein the recombinant microorganism has an enhanced O-phosphoserine production capacity compared to endogenous O-phosphoserine production capacity.

3. The microorganism according to claim 1, wherein the MntH protein comprises the amino acid sequence of SEQ ID NO:

1.

4. The microorganism according to claim 1, wherein the polynucleotide encoding the MntH protein comprises the nucleotide sequence of SEQ ID NO:

2.

5. The microorganism according to claim 1, wherein the activity of one or more selected from NADH:quinone oxidoreductase and O-phosphoserine export protein is further enhanced compared with endogenous activity.

6. The microorganism according to claim 1, wherein the recombinant microorganism belongs to the genus Escherichia.

7. A method for producing O-phosphoserine, the method comprising culturing a recombinant microorganism producing O-phosphoserine in a culture medium, wherein the activity of the MntH protein is enhanced compared to endogenous activity in the recombinant microorganism.

8. The method of claim 7, wherein the method further comprises recovering O-phosphoserine from the culture medium or microorganism.

9. The method of claim 7, wherein the MntH protein comprises the amino acid sequence of SEQ ID NO:

1.

10. The method of claim 7, wherein the polynucleotide encoding the MntH protein comprises the nucleotide sequence of SEQ ID NO:

2.

11. A method for producing cysteine ​​or a derivative thereof, the method comprising: a) Culturing recombinant microorganisms that produce O-phosphoserine in a medium for producing O-phosphoserine or in a medium containing O-phosphoserine, wherein the activity of MntH is enhanced in the recombinant microorganisms compared with endogenous activity. and b) In the presence of O-phosphoserine hydrogen sulfide hydrolase (OPSS) or a microorganism containing the enzyme, react the O-phosphoserine produced in step a) or a culture medium containing it with the sulfide.

12. The method according to claim 11, wherein the sulfide is one or more selected from the group consisting of Na2S, NaSH, (NH4)2S, H2S and Na2S2O3.

13. The method of claim 11, wherein the MntH protein comprises the amino acid sequence of SEQ ID NO:

1.

14. The method of claim 11, wherein the polynucleotide encoding the MntH protein comprises the nucleotide sequence of SEQ ID NO:

2.

15. Use of recombinant microorganisms producing O-phosphoserine in the production of O-phosphoserine, cysteine ​​or cysteine ​​derivatives, wherein the activity of MntH protein is enhanced compared to endogenous activity in said recombinant microorganisms.

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