WO2022055192A1 - 신규 o-포스포세린 배출 단백질 및 이를 이용한 o-포스포세린, 시스테인 및 이의 유도체의 생산 방법 - Google Patents
신규 o-포스포세린 배출 단백질 및 이를 이용한 o-포스포세린, 시스테인 및 이의 유도체의 생산 방법 Download PDFInfo
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- WO2022055192A1 WO2022055192A1 PCT/KR2021/011994 KR2021011994W WO2022055192A1 WO 2022055192 A1 WO2022055192 A1 WO 2022055192A1 KR 2021011994 W KR2021011994 W KR 2021011994W WO 2022055192 A1 WO2022055192 A1 WO 2022055192A1
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- WIPO (PCT)
- Prior art keywords
- phosphoserine
- activity
- mdth
- cysteine
- producing
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/12—Methionine; Cysteine; Cystine
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- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01095—Phosphoglycerate dehydrogenase (1.1.1.95)
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- C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
- C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
- C12Y205/01065—O-Phosphoserine sulfhydrylase (2.5.1.65)
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- C12Y206/00—Transferases transferring nitrogenous groups (2.6)
- C12Y206/01—Transaminases (2.6.1)
- C12Y206/01052—Phosphoserine transaminase (2.6.1.52)
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03003—Phosphoserine phosphatase (3.1.3.3)
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Definitions
- the present application relates to a novel O-phosphoserine excreting protein and a method for producing O-phosphoserine, cysteine and derivatives of cysteine using the same.
- L-cysteine is an important amino acid for sulfur metabolism in all living things. It is used not only for the synthesis of in vivo proteins such as hair keratin, glutathione, biotin, methionine and other sulfur-containing metabolites, but also as a precursor for coenzyme A biosynthesis. do.
- a method of producing L-cysteine using a microorganism 1) A method of biologically converting D,L-ATC (D,L-2-amino-2-thiazoline-4-carboxylate) using a microorganism, 2) Direct fermentation method for producing L-cysteine using E. coli (European Patent Registration EP0885962B; Wada M and Takagi H, Appl. Microbiol.
- O-phosphoserine using microorganisms
- O-phosphoserine hereinafter “OPS”
- OPSS O-phosphoserine sulfhydrylase
- the present inventors identified an appropriate excretion factor that allows O-phosphoserine produced in the OPS-producing strain to be smoothly discharged out of the cell, and as a result of earnest efforts to increase the production of OPS, the present application by discovering a novel OPS excreting protein was completed.
- One object of the present application is to provide a recombinant microorganism producing O-phosphoserine, in which the activity of the mdtH protein is enhanced compared to the intrinsic activity.
- Another object of the present application is to provide a method for producing O-phosphoserine using the O-phosphoserine-producing recombinant microorganism of the present application.
- Another object of the present application is to provide a method for producing cysteine or a derivative thereof using the O-phosphoserine-producing recombinant microorganism of the present application.
- the present application provides an O-phosphoserine-producing recombinant microorganism in which the activity of the mdtH protein is enhanced compared to the intrinsic activity.
- OPS O-phosphoserine
- OPSS OPS sulfhydrylase
- the recombinant microorganism of the present application may have enhanced O-phosphoserine excretion activity compared to its intrinsic activity.
- the mdtH protein of the present application may have an activity of releasing O-phosphoserine out of cells, and this activity was first identified in the present application.
- the mdtH protein of the present application may be a membrane protein, and the mdtH protein of the present application may be derived from Escherichia coli .
- mdtH protein of the present application may be a transporter belonging to a major facilitator superfamily (MFS).
- MFS major facilitator superfamily
- the mdtH protein of the present application may be a protein comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence exhibiting 95% or more identity with SEQ ID NO: 1 while having O-phosphoserine excretion activity.
- the protein comprising the amino acid sequence showing 95% or more identity to the amino acid sequence of SEQ ID NO: 1 is the same as or equivalent to the mdtH protein comprising the amino acid sequence of SEQ ID NO: 1 If it is a protein that exhibits OPS excretion activity, including without limitation do.
- amino acid sequence showing 95% or more identity to the amino acid sequence of SEQ ID NO: 1 is 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.75 to the amino acid sequence of SEQ ID NO: 1 % or more, and less than 100% identity.
- the mdtH protein of the present application is a protein variant in which some sequences are deleted, modified, substituted, conservatively substituted or added in the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence showing 95% or more identity with the amino acid sequence of SEQ ID NO: 1 Also may be included within the scope of the present application as long as it has substantially O-phosphoserine excretion activity.
- the mdtH protein of the present application has the amino acid sequence of SEQ ID NO: 1 or O-phosphoserine excretion activity while adding meaningless sequences before and after the amino acid sequence having 95% or more identity with SEQ ID NO: 1 or naturally occurring mutations , or a latent mutation thereof may be apparent to those skilled in the art.
- the term 'homology' or 'identity' refers to the degree of similarity between two given amino acid sequences or nucleotide sequences and may be expressed as a percentage.
- the terms homology and identity can often be used interchangeably.
- Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially homologous or identical sequences are generally capable of hybridizing with all or part of a sequence under moderate or high stringent conditions. It is apparent that hybridization also includes hybridization with a polynucleotide containing a common codon in a polynucleotide or a codon in consideration of codon degeneracy.
- a GAP program can be defined as the total number of symbols in the shorter of two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids).
- Default parameters for the GAP program are: (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty 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 end gaps.
- OPS orthophosphoserine
- OPS-producing recombinant microorganism refers to a microorganism having a naturally weak OPS-producing ability or a parent strain without OPS-producing ability naturally or artificially genetically modified to give OPS-producing ability. It may mean microorganisms.
- OPS-producing recombinant microorganism may be used interchangeably with "a microorganism having an OPS-producing ability”.
- the production of OPS may be increased compared to the wild-type or pre-modified microorganism. This is because the wild-type or pre-modified microorganism cannot produce OPS or can produce a trace amount even if it produces OPS, whereas the microorganism of the present application can increase the OPS-producing ability by enhancing the activity of the mdtH protein.
- the term “enhancement” of a polypeptide activity means that the activity of the polypeptide is increased compared to the intrinsic activity.
- the reinforcement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase.
- activation, enhancement, upregulation, overexpression, and increase may include all of those exhibiting an activity that was not originally possessed, or exhibiting an improved activity compared to intrinsic activity or activity before modification.
- intrinsic activity refers to the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. It can be used interchangeably.
- Enhancement, “upregulation”, “overexpression” or “increase” of the activity of a polypeptide compared to the intrinsic activity means that the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation. And / or concentration (expression amount) means improved compared to.
- the enrichment can be achieved by introducing an exogenous polypeptide, or by enhancing the activity and/or concentration (expression amount) of an endogenous polypeptide. Whether or not the activity of the polypeptide is enhanced can be confirmed from the increase in the level of activity, expression level, or the amount of product excreted from the polypeptide.
- the enhancement of the activity of the polypeptide can be applied by various methods well known in the art, and is not limited as long as it can enhance the activity of the target polypeptide compared to the microorganism before modification. Specifically, it may be one using genetic engineering and/or protein engineering well known to those skilled in the art, which is a routine method of molecular biology, but is not limited thereto (eg, Sitnicka et al. Functional Analysis of Genes. Advances in Cell). Biology 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).
- modification of the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide eg, modification of the polynucleotide sequence of the polypeptide gene to encode a polypeptide that has been modified to enhance the activity of the polypeptide;
- the increase in the intracellular copy number of the polynucleotide encoding the polypeptide is achieved by introduction of a vector, to which the polynucleotide encoding the polypeptide is operably linked, which can replicate and function independently of the host, into a host cell.
- the polynucleotide encoding the polypeptide may be achieved by introducing one copy or two or more copies into a chromosome in a host cell.
- the introduction into the chromosome may be performed by introducing a vector capable of inserting the polynucleotide into the chromosome in the host cell into the host cell, but is not limited thereto.
- the vector is the same as described above.
- Replacing the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence with strong activity is, for example, deletion, insertion, non-conservative or Conservative substitution or a combination thereof may result in a mutation in the sequence, or replacement with a sequence having a stronger activity.
- the expression control region is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence for regulating the termination of transcription and translation. As an example, it may be to replace the original promoter with a strong promoter, but is not limited thereto.
- Examples of known strong promoters include cj1 to cj7 promoter (US Patent US 7662943 B2), 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 US 10584338 B2), O2 promoter (US Patent US 10273491 B2), tkt promoter, yccA promoter, etc., but is not limited thereto.
- the modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a nucleotide sequence encoding another start codon having a higher expression rate of the polypeptide compared to the intrinsic start codon. It may be a substitution, but is not limited thereto.
- the modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above may include deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance activity of the polypeptide; A combination thereof may result in sequence mutation, or replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity or an amino acid sequence or polynucleotide sequence improved to increase activity, but is not limited thereto.
- the replacement may be specifically performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto.
- the vector used may further include a selection marker for confirming whether or not the chromosome is inserted. The selection marker is the same as described above.
- the introduction of the foreign polynucleotide exhibiting the activity of the polypeptide may be introduction of the foreign polynucleotide encoding the polypeptide exhibiting the same/similar activity as the polypeptide into a host cell.
- the foreign polynucleotide is not limited in origin or sequence as long as it exhibits the same/similar activity as the polypeptide.
- the method used for the introduction can be performed by appropriately selecting a known transformation method by those skilled in the art, and the introduced polynucleotide is expressed in a host cell to generate a polypeptide and increase its activity.
- Codon optimization of the polynucleotide encoding the polypeptide is codon-optimized so that the transcription or translation of the endogenous polynucleotide is increased in the host cell, or the transcription and translation of the foreign polynucleotide is optimized in the host cell. It may be that its codons are optimized so that the
- Selecting an exposed site by analyzing the tertiary structure of the polypeptide and modifying or chemically modifying it for example, compares the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored to determine the degree of sequence similarity. Accordingly, it may be to determine a template protein candidate, check the structure based on this, and select an exposed site to be modified or chemically modified and modified or modified.
- Such enhancement of polypeptide activity is to increase the activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type or pre-modified microbial strain, or increase the amount of product produced from the polypeptide.
- the present invention is not limited thereto.
- the term "vector” refers to a DNA comprising a nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. preparations may be included.
- the expression control 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 a sequence regulating the termination of transcription and translation. After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.
- the vector used in the present application is not particularly limited, and any vector known in the art may be used.
- Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages.
- pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pDZ-based, pBR-based, and pUC-based plasmid vectors may be used.
- pBluescript II-based, pGEM-based, pTZ-based, pCL-based, pET-based and the like can be used.
- pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.
- a polynucleotide encoding a target polypeptide may be inserted into a chromosome through a vector for intracellular chromosome insertion.
- the insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto.
- It may further include a selection marker (selection marker) for confirming whether the chromosome is inserted.
- the selection marker is used to select cells transformed with the vector, that is, to determine whether a target nucleic acid molecule is inserted, and selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface polypeptide expression. Markers to be given can be used. In an environment treated with a selective agent, only the cells expressing the selectable marker survive or exhibit other expression traits, so that the transformed cells can be selected.
- the term “transformation” refers to introducing a vector including 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 the chromosome of the host cell or located extrachromosomally, as long as they can be expressed in the host cell.
- the polynucleotide includes DNA and/or RNA encoding a target polypeptide.
- the polynucleotide may be introduced in any form as long as it can be introduced and expressed into a host cell.
- the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression.
- the expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal.
- the expression cassette may be in the form of an expression vector capable of self-replication.
- the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.
- operably linked means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target variant of the present application and the polynucleotide sequence are functionally linked.
- the recombinant microorganism of the present application includes a microorganism into which the gene/polynucleotide encoding the mdtH protein is introduced, a microorganism in which the intracellular copy number of the polynucleotide encoding the mdtH protein is increased, and a polynucleotide encoding the mdtH protein It may be a microorganism transformed with a vector that is there is.
- the gene encoding the mdtH protein may be an mdtH gene.
- the polynucleotide encoding the mdtH protein may be a polynucleotide including the nucleotide sequence of SEQ ID NO: 2.
- the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 can be obtained from GenBank of NCBI, which is a known database.
- the protein/polypeptide comprising the amino acid sequence of the specific SEQ ID NO: or the amino acid sequence described in the specific SEQ ID NO: of the present application is a protein/polypeptide having the amino acid sequence of the specific SEQ ID NO: or the amino acid sequence described in the specific SEQ ID NO: It may be a protein/polypeptide consisting essentially of an amino acid sequence or an amino acid sequence set forth in a specific SEQ ID NO:
- the polynucleotide comprising the nucleotide sequence of the specific SEQ ID NO: or the nucleotide sequence described in the specific SEQ ID NO: of the present application is a polynucleotide having the nucleotide sequence of the specific SEQ ID NO: or the nucleotide sequence described in the specific SEQ ID NO: It may be a polynucleotide consisting of/consisting essentially of a base sequence or a base sequence described in a specific SEQ ID NO.
- the type of the microorganism of the present application is not particularly limited as long as it can produce OPS, and both prokaryotic and eukaryotic cells are possible, but specifically prokaryotic cells.
- Escherichia genus Erwinia genus, Serratia genus, Providencia genus, Corynebacterium genus, and Brevibacterium belonging to the genus It may include a microbial strain, specifically, a microorganism of the genus Escherichia, more specifically Escherichia coli , but may be, but is not limited thereto.
- the recombinant microorganism of the present application may additionally have a weakened activity of phosphoserine phosphatase (SerB) compared to its intrinsic activity.
- SerB phosphoserine phosphatase
- the SerB Since the SerB has an activity of converting OPS to L-serine (L-serine), the microorganism mutated to weaken the SerB activity has the characteristic of accumulating OPS and can be usefully used in the production of OPS.
- the SerB may be a protein including the amino acid sequence shown in SEQ ID NO: 3, but is not limited thereto.
- the SerB includes an amino acid sequence that is 80% or more, specifically 90% or more, more specifically 95% or more, more specifically 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 3 as long as it exhibits SerB activity can, but is not limited to.
- the polynucleotide encoding the SerB may have a nucleotide sequence encoding the amino acid set forth in SEQ ID NO: 3.
- various modifications are made to the coding region within the range that does not change the amino acid sequence of the variant of the present application. can be done
- the polynucleotide encoding the SerB may include, for example, the nucleotide sequence of SEQ ID NO: 4, and the identity thereof is 80% or more, specifically 90% or more, more specifically 95% or more, more specifically 99% or more. It may include more than one nucleotide sequence, but is not limited thereto.
- the term "weakening" of a polypeptide is a concept that includes both reduced or no activity compared to intrinsic activity.
- the attenuation may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, attenuation, and the like.
- the attenuation is when the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide possessed by the original microorganism due to mutation of the polynucleotide encoding the polypeptide, etc.
- the overall polypeptide activity level and/or concentration (expression amount) in the cell is lower than that of the native strain due to (translation) inhibition, etc., when the expression of the polynucleotide is not made at all, and/or when the expression of the polynucleotide is Even if there is no activity of the polypeptide, it may also be included.
- the "intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain, wild-type or unmodified microorganism before transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification”. "Inactivation, deficiency, reduction, downregulation, reduction, attenuation” of the activity of a polypeptide compared to the intrinsic activity means that the activity of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation is lowered.
- Attenuation of the activity of the polypeptide may be performed by any method known in the art, but is not limited thereto, and may be achieved by application of various methods well known in the art (eg, 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.).
- the attenuation of the polypeptide of the present application is
- an antisense oligonucleotide eg, antisense RNA
- an antisense oligonucleotide that complementarily binds to the transcript of said gene encoding the polypeptide
- deletion of a part or all of the gene encoding the polypeptide may be the removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacement with a polynucleotide in which some nucleotides are deleted, or replacement with a marker gene.
- the above 2) modification of the expression control region is deletion, insertion, non-conservative or conservative substitution or a combination thereof, resulting in mutation in the expression control region (or expression control sequence), or weaker replacement with an active sequence.
- the expression control 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.
- the base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a base encoding another start codon having a lower polypeptide expression rate than the intrinsic start codon It may be substituted with a sequence, but is not limited thereto.
- the modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above deletes, inserts, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to weaken the activity of the polypeptide. Or a combination thereof may result in sequence mutation, or replacement with an amino acid sequence or polynucleotide sequence improved to have weaker activity, or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto.
- the expression of a gene may be inhibited or attenuated, but is not limited thereto.
- antisense oligonucleotide eg, antisense RNA
- antisense RNA an antisense oligonucleotide that complementarily binds to the transcript of the gene encoding the polypeptide
- Weintraub, H. et al. Antisense-RNA as a molecular tool. for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].
- RTE reverse transcription engineering
- Modification of part or all of the polynucleotide in the microorganism of the present application is (a) homologous recombination using a vector for chromosome insertion in the microorganism or genome correction using engineered nuclease (eg, CRISPR-Cas9) and/or (b) It may be induced by light and/or chemical treatments such as, but not limited to, ultraviolet and radiation.
- the method for modifying part or all of the gene may include a method by DNA recombination technology.
- a part or all of the gene may be deleted.
- the injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.
- the recombinant microorganism of the present application is additionally phosphoglycerate dehydrogenase (phosphoglycerate dehydrogenase, SerA), phosphoserine aminotransferase (phosphoserine aminotransferase, SerC), or any one of a combination of the activity is enhanced compared to the intrinsic activity it may have been
- the SerA is a protein having an activity of converting 3-phosphoglycerate to 3-phospho-hydroxypymvate, and the SerC is 3-phosphohydroxypymvate. It is a protein having the activity of converting rubate into OPS. Therefore, the microorganisms with enhanced SerA or/and SerC activity can be usefully used as OPS-producing strains.
- the SerA is not limited thereto, but may be a protein comprising the amino acid sequence of SEQ ID NO: 5 or 6.
- SEQ ID NO: 5 is a sequence of wild-type SerA
- SEQ ID NO: 6 is a sequence of a SerA mutant in which feedback to serine is canceled.
- the SerA is 80% or more, specifically 90% or more, more specifically 95% of the amino acid sequence set forth in SEQ ID NO: 5 or 6 Above, more specifically, it may include an amino acid sequence that is 99% or more identical, but is not limited thereto.
- the SerA mutant in which the feedback to serine is released refers to a case in which the activity is maintained or enhanced from feedback inhibition by serine or glycine by introducing a mutation such as insertion or substitution into the SerA-encoding gene, SerA variants with abolished feedback to the serine are already well known (Grant GA et al., J. Biol. Chem., 39: 5357-5361, 1999; Grant GA et al., Biochem., 39: 7316- 7319, 2000; Grant GA et al., J. Biol. Chem., 276:17844-17850, 2001; Peters-Wendisch P et al., Appl. Microbiol. BiotechnoL, 60:37-441, 2002; European Patent EP 0943687 B).
- the polynucleotide encoding the wild-type SerA or the SerA variant in which feedback for serine is canceled may include a nucleotide sequence encoding any one of the amino acid sequences set forth in SEQ ID NOs: 5 to 6, but is not limited thereto.
- various modifications may be made to the coding region within the range that does not change the amino acid sequence of the polypeptide due to codon degeneracy or in consideration of codons preferred by the organism in which the polypeptide is to be expressed.
- the polynucleotide encoding the wild-type SerA or the SerA variant in which feedback to serine is canceled may be, for example, a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 7 or 8, and the homology thereof is 80% or more, specifically It may be a polynucleotide comprising a nucleotide sequence of 90% or more, more specifically 95% or more, and more specifically 99% or more, but is not limited thereto.
- the SerC may be, for example, a protein comprising the amino acid sequence set forth in SEQ ID NO: 9, but is not limited thereto.
- it may include an amino acid sequence that is 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 9. However, it is not limited thereto.
- the polynucleotide encoding the SerC may include a nucleotide sequence encoding the amino acid set forth in SEQ ID NO: 9.
- various modifications can be made in the coding region within the range that does not change the amino acid sequence of the polypeptide due to codon degeneracy or in consideration of codons preferred by the organism in which the polypeptide is to be expressed.
- the SerC-encoding polynucleotide may include, for example, the nucleotide sequence of SEQ ID NO: 10, and the homology thereof is 80% or more, specifically 90% or more, more specifically 95% or more, more specifically 99% or more. It may include a nucleotide sequence, but is not limited thereto.
- the recombinant microorganism of the present application may be a microorganism in which the ability to further reduce the influx or degradation of OPS into cells.
- the present application provides a method for producing O-phosphoserine, comprising the step of culturing a recombinant microorganism producing O-phosphoserine in which the activity of the mdtH protein is enhanced compared to the intrinsic activity. .
- the mdtH protein, intrinsic activity, enrichment, O-phosphoserine and microorganisms are the same as described above.
- the term "cultivation” means growing the microorganism in an appropriately controlled environmental condition.
- the culture process of the present application may be made according to a suitable medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain.
- the culture may be a batch type, a continuous type, and a fed-batch type, but is not limited thereto.
- Glycine may be provided in the form of purified glycine, yeast extract containing glycine, tryptone, and the concentration contained in the culture medium may be usually 0.1 to 10 g/L, specifically 0.5 to 3 g/L.
- serine may be provided in the form of purified serine, yeast extract containing serine, tryptone, etc., and the concentration contained in the culture medium may be usually 0.1 to 5 g/L, specifically 0.1 to 1 g/L.
- Carbon sources included in the medium include glucose, sucrose, lactose, fructose, maltose, starch, sugars and carbohydrates such as cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, fatty acids such as stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid, and these substances may be used individually or as a mixture, but are not limited thereto.
- sugars and carbohydrates such as cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, fatty acids such as stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid, and these substances may be used individually or as a mixture, but are not limited thereto.
- organic nitrogen sources such as peptone, yeast extract, broth, malt extract, corn steep liquor and soybean wheat and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, anmonium carbonate and ammonium nitrate are included. and these nitrogen sources may be used alone or in combination, but are not limited thereto.
- potassium hydrogen phosphate dipotassium hydrogen phosphate and the corresponding sodium-containing salt may be included, but the present invention is not limited thereto.
- the medium may contain a metal salt such as magnesium sulfate or iron sulfate, and in addition, amino acids, vitamins and appropriate precursors may be included. These media or precursors may be added to the culture in a batch or continuous manner, but is not limited thereto.
- chemicals such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture.
- an antifoaming agent such as fatty acid polyglycol ester can be used to suppress foaming.
- oxygen or oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen or carbon dioxide gas may be injected with or without gas to maintain anaerobic and microaerobic conditions.
- the temperature of the culture may be usually 25°C to 40°C, specifically 30°C to 35°C.
- the incubation period of the culture may be continued until the desired production of useful substances is obtained, and specifically may be 10 to 100 hours. However, it is not limited to these examples.
- the production method of O-phosphoserine of the present application includes the steps of preparing the microorganism of the present application, preparing a medium for culturing the microorganism, or a combination thereof (regardless of order, in any order), for example , prior to the culturing step, may be further included.
- the method for producing O-phosphoserine of the present application may further include recovering O-phosphoserine from the medium according to the culture (the culture medium) or the microorganism of the present application. The recovering step may be further included after the culturing step.
- the method for recovering the O-phosphoserine is a method of culturing the microorganism of the present application, for example, a desired O-phosphoserine using a suitable method known in the art according to a batch, continuous or fed-batch culture method, etc. It may be to collect.
- centrifugation, filtration, treatment with a crystallized protein precipitant salting out method
- extraction ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity
- chromatography such as island chromatography, HPLC, or a combination thereof
- a desired O-phosphoserine may be recovered from a medium or a microorganism using a suitable method known in the art.
- the O-phosphoserine production method of the present application may additionally include a purification step.
- the purification may be performed using a suitable method known in the art.
- the recovery step and the purification step are performed continuously or discontinuously, regardless of the order, or simultaneously or in one step It may be integrated and performed, but is not limited thereto.
- variants, polynucleotides, vectors, strains, etc. are as described in the other aspects above.
- the present application relates to: a) O-phosphoserine-producing recombinant microorganisms in which the activity of mdtH protein is enhanced compared to the intrinsic activity is cultured in a medium, and a medium containing O-phosphoserine or O-phosphoserine producing; And b) in the presence of O-phosphoserine sulfliydrylase (O-phosphoserine sulfliydrylase, OPSS) or a microorganism comprising the same, reacting the O-phosphoserine produced in step a) or a medium containing the same with sulfide It provides a method for producing cysteine or a derivative thereof, comprising a.
- O-phosphoserine sulfliydrylase O-phosphoserine sulfliydrylase
- the term “comprising” a specific protein means a state in which a specific protein of interest is introduced into the microorganism or expressed in the microorganism.
- the mdtH protein, intrinsic activity, enrichment, and O-phosphoserine microorganisms are as described above.
- derivative refers to a similar compound obtained by chemically changing a part of a certain compound, and usually refers to a compound in which a hydrogen atom or a specific atomic group is substituted with another atom or atomic group.
- cyste derivative refers to a compound in which a hydrogen atom or a specific atomic group of cysteine is substituted with another atom or atomic group.
- it may be a form in which another atom or group is attached to the nitrogen atom of the amine group (-NH2) of cysteine or the sulfur atom of the thiol group (-SH), for example, 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-
- cysteine As long as cysteine is produced according to the method of the present application, the conversion to a cysteine derivative may be easily converted into various cysteine derivatives by a method well known in the art.
- the method for producing the cysteine derivative may further include the step of converting the cysteine produced in step b) into a cysteine derivative, for example, by reacting cysteine with an acetylation agent to form NAC ( N-acetylcysteine) or by reacting cysteine with haloacetic acid under basic conditions to synthesize S-Carboxymetylcysteine (SCMC), but is not limited thereto.
- NAC N-acetylcysteine
- SCMC S-Carboxymetylcysteine
- the cysteine derivative is mainly used as a pharmaceutical raw material as an antitussive, a cough reliever, bronchitis, bronchial asthma and sore throat, but is not limited thereto.
- OPS O-phosphoserine sulfhydrylase
- the OPSS includes not only the wild-type OPSS protein, but also a mutant protein in which some of the polynucleotide sequences encoding the OPSS are deleted, substituted, or added, and exhibiting an activity equal to or higher than that of the wild-type OPSS protein. and OPSS protein disclosed in European Patent Application Publication No. EP 2444481 and US Patent No. 9127324 and variant proteins thereof may also be included.
- the sulfide is provided in the form of a liquid or gas due to differences in pH, pressure, and solubility, as well as a solid commonly used in the art, so that sulfide (S 2- ), thiosulfate (thiosulfate, S 2 0 3 ) If it is a sulfide that can be converted to a thiol group (SH group) in the form of 2- ), etc., it can be used without limitation. Specifically, Na 2 S, NaSH, H 2 S, (NH 4 ) 2 S, or Na 2 S 2 0 3 that provides a thiol group to the OPS may be used, but is not limited thereto.
- the reaction is a reaction for preparing one cysteine or cysteine derivative by providing one thiol group to one OPS reactor, and the amount of sulfide added in the reaction may be 0.1 to 3 times the molar concentration of OPS, specifically, 1 to 2 It may be a ship, but is not limited thereto.
- the present application may further include the step of recovering the cysteine produced through the reaction step.
- the desired cysteine may be isolated and purified from the reaction solution and collected using a suitable reaction known in the art.
- the present application provides a use for producing a derivative of O-phosphoserine, cysteine or cysteine of a recombinant microorganism producing O-phosphoserine in which the activity of the mdtH protein is enhanced compared to the intrinsic activity.
- the present application provides a use for excreting O-phosphoserine of mdtH protein from a microorganism.
- the mdtH protein, intrinsic activity, enrichment, O-phosphoserine, cysteine, cysteine derivatives and microorganisms are the same as described above.
- the screening-based strain is an OPS-producing strain mutated to weaken the activity of endogenous phosphoserine phosphatase (serB) in W3110, CA07-0012 (KCCM11212P, European Patent Publication EP 2444481; US Patent Publication No. 2012-0190081) ) is a strain named
- CA07-0012 was cultured in a medium containing OPS to establish optimal screening conditions showing growth inhibition.
- the W3110 genome library plasmid was transformed into CA07-0012 by electroporation (van der Rest et al. 1999), and colonies in which growth degradation was released in the medium condition with an excess of OPS were selected. Plasmids were obtained from the selected colonies and nucleotide sequences were analyzed through sequencing techniques. From this, two types of E. coli membrane proteins involved in canceling growth degradation under the condition of adding excess OPS were identified.
- the gene encoding the E. coli membrane protein was identified as an mdtH transporter belonging to the major facilitator superfamily (MFS).
- pCL_Ptrc-yhhS To prepare pCL_Ptrc-yhhS, pCL_Ptrc-gfp (WO 2016/024771 A1) was used as a template, and PCR was performed using SEQ ID NO: 11 and SEQ ID NO: 12 to obtain a Ptrc DNA fragment.
- the yhhS DNA fragment was obtained by PCR with SEQ ID NO: 13 and SEQ ID NO: 14 using W3110 as a template.
- the amplified fragment was subjected to IST with the pCL1920 vector treated with restriction enzymes XbaI and HindIII to obtain pCL_Ptrc-yhhS.
- IST DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix
- pCL_Ptrc-mdtH also used pCL_Ptrc-gfp as a template, but Ptrc DNA fragments were obtained using SEQ ID NOs: 15 and 16.
- An mdtH DNA fragment was obtained using SEQ ID NO: 17 and SEQ ID NO: 18 using W3110 as a template, and pCL_Ptrc-mdtH was prepared through IST in the same manner as pCL_Ptrc-yhhS.
- pCL_Ptrc-yfaV was also cloned in the same manner as in the production of the two plasmids, and the Ptrc DNA fragment was obtained using pCL_Ptrc-gfp and the yfaV DNA fragment using W3110 as a template.
- SEQ ID NO: 19 and SEQ ID NO: 20 were used for Ptrc, and PCR was performed using SEQ ID NO: 21 and SEQ ID NO: 22 for yfaV.
- Example 3 mdtH MFS transporter-enhanced strain production and OPS production capacity evaluation
- Example 2 The three types of plasmid prepared in Example 2 were each introduced into the OPS-producing strain CA07-0012 to prepare a strain, and the production ability of OPS was evaluated.
- OPS production ability is increased by enhancing the activities of serA (3-phosphoglycerate dehydrogenase) and serC (3-phosphoserine aminotransferase), which are OPS biosynthetic pathways.
- serA 3-phosphoglycerate dehydrogenase
- serC 3-phosphoserine aminotransferase
- pCL_Ptrc-serA*C was prepared to construct a negative control plasmid into which serA and serC were introduced.
- a Ptrc DNA fragment was obtained by using SEQ ID NO: 23 and SEQ ID NO: 24 and using the template as pCL_Ptrc-gfp.
- pC_Prmf-serA*C (WO 2016/024771 A1) as a template, PCR was performed using SEQ ID NOs: 25 and 26 to obtain a serA*C DNA fragment.
- the amplified fragment was subjected to IST with a pCL1920 vector treated with XbaI and HindIII to obtain pCL_Ptrc-serA*C.
- SEQ ID NOs: 27 and 28 were used to construct pCL_Ptrc-serA*C_Ptrc-yhhS, and a template PCR was performed using the pCL_Ptrc-yhhS prepared above. From this, a Ptrc-yhhS DNA fragment was obtained. The amplified fragment was subjected to IST with pCL_Ptrc-serA*C vector treated with HindIII restriction enzymes to obtain pCL_Ptrc-serA*C_Ptrc-yhhS.
- pCL_Ptrc-serA*C_Ptrc-mdtH and pCL_Ptrc-serA*C_Ptrc-yfaV were also constructed in the same manner as pCL_Ptrc-serA*C_Ptrc-yhhS.
- SEQ ID NO: 29 and SEQ ID NO: 30 a Ptrc-mdtH DNA fragment was obtained using pCL_Ptrc-mdtH as a template, and a Ptrc-yfaV DNA fragment was obtained using pCL_Ptrc-yfaV as a template using SEQ ID NO: 31 and SEQ ID NO: 32 did.
- the OPS-producing ability was confirmed by introducing the prepared plasmid into CA07-0022, an OPS-producing strain, and the results are shown in Table 5 below.
- a strain in which the autologous promoter is substituted with the trc promoter was prepared and the production ability of O-phosphoserine was evaluated in order to check whether the excretion ability is improved.
- the method of introducing the trc promoter into the E. coli chromosome was prepared by the following method commonly used.
- a pSKH130 vector having an R6K replicon that is dependent on PI protein (pir gene) and into which a sacB (Levansucrase) gene is introduced was used.
- the vector contains a kanamycin resistance gene, which was used as a selection marker for strain construction.
- the antibiotic was removed from the medium with sucrose to prepare a strain.
- a pSKH130_Ptrc-mdtH vector was constructed, and the primer sequences used are shown in Table 6 below.
- sequence name primer gene vector 33 mdtHUP_F CAGGAATTCGATATCTAATCTCTTTTTCGTCCGGG mdtH pSKH130_mdtHUP_Ptrc-mdtH 34 mdtHUP_R GAGTTGCAGCAAGCGTTCCCCTCCCGGGAAATAAA 35 Ptrc-mdtH_F TTCCCGGGAGGGGAACGCTTGCTGCAACTCTCTCTCA 36 Ptrc-mdtH_R GACTAGCGTGATATCCAGACCAGGCGAAAGTCGTA 37 Ptrc-mdtH_conf_F CACCGCTGCGTTTATTGT 38 Ptrc-mdtH_conf_R AAACGCTTGTCACGCATCA
- pSKH130_Ptrc-mdtH W3110 was used as a template, and PCR was performed with SEQ ID NO: 33 and SEQ ID NO: 34 to obtain an mdtHUP DNA fragment.
- SEQ ID NO: 35 and SEQ ID NO: 36 PCR was performed with the pCL_Ptrc-mdtH prepared above as a template to obtain a DNA fragment of Ptrc-mdtH.
- the amplified fragments were subjected to IST with the pSKH130 vector treated with EcoRV restriction enzymes to obtain pSKH_mdtHUP_Ptrc-mdtH.
- the obtained plasmid was transformed into strain CA7-0022 by electroporation.
- OPS is a substance containing phosphate and has a chemically and structurally similar form to 3-phosphoglycerate (hereinafter referred to as 3PG), so it is assumed that the OPS emitter may emit 3PG, 3PG production capacity was also measured in the strain fortified OPS emitter. 3PG was measured using a high performance liquid chromatography (HPLC) instrument, and the 3PG production capacity is shown in Table 8.
- HPLC high performance liquid chromatography
- strain name OD562nm OPS (g/L) 3PG (g/L) CA07-0022/pCL_Ptrc-serA*C 45.0 3.5 0.2 CA07-0022/pCL_Ptrc-serA*C_Ptrc-yhhS 35.1 7.6 2.6 CA07-0022/pCL_Ptrc-serA*C_Ptrc-mdtH 30.0 7.8 0.3 CA07-0022/pCL_Ptrc-serA*C_Ptrc-yfaV 25.9 3.2 0.3
- 3PG was accumulated in the yhhS-enhanced strain, but 3PG was not increased in the mdtH membrane protein-enhanced strain. That is, the strain into which mdtH was introduced had an increased OPS excretion ability, and it was also found that the OPS-specific excretion ability was increased.
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Abstract
Description
서열번호 | 서열명 | 프라이머 | 유전자 | 벡터 |
11 | yhhS_ Ptrc_F | CGGGGATCCTCTAGACGCTTGCTGCAACTCTCTCA | yhhS | pCL_Ptrc-yhhS |
12 | yhhS_ Ptrc_R | TACGGGTTCGGGcatGATATCTTTCCTGTGTGAAA | ||
13 | yhhS_F | CACAGGAAAGATATCatgCCCGAACCCGTAGCCGA | ||
14 | yhhS_R | GATTACGCCAAGCTTttaAGATGATGAGGCGGCCT | ||
15 | mdtH_ Ptrc_F | CGGGGATCCTCTAGACGCTTGCTGCAACTCTCTCA | mdtH | pCL_Ptrc-mdtH |
16 | mdtH_ Ptrc_R | CGACACGCGGGAcatGATATCTTTCCTGTGTGAAA | ||
17 | mdtH_F | CACAGGAAAGATATCatgTCCCGCGTGTCGCAGGC | ||
18 | mdtH_R | GATTACGCCAAGCTTtcaGGCGTCGCGTTCAAGCA | ||
19 | yfaV_ Ptrc_F | CGGGGATCCTCTAGACGCTTGCTGCAACTCTCTCA | yfaV | pCL_Ptrc-yfaV |
20 | yfaV_ Ptrc_R | CAAAGCGGTGCTcatGATATCTTTCCTGTGTGAAA | ||
21 | yfaV_F | CACAGGAAAGATATCatgAGCACCGCTTTGCTTGA | ||
22 | yfaV_R | GATTACGCCAAGCTTttaATGATGTGCCACGTCGG |
배지성분 | 조제량 |
포도당 | 40g |
KH2PO4(KP1) | 6g |
(NH4)2SO4 | 17g |
MgSO4.7H2O | 1g |
MnSO4.4H2O | 5mg |
FeSO4.7H2O | 10mg |
L-글리신 | 2.5g/L |
호모액기스 | 3g/L |
CaCO3 | 30g/L |
pH | 6.8 |
균주명 | OD562nm | 소모당(g/L) | O-포스포세린(g/L) |
CA07-0012/pCL1920 | 50.6 | 40 | 1.3 |
CA07-0012/pCL_Ptrc-yhhS | 45.1 | 40 | 2.3 |
CA07-0012/pCL_Ptrc-mdtH | 38.2 | 40 | 2.4 |
CA07-0012/pCL_Ptrc-yfaV | 39.5 | 40 | 1.3 |
서열번호 | 서열명 | 프라이머 | 유전자 | 벡터 |
23 | serA*,serC_ Ptrc_F | CGGGGATCCTCTAGAGGTACCCGCTTGCTGCAACT | serA*,serC | pCL_Ptrc- serA*C |
24 | serA*,serC_ Ptrc_R | CGATACCTTTGCCATGATATCTTTCCTGTGTGAAA | ||
25 | serA*,serC_F | CACAGGAAAGATATCATGGCAAAGGTATCGCTGGA | ||
26 | serA*,serC_R | GATTACGCCAAGCTTTTAACCGTGACGGCGTTCGA | ||
27 | serA*,serC,yhhS_ Ptrc-yhhS_F | CACGGTTAAAAGCTTCGCTTGCTGCAACTCTCTCA | serA*,serC,yhhS | pCL_Ptrc-serA*C_ Ptrc-yhhS |
28 | serA*,serC,yhhS_ Ptrc-yhhS_R | GATTACGCCAAGCTTttaAGATGATGAGGCGGCCT | ||
29 | serA*,serC,mdtH_ Ptrc-mdtH_F | CACGGTTAAAAGCTTCGCTTGCTGCAACTCTCTCA | serA*,serC,mdtH | pCL_Ptrc-serA*C_ Ptrc-mdtH |
30 | serA*,serC,mdtH_ Ptrc-mdtH_R | GATTACGCCAAGCTTtcaGGCGTCGCGTTCAAGCA | ||
31 | serA*,serC,mdtH_ Ptrc-yfaV_F | CACGGTTAAAAGCTTCGCTTGCTGCAACTCTCTCA | serA*,serC,yfaV | pCL_Ptrc-serA*C_ Ptrc-yfaV |
32 | serA*,serC,mdtH_ Ptrc-yfaV_R | GATTACGCCAAGCTTttaATGATGTGCCACGTCGG |
균주명 | OD562nm | 소모당(g/L) | O-포스포세린(g/L) |
CA07-0022/pCL_Ptrc-serA*C(serA*(G336V)-(RBS)serC | 45.0 | 40 | 3.5 |
CA07-0022/pCL_Ptrc-serA*C_Ptrc-yhhS | 35.1 | 40 | 7.6 |
CA07-0022/pCL_Ptrc-serA*C_Ptrc-mdtH | 30.0 | 40 | 7.8 |
CA07-0022/pCL_Ptrc-serA*C_Ptrc-yfaV | 25.9 | 40 | 3.2 |
서열번호 | 서열명 | 프라이머 | 유전자 | 벡터 |
33 | mdtHUP_F | CAGGAATTCGATATCTAATCTCTTTTTCGTCCGGG | mdtH | pSKH130_mdtHUP_Ptrc-mdtH |
34 | mdtHUP_R | GAGTTGCAGCAAGCGTTCCCCTCCCGGGAAATAAA | ||
35 | Ptrc-mdtH_F | TTCCCGGGAGGGGAACGCTTGCTGCAACTCTCTCA | ||
36 | Ptrc-mdtH_R | GACTAGCGTGATATCCAGACCAGGCGAAAGTCGTA | ||
37 | Ptrc-mdtH_conf_F | CACCGCTGCGTTTATTGT | ||
38 | Ptrc-mdtH_conf_R | AAACGCTTGTCACGCATCA |
균주명 | OD562nm | 소모당(g/L) | O-포스포세린(g/L) |
CA07-0022/pCL_Ptrc-serA*C | 42.4 | 40 | 3.5 |
CA07-0022::Ptrc-mdtH/pCL_Ptrc-serA*C | 38.2 | 40 | 4.0 |
균주명 | OD562nm | OPS(g/L) | 3PG(g/L) |
CA07-0022/pCL_Ptrc-serA*C | 45.0 | 3.5 | 0.2 |
CA07-0022/pCL_Ptrc-serA*C_Ptrc-yhhS | 35.1 | 7.6 | 2.6 |
CA07-0022/pCL_Ptrc-serA*C_Ptrc-mdtH | 30.0 | 7.8 | 0.3 |
CA07-0022/pCL_Ptrc-serA*C_Ptrc-yfaV | 25.9 | 3.2 | 0.3 |
Claims (13)
- mdtH 단백질의 활성이 내재적 활성에 비하여 강화된, O-포스포세린 생산 재조합 미생물.
- 제1항에 있어서, 상기 재조합 미생물은 O-포스포세린 배출 활성이 내재적 활성에 비하여 강화된, 미생물.
- 제1항에 있어서, 상기 mdtH 단백질은 서열번호 1의 아미노산 서열 또는 O-포스포세린 배출 활성을 가지면서 상기 서열번호 1과 95% 이상의 동일성을 갖는 아미노산 서열을 포함하는 것인, 미생물.
- 제1항에 있어서, 상기 재조합 미생물은 추가로 포스포세린 포스파타아제(phosphoserine phosphatase, SerB)의 활성이 내재적 활성에 비해 약화된, 미생물.
- 제1항에 있어서, 상기 재조합 미생물은 추가로 포스포글리세라이트 디하이드로게나제(phosphoglycerate dehydrogenase, SerA), 포스포세린 아미노트랜스퍼라제(phosphoserine aminotransferase, SerC) 또는 이의 조합 중 어느 하나의 활성이 내재적 활성에 비해 강화된, 미생물.
- 제1항에 있어서, 상기 재조합 미생물은 에스케리키아 속(the genus of Escherichia)인, 미생물.
- mdtH 단백질의 활성이 내재적 활성에 비하여 강화된 O-포스포세린 생산 재조합 미생물을 배지에서 배양하는 단계를 포함하는, O-포스포세린의 생산 방법.
- 제7항에 있어서, 상기 방법은 배양된 배지 또는 미생물에서 O-포스포세린을 회수하는 단계를 추가로 포함하는, O-포스포세린의 생산 방법.
- a) mdtH 단백질의 활성이 내재적 활성에 비하여 강화된 O-포스포세린 생산 재조합 미생물을 배지에서 배양하여 O-포스포세린 또는 O-포스포세린을 포함하는 배지를 생산하는 단계; 및b) O-포스포세린 설프하이드릴라아제(O-phosphoserine sulfliydrylase, OPSS) 또는 이를 포함하는 미생물의 존재 하에, 상기 a) 단계에서 생산된 O-포스포세린 또는 이를 포함하는 배지를 황화물과 반응시키는 단계를 포함하는, 시스테인 또는 이의 유도체의 생산 방법.
- 제9항에 있어서, 상기 시스테인 유도체의 생산 방법은 상기 b) 단계에서 생성된 시스테인을 시스테인 유도체로 전환시키는 단계를 추가로 포함하는, 시스테인 또는 이의 유도체의 생산 방법.
- 제9항에 있어서, 상기 황화물은 Na2S, NaSH, (NH4)2S, H2S 및 Na2S2O3로 이루어지는 군으로부터 선택되는 하나 이상인, 시스테인 또는 이의 유도체의 생산 방법.
- mdtH 단백질의 활성이 내재적 활성에 비하여 강화된 O-포스포세린 생산 재조합 미생물의 O-포스포세린, 시스테인 또는 시스테인의 유도체 생산 용도.
- mdtH 단백질의 O-포스포세린을 미생물로부터 배출하는 용도.
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US18/023,462 US20230313243A1 (en) | 2020-09-09 | 2021-09-06 | Novel O-Phosphoserine Export Protein and Methods for Producing O-Phosphoserine, Cysteine, and Cysteine Derivative Using Same |
BR112023004005A BR112023004005A2 (pt) | 2020-09-09 | 2021-09-06 | Micro-organismo recombinante para produzir o-fosfoserina e seu uso, método para produzir o-fosfoserina e para produzir cisteína ou derivados da mesma, e uso de uma proteína mdth |
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AU2021340470A AU2021340470A1 (en) | 2020-09-09 | 2021-09-06 | Novel O-phosphoserine export protein and methods for producing o-phosphoserine, cysteine, and cysteine derivative using same |
MX2023002722A MX2023002722A (es) | 2020-09-09 | 2021-09-06 | Proteina de exportacion de o-fosfoserina novedosa y procedimientos para producir o-fosfoserina, cisteina, y derivado de cisteina usando la misma. |
CA3191495A CA3191495A1 (en) | 2020-09-09 | 2021-09-06 | Novel o-phosphoserine export protein and methods for producing o-phosphoserine, cysteine, and cysteine derivative using same |
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CA3191495A1 (en) | 2022-03-17 |
JP2023540374A (ja) | 2023-09-22 |
KR102414743B1 (ko) | 2022-06-29 |
CN116568701A (zh) | 2023-08-08 |
AR123451A1 (es) | 2022-11-30 |
AU2021340470A1 (en) | 2023-04-06 |
EP4198130A1 (en) | 2023-06-21 |
BR112023004005A2 (pt) | 2023-04-04 |
MX2023002722A (es) | 2023-03-28 |
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US20230313243A1 (en) | 2023-10-05 |
EP4198130A4 (en) | 2024-01-17 |
ZA202303414B (en) | 2024-07-31 |
KR20220033316A (ko) | 2022-03-16 |
TW202216741A (zh) | 2022-05-01 |
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