O-phosphoserine-producing microorganisms and methods for producing O-phosphoserine or L-cysteine ​​using the same

Abstract

The present application relates to a microorganism having weakened erythronate-4-phosphate dehydrogenase protein activity; a method for producing O-phosphoserine, cysteine ​​and cysteine ​​derivatives using the same; a composition for producing O-phosphoserine containing the microorganism; and use of the microorganism for producing O-phosphoserine, cysteine ​​and cysteine ​​derivatives.

Description

Technical Field

[0001] This application relates to a microorganism with weakened activity of Erythronate-4-phosphate dehydrogenase protein; a method for producing O-phosphoserine, cysteine and derivatives of cysteine using the same; a composition for producing O-phosphoserine containing the microorganism; and uses of the microorganism for producing O-phosphoserine, cysteine and derivatives of cysteine.

Background Art

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

[0003] Known methods for producing L-cysteine using microorganisms include: 1) a method of biologically converting D, L-ATC (D, L-2-aminothiazoline-4-carboxylic acid) using microorganisms; 2) a direct fermentation method for producing L-cysteine using Escherichia coli (European registered patent EP0885962B; Wada M and Takagi H, Appl. Microbiol. Biochem., 73:48-54, 2006); 3) a method of fermentatively producing OPS using microorganisms and then reacting it with sulfide under the catalysis of O-phosphoserine sulfhydrylase (OPSS) to convert it into L-cysteine (US registered publication US 8557549 B2).

[0004] At that time, in order to produce cysteine in high yield by the above method 3), it is necessary to overproduce the precursor O-phosphoserine (OPS).

Prior Art Documents

Patent Documents

[0005] [License 1] European Registration Permit EP0885962B [License 2] US Registry Bulletin US 8557549 B2 [License 3] US Registry Permit US 7662943 B2 [License 4] US Registry Permit US 10584338 B2 [Patent Document 5] US Registry Permit US 10273491 B2 [License 6] U.S. Open Gazette No. 2012-0190081 [License 7] US Registry Bulletin US 9127324 B2 [License 8] U.S. Patent and Trademark No. 2020-0048619 [Non-licensed literature]

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

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Summary of the Invention

Problems to be Solved by the Invention

[0007] The inventors of the present invention have developed a microorganism that produces O-phosphoserine with weakened activity of erythronate-4-phosphate dehydrogenase protein, a method for producing O-phosphoserine using the microorganism, a method for producing cysteine or its derivative using the microorganism, and a composition for producing O-phosphoserine containing the microorganism, and completed this application.

Means for Solving the Problems

[0008] One object of the present application is to provide a microorganism that produces O-phosphoserine with weakened activity of erythronate-4-phosphate dehydrogenase (Erythronate-4-phosphate dehydrogenase) protein.

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

[0010] Another object of the present application is to provide a method for producing cysteine or its derivative, including: a) culturing the microorganism of the present application in a medium to produce O-phosphoserine or a medium containing the same; and b) contacting O-phosphoserine sulfhydrylase (O-phosphoserine sulfliydrylase, OPSS) or a microorganism expressing the same, the O-phosphoserine or the medium containing the same produced in step a) with a sulfide.

[0011] Another object of the present application is to provide a composition for producing O-phosphoserine containing the microorganism of the present application.

[0012] Another object of the present application is to provide the use of the microorganism of the present application for producing O-phosphoserine, cysteine or a derivative of cysteine.

Effects of the Invention

[0013] This application describes a method for efficiently producing O-phosphoserine using microorganisms that produce O-phosphoserine with weakened erythronate-4-phosphate dehydrogenase protein activity compared to endogenous activity. Furthermore, when producing cysteine ​​and its derivatives from the produced O-phosphoserine through an enzymatic conversion reaction, this method allows for more environmentally friendly and efficient cysteine ​​production than chemical synthesis methods. [Modes for carrying out the invention]

[0014] This can be explained in more detail as follows: On the other hand, each description and embodiment disclosed in this application can also be applied to each other different descriptions and embodiments. That is, all combinations of the diverse elements disclosed in this application fall within the scope of this application. Furthermore, the categories of this application are not considered to be limited by the specific descriptions described below.

[0015] Furthermore, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated as references in their entirety into this specification to more clearly explain the level of the technical field to which this application belongs and the content of this application.

[0016] One aspect of this application provides a microorganism that produces O-phosphoserine with weakened erythronate-4-phosphate dehydrogenase protein activity.

[0017] In this application, the term "erythronate-4-phosphate dehydrogenase" is used interchangeably with "PdxB" in this application. Specifically, the erythronate-4-phosphate dehydrogenase is publicly known in the art, and its protein and gene sequences can be obtained from publicly known databases, such as NCBI's GenBank. More specifically, the erythronate-4-phosphate dehydrogenase may have and / or contain the amino acid sequence described in Sequence ID No. 1, or be composed of the amino acid sequence.

[0018] Furthermore, the erythronate-4-phosphate dehydrogenase of this application may include amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the amino acid sequence described in Sequence ID No. 1. It is also obvious that erythronate-4-phosphate dehydrogenase having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added is also included within the scope of this application, as long as it has such homology or identity and exhibits the efficacy corresponding to the erythronate-4-phosphate dehydrogenase of this application.

[0019] Even if this application describes a polypeptide or protein containing an amino acid sequence described by a specific sequence number, a polypeptide or protein consisting of an amino acid sequence described by a specific sequence number, or a polypeptide or protein having an amino acid sequence described by a specific sequence number, it is obvious that proteins having amino acid sequences in which some sequences are deleted, modified, substituted, conserved substituted, or added may also be used in this application, as long as they have the same or equivalent activity as the polypeptide consisting of the amino acid sequence of the said sequence number. For example, this includes cases where the N-terminus and / or C-terminus of the amino acid sequence have added sequences that do not alter the function of the protein, naturally occurring mutations, silent mutations, or conserved substitutions.

[0020] For example, the amino acid sequence may have additions or deletions of sequences that do not alter the function of the erythronate-4-phosphate dehydrogenase of this application, spontaneous mutations, silent mutations, or conservative substitutions at its N-terminus, C-terminus, and / or within it.

[0021] In this application, the term "conservative substitution" means the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartate; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Furthermore, amino acids can be classified into those with electrically charged side chains and those with uncharged side chains. Amino acids with electrically charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino acids with uncharged side chains can be further classified into nonpolar amino acids and polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conservative substitutions have little to no effect on the activity of the resulting polypeptide. Typically, conservative substitutions have little to no effect on the activity of a protein or polypeptide.

[0022] Furthermore, erythronate-4-phosphate dehydrogenases may include amino acid deletions or additions that have minimal impact on the polypeptide's properties and secondary structure. For example, a polypeptide can be conjugated with a protein N-terminal signal (or leader) sequence involved in protein transfer co-translationally or post-translationally. The polypeptide can also be conjugated with other sequences or linkers to enable the polypeptide to be identified, purified, or synthesized.

[0023] In this application, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or base sequences, and may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0024] The homology or identity of sequences of conserved polynucleotides or polypeptides is determined by standard sequencing algorithms, and a default gap penalty established by the program used is available. Substantially homologous or identical sequences can generally be hybridized, in whole or in part, with other sequences under moderate to high stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing codons in general or codon degeneracy in polynucleotides.

[0025] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms such as the "FASTA" program with default parameters, for example, 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), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] [ET AL, J MOLEC BIOL 215]):403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.] Academic Press, San (Including Diego, 1994, and [CARILLO ETA / .] (1988) SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Databases.

[0026] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443, as is publicly known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as the total number of symbols in the shorter of two sequences divided by the number of similarly sequenced symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program may include: (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14:6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0027] In this application, the term "corresponding to" refers to an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar, identical, or homologous to a residue listed in the polypeptide. Identifying the amino acid at the corresponding position may also mean determining a specific amino acid in a sequence that references a particular sequence. As used in this application, "corresponding region" generally refers to a similar or corresponding position in a related protein or reference protein.

[0028] For example, any amino acid sequence can be aligned with sequence number 1, and based on this, each amino acid residue in the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in sequence number 1. For example, a sequence alignment algorithm such as the one described in this application can be used to verify the position of amino acids, or the position where deformations such as substitution, insertion, or deletion occur, by comparing them with a query sequence (also called a "reference sequence").

[0029] For such sorting, one can use, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) or the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277), but is not limited to these. Any sequence sorting program or pairwise sequence comparison algorithm known in this field can be used appropriately.

[0030] In this application, the term "O-phosphoserine (OPS)" refers to a phosphoric acid ester of serine, which is a component of various proteins. OPS is a precursor of L-cysteine ​​and is converted to cysteine ​​by reaction with sulfides under the catalysis of OPS sulfhydrylase (OPSS), but is not limited to this (U.S. Patent Publication US 8557549 B2).

[0031] In this application, the term "microorganism producing O-phosphoserine" means a microorganism that naturally possesses the ability to produce O-phosphoserine or a microorganism in which the ability to produce O-phosphoserine has been conferred to a parent strain that previously lacked the ability to produce O-phosphoserine. For the purposes of this application, the O-phosphoserine-producing microorganism is characterized by weakened activity of the PdxB protein and increased ability to produce the desired O-phosphoserine, and may be, but is not limited to, a genetically modified microorganism or a recombinant microorganism.

[0032] In this application, the terms "strain" or "microorganism" include all wild-type microorganisms and naturally occurring or artificially genetically modified microorganisms, and are microorganisms in which a particular mechanism is weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of the activity of endogenous genes, and may include microorganisms that undergo genetic modification for the production of the target polypeptide, protein, or product.

[0033] The microorganisms of this application may also be microorganisms that have the ability to produce O-phosphoserine.

[0034] The microorganisms of this application may be, but are not limited to, microorganisms that naturally possess the ability to produce O-phosphoserine, or microorganisms in which the ability to produce O-phosphoserine has been conferred to a parent strain that does not possess the ability to produce O-phosphoserine.

[0035] For example, the strain of this application may be a recombinant strain in which the PdxB protein activity is weakened compared to the endogenous activity in a naturally occurring wild-type microorganism or an O-phosphoserine-producing microorganism, and the O-phosphoserine production capacity is increased. The recombinant strain with increased O-phosphoserine production capacity may be a microorganism in which the O-phosphoserine production capacity is increased compared to a naturally occurring wild-type microorganism or a non-mutant microorganism having endogenous activity of the PdxB protein.

[0036] The microorganisms of this application may be, but are not limited to, microorganisms in which the start codon of the polynucleotide encoding erythronate-4-phosphate dehydrogenase is GTG or ATG and thus have increased O-phosphoserine production capacity. For example, the recombinant strain with increased production capacity may have an O-phosphoserine production capacity of about 1% or more compared to the parent strain before mutation or a non-mutated microorganism with intrinsic activity of the PdxB protein. Specifically, about 1% or more, about 10% or more, about 20% or more, about 29% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 101% or more, and about 10% or more. The increase may be 2% or more, approximately 103% or more, approximately 104% or more, approximately 105% or more, approximately 106% or more, or approximately 107% or more (there are no special restrictions on the upper limit; for example, it may be approximately 300% or less, approximately 200% or less, approximately 150% or less, approximately 110% or less, approximately 109% or less, or approximately 108% or less), but it is not limited to this as long as it has a positive increase compared to the productivity of the parent strain or non-mutated microorganism before mutation. In other examples, the recombinant strains with increased production capacity are those in which the O-phosphoserine production capacity is increased by approximately 1.01 times or more, approximately 1.02 times or more, approximately 1.03 times or more, approximately 1.04 times or more, approximately 1.05 times or more, approximately 1.06 times or more, or approximately 1.07 times or more (there is no special limit on the upper limit; for example, it may be approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, approximately 2 times or less, or approximately 1.5 times or less), but are not limited to these.

[0037] In this application, the term "non-myxoid microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but means the wild-type or natural-type strain itself, or a strain before its characteristics are altered by genetic mutations due to natural or artificial factors. For example, the non-myxoid microorganism means a strain in which the erythronate-4-phosphate dehydrogenase protein activity described herein is not weakened compared to its endogenous activity, or before it is weakened. The term "non-myxoid microorganism" may be used interchangeably with "pre-deformation strain," "pre-deformation microorganism," "non-mutant strain," "non-myxoid strain," "non-mutant microorganism," or "reference microorganism."

[0038] As another example of this application, the microorganisms of this application may be microorganisms capable of producing O-phosphoserine, and the type is not particularly limited. The microorganisms of this application may be either prokaryotic or eukaryotic cells, but more specifically, they may be prokaryotic cells. The prokaryotic cells may include, for example, microbial strains belonging to the genera Escherichia, Erwinia, Seratia, Providencia, Corynebacterium, and Brevibacterium, and more specifically may be, but are not limited to, microorganisms of the genus Escherichia, and more specifically Escherichia coli. In particular, in the case of the Escherichia microorganisms of this application, OPS and L-serine can be produced through 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). The “non-mutant microorganism” may be, but is not limited to, a microorganism in which the start codon of the polynucleotide encoding erythronate-4-phosphate dehydrogenase is ATG or GTG. More specifically, the “non-mutant microorganism” may be a microorganism containing the polynucleotide consisting of Sequence ID No. 2. In this application, the term "weakening" of polypeptide activity encompasses all concepts of reduced or absent activity compared to endogenous activity. This weakening may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decline, reduce, and attenuation.

[0039] The aforementioned weakening may include cases where the activity of the polypeptide itself is reduced or eliminated compared to the polypeptide activity originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide, cases where the overall polypeptide activity and / or concentration (expression level) in the cell is lower than that of the natural strain due to inhibition of the expression of the gene encoding the polynucleotide or inhibition of translation into the polypeptide, cases where the polynucleotide is not expressed at all, and / or cases where the polypeptide is not active despite the expression of the polynucleotide. The "intrinsic activity" refers to the activity of a specific polypeptide that was originally possessed by the parent strain, wild type, or non-myxoid microorganism before the trait change due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before the change." When polypeptide activity is "inactivated, deficient, reduced, downregulated, decreased, or attenuated" compared to its intrinsic activity, it means that it has decreased compared to the activity of a specific polypeptide that was originally possessed by the parent strain or non-myxoid microorganism before the trait change.

[0040] The weakening of the activity of such polypeptides can be achieved by any method known in the art, but is not limited to these methods, and can be achieved by applying a variety of well-known methods in the field (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

[0041] Specifically, the weakening of polypeptide activity in this application is, 1) Deletion of all or part of the gene encoding the polypeptide; 2) Modification of the gene expression regulatory region (or gene expression regulatory sequence) so that the expression of the gene encoding the polypeptide is reduced; 3) Modification of the amino acid sequence constituting the polypeptide so as to remove or weaken the polypeptide's activity (e.g., removal / substitution / addition of one or more amino acids in the amino acid sequence); 4) Modification of the gene sequence encoding the polypeptide so as to remove or weaken the polypeptide's activity (for example, removal / substitution / addition of one or more nucleic acid bases on the nucleic acid sequence of the polypeptide gene so as to encode a polypeptide that has been modified so as to remove or weaken the polypeptide's activity); 5) Modifications of the nucleotide sequence encoding the start codon or 5'-UTR region of a polypeptide-encoding gene transcript; 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in a polypeptide-encoding gene to form a secondary structure that cannot be attached to a ribosome; 8) Addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the polypeptide-coding gene sequence (reverse transcription engineering, RTE); or 9) A combination of two or more selected from items 1) to 8) above is also acceptable, but is not particularly limited thereto.

[0042] for example, The deletion of part or all of the gene encoding the polypeptide described in 1) above may be the removal of the entire polynucleotide encoding the endogenous target polypeptide within the chromosome, replacement with a polynucleotide in which some nucleotides are deleted, or replacement with a marker gene.

[0043] Furthermore, the modification of the expression regulatory region (or expression regulatory sequence) described in 2) above may involve deletion, insertion, non-conservative or conservative substitution, or a combination thereof, resulting in a mutation on the expression regulatory region (or expression regulatory sequence), or replacement with a sequence having weaker activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and decoding.

[0044] Furthermore, the modifications of the amino acid sequence or polynucleotide sequence described in 3) and 4) above may be, but are not limited to, deletion, insertion, non-conservative or conservative substitution, or combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, thereby causing mutations in the sequence, or replacement with an amino acid sequence or polynucleotide sequence modified to have weaker activity or an amino acid sequence or polynucleotide sequence modified to have no activity, in order to weaken the activity of the polypeptide. For example, gene expression can be inhibited or weakened by introducing mutations within the polynucleotide sequence to form a termination codon, but is not limited to this.

[0045] Furthermore, the base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide (5) may, for example, be replaced with a base sequence encoding another start codon with a lower polypeptide expression rate compared to the endogenous start codon, but is not limited to these.

[0046] The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide (6) can be done by referring to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol.1(1) 1986].

[0047] 7) In order to form a secondary structure that ribosomes cannot attach to, the addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in the polypeptide-encoding gene may make mRNA translation impossible or reduce its rate.

[0048] The addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE) may be performed by creating complementary antisense nucleotides in the transcript of the polypeptide-encoding gene to weaken its activity.

[0049] Specifically, the microorganisms producing O-phosphoserine in this application may, but are not limited to, those having weakened erythronate-4-phosphate dehydrogenase protein activity compared to its endogenous activity.

[0050] Specifically, the start codon of the polypeptide-coding gene transcript can be altered to weaken the activity of the erythronate-4-phosphate dehydrogenase protein.

[0051] In this application, the term "initiation codon" refers to the three nucleotides that correspond to the translation disclosure site when the coding sequence of mRNA (messenger RNA) is translated into a protein.

[0052] In this application, the microorganism of this application may be, but is not limited to, a microorganism in which the start codon of the polynucleotide encoding erythronate-4-phosphate dehydrogenase is TTG or CTG.

[0053] In this application, the term "polynucleotide" means a polymer of nucleotides in which nucleotide units (monomers) are covalently linked in a long chain, and is a DNA or RNA chain of a certain length or longer, and more specifically, a polynucleotide fragment that codes for the aforementioned variant.

[0054] More specifically, the microorganism of this application may be a microorganism containing the polynucleotides comprising SEQ ID NO: 3 and SEQ ID NO: 4.

[0055] The polynucleotides of this application may undergo various modifications to the coding region, taking into account the degeneracy of the codons or the preferred codons in the organisms that intend to express the variants of this application, while not altering the amino acid sequence of the variants of this application. Specifically, the polynucleotides of this application may, but are not limited to, having or containing a nucleotide sequence that has 70% or more, 75% or more, 6% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or consisting of a nucleotide sequence that has 70% or more, 75% or more, 6% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0056] Furthermore, the polynucleotides of this application are not limited to any probes produced from known gene sequences, such as sequences that can hybridize under stringent conditions with complementary sequences to all or part of the polynucleotide sequences of this application. The “stringent conditions” refer to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; FMAusubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, we can list conditions in which polynucleotides with high homology or identity are hybridized with each other, with homology or identity levels of 70% or more, 75% or more, 6% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and polynucleotides with lower homology or identity are not hybridized. Alternatively, we can list conditions in which the polynucleotides are washed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of normal Southern hybridization: 60°C, 1xSSC, 0.1% SDS, more specifically 60°C, 0.1xSSC, 0.1% SDS, or more specifically 68°C, 0.1xSSC, 0.1% SDS.

[0057] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. The term “complementary” is used to describe the relationships between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of this application may also include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary throughout the sequence.

[0058] Specifically, polynucleotides homologous or identical to the polynucleotides of this application can be detected using hybridization conditions that include a hybridization step at a Tm value of 55°C, and under the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0059] The appropriate stringency for hybridizing the aforementioned polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (e.g., J. Sambrook et al., ibid.).

[0060] Specifically, the microorganisms that produce O-phosphoserine in this application may, but are not limited to, microorganisms in which the erythronate-4-phosphate dehydrogenase protein activity has not been inactivated.

[0061] More specifically, the microorganisms producing O-phosphoserine in this application may, but are not limited to, microorganisms that are not deficient in the polynucleotide encoding erythronate-4-phosphate dehydrogenase.

[0062] For the purposes of this application, the microorganism may, but is not limited to, a microorganism having weakened erythrone-4-phosphate dehydrogenase protein activity compared to its endogenous activity, due to the inclusion of an expression vector for expressing the mutant polynucleotide in the host.

[0063] The vector of this application may include a DNA product comprising a polynucleotide sequence encoding the target polypeptide, operably linked to a suitable regulatory region (or regulatory sequence) so as to enable the expression of the target polypeptide in a suitable host. The regulatory region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences regulating the termination of transcription and decoding. After being transformed into a suitable host cell, the vector may replicate or function independently of the host genome, or it may be integrated into the genome itself.

[0064] The vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pSK, pSKH, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pSK, pSKH130, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0065] As an example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a chromosome insertion vector within the cell. The insertion of the polynucleotide into the chromosome may be carried out by any method known in the art, such as homologous recombination, but is not limited to these methods. The method may further include a selection marker for confirming the presence or absence of the chromosome insertion. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the target nucleic acid molecule insertion, and may be a marker that confers a selectable phenotype, such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of a surface polypeptide. Transformed cells can be selected because, in an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes.

[0066] In this application, the term "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them, regardless of whether they are inserted into or outside the chromosomes of the host cell, as long as they can be expressed in the host cell. The polynucleotide also includes DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the host cell in any form that can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene structure containing all the elements necessary for its expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to the sequences necessary for expression in the host cell, but is not limited thereto.

[0067] Furthermore, the term "operably linked" in the foregoing means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target variant of this application.

[0068] Modification of some or all of the polynucleotides in the microorganisms of this application may be induced by (a) homologous recombination using a chromosome insertion vector within the microorganism or genome editing using an engineered nuclease (e.g., CRISPR-Cas9) and / or (b) light and / or chemical treatment such as ultraviolet light and radiation. The method for modifying some or all of the genes may include methods using DNA recombination techniques. For example, deletion of some or all of the genes may be achieved by injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination. The injected nucleotide sequence or vector may include, but is not limited to, a dominant selection marker.

[0069] For example, various methods known in the art may be used to replace the start codon of a target gene with a TTG or CTG on the chromosome of the microorganism of this application. For instance, the start codon sequence of pdxB, which is endogenous in the microorganism, can be replaced on the chromosome, or alternatively, the gene with the substituted start codon sequence can be introduced into the microorganism in the form of a plasmid.

[0070] The O-phosphoserine-producing microorganism of this application may further have enhanced YhhS protein activity compared to its endogenous activity.

[0071] In this application, the term "YhhS" refers to a polypeptide exhibiting O-phosphoserine (OPS) efflux activity, specifically a membrane protein having the activity to efflux OPS to the extracellular space. In this application, YhhS may also be a YhhS MFS (major facilitator superfamily) transporter, which is a membrane protein having the activity to efflux OPS to the extracellular space. YhhS has been identified as a protein exhibiting OPS efflux activity from Escherichia coli whose growth inhibition has been lifted under conditions where an excess of OPS is present.

[0072] Specifically, the YhhS may be used in combination with the YhhS MFS transporter. In this application, the amino acid sequence of the YhhS can be obtained from the NCBI GenBank, a known database. The YhhS of this application may be a protein having or containing the amino acid sequence described in SEQ ID NO: 7, or a protein consisting of or essentially comprising the amino acid sequence described in SEQ ID NO: 7, but is not limited thereto. Furthermore, the YhhS of this application may have or contain an amino acid sequence that is homologous or identical to the amino acid sequence described in SEQ ID NO: 7 by at least 70%, 80%, 90%, 95%, or 99%, insofar as it exhibits YhhS activity. In addition, the YhhS of this application consists of or essentially comprises an amino acid sequence that is homologous or identical to the amino acid sequence described in SEQ ID NO: 7 by at least 70%, 80%, 90%, 95%, or 99%, insofar as it exhibits YhhS activity. Furthermore, the polynucleotide encoding YhhS may have or include a base sequence encoding the amino acid sequence described in Sequence ID No. 8. In addition, the polynucleotide encoding YhhS may consist of or be required to consist of a base sequence encoding the amino acid sequence described in Sequence ID No. 7. The polynucleotide encoding YhhS of this application may have various modifications to its coding region, within the limits that do not alter the amino acid sequence of the YhhS protein, due to codon degeneracy or considering the preferred codon in the organism intended to express the YhhS protein. The polynucleotide encoding YhhS of this application may have or include a base sequence that has at least 70%, 80%, 90%, 95%, or 99% or more, and less than 100%, homology or identity with the base sequence of Sequence ID No. 8. In addition, the polynucleotide encoding YhhS of this application consists of, but is not limited to, a nucleotide sequence having at least 70%, 80%, 90%, 95%, or 99% and less than 100% homology or identity with the nucleotide sequence of Sequence ID No. 8.

[0073] The O-phosphoserine-producing microorganisms of this application may further have the protein activity of YhhS, phosphoserine aminotransferase (SerC), or a combination thereof introduced or enhanced.

[0074] Since the aforementioned SerC has the activity to convert the aforementioned 3-phosphohydroxypirubate to O-phosphoserine, microorganisms mutated to introduce or enhance the SerC activity have the characteristic of accumulating O-phosphoserine and can be usefully used for the production of O-phosphoserine. The SerC of this application may be, but is not limited to, a protein having or containing the amino acid sequence described in SEQ ID NO: 9, or a protein consisting of or essentially comprising the amino acid sequence described in SEQ ID NO: 9. Furthermore, the SerC of this application may have or contain an amino acid sequence that has at least 70%, 80%, 90%, 95%, or 99% homology or identity with the amino acid sequence described in SEQ ID NO: 9, insofar as it exhibits the activity of SerC. In addition, the SerC of this application consists of or essentially comprises an amino acid sequence that has at least 70%, 80%, 90%, 95%, or 99% homology or identity with the amino acid sequence described in SEQ ID NO: 9. Furthermore, the polynucleotide encoding SerC may have or include a base sequence encoding the amino acid sequence described in Sequence ID No. 10. In addition, the polynucleotide encoding SerC may consist of or be required to consist of a base sequence encoding the amino acid sequence described in Sequence ID No. 9. The polynucleotide encoding SerC of this application may have various modifications to its coding region, within the limits that do not alter the amino acid sequence of the SerC protein, due to codon degeneracy or considering the preferred codon in the organism that intends to express the SerC protein. The polynucleotide encoding SerC of this application may have or include a base sequence that has at least 70%, 80%, 90%, 95%, or 99% or more, and less than 100%, homology or identity with the base sequence of Sequence ID No. 10. In addition, the polynucleotide encoding SerC of this application consists of, but is not limited to, a nucleotide sequence having at least 70%, 80%, 90%, 95%, or 99% and less than 100% homology or identity with the nucleotide sequence of Sequence ID No. 10.

[0075] The O-phosphoserine-producing microorganism of this application may further have weakened phosphoserine phosphatase (SerB) activity compared to its endogenous activity.

[0076] Since the aforementioned SerB has the activity to convert O-phosphoserine to L-serine, microorganisms mutated to weaken the SerB activity have the characteristic of accumulating O-phosphoserine and can be usefully used for the production of O-phosphoserine. The SerB of this application may have, or contain, the amino acid sequence described in SEQ ID NO: 5, or consist of, or be an essential component of, the amino acid sequence described in SEQ ID NO: 5, but is not limited thereto. Furthermore, the SerB of this application may have, or contain, an amino acid sequence that has at least 70%, 80%, 90%, 95%, or 99% homology or identity with the amino acid sequence described in SEQ ID NO: 5, insofar as it exhibits SerB activity. In addition, the SerB of this application consists of, or is essential component of, an amino acid sequence that has at least 70%, 80%, 90%, 95%, or 99% homology or identity with the amino acid sequence described in SEQ ID NO: 5, but is not limited thereto. Furthermore, the polynucleotide encoding SerB may have or include a base sequence encoding the amino acid sequence described in Sequence ID No. 6. In addition, the polynucleotide encoding SerB may consist of or be required to consist of a base sequence encoding the amino acid sequence described in Sequence ID No. 5. The polynucleotide encoding SerB of this application may have various modifications to its coding region, within the limits that do not alter the amino acid sequence of the SerB protein, due to codon degeneracy or considering the preferred codon in the organism that intends to express the SerB protein. The polynucleotide encoding SerB of this application may have or include a base sequence that has at least 70%, 80%, 90%, 95%, or 99% or more, and less than 100%, homology or identity with the base sequence of Sequence ID No. 6. Furthermore, the polynucleotide encoding SerB of this application consists of, but is not limited to, a nucleotide sequence having at least 70%, 80%, 90%, 95%, or 99% and less than 100% homology or identity with the nucleotide sequence of Sequence ID No. 6.

[0077] In this application, the term "enhancement" of polypeptide activity means that the activity of a polypeptide increases compared to its endogenous activity. This enhancement may be used interchangeably with terms such as activation, upregulation, overexpression, and increase. Here, activation, enhancement, upregulation, overexpression, and increase can all include exhibiting activity that was not originally present, or exhibiting improved activity compared to the endogenous activity or pre-mutation activity. "Endogenous activity" means the activity of a specific polypeptide that was originally present in the parent strain or non-mutant microorganism before the trait change, in cases where the trait has changed due to a genetic mutation caused by natural or artificial factors. This may be used interchangeably with "pre-mutation activity." "Enhancement," "upregulation," "overexpression," or "increase" of polypeptide activity compared to its endogenous activity means that the activity and / or concentration (expression level) of a specific polypeptide that was originally present in the parent strain or non-mutant microorganism before the trait change is improved.

[0078] The aforementioned enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. Whether or not the polypeptide's activity has been enhanced can be confirmed by an increase in the polypeptide's activity level, expression level, or the amount of product excreted from the polypeptide.

[0079] The enhancement of the activity of the polypeptide can be achieved by applying a variety of methods well known in the field, and is not limited as long as it can enhance the activity of the target polypeptide compared to the microorganism before deformation. Specifically, this may involve, but is not limited to, the use of gene engineering and / or protein engineering, which are routine methods in molecular biology and are well known to ordinary technicians in the field (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.).

[0080] Specifically, the strengthening of the polypeptide in this application is 1) Increase in the intracellular copy number of polynucleotides encoding polypeptides; 2) Modification of gene expression regulatory regions on chromosomes that encode polypeptides (e.g., occurrence of mutations within the regulatory region, replacement with a more active sequence, or insertion of a more active sequence); 3) Modifications of the nucleotide sequence encoding the start codon or 5'-UTR region of a polypeptide-encoding gene transcript; 4) Modification of the amino acid sequence of the polypeptide to enhance polypeptide activity; 5) Modification of the polynucleotide sequence encoding the polypeptide so as to enhance polypeptide activity (for example, modification of the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified to enhance polypeptide activity); 6) Introduction of a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding it; 7) Codon optimization of polynucleotides encoding polypeptides; 8) Analyze the tertiary structure of the polypeptide, select exposed areas to deform or chemically modify; or 9) A combination of two or more selected from items 1) to 8) above is also acceptable, but is not particularly limited thereto.

[0081] More specifically, The increase in the intracellular copy number of the polynucleotide encoding the polypeptide described in 1) above may be achieved by introducing into the host cell a vector that can replicate and function independently of the host, on which the polynucleotide encoding the polypeptide is operably linked. Alternatively, it may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into the chromosomes within the host cell. The introduction into the chromosomes can be performed by introducing into the host cell a vector that can insert the polynucleotide into the chromosomes within the host cell, but is not limited to these methods. The vector is as described above.

[0082] The replacement of the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the polypeptide with a more potent sequence may, for example, involve the generation of a sequence mutation by deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacement with a sequence having stronger activity, in order to further enhance the activity of the expression regulatory region. The expression regulatory region may include, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences that regulate the termination of transcription and decoding. For example, the original promoter may be replaced with a potent promoter, but is not limited to these.

[0083] Examples of well-known powerful promoters include, but are not limited to, the CJ1-CJ7 promoters (US Patent No. 7662943 B2), the lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent No. 10584338 B2), O2 promoter (US Patent No. 10273491 B2), tkt promoter, and yccA promoter.

[0084] The sequence modification encoding the start codon or 5'-UTR region of the polypeptide-encoding gene transcript described in 3) above may, but is not limited to, substitution with a sequence encoding another start codon that has a higher polypeptide expression rate compared to the endogenous start codon.

[0085] The modifications of the amino acid sequence or polynucleotide sequence described in 4) and 5) above may be, but are not limited to, deletion, insertion, non-conservative or conservative substitution, or combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, thereby causing a sequence mutation, or replacement with an improved amino acid sequence or polynucleotide sequence that has stronger activity or an improved amino acid sequence or polynucleotide sequence that has increased activity, in order to enhance the activity of the polypeptide. Specifically, the replacement can be carried out by inserting a polynucleotide into the chromosome by homologous recombination, but is not limited to this. The vector used in this case may further include a selection marker for confirming the presence or absence of chromosomal insertion. The selection marker is as described above.

[0086] The introduction of a foreign polynucleotide exhibiting polypeptide activity (6) above may be the introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same or similar activity as the polypeptide into the host cell. The foreign polynucleotide is not restricted in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be appropriately selected by those skilled in the art from known transformation methods, and the polypeptide may be generated and its activity increased by the expression of the introduced polynucleotide in the host cell.

[0087] The codon optimization of the polynucleotide encoding the polypeptide described in 7) above may be codon optimization of the endogenous polynucleotide so that transcription or translation increases in the host cell, or the codon of the exogenous polynucleotide so that optimized transcription or translation occurs in the host cell.

[0088] 8) Analyzing the tertiary structure of a polypeptide and selecting exposed sites to deform or chemically modify may, for example, involve comparing the sequence information of the polypeptide to be analyzed with a database containing sequence information of known proteins to determine candidate template proteins according to the degree of sequence similarity, confirming the structure based on that, and selecting exposed sites to deform or chemically modify.

[0089] Such enhancement of polypeptide activity may, but is not limited to, an increase in the activity or concentration expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type or pre-deformation microbial strain, or an increase in the amount of product produced from said polypeptide.

[0090] Furthermore, the microorganisms may also be microorganisms whose ability to infiltrate and decompose OPS into cells is further reduced.

[0091] The information concerning OPS-producing microorganisms as described above may be used as reference material in this application, but is not limited to, information disclosed in U.S. Register US 8557549 B2 or U.S. Publication No. 2012-0190081, etc., in addition to the information described above.

[0092] Another aspect of this application is to provide a method for producing O-phosphoserine, which includes the step of culturing the microorganism of this application in a culture medium.

[0093] The method for producing O-phosphoserine according to this application may include the step of culturing the microorganism of this application in a culture medium.

[0094] In this application, the term "culture" means growing the microorganisms of this application under appropriately controlled environmental conditions. The culture process of this application can be carried out according to suitable culture media and culture conditions known in the art. Such a culture process can be easily adapted and used by those skilled in the art depending on the selected strain. Specifically, the culture may be batch, continuous, or fed-batch, but is not limited to these.

[0095] In this application, the term "culture medium" means a substance mixed primarily with nutrients necessary for culturing the microorganisms of this application, supplying nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganisms of this application can be any culture medium used for culturing ordinary microorganisms without any special restrictions, but the microorganisms of this application can be cultured under aerobic conditions in an ordinary culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, while adjusting the temperature, pH, etc.

[0096] In this application, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration can also be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and other appropriate amounts of carbon sources can be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited to these uses.

[0097] The nitrogen sources used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn maceration liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products. These nitrogen sources may be used individually or in combination of two or more, and are not limited to these uses.

[0098] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds such as sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate may be used, and other components such as amino acids, vitamins, and / or appropriate precursors may also be included. These components or precursors can be added to the culture medium in batches or continuously, but are not limited to these methods.

[0099] The culture medium may contain metal salts such as magnesium sulfate or iron sulfate, and may also contain amino acids, vitamins, and suitable precursors. These culture media or precursors may be added to the culture in batches or continuously, but are not limited to these methods.

[0100] As an example, culturing recombinant microorganisms in which SerB activity is weakened compared to endogenous activity induces serine requirements in the microorganisms, and the culture medium may further contain glycine or serine. Glycine can be provided in the form of purified glycine, yeast extract containing glycine, or tryptone, and the concentration in the culture medium is usually 0.1 to 10 g / L, specifically 0.5 to 3 g / L. Similarly, serine may be provided in the form of purified serine, yeast extract containing serine, or tryptone, and the concentration in the culture medium is usually 0.1 to 5 g / L, specifically 0.1 to 1 g / L.

[0101] Furthermore, during the cultivation of the microorganisms of this application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture medium in an appropriate manner to adjust the pH of the culture medium. In addition, during cultivation, antifoaming agents such as fatty acid polyglycol esters can be used to suppress the formation of bubbles. Furthermore, in order to maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas can be injected into the culture medium, or in order to maintain an anaerobic and microaerobic state, no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected, but these are not limited to these.

[0102] In the culture described in this application, the culture temperature can be maintained at 20-45°C, specifically 25-40°C, and the culture can be performed for approximately 10-160 hours, but is not limited to this.

[0103] The O-phosphoserine produced by the culture described in this application is either secreted into the culture medium or remains within the cells.

[0104] The method for producing O-phosphoserine according to this application may further include, for example, a step of preparing the microorganism of this application, a step of preparing a culture medium for culturing the microorganism, or a combination thereof (in any order), for example, before the culturing step.

[0105] The method for producing O-phosphoserine according to this application may further include a step of recovering O-phosphoserine from the culture medium (the culture medium in which the culture was performed) or microorganisms. The recovery step may further include a step after the culture step.

[0106] The aforementioned recovery may involve collecting the target O-phosphoserine using appropriate methods known in the art, such as the microorganism culture methods of this application, for example, batch, continuous, or fed-batch culture methods. For example, various chromatography methods such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof can be used to recover the target O-phosphoserine from the culture medium or microorganism using appropriate methods known in the art.

[0107] Furthermore, the O-phosphoserine production method of this application may further include a purification step. The purification can be carried out using appropriate methods known in the art. For example, if the O-phosphoserine production method of this application includes both a recovery step and a purification step, the recovery step and the purification step can be carried out sequentially or discontinuously, regardless of the procedure, or simultaneously or integrated into a single step, but are not limited thereto.

[0108] In the method of this application, "microorganisms," "O-phosphoserine," etc., are as described in the other embodiments above.

[0109] Another aspect of this application provides a method for producing cysteine ​​or a derivative thereof, comprising the steps of: a) culturing the microorganism of this application in a culture medium to produce O-phosphoserine or a culture medium containing the same; b) contacting O-phosphoserine sulfliydrylase (OPSS) or a microorganism expressing the same with the O-phosphoserine or culture medium containing the same produced in step a) and a sulfide.

[0110] In the method of this application, "microorganisms," "O-phosphoserine," etc., are as described in the other embodiments above.

[0111] Specifically, the method may be a method for producing cysteine ​​or a derivative thereof, comprising the steps of: culturing an O-phosphoserine-producing microorganism in which the erythronate-4-phosphate dehydrogenase protein activity is weakened compared to its endogenous activity in a culture medium to produce O-phosphoserine or a culture medium containing it; and reacting O-phosphoserine sulfhydrylase or a microorganism expressing it, or the O-phosphoserine or culture medium containing it produced in the above step, with a sulfide.

[0112] In this application, the term "derivative" refers to a similar compound obtained by chemically altering a part of a compound, and usually means a compound in which a hydrogen atom or a specific group of atoms in the compound is substituted with another atom or group of atoms.

[0113] In this application, the term "cysteine ​​derivative" means a compound in which a hydrogen atom or a specific group of atoms of cysteine ​​is substituted with another atom or group of atoms. Examples include, but are not limited to, a form in which another atom or group of atoms is attached to the nitrogen atom of the amine group (-NH2) or the sulfur atom of the thiol group (-SH) of cysteine. Examples include, but are not limited to, NAC (N-acetylcysteine), SCMC (S-Carboxymetylcysteine), BOC-CYS(ME)-OH, (R)-S-(2-Amino-2-carboxyethyl)-L-homocysteine, (R)-2-Amino-3-sulfopropionic acid, D-2-Amino-4-(ethylthio)butyric acid, 3-sulfino-L-alanine, Fmoc-Cys(Boc-methyl)-OH, Seleno-L-cystine, S-(2-Thiazolyl)-L-cysteine, S-(2-Thienyl)-L-cysteine, S-(4-Tolyl)-L-cysteine.

[0114] As long as cysteine ​​is produced by the method of this application, it may be possible to easily convert it into a variety of cysteine ​​derivatives using methods widely known in the art.

[0115] Specifically, the method for producing the cysteine ​​derivative may further include a step of converting the cysteine ​​generated in step b) into a cysteine ​​derivative. For example, this could involve synthesizing NAC (N-acetylcysteine) by reacting cysteine ​​with an acetylation agent, or synthesizing SCMC (S-Carboxymetylcysteine) by reacting cysteine ​​with haloacetic acid under basic conditions, but is not limited to these.

[0116] The cysteine ​​derivatives may be used primarily as pharmaceutical raw materials for antitussives, cough relievers, and therapeutic agents for bronchitis, bronchial asthma, and pharyngitis, but are not limited to these uses.

[0117] In this application, the term "O-phosphoserine sulfhydrylase (OPSS)" refers to an enzyme that catalyzes the reaction in which O-phosphoserine is converted to cysteine ​​by providing a thiol group (SH group) to O-phosphoserine. This enzyme was first identified in Aeropymm pernix, Mycobacterium tuberculosis, Mycobacterium megmatics, and Trichomonas vaginalis (Mino K and Ishikawa K, FEBSletters, 551:133-138, 2003; Bums KE et al. J.Am.Chem.Soc, 127:11602-11603, 2005). Furthermore, the O-phosphoserine sulfhydrase includes not only wild-type O-phosphoserine sulfhydrase, but also mutants in which a portion of the polynucleotide sequence encoding the O-phosphoserine sulfhydrase is deleted, substituted, or added, and which exhibit activity equivalent to or greater than that of wild-type O-phosphoserine sulfhydrase. It may also include all O-phosphoserine sulfhydrases and their mutants disclosed in U.S. Registry Publication US 8557549 B2 and U.S. Registry Publication US 9127324 B2.

[0118] The aforementioned sulfides are provided not only as solids commonly used in the art, but also in liquid or gaseous form depending on differences in pH, pressure, and solubility, and are sulfides (sulfide, S 2- ), thiosulfate (S2O3 2-Any sulfide that can be converted to a thiol group (SH group) in a form such as ) can be used without limitation. Specifically, Na2S, NaSH, H2S, (NH4)2S and Na2S2O3 that provide a thiol group to O-phosphoserine can be used, but are not limited to these. The above reaction is a reaction that provides one thiol group to one O-phosphoserine reactive group to produce one cysteine ​​or cysteine ​​derivative, and the amount of sulfide added during the above reaction may be 0.1 to 3 times the molar concentration of O-phosphoserine, specifically 1 to 2 times, but is not limited to these.

[0119] Furthermore, this application may include a step of recovering the cysteine ​​produced through the reaction steps. In this case, the desired cysteine ​​can be separated and purified from the reaction solution and collected using a suitable reaction known in the art.

[0120] Another aspect of this application is to provide a composition for O-phosphoserine production containing a microorganism in which the erythronate-4-phosphate dehydrogenase protein activity of this application is weakened compared to its endogenous activity.

[0121] The composition of this application may further contain any suitable excipients commonly used in compositions for the production of O-phosphoserine, such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0122] In the composition of this application, "microorganisms" and "O-phosphoserine," etc., are as described in the other embodiments above.

[0123] Another aspect of this application is to provide applications for the production of O-phosphoserine, cysteine, or cysteine ​​derivatives in microorganisms in which the erythronate-4-phosphate dehydrogenase protein activity of this application is weakened compared to its endogenous activity.

[0124] The present application will be described in more detail below through experimental examples. However, the following embodiments are merely preferred embodiments for illustrating the present application and are not intended to limit the scope of the rights of this application. On the other hand, technical matters not described herein can be easily understood and performed by a person of ordinary skill who is skilled in the art of this application or a similar art.

[0125] Example 1: Production of YhhS-enhanced strains and evaluation of OPS production capacity. 1-1. Plasmid construction for enhancing yhhS expression Using the chromosomal DNA of the wild-type Escherichia coli (ATCC27325) as a template, a gene fragment of the upstream region of the wild-type promoter of the yhhS gene, which undergoes homologous recombination on the chromosome, was obtained using the primer pair of SEQ ID NO: 11 and SEQ ID NO: 12. Furthermore, using pCL_Ptrc-gfp (WO2016024771A1) as a template, the Ptrc promoter was obtained using the primer pair of SEQ ID NO: 13 and SEQ ID NO: 14. Additionally, using the chromosomal DNA of Escherichia coli (ATCC27325) as a template, a gene fragment of the downstream region of the wild-type promoter of the yhhS gene, which undergoes homologous recombination on the chromosome, was obtained using the primer pair of SEQ ID NO: 15 and SEQ ID NO: 16. The primer sequences used are shown in Table 1 below.

[0126] [Table 1]

[0127] To obtain the aforementioned fragments, Solg™ Pfu-X DNA polymerase was used, and PCR amplification was performed under the following conditions: denaturation at 95°C for 2 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization reaction at 72°C for 5 minutes. The upstream fragment of the yhhS promoter, the Ptrc promoter fragment, and the downstream fragment of the yhhS promoter obtained in the above process were cloned together with the chromosome transformation vector pSKH130 (US Patent Publication No. 2020-0048619), which had been cleaved with EcoRV restriction enzyme, using an infusion cloning kit (Clontech Laboratories, Inc.) to obtain a recombinant plasmid, which was named pSKH_Ptrc-yhhS. The aforementioned pSKH130 vector is a vector containing a PI protein (pir gene)-dependent R6K replicon, a SacB (Levansucrase) gene, and a kanamycin resistance gene.

[0128] 1-2. Production of YhhS-expressing enhanced bacterial strains Using the endogenous phosphoserine phosphatase (SerB)-deficient strain CA07-0012 (KCCM 11121P, US registration publication US 8557549 B2), we decided to enhance the expression of the OPS-efflux protein YhhS (SEQ ID NO: 7) to further improve OPS efflux capacity.

[0129] Specifically, the pSKH_Ptrc-yhhS prepared in Example 1-1 was transformed into CA07-0012, a host with weakened OPS resolution, using electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). After obtaining the desired strain using R6K and kanamycin in a primary crossover, a secondary crossover process was carried out in a sucrose-containing medium to remove the kanamycin resistance gene and obtain a strain in which the Ptrc promoter sequence was inserted into the end of the wild-type promoter sequence of the yhhS gene. The insertion of the Ptrc promoter sequence was confirmed by PCR amplification and genome sequencing using primer pairs of SEQ ID NO: 17 and SEQ ID NO: 18 (Table 2), which can amplify the external regions of the homologous recombination upstream and downstream regions, respectively. A bacterial strain (CA07-0012::Ptrc-yhhS) in which the promoter sequence was inserted using CA07-0012 as the host was named CA07-4821.

[0130] [Table 2]

[0131] 1-3. Evaluation of OPS production capacity of YhhS-enhanced strains To evaluate the phosphoserine (O-phosphoserine) production capacity of the YhhS-expressing enhanced strains prepared in Examples 1-2, the following culture media (Table 3) were used for evaluation.

[0132] [Table 3]

[0133] Specifically, each bacterial strain was inoculated onto LB solid medium and then cultured overnight in an incubator at 33°C. The strains cultured overnight in LB solid medium were then inoculated into 25 mL of the titer medium shown in Table 3, and cultured in an incubator at 33°C and 200 rpm for 48 hours. The results are shown in Table 4.

[0134] [Table 4]

[0135] As shown in Table 4, we confirmed that the OPS production capacity of CA07-4821, an enhanced YhhS expression strain, was approximately 127% of that of the parent strain.

[0136] Example 2: Production of a SerC-enhanced strain and evaluation of its OPS production capacity. 2-1. Plasmid construction for enhancing SerC expression To insert the endogenous serC gene and its promoter into the maeB position on the host genome, a gene fragment of the wild-type promoter upstream region of the maeB gene, where homologous recombination occurs on the chromosome, was obtained using the chromosomal DNA of wild-type Escherichia coli (ATCC27325) as a template and the primer pair of SEQ ID NO: 19 and SEQ ID NO: 20. Furthermore, the serC gene and its wild-type promoter region were obtained using the chromosomal DNA of wild-type Escherichia coli (ATCC27325) as a template and the primer pair of SEQ ID NO: 21 and SEQ ID NO: 22. Additionally, a gene fragment of the wild-type promoter downstream region of the maeB gene, where homologous recombination occurs on the chromosome, was obtained using the chromosomal DNA of Escherichia coli (ATCC27325) as a template and the primer pair of SEQ ID NO: 23 and SEQ ID NO: 24.

[0137] The primer sequences used here are shown in Table 5 below.

[0138] [Table 5]

[0139] To obtain the aforementioned fragments, Solg™ Pfu-X DNA polymerase was used. PCR amplification was performed under the following conditions: denaturation at 95°C for 2 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes. The upstream fragment of the maeB gene region, the fragment of the serC gene region containing the wild-type promoter, and the downstream fragment of the maeB promoter obtained in the above process were cloned together with the chromosome transformation vector pSKH130, which had been cleaved with EcoRV restriction enzyme, using an infusion cloning kit (Clontech Laboratories, Inc.) to obtain a recombinant plasmid, which was named pSKH△maeB::Pn_serC. To understand the effect of the deleted maeB gene on OPS production capacity due to the additional introduction of the serC gene on the host genome, a plasmid containing the nucleotide sequence for maeB deletion was constructed. Due to the maeB gene deficiency, a gene fragment of the wild-type promoter upstream region of the maeB gene, which undergoes homologous recombination on the chromosome, was obtained using the chromosomal DNA of the wild-type Escherichia coli (ATCC27325) as a template and the primer pair of SEQ ID NO: 25 and SEQ ID NO: 26. Additionally, a gene fragment of the wild-type promoter downstream region of the maeB gene, which undergoes homologous recombination on the chromosome, was obtained using the chromosomal DNA of Escherichia coli (ATCC27325) as a template and the primer pair of SEQ ID NO: 27 and SEQ ID NO: 28.

[0140] The primer sequences used here are shown in Table 6 below.

[0141] [Table 6]

[0142] To obtain the aforementioned fragments, Solg™ Pfu-X DNA polymerase was used. PCR amplification was performed under the following conditions: denaturation at 95°C for 2 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes. The upstream fragment of the maeB gene region and the downstream fragment of the maeB promoter obtained in the above process were cloned together with the chromosome transformation vector pSKH130, which had been cleaved with EcoRV restriction enzyme, using an infusion cloning kit (Clontech Laboratories, Inc.) to obtain a recombinant plasmid, which was named pSKH△maeB.

[0143] 2-2. Production of SerC-expressing enhanced bacterial strains The pSKH△maeB::Pn_serC prepared in Example 2-1 was transformed into CA07-4821 prepared in Example 1-2 using electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). After a secondary crossover process, a strain was obtained in which the serC gene sequence containing the wild-type promoter was inserted at the position of the maeB gene sequence. The insertion of the serC gene sequence containing the wild-type promoter was confirmed by PCR amplification and genome sequencing using primer pairs of SEQ ID NOs. 29 and 30, which can amplify the external regions of the homologous recombination upstream and downstream regions, respectively. The strain in which the above sequence was inserted using CA07-4821 from Example 1-2 as the host was named CA07-4881.

[0144] Furthermore, in order to eliminate maeB on the host genome, the pSKH△maeB prepared in Example 2-1 was transformed into CA07-4821 prepared in Example 1-2 using electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), and a strain with a deleted maeB gene sequence was obtained through a secondary cross-reaction process. The deletion of the maeB gene sequence was confirmed by PCR amplification and genome sequencing using primer pairs of SEQ ID NOs. 29 and 30, which can amplify the external regions of the homologous recombination upstream and downstream regions, respectively. The strain with a deleted maeB sequence using CA07-4821 from Example 1-2 as the host was named CA07-4882.

[0145] The primer sequences used here are shown in Table 7 below.

[0146] [Table 7]

[0147] 2-3. Evaluation of OPS production capacity of SerC-enhanced strains To evaluate the phosphoserine (O-phosphoserine) production capacity of CA07-4881, CA07-4882, and the control groups CA07-0012 and CA07-4821 prepared in Example 2-2, the evaluation was carried out using the culture medium from Example 1-3 (Table 3).

[0148] Specifically, each bacterial strain was streaked onto LB solid medium and then incubated overnight in an incubator at 33°C. The strains cultured overnight in LB solid medium were then inoculated into 25 mL of the titer medium shown in Table 3, and incubated at 33°C at 200 rpm for 48 hours. The results are shown in Table 8.

[0149] [Table 8]

[0150] As shown in Table 8, in the case of CA07-4881, in which serC was enhanced through the additional introduction of serC on the host genome, we confirmed that the OPS production capacity was approximately 144% higher than that of the parent strain CA07-4821. On the other hand, we confirmed that there was no difference in OPS production capacity between the parent strain (CA07-4821) and the maeB-deficient strain CA07-4882, which lacks maeB.

[0151] Example 3: Production of strains with weakened or deficient pdxB expression and evaluation of OPS production capacity 3-1. Plasmid construction for weakening or deleting pdxB expression To reduce the expression level of the pdxB gene, we decided to either replace the start codon of the pdxB gene or construct a plasmid for pdxB gene deletion.

[0152] Specifically, we decided to replace the start codon sequence (gtg) of pdxB (SEQ ID NO: 2) with ttg~ctg. First, in order to replace the start codon sequence (gtg) with ttg, we used the chromosomal DNA of the wild species Escherichia coli (ATCC27325) as a template and obtained a gene fragment of the upstream region of the pdxB gene in which homologous recombination occurs on the chromosome, containing a mutation (ttg) in the wild-type start codon sequence, using primer pairs of SEQ ID NO: 31 and SEQ ID NO: 32.

[0153] Furthermore, using Escherichia coli (ATCC27325) chromosomal DNA as a template, we obtained a gene fragment of the downstream region of the pdxB gene, which undergoes homologous recombination on the chromosome, containing a mutation (ttg) in the wild-type start codon sequence, using primer pairs of SEQ ID NO: 33 and SEQ ID NO: 34.

[0154] Next, in order to replace the start codon sequence (gtg) with ctg, we used Escherichia coli (ATCC27325) chromosomal DNA as a template and obtained a gene fragment of the upstream region of the pdxB gene in which homologous recombination occurs on the chromosome, containing a mutation (ctg) in the wild-type start codon sequence using primer pairs of SEQ ID NO: 31 and SEQ ID NO: 35.

[0155] Furthermore, using Escherichia coli (ATCC27325) chromosomal DNA as a template, we obtained a gene fragment of the downstream region of the pdxB gene, which undergoes homologous recombination on the chromosome, containing a mutation (ctg) in the wild-type start codon sequence, using primer pairs of SEQ ID NO: 36 and SEQ ID NO: 34.

[0156] On the other hand, to promote pdxB deletion, we obtained a gene fragment of the upstream region of the pdxB gene, which undergoes homologous recombination on the chromosome, using Escherichia coli (ATCC27325) chromosomal DNA as a template and primer pairs of SEQ ID NO: 37 and SEQ ID NO: 38. We also obtained a gene fragment of the downstream region of the pdxB gene, which undergoes homologous recombination on the chromosome, using primer pairs of SEQ ID NO: 39 and SEQ ID NO: 40, also using Escherichia coli (ATCC27325) chromosomal DNA as a template.

[0157] The primer sequences used here are shown in Table 9 below. Specifically, in the names of Sequence ID No. 31 and Sequence ID No. 34, n represents either t or c.

[0158] [Table 9]

[0159] To obtain the aforementioned fragments, Solg™ Pfu-X DNA polymerase was used, and PCR amplification was performed under the following conditions: denaturation at 95°C for 2 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization reaction at 72°C for 5 minutes.

[0160] The upstream fragment containing the pdxB start codon mutation and the downstream fragment containing the pdxB start codon mutation obtained in the above process were cloned together with the chromosome transformation vector pSKH130, which had been cleaved with EcoRV restriction enzyme, using an infusion cloning kit (Clontech Laboratories, Inc.) to obtain recombinant plasmids, which were named pSKH_pdxB(ttg) and pSKH_pdxB(ctg), respectively.

[0161] Meanwhile, the upstream and downstream fragments for pdxB deficiency obtained in the above process were cloned together with the chromosome transformation vector pSKH130, which had been cleaved with EcoRV restriction enzyme, using an infusion cloning kit (Clontech Laboratories, Inc.) to obtain a recombinant plasmid, which was named pSKH△pdxB.

[0162] 3-2. Production of strains with weakened or deficient pdxB expression. The pSKH_pdxB(ttg) and pSKH_pdxB(ctg) strains prepared in Example 3-1 were transformed into CA07-4881 prepared in Example 2-2 using electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). After a secondary crossover, strains were obtained in which the wild-type start codon sequence of the pdxB gene was replaced with ttg or ctg. The exchange of the pdxB start codon sequence was confirmed by PCR amplification and genome sequencing using primer pairs of SEQ ID NOs. 41 and 42, which can amplify the external regions of the homologous recombination upstream and downstream regions, respectively. The strain in which the start codon sequence was replaced with ttg, using CA07-4881 from Example 2-2 as the host, was named CA07-4883, and the strain in which the start codon sequence was replaced with ctg was named CA07-4884.

[0163] On the other hand, the pSKH△pdxB(ttg) prepared in Example 3-1 was transformed into CA07-4881 prepared in Example 2-2 using electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), and a strain lacking the pdxB gene was obtained through a secondary cross-reaction. The absence of the pdxB base sequence was confirmed by PCR amplification and genome sequencing using primer pairs of SEQ ID NOs. 41 and 42, which can amplify the external regions of the homologous recombination upstream and downstream regions, respectively. The strain lacking the pdxB base sequence, using CA07-4881 from Example 2-2 as the host, was named CA07-4886.

[0164] The primer sequences used here are shown in Table 10 below.

[0165] [Table 10]

[0166] 3-3. Evaluation of OPS production capacity of strains with weakened or deficient pdxB expression. To evaluate the phosphoserine (O-phosphoserine) production capacity of the CA07-4883, CA07-4884, and CA07-4886 strains prepared in Example 3-2, and the control group CA07-0012, CA07-4821, CA07-4881, and CA07-4882, the evaluation was carried out using the culture media from Example 1-3 (Table 3).

[0167] Specifically, each bacterial strain was streaked onto LB solid medium and then incubated overnight in an incubator at 33°C. The strains cultured overnight in LB solid medium were then inoculated into 25 mL of the titer medium shown in Table 3, and incubated at 33°C at 200 rpm for 48 hours. The results are shown in Table 11.

[0168] [Table 11]

[0169] As shown in Table 11, the OPS production capacity of CA07-4883 and CA07-4884, in which the pdxB start codon was replaced with ttg or ctg, respectively, was confirmed to be approximately 103% and 107% of that of the parent strain (CA07-4881). On the other hand, the OPS production capacity of CA07-4886, a strain lacking pdxB, was confirmed to be lower than that of the parent strain (CA07-4881).

[0170] From the above description, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified and altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

Claims

1. A recombinant microorganism having increased O-phosphoserine production capacity compared to a non-mutated microorganism in which the start codon of the polynucleotide encoding the erythronate-4-phosphate dehydrogenase protein is not substituted, wherein the erythronate-4-phosphate dehydrogenase protein (polypeptide) activity is weakened compared to the endogenous activity by substitution of the start codon of the polynucleotide encoding the erythronate-4-phosphate dehydrogenase protein.

2. The microorganism according to claim 1, wherein the start codon of the polynucleotide encoding erythronate-4-phosphate dehydrogenase is substituted with TTG or CTG.

3. The microorganism according to claim 2, wherein the microorganism has increased O-phosphoserine production capacity compared to a non-mutant microorganism in which the start codon of the polynucleotide encoding erythronate-4-phosphate dehydrogenase is GTG or ATG.

4. The microorganism according to claim 1, wherein the YhhS protein (polypeptide) activity is further enhanced compared to the intrinsic activity by enhancing YhhS expression.

5. The microorganism according to claim 1, wherein the microorganism has a deficiency in endogenous phosphoserine phosphatase (SerB), resulting in further weakened phosphoserine phosphatase (polypeptide) activity compared to its endogenous activity.

6. The microorganism according to claim 1, wherein the microorganism has been further enhanced by introducing or enhancing the protein (polypeptide) activity of YhhS, phosphoserine aminotransferase, or a combination thereof, compared to its endogenous activity, by enhancing YhhS expression and phosphoserine aminotransferase (SerC) expression.

7. The microorganism according to claim 1, wherein the microorganism is a microorganism of the genus Escherichia.

8. The microorganism according to claim 7, wherein the microorganism of the genus Escherichia is Escherichia coli.

9. A method for producing O-phosphoserine, comprising the step of culturing the microorganism described in claim 1 in a culture medium.

10. A method for producing cysteine ​​or a derivative thereof, a) The step of culturing the microorganism described in claim 1 in a culture medium to produce O-phosphoserine or a culture medium containing the same; b) a step of contacting O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing the same with the O-phosphoserine or culture medium containing the same produced in step a) above and a sulfide; Cysteine ​​derivatives include NAC (N-acetylcysteine), SCMC (S-Carboxymethylcysteine), BOC-CYS(ME)-OH, (R)-S-(2-Amino-2-carboxyethyl)-L-homocysteine, (R)-2-Amino-3-sulfopropionic acid, and D-2-Amino-4-(ethylthio)butyric A method selected from the group consisting of acid, 3-sulfino-L-alanine, Fmoc-Cys(Boc-methyl)-OH, Seleno-L-cysteine, S-(2-Thiazolyl)-L-cysteine, S-(2-Thienyl)-L-cysteine, and S-(4-Tolyl)-L-cysteine.

11. The use of the microorganism described in claim 1 in the production of O-phosphoserine, cysteine, or a derivative thereof, Cysteine ​​derivatives include NAC (N-acetylcysteine), SCMC (S-Carboxymethylcysteine), BOC-CYS(ME)-OH, (R)-S-(2-Amino-2-carboxyethyl)-L-homocysteine, (R)-2-Amino-3-sulfopropionic acid, and D-2-Amino-4-(ethylthio)butyric Use of a substance selected from the group consisting of acid, 3-sulfino-L-alanine, Fmoc-Cys(Boc-methyl)-OH, Seleno-L-cysteine, S-(2-Thiazolyl)-L-cysteine, S-(2-Thienyl)-L-cysteine, and S-(4-Tolyl)-L-cysteine.

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