Provided are a recombinant microorganism in which expression of NADH:quinone oxidoreductase is regulated, and a method for producing o-phosphoserine, cysteine and derivatives thereof using the same.

Abstract

To provide a microorganism for excessively producing O-phosphoserine (hereinafter referred to as "OPS") which is a precursor for producing cysteine in high yield by a method for producing the OPS by fermentation using the microorganism and then reacting the OPS with a sulfide under the catalytic action of O-phosphoserine sulfhydrylase to convert the OPS into L-cysteine.SOLUTION: Provided is a recombinant microorganism of the genus Escherichia producing O-phosphoserine by enhancing the activity of NADH:quinone oxidoreductase.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present application relates to a recombinant microorganism in which the expression of NADH:quinone oxidoreductase is regulated, and a method for producing O-phosphoserine, cysteine, and cysteine ​​derivatives 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] Methods for producing L-cysteine ​​using microorganisms include: 1) a method for biologically converting D,L-ATC (D,L-2-aminothiazoline-4-carboxylic acid) using microorganisms; 3) a direct fermentation method for producing L-cysteine ​​using Enterococcus coli (Patent Document 1, Non-Patent Document 1), 4) a method for producing O-phosphoserine (hereinafter referred to as "OPS") by fermentation using microorganisms, Then, O-phosphoserine sulfhydrylase (hereinafter referred to as A method for converting L-cysteine ​​into L-cysteine ​​by reacting it with a sulfide under the catalytic action of an OPSS (Patent Document 2) is known.

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

[0005] [Patent Document 1] European Patent No. 0885962 [Patent Document 2] US Patent Application Publication No. 2012 / 0190081 [Patent Document 3] U.S. Patent No. 7,662,943 [Patent Document 4] Korean Patent No. 1381048 [Patent Document 5] U.S. Patent No. 9,127,324 [Patent Document 6] US Patent Application Publication No. 2020 / 0048619 [Patent Document 7] U.S. Patent No. 10,323,262 [Patent Document 8] US Patent Application Publication No. 2017 / 0247727 [Non-patent literature]

[0006] [Non-Patent Document 1] Wada M and Takagi H, Appl. Microbiol. Biochem., 73:48-54, 2006 [Non-patent document 2] www.biocyc.org [Non-patent document 3] Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90, 5873 (1993) [Non-patent document 4] Methods Enzymol., 183, 63, 1990 [Non-patent document 5] http: / / www.ncbi.nlm.nih.gov [Non-patent document 6] J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 [Non-Patent Document 7] FM Ausubel et al., Current Protocols in Molecular Biology [Non-licensed document 8] Ahmed Zahoor, Computational and structural biotechnology journal, vol 3, 2012 October

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

[0007] An objective of the present application is to provide a recombinant microorganism in which the expression of NADH:quinone oxidoreductase is regulated, and a method for producing O-phosphoserine, cysteine, and cysteine ​​derivatives using the same. [Means for solving the problem]

[0008] An object of the present application is to provide a recombinant Escherichia microorganism having enhanced NADH:quinone oxidoreductase activity and the ability to produce O-phosphoserine.

[0009] Another object of the present application is to provide a method for producing O-phosphoserine, which includes a step of culturing the O-phosphoserine-producing microorganism of the present application in a medium.

[0010] Furthermore, the present application provides a method for producing O-phosphoserine or a medium containing O-phosphoserine by culturing an O-phosphoserine-producing microorganism having enhanced activity of NADH:quinone oxidoreductase in a medium; and b) culturing the O-phosphoserine produced in step a) in the presence of O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing O-phosphoserine sulfhydrylase. and reacting phosphoserine or a medium containing phosphoserine with sulfide. [Effects of the Invention]

[0011] The OPS-producing microorganism of the present invention, which has enhanced activity of the NADH:quinone oxidoreductase, can produce OPS with high efficiency. DETAILED DESCRIPTION OF THE INVENTION

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

[0013] One aspect of the present application is to enhance the activity of NADH:quinone oxidoreductase and to produce O-phospholipids. A microorganism that produces sphoserin is provided.

[0014] In the present application, "O-phosphoserine (hereinafter referred to as "OPS")" means serine. It is a phosphoric acid ester of phosphorus and is a component of various proteins. OPS is a precursor of L-cysteine ​​and is converted to cysteine ​​by reacting with sulfide under the catalytic action of O-phosphoserine sulfhydrylase (OPS sulfhydrylase, OPSS). However, the present invention is not limited to this (Patent Document 2).

[0015] In the present application, "NADH:quinone oxidoreductase (hereinafter referred to as "Nuo")" refers to an enzyme that oxidizes NADH in the electron transport system of a microorganism to produce NADH. This refers to an enzyme that reduces membrane quinone. This enzyme protein is also called NADH dehydrogenase-1 (NDH-1). The gene encoding the nuoABCEFGHIJKLMN gene is, for example, but not limited to, the nuoABCEFGHIJKLMN gene cluster. The nuoABCEFGHIJKLMN gene cluster constitutes the nuo operon, and its expression can be regulated by a promoter and a ribosome binding site polynucleotide upstream of the operon. In this application, the term "nuoABCEFGHIJKLMN gene" is also used interchangeably with "gene encoding NADH:quinone oxidoreductase," "nuoABCEFGHIJKLMN gene," "nuo operon," and "nuo gene."

[0016] In this application, the term "operon" refers to a functional unit of DNA containing a group of genes whose expression is regulated by one expression control sequence, specifically one promoter. The mRNA transcribed by the operon may be a polycistronic mRNA in which one mRNA molecule encodes one or more proteins, and one mRNA molecule encodes one protein. It may be a monocistronic mRNA encoding a protein. stomach.

[0017] Nuo is a complex of 13 subunit proteins (NuoA, The translation of each subunit protein (NuoB, NuoC, NuoE, NuoF, NuoG, NuoH, NuoI, NuoJ, NuoK, NuoL, NuoM, NuoN) uses two types of operons, nuoABCEFGHIJKL and nuoMN, as templates. The structure of the nuo operon can be confirmed in EcoCyc (Non-Patent Document 2) (Registration Number: EG12082). The nuo operon is known to contain a structural gene and an expression regulatory region. The structure of the nuo operon The term "expression regulatory region" refers to a site located upstream of the structural genes constituting the nuo operon and capable of regulating the expression of the structural genes. The expression regulatory region of the nuo operon may include a promoter (nuoA promoter and / or nuoM promoter) excluding the structural genes and an operator, and specifically may include a promoter.

[0018] The nuo operon may comprise a base sequence encoding an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the amino acid sequences represented by SEQ ID NOs: 26 to 38. Specifically, the nuo operon may comprise a structural gene sequence encoding the amino acid sequence of SEQ ID NOs: 26 to 38, or an amino acid sequence having homology or identity thereto and corresponding function, and an expression regulatory region that regulates the expression of the structural gene sequence. The sequences of SEQ ID NOs: 26 to 38 can be confirmed in the publicly known database NCBI Genbank.

[0019] Specifically, the nuo operon has the base sequence of SEQ ID NO: 1 and / or a sequence identical to SEQ ID NO: 1. The base sequence may have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with the nuo operon. Needless to say, the present application also includes base sequences in which a portion of the sequence has been deleted, modified, substituted, or added, as long as the base sequence has such homology or identity and functions equivalent to the nuo operon.

[0020] As used herein, "homology" and "identity" refer to the degree to which two given amino acid or nucleotide sequences are related, expressed as a percentage. Homology and identity are often used interchangeably.

[0021] Conserved polynucleotide or polypeptide sequence homology or identity Identity may be determined by standard alignment algorithms, with default gap penalties established by the program being used. Substantially homologous or identical sequences are detected under moderate or high stringency conditions. Generally, a polynucleotide hybridizes under stringent conditions to at least about 50%, 60%, 70%, 80%, or 90% or more of the entire sequence or length, even if the polynucleotide has degenerate codons in place of certain codons.

[0022] The homology or identity of the polypeptide or polynucleotide sequences can be determined, for example, using the literature algorithm BLAST [see Non-Patent Document 3] or FASTA by Pearson (see Non-Patent Document 4). Based on such an algorithm, BLAST, programs called BLASTN and BLASTX have been developed (see Non-Patent Document 5). Furthermore, whether any amino acid or polynucleotide sequence has homology, similarity, or identity can be confirmed by comparing the sequences in a hybridization experiment under defined stringent conditions, and suitable defined hybridization conditions are within the skill of the art and can be determined by methods well known to those skilled in the art (e.g., Non-Patent Documents 6 and 7).

[0023] The microorganisms of the present application are not particularly limited in type as long as they are capable of producing OPS, and may be either prokaryotic or eukaryotic cells, specifically prokaryotic cells. Examples of prokaryotic cells include microorganisms belonging to the genera Escherichia, Erwinia, Seratia, Providencia, Corynebacterium, and Brevibacterium. Examples of suitable microorganisms include, but are not limited to, Escherichia microorganisms, more specifically Escherichia coli. For example, Escherichia microorganisms can produce OPS and L-serine using SerA, SerC, and SerB, which are enzymes in the L-serine biosynthetic pathway (Non-Patent Documents 8, 9, and 10).

[0024] In the present application, the term "microorganism capable of producing O-phosphoserine" refers to a microorganism naturally capable of producing O-phosphoserine, or a microorganism in which O-phosphoserine-producing ability has been imparted to a parent strain lacking O-phosphoserine-producing ability. Specifically, the microorganism may be a microorganism that has been genetically modified naturally or artificially to enhance Nuo activity and produce O-phosphoserine. For the purposes of the present application, the O-phosphoserine-producing microorganism may be any microorganism that produces O-phosphoserine and whose Nuo activity has been enhanced by the method disclosed in the present application. In the present application, the term "microorganism capable of producing O-phosphoserine" is used interchangeably with "O-phosphoserine-producing microorganism" and "microorganism having O-phosphoserine-producing ability."

[0025] In one embodiment, the microorganism producing O-phosphoserine of the present application is a genetically modified microorganism or a microorganism in which the activity of Nuo is enhanced, thereby improving the ability to produce the target O-phosphoserine. Examples include, but are not limited to, recombinant microorganisms.

[0026] In the present application, "enhancing the activity" of a protein means improving the activity of the protein compared to its endogenous activity. The term "endogenous activity" refers to the activity of a specific protein inherently possessed by a parent strain or unmodified microorganism prior to the phenotypic change, when the trait is altered through genetic mutation due to natural or artificial factors. This term is used interchangeably with "activity prior to modification." "Enhancing" a protein's activity compared to its endogenous activity means improving the activity of a specific protein relative to the activity inherently possessed by a parent strain or unmodified microorganism prior to the phenotypic change. For example, the parent strain may be Escherichia coli (ATCC27325). For another example, the parent strain may be a strain modified to improve its OPS-producing ability, such as, but not limited to, CA07-0012 (KCCM11212P; Patent Document 2) or CA07-4821.

[0027] The aforementioned "improvement of activity" may be achieved by introducing a foreign protein or by enhancing the activity of an endogenous protein, specifically by enhancing the activity of an endogenous protein. Whether the activity of the protein has been enhanced can be confirmed by measuring the level of activity, expression level, or an increase in the amount of a product produced from the protein.

[0028] Various methods well known in the art can be applied to enhance the activity of the target protein, and any method can be used as long as it can enhance the activity of the target protein compared to the unmodified microorganism. Specifically, the method includes, but is not limited to, conventional methods in molecular biology that use genetic engineering and / or protein engineering well known to those skilled in the art (e.g., Non-Patent Documents 11 and 12).

[0029] In the present application, the protein whose activity is to be enhanced, i.e., the target protein, is Nuo, but it may be any protein that is encoded by the nuo operon and consumes NADH or consumes NADH to form proton motive force.

[0030] In the present application, enhancing Nuo activity includes enhancing the activity of at least one of the subunit proteins that constitute the Nuo protein complex.

[0031] Specifically, the enhancement of protein activity in the present application is achieved by, but is not limited to, 1) increasing the intracellular copy number of a gene encoding the protein, 2) modifying an expression regulatory sequence of a gene on a chromosome encoding the protein, 3) modifying the nucleotide sequence of the start codon or 5'UTR region of a transcript of a gene encoding the protein, 4) modifying the amino acid sequence to enhance protein activity, 5) modifying the polynucleotide sequence encoding the protein to enhance protein activity (for example, modifying the gene sequence encoding the protein to encode a protein modified to enhance activity), 6) introducing an exogenous polynucleotide that exhibits protein activity, or a codon-optimized mutant polynucleotide of the polynucleotide, 7) optimizing the codons of a polynucleotide encoding the protein, 8) analyzing the tertiary structure of the protein and selectively modifying or chemically modifying exposed portions, or 9) a combination of two or more selected from 1) to 8) above.

[0032] More specifically, the above 1) increasing the intracellular copy number of a gene encoding a protein can be achieved by any method known in the art, for example, by introducing into a host cell a vector to which a gene encoding the protein is operably linked, the vector replicating and functioning independently of the host. Alternatively, the gene can be operably linked to a vector capable of inserting the gene into a chromosome of the host cell, but this is not limited to these methods.

[0033] In this application, the term "vector" refers to a DNA product comprising a polynucleotide sequence encoding a target protein operably linked to an expression control sequence so as to enable the target protein to be expressed in a suitable host. The expression control sequence includes a promoter that initiates transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that regulates the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate or function independently of the host genome, or can be integrated into the genome itself.

[0034] 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. Examples of plasmid vectors that can be used include pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pSK, pSKH, and pET. Specifically, pCL, pSK, pSKH130, pDZ, pACYC177, pACYC184, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0035] The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination.

[0036] In this application, "transformation" means introducing a recombinant vector containing a polynucleotide encoding a target protein into a host cell, thereby expressing the protein encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide that is expressed in the host cell, regardless of whether it is located by insertion into the chromosome of the host cell or extrachromosomally. The transformation method may be any method for introducing nucleic acid into a cell, and can be carried out by selecting a standard technique suitable for the host cell, as known in the art. For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (Ca These include, but are not limited to, (Cl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0037] Furthermore, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence or expression regulatory region that initiates and mediates transcription of the polynucleotide encoding the target protein of the present application. Operable linking can be achieved using recombinant DNA techniques known in the art, and site-specific DNA cleavage and ligation can be achieved using cleavage and ligation enzymes known in the art, but is not limited to these.

[0038] The 2) modification of the gene expression regulatory sequence on the chromosome encoding the protein can be performed by any method known in the art, for example, by deletion, insertion, non-conservative or conservative substitution of a nucleic acid sequence, or a combination thereof, so that the activity of the expression regulatory sequence is further enhanced. This may be achieved by generating a mutation in the sequence, substituting a nucleic acid sequence with a more active one, or inserting the nucleic acid sequence. The expression control sequence includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence controlling the termination of transcription and translation, etc. Specifically, this method is achieved by, but is not limited to, inserting a strong heterologous promoter after the native promoter.

[0039] Examples of known strong promoters include the CJ1 to CJ7 promoters (Patent Document 3), the lac promoter, the Trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, and the rmf promoter, but are not limited to these, and any promoter that can be substituted with a stronger promoter than the endogenous activity may be used.

[0040] The nucleotide sequence of the start codon or 5'UTR region of the gene transcript encoding the protein (3) can be modified by any method known in the art, for example, by substituting the endogenous start codon of the protein with another start codon that results in a higher protein expression rate than the endogenous start codon, but is not limited to this.

[0041] The amino acid sequences or polynucleotide sequences of 4) and 5) above may be modified by any method known in the art, for example, by generating mutations in the expression regulatory sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to further enhance the activity of the polynucleotide sequence, or by substituting an improved polynucleotide sequence with one having higher activity. Specifically, the substitution is performed by inserting the gene into a chromosome by homologous recombination, but is not limited thereto.

[0042] The vector used here may further contain a selection marker to confirm whether it has been inserted into the chromosome. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether the gene to be introduced has been inserted. Markers that confer selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, and expression of surface proteins are used, but are not limited to these. In an environment treated with a selective agent, the cells that express the selection marker are transformed. Transformed cells can be selected because only those cells that express the gene will survive or exhibit a different phenotype.

[0043] Introduction of an exogenous polynucleotide exhibiting the activity of the protein (6) may be carried out by any method known in the art, for example, by introducing into a host cell an exogenous polynucleotide encoding a protein exhibiting the same or similar activity as the protein, or a codon-optimized mutant polynucleotide thereof. The exogenous polynucleotide may be of any origin or sequence, as long as it exhibits the same or similar activity as the protein. Furthermore, the codons of the introduced exogenous polynucleotide may be optimized before introduction into a host cell so that optimized transcription and translation occur in the host cell. The introduction can be carried out by a person skilled in the art using a known transformation method appropriately selected, and the introduced polynucleotide is expressed in the host cell as described above, resulting in the production of the protein and its activity being improved.

[0044] 7) Optimizing the codons of a polynucleotide encoding a protein may be performed by optimizing the codons of an endogenous polynucleotide so that transcription or translation is increased in the host cell, or by optimizing the codons of an exogenous polynucleotide so that optimized transcription or translation is performed in the host cell.

[0045] 8) Analyzing the tertiary structure of a protein and selecting and altering or chemically modifying an exposed portion may be carried out, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins based on the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and altering or modifying the exposed portion to be altered or chemically modified.

[0046] In one embodiment, the enhancement of the protein activity may be at least one of 1) and 2).

[0047] In any of the above-described embodiments, the activity of the NADH:quinone oxidoreductase of the present application may be enhanced by increasing expression of the nuo operon. In any of the above-described embodiments, the activity of the NADH:quinone oxidoreductase may be enhanced by including a gene expression regulatory sequence with enhanced activity upstream of the gene encoding it. Specifically, the upstream of the gene encoding the NADH:quinone oxidoreductase may be upstream of the nuoA gene. In one embodiment, the activity of the NADH:quinone oxidoreductase may be enhanced by modifying an expression regulatory sequence present in the nuo operon encoding the Nuo protein complex to enhance expression of one or more structural genes present in the operon, for example, two or more, three or more, or all of the structural gene sequences. Specifically, the modification of the expression regulatory sequence is achieved by inserting an additional gene expression regulatory sequence with enhanced activity between the endogenous promoter of the nuo operon and the nuoA gene. For example, the gene expression regulatory sequence may be a promoter, but is not limited to this.

[0048] Such enhancement of protein activity may include, but is not limited to, an improvement in the activity or concentration of the corresponding protein compared to the activity or concentration of the protein expressed in a wild-type or unmodified microbial strain, or an increase in the amount of a product produced from the protein.

[0049] In this application, the terms "pre-modification strain" and "pre-modification microorganism" do not exclude strains containing mutations that may occur naturally in microorganisms, but refer to the natural strain itself or a strain before its traits are changed due to genetic mutation caused by natural or artificial factors. In this application, the trait change may be an enhancement of Nuo activity. The terms "pre-modification strain" and "pre-modification microorganism" are also used interchangeably with "non-mutated strain," "non-modified strain," "non-mutated microorganism," "non-modified microorganism," or "reference microorganism."

[0050] The microorganisms of the present application may be further modified to enhance their ability to produce OPS and / or their ability to export OPS to the outside of the cell, or may be modified to enhance their ability to degrade OPS and / or their ability to influx OPS.

[0051] Examples of modifications that enhance OPS production and / or extracellular export ability, or that enhance OPS degradation and / or influx ability, include, but are not limited to, attenuating the activity of phosphoserine phosphatase (SerB), enhancing the activity of phosphoserine export protein (YhhS), or a combination of these modifications. In the present application, "attenuating" a polypeptide is a concept that encompasses all instances of reduced activity compared to endogenous activity and the absence of activity. The term "attenuating" is also used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0052] The attenuation may include a case in which the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by the microorganism due to a mutation in the polynucleotide encoding the polypeptide. and inhibiting the expression of the gene encoding the polynucleotide and inhibiting translation into the polypeptide, thereby decreasing the overall level and / or concentration of polypeptide activity in the cell. This includes at least one of the following: a decrease in the expression level (expression level) compared to a native strain, complete absence of expression of the polynucleotide, and absence of polypeptide activity even if the polynucleotide is expressed. The term "endogenous activity" refers to the activity of a specific polypeptide that was originally possessed by a parent strain, wild-type, or unmodified microorganism before the trait change, when the trait is changed due to genetic mutation caused by natural or artificial factors. This term is used interchangeably with "activity before modification." "Inactivation," "absence," "reduction," "down-regulation," "decrease," or "attenuation" of a polypeptide activity compared to the endogenous activity means a decrease in the activity of a specific polypeptide compared to the activity of a parent strain or unmodified microorganism before the trait change.

[0053] Such attenuation of the activity of a polypeptide can be achieved by applying various methods well known in the art, including, but not limited to, those described in Non-Patent Documents 12 and 13.

[0054] Specifically, the attenuation of a polypeptide of the present application can be achieved by 1) deleting all or part of a gene encoding the polypeptide, 2) modifying an expression regulatory region (or expression regulatory sequence) so as to reduce the expression of a gene encoding the polypeptide, 3) modifying an amino acid sequence constituting the polypeptide so as to delete or attenuate the activity of the polypeptide (for example, by deleting / substituting / adding one or more amino acids in the amino acid sequence), or 4) modifying a gene sequence encoding the polypeptide so as to delete or attenuate the activity of the polypeptide (for example, by modifying the gene sequence encoding the polypeptide so as to encode a polypeptide modified so as to delete or attenuate the activity of the polypeptide). 5) modifying the base sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide; 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding the polypeptide; 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the polypeptide-encoding gene so that a secondary structure that prevents ribosome attachment is formed; 8) adding a promoter to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide so that reverse transcription occurs (reverse transcription engineering, RTE), or 9) a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0055] For example, 1) deleting a part or all of the gene encoding the polypeptide may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, or by substituting a polynucleotide with a partial deletion of nucleotides or a marker gene.

[0056] Furthermore, the modification of the expression regulatory region (or expression regulatory sequence) described above in 2) may be carried out by generating a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting a sequence having a lower activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0057] Furthermore, the base sequence encoding the start codon or 5'UTR region of the gene transcription product encoding the polypeptide (5) can be modified, for example, by substituting it with a base sequence encoding another start codon that has a lower polypeptide expression rate than the endogenous start codon, but this is not limited to this.

[0058] Furthermore, modifying the amino acid sequence or polynucleotide sequence of 3) and 4) above can be performed by, but is not limited to, generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to attenuate the activity of the polypeptide, or by substituting an amino acid sequence or polynucleotide sequence that has been improved to have lower activity or to eliminate activity. For example, gene expression can be inhibited or attenuated by, but is not limited to, introducing a mutation into a polynucleotide sequence to form a stop codon.

[0059] 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcription product encoding the polypeptide may be carried out, for example, as described in Non-Patent Document 14.

[0060] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide so that a secondary structure that prevents ribosome attachment is formed may be achieved by disabling or slowing down mRNA translation.

[0061] 8) Adding a promoter to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide so as to reverse transcribe it (reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the gene transcript encoding the polypeptide to attenuate its activity.

[0062] Because SerB of the present application has the activity of converting OPS to L-serine, microorganisms mutated to attenuate the SerB activity are characterized by accumulating OPS and are useful for producing OPS. SerB of the present application may be a protein having the amino acid sequence set forth in SEQ ID NO: 2, a protein containing the amino acid sequence, a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, or a protein essentially consisting of the amino acid sequence, but is not limited to these. Furthermore, SerB of the present application may have or contain an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 2, so long as it exhibits SerB activity. Furthermore, SerB of the present application may be 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: 2, or may essentially consist of the amino acid sequence, but is not limited to these. Furthermore, the polynucleotide encoding SerB may have a nucleotide sequence 3 encoding the amino acid sequence represented by SEQ ID NO: 2, or may include said nucleotide sequence. Furthermore, the polynucleotide encoding SerB may consist of a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2, or may essentially consist of said nucleotide sequence. The polynucleotide encoding SerB of the present application may undergo various modifications in the coding region, taking into account codon degeneracy or codons preferred in the organism in which the SerB protein is to be expressed, as long as the amino acid sequence of the SerB protein is not changed. The polynucleotide encoding SerB of the present application may have a nucleotide sequence that is at least 70%, 80%, 90%, 95%, or 99% homologous or identical to, and less than 100%, the nucleotide sequence of SEQ ID NO: 3, or may include said nucleotide sequence.Furthermore, the polynucleotide encoding SerB of the present application has a homology or identity of at least 70%, 80%, 90%, 95%, or 99% or more, and less than 100%, to the base sequence of SEQ ID NO:3. The nucleic acid sequence may be a sequence consisting of a base sequence other than the above, or may be a sequence consisting essentially of the above base sequence, but is not limited thereto.

[0063] Because the YhhS of the present application has the activity of excreting OPS, microorganisms mutated to enhance the YhhS activity are characterized by their ability to excrete OPS and are useful for producing OPS. The YhhS of the present application may be, but is not limited to, a protein having the amino acid sequence set forth in SEQ ID NO: 4, a protein containing the amino acid sequence, a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4, or a protein essentially consisting of the amino acid sequence. Furthermore, the YhhS of the present application may have or contain an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 4, as long as it exhibits YhhS activity. Furthermore, the YhhS of the present application may be, but is not limited to, an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 4, or a protein essentially consisting of the amino acid sequence. Furthermore, the polynucleotide encoding YhhS may have or contain a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 4. Furthermore, the polynucleotide encoding YhhS may consist of or essentially consist of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 4. The polynucleotide encoding YhhS of the present application can undergo various modifications in the coding region, taking into account codon degeneracy or codons preferred in the organism in which the YhhS protein is to be expressed, as long as the amino acid sequence of the YhhS protein is not altered. The polynucleotide encoding YhhS of the present application may have or contain a nucleotide sequence that is at least 70%, 80%, 90%, 95%, or 99% homologous or identical to, but less than 100%, the nucleotide sequence of SEQ ID NO: 5.Furthermore, the polynucleotide encoding YhhS of the present application may consist of a base sequence that has at least 70%, 80%, 90%, 95%, or 99% or more homology or identity to the base sequence of SEQ ID NO: 5, but less than 100%, or may be essentially composed of the base sequence, but is not limited to these.

[0064] In one embodiment, the microorganism modified to enhance OPS production and / or extracellular export ability or to enhance OPS degradation and / or influx ability is CA07-0012 (KCCM11212P; Patent Document 2) or CA07-4821, but is not limited thereto. In addition to the above, the OPS-producing microorganisms disclosed in Patent Documents 2 and 4 are used as reference materials for this application, but are not limited thereto.

[0065] Another aspect of the present application provides a method for producing OPS, comprising culturing in a medium an OPS-producing microorganism having enhanced NADH:quinone oxidoreductase activity.

[0066] The microorganisms are as described above.

[0067] The term "culturing" in the present application means growing the microorganism under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using a suitable medium and culture conditions known in the art. Those skilled in the art can easily adjust such a culturing process depending on the selected strain. Specifically, the culturing may be batch, continuous, or fed-batch culture, but is not limited thereto.

[0068] The culture medium for culturing the microorganism may further contain glycine or serine. Serine is supplied in the form of purified glycine, yeast extract containing glycine, or tryptone, and its concentration in the culture medium is usually 0.1 to 10 g / L, specifically 0.5 to 3 g / L. Serine is supplied in the form of purified serine, yeast extract containing serine, or tryptone, and its concentration in the culture medium is usually 0.1 to 5 g / L, specifically 0.1 to 1 g / L.

[0069] Carbon sources contained in the medium include, but are not limited to, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose, fats and oils such as soybean oil, sunflower oil, castor oil, and coconut oil, fatty acids such as palmitic acid, stearic acid, and linoleic acid, alcohols such as glycerin and ethanol, and organic acids such as acetic acid. These substances can be used alone or in combination.

[0070] Nitrogen sources contained in the medium include, but are not limited to, organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal, and urea, and inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. These nitrogen sources can be used alone or in combination.

[0071] Phosphorus sources contained in the medium include, but are not limited to, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding sodium-containing salts.

[0072] The medium may also contain metal salts such as magnesium sulfate and iron sulfate, as well as amino acids, vitamins, suitable precursors, etc. These media or precursors can be added to the culture in a batch or continuous manner, but are not limited thereto.

[0073] The pH of the culture can be adjusted by adding chemicals such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in a suitable manner during cultivation. Furthermore, foam formation can be suppressed during cultivation using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, oxygen or an oxygen-containing gas may be injected into the culture to maintain an aerobic state, while anaerobic and microaerobic states can be maintained without gas injection or by injecting nitrogen, hydrogen, or carbon dioxide gas. The temperature of the culture is usually 25°C to 40°C, specifically 30°C to 35°C. The cultivation period of the culture is continued until the desired amount of useful substance is produced, specifically 10 to 100 hours. However, the cultivation period is not limited to these.

[0074] The present application further includes, but is not limited to, the step of preparing a medium prior to the culturing step in the method of the present application.

[0075] The method may further include a step of recovering OPS from the culture medium or the microorganism used for the culture. The recovery step may be further included after the culturing step.

[0076] The method for recovering OPS of the present application may involve recovering the target OPS from the culture medium using a suitable method known in the art, depending on the culture method. For example, The desired OPS can be recovered from the culture medium or the microorganism using any suitable method known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization, or HPLC.

[0077] The recovery step may also include a purification step, and can be carried out using a suitable method known in the art. Thus, as described above, the recovered OPS may be in a purified form or may be a microbial fermentation broth containing OPS. Furthermore, OPS can be efficiently recovered by adding a suitable method known in the art before or after the culture step or before or after the recovery step.

[0078] Yet another aspect of the present application provides a method for producing cysteine ​​or a derivative thereof, comprising: a) the steps of culturing an OPS-producing microorganism having enhanced NADH:quinone oxidoreductase activity in a medium to produce OPS or a medium containing OPS; and b) the steps of reacting the OPS produced in step a) or the medium containing OPS with sulfide in the presence of O-phosphoserine sulfliydrylase (OPSS) or a microorganism expressing OPS.

[0079] The steps a) and b) are not necessarily limited to being performed consecutively or in order, and there may be no time interval between the steps, and the steps may be performed simultaneously, or may be performed with an interval of several seconds, several minutes, several hours, or several days.

[0080] Specifically, the method for producing cysteine ​​or a derivative thereof includes the steps of: culturing an OPS-producing microorganism with enhanced Nuo activity in a medium to produce OPS or a medium containing OPS; and reacting the OPS produced in the above step or the medium containing OPS with sulfide in the presence of O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing OPS. The enhanced activity of NADH:quinone oxidoreductase and the O-phosphoserine-producing microorganism are as described above.

[0081] 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.

[0082] The term "cysteine ​​derivative" as used herein refers to a compound in which a hydrogen atom or a specific atomic group of cysteine ​​is substituted with another atom or atomic group. Examples include a form in which another atom or atomic group is bound to the nitrogen atom of the amino group (-NH2) or the sulfur atom of the thiol group (-SH) of cysteine, such as N-acetylcysteine ​​(NAC), S-carboxymethylcysteine ​​(SCMC), BOC-CYS(ME)-OH, (R)-S-(2-Amino-2-carboxyethyl)-L-homocysteine, (R)-2-Amino-3-sulfopropionic acid, D-2-Amino-4-(ethylt hio)butyric acid, 3-sulfino-L-alanine, Fmo Examples include, but are not limited to, c-Cys(Boc-methyl)-OH, Seleno-L-cystine, S-(2-Thiazolyl)-L-cysteine, S-(2-Thienyl)-L-cysteine, and S-(4-Tolyl)-L-cysteine.

[0083] If cysteine ​​is produced by the method of the present application, it may be easily converted into various cysteine ​​derivatives by methods well known in the art.

[0084] Specifically, the method for producing a cysteine ​​derivative further includes a step of converting the cysteine ​​produced in step b) into a cysteine ​​derivative, for example, by reacting cysteine ​​with an acetylation agent to synthesize NAC (N-acetylcysteine). or reacting cysteine ​​with haloacetic acid under basic conditions to produce SC MC (S-Carboxymetylcysteine) is synthesized, but is not limited to these.

[0085] The cysteine ​​derivatives are mainly used as pharmaceutical raw materials in antitussives, cough suppressants, and therapeutic agents for bronchitis, bronchial asthma, sore throat, etc., but are not limited thereto.

[0086] In this application, "O-phosphoserine sulfhydrylase (OPSS)" refers to an enzyme that catalyzes the reaction of converting OPS to cysteine ​​by donating a thiol group (SH group) to OPS. This enzyme has been found to be produced by Aeropyrum pernix, Mycobacterium tuberculosis, Mycobacterium smegmatis, and Trichomonas vaginalis (Non-Patent Documents 15, 16). The OPSS protein includes not only the wild-type OPSS protein but also mutant proteins that have a partial deletion, substitution, or addition of the polynucleotide sequence encoding the OPSS protein and exhibit biological activity equivalent to or greater than that of the wild-type OPSS protein, including all of the OPSS proteins and their mutant proteins disclosed in Patent Documents 2 and 5.

[0087] The sulfide may be supplied not only in the form of a solid commonly used in the art, but also in the form of a liquid or gas depending on differences in pH, pressure, and solubility. 2- ),blood Thiosulfate (S203 2-Any sulfide that can be converted into a thiol group (SH group) in the form of, for example, Na2S, NaSH, H2S, (NH4)2S, NaSH, and Na2S2O3, which donate a thiol group to OPS, may be used. Specific examples include, but are not limited to, Na2S, NaSH, H2S, (NH4)2S, NaSH, and Na2S2O3, which donate a thiol group to OPS. The reaction is a reaction in which one thiol group is donated to one OPS functional group to produce one cysteine ​​or cysteine ​​derivative. The amount of sulfide added in the reaction is 0.1 to 3 times, specifically 1 to 2 times, the molar concentration of OPS, but is not limited to these.

[0088] Yet another aspect of the present application provides a method for producing an O-phosphoserine-producing microorganism, the method comprising the step of modifying an Escherichia microorganism so that the activity of NADH:quinone oxidoreductase is enhanced.

[0089] Another object of the present application is to provide a use of a microorganism having enhanced NADH:quinone oxidoreductase activity for producing O-phosphoserine, cysteine, or a derivative thereof.

[0090] NADH:quinone oxidoreductase, its activity enhancement, microorganisms, etc. have been described above. [Example]

[0091] The present application will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present application and are not intended to limit the present application. This will be apparent to those skilled in the art. [Example]

[0092] Evaluation of OPS production ability of strains with mutations in the Nuo expression regulatory region Previous studies have shown that the average transcription levels of the nuoA, rmf, and idi genes in OPS-producing host strains are 8986, 32205, and 631, respectively. Specific values ​​for each cultivation interval are shown in Table 1.

[0093] [Table 1]

[0094] The average transcription level of rmf was 3.6 times that of nuoA, and the average transcription level of idi was 0.07 times that of nuoA, indicating that the rmf promoter is a stronger promoter than the nuoA promoter, while the idi promoter is a weaker promoter.

[0095] Therefore, we decided to regulate the expression of NADH:quinone oxidoreductase by inserting the rmf promoter and idi promoter, which have different activities, into the nuo operon (sequence number 1) in OPS-producing microorganisms and compare the OPS production ability of the microorganisms.

[0096] 1-1: Construction of a plasmid to enhance the Nuo expression regulatory region The chromosomal DNA of E. coli ATCC27325 was used as a template, and SEQ ID NO: 14 and SEQ ID NO: A gene fragment from the upstream region of the wild-type promoter of the nuo gene was obtained using a primer pair having the nucleotide sequences of SEQ ID NO: 15, and a gene fragment from the downstream region of the promoter was obtained using a primer pair having the nucleotide sequences of SEQ ID NO: 18 and SEQ ID NO: 19. In addition, the promoter region of the rmf gene was obtained using a primer pair having the nucleotide sequences of SEQ ID NO: 16 and SEQ ID NO: 17, using the chromosomal DNA of E. coli ATCC27325 as a template.

[0097] To obtain the fragment, PCR was performed using Solg™ Pfu-X DNA polymerase as the polymerase under the following PCR conditions: denaturation at 95°C for 2 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 60 seconds, followed by polymerization at 72°C for 5 minutes.

[0098] The upstream and downstream fragments of the nuo promoter and the rmf promoter fragment obtained above were cloned into the chromosomal transformation vector pSKH130 (SEQ ID NO: 39, Patent Document 6) cleaved with EcoRV restriction enzyme using an in-fusion cloning kit (Clontech Laboratories, Inc.) to obtain a recombinant plasmid designated pSKH130_Prmf-nuoA.

[0099] 1-2: Construction of a plasmid to weaken the Nuo expression regulatory region The chromosomal DNA of E. coli ATCC27325 was used as a template, and SEQ ID NO: 14 and SEQ ID NO: A gene fragment from the upstream region of the wild-type promoter of the nuo gene was obtained using a primer pair having the nucleotide sequences of SEQ ID NO: 20, and a gene fragment from the downstream region of the promoter was obtained using a primer pair having the nucleotide sequences of SEQ ID NO: 23 and SEQ ID NO: 19. In addition, the promoter region of the idi gene was obtained using a primer pair having the nucleotide sequences of SEQ ID NO: 21 and SEQ ID NO: 22, using the chromosomal DNA of E. coli ATCC27325 as a template.

[0100] PCR was carried out under the same PCR conditions as in Example 1-1 to obtain the fragment. Using the fragment, cloning was carried out in the same manner as in Example 1-1 to obtain a recombinant plasmid. It was named SKH130_Pidi-nuoA.

[0101] The primer sequences used in Examples 1-1 and 1-2 are shown in Table 2.

[0102] [Table 2]

[0103] 1-3: Construction of strains with mutants in the Nuo expression regulatory region The pSKH130_Prmf-nuoA prepared in Example 1-1 was electroporated (Non-Patent Document 17) to transform CA07-0012 (KCCM11212P, Patent Document 2), which has the ability to produce OPS. Subsequently, a secondary crossover process was carried out to obtain strain CA07-4826, in which the promoter sequence of the rmf gene was inserted at the end of the wild-type promoter sequence of the nuo gene.

[0104] Specifically, the pSKH130 vector contains the R6K replicon, SacB (Levansucrase) gene, and kanamycin resistance gene, which are dependent on the PI protein (pir gene). Therefore, R6K and kanamycin were used in the first crossover. The desired strain is isolated by filtration, and then the antibiotic is removed from the sucrose-containing medium. The strain was prepared.

[0105] It was confirmed by PCR using the primer pair of SEQ ID NO: 24 and SEQ ID NO: 25 and genome sequencing that the rmf promoter nucleotide sequence was inserted into CA07-4826.

[0106] Similarly, pSKH130_Pidi-nuoA prepared in Example 1-2 was transformed by electroporation (Non-Patent Document 17), followed by a secondary crossover process to obtain strain CA07-4827, in which the promoter sequence of the idi gene was inserted at the end of the wild-type promoter sequence of the nuo gene. PCR and genome sequencing using the primer pair of SEQ ID NOs: 24 and 25 confirmed that the idi promoter sequence had been inserted into strain CA07-4827. The primer sequences used are shown in Table 3.

[0107] [Table 3]

[0108] 1-4: Comparison of OPS production ability between nuo operon-enhanced and -weakened strains using titer medium The O-phosphoserine (hereinafter referred to as "OPS") production abilities of the two strains CA07-4826 and CA07-4827 prepared in Examples 1-3 and the control strain CA07-0012 were evaluated. For the evaluation, the following media (Table 4) were used.

[0109] [Table 4]

[0110] Specifically, each strain was smeared onto LB solid medium and then cultured overnight in an incubator at 33°C. The strains cultured overnight on LB solid medium were inoculated into 25 mL of the titer medium shown in Table 3, which was then cultured in an incubator at 33°C and 200 rpm for 48 hours. The concentrations of OPS produced as a result are shown in Table 5.

[0111] [Table 5]

[0112] CA07-4826, in which the nuo operon is enhanced by the rmf promoter, exhibits OPS production capacity that is approximately 12.8% higher than that of the parent strain, while CA07-4827, in which the nuo operon is weakened by the idi promoter, exhibits OPS production capacity that is approximately 79.5% lower than that of the parent strain. [Example]

[0113] Evaluation of OPS production ability by enhancing the nuo operon in strains with improved OPS production ability In the strain with enhanced OPS efflux, enhancing the Nuo operon further increased OPS efflux. To confirm whether the OPS production ability would be improved, the nuo operon was further enhanced in a strain in which the OPS-exporting protein YhhS (SEQ ID NO: 4, Patent Document 7) was enhanced, and the OPS production ability was evaluated.

[0114] 2-1: Construction of a plasmid to enhance YhhS expression in OPS-producing strains The chromosomal DNA of E. coli ATCC27325 was used as a template, and the sequences of SEQ ID NO: 6 and SEQ ID NO: A gene fragment from the upstream region of the wild-type promoter of the yhhS gene was obtained using a primer pair having the nucleotide sequence of SEQ ID NO: 7, and a gene fragment from the downstream region of the wild-type promoter of the yhhS gene was obtained using a primer pair having the nucleotide sequences of SEQ ID NO: 8 and SEQ ID NO: 9. Furthermore, the trc promoter (Ptrc) was obtained using pCL_Ptrc-gfp (Patent Document 8) as a template and the primer pair of SEQ ID NO: 8 and SEQ ID NO: 9. The primer sequences used here are shown in Table 6.

[0115] [Table 6]

[0116] Solg™ Pfu-X DNA polymerase was used to obtain the fragment. PCR amplification was performed under the following conditions: denaturation at 95°C for 2 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 60 seconds, followed by polymerization at 72°C for 5 minutes.

[0117] The upstream and downstream fragments of the yhhS promoter and the trc promoter fragment obtained above were cloned into the chromosomal transformation vector pSKH130 cleaved with EcoRV restriction enzyme using an in-fusion cloning kit (Clontech Laboratories, Inc.) to obtain a recombinant plasmid designated pSKH130_Ptrc-yhhS.

[0118] 2-2: Preparation of YhhS-enriched strain The pSKH130_Ptrc-yhhS prepared in Example 2-1 was transformed into CA07-0012 by electroporation (Non-Patent Document 17), and then a secondary crossover process was performed to obtain strain CA07-4821, in which the trc promoter sequence was inserted at the end of the wild-type promoter sequence of the yhhS gene. PCR and genome sequencing using the primer pair of SEQ ID NOs: 12 and 13 (Table 7) confirmed that the trc promoter sequence had been inserted into strain CA07-4821.

[0119] [Table 7]

[0120] 2-3: Evaluation of OPS production ability of YhhS-enriched strains using titer medium To evaluate the OPS-producing ability of the strain CA07-4821 prepared in Example 2-2 and its parent strain CA07-0012, the evaluation was carried out in the same manner as in Example 1-4 using the medium (Table 4).

[0121] As a result, it was confirmed that CA07-4821 had an OPS production capacity that was approximately 26.7% higher than that of CA07-0012, as shown in Table 8.

[0122] [Table 8]

[0123] 2-4: Construction of a strain with enhanced YhhS and nuo operons The pSKH130_Prmf-nuoA prepared in Example 1-1 was electroporated (Non-Patent Document 17) and transformed into CA07-4821 prepared in Example 2-3, and then a secondary crossover process was carried out to obtain strain CA07-4828 in which the promoter base sequence of the rmf gene was inserted at the end of the wild-type promoter base sequence of the nuo gene.

[0124] The strain CA07-4828 was confirmed to have the rmf promoter sequence inserted therein by PCR using the primer pair of SEQ ID NO: 24 and SEQ ID NO: 25 and genome sequencing.

[0125] 2-5: Evaluation of OPS production ability of strains with enhanced YhhS and nuo operons using titer medium To evaluate the OPS-producing ability of CA07-4828, evaluation was performed in the same manner as in Examples 1-4, using CA07-4821 as a control. The results are shown in Table 9.

[0126] [Table 9]

[0127] CA07-4828, in which both the YhhS and nuo operons were enhanced, showed an approximately 2.4% increase in OPS production compared to CA07-4821, in which only YhhS was enhanced. This confirms that even in strains with enhanced OPS production, enhancement of the nuo operon further increases OPS production.

[0128] From the above description, a person skilled in the art of the present application will understand that the present application is based on the technical idea It will be understood that the present invention can be embodied in other specific forms without changing the essential features or characteristics. It should be understood that the above-described embodiments are merely illustrative and not limiting. This application should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalents.

Claims

1. A recombinant Escherichia microorganism having enhanced activity of NADH:quinone oxidoreductase and the ability to produce O-phosphoserine.

2. The microorganism according to claim 1 , wherein the enhanced activity of the NADH:quinone oxidoreductase is increased expression of the nuo operon.

3. The microorganism according to claim 1, wherein the activity of the NADH:quinone oxidoreductase is enhanced by including a gene expression regulatory sequence with enhanced activity upstream of the gene encoding the NADH:quinone oxidoreductase.

4. The microorganism according to claim 3 , wherein the upstream of the gene encoding the NADH:quinone oxidoreductase is the upstream of the nuoA gene.

5. The microorganism according to claim 1, further comprising an attenuated activity of phosphoserine phosphatase (SerB).

6. The microorganism according to claim 1, further comprising enhanced activity of an O-phosphoserine efflux protein (YhhS).

7. The microorganism of claim 1 , wherein the microorganism is Escherichia coli.

8. A method for producing O-phosphoserine, comprising the step of culturing the microorganism according to any one of claims 1 to 7 in a medium.

9. 9. The method for producing O-phosphoserine according to claim 8, further comprising the step of recovering O-phosphoserine from the culture medium or the microorganism.

10. a) culturing an O-phosphoserine-producing microorganism having enhanced NADH:quinone oxidoreductase activity in a medium to produce O-phosphoserine or a medium containing O-phosphoserine; b) reacting the O-phosphoserine produced in step a) or a medium containing the O-phosphoserine with sulfide in the presence of O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing the O-phosphoserine.

11. 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 10, wherein the cysteine ​​or derivative thereof is at least one selected from the group consisting of:

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