WO2021112469A1 - 신규한 분지쇄 아미노산 아미노트랜스퍼라제 변이체 및 이를 이용한 류신 생산방법 - Google Patents
신규한 분지쇄 아미노산 아미노트랜스퍼라제 변이체 및 이를 이용한 류신 생산방법 Download PDFInfo
- Publication number
- WO2021112469A1 WO2021112469A1 PCT/KR2020/016675 KR2020016675W WO2021112469A1 WO 2021112469 A1 WO2021112469 A1 WO 2021112469A1 KR 2020016675 W KR2020016675 W KR 2020016675W WO 2021112469 A1 WO2021112469 A1 WO 2021112469A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- amino acid
- variant
- leucine
- microorganism
- protein
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
-
- 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
- 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
-
- 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
-
- 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
- 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)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y206/00—Transferases transferring nitrogenous groups (2.6)
- C12Y206/01—Transaminases (2.6.1)
- C12Y206/01042—Branched-chain-amino-acid transaminase (2.6.1.42)
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/15—Corynebacterium
Definitions
- the present application relates to a novel branched chain amino acid aminotransferase variant and a leucine production method using the same.
- Branched-chain amino acids refer to three types of valine, leucine, and isoleucine, and are known to be mainly metabolized in muscles and used as an energy source during activity. As branched chain amino acids are known to play an important role in maintaining and increasing muscle during activity, their usage is increasing. In particular, leucine is a kind of essential amino acid and is widely used in medicine, food, feed additive, and industrial medicine.
- the production of leucine using microorganisms is mainly made through microorganisms of the genus Escherichia or microorganisms of the genus Corynebacterium (US 2020-0032305 A1), and goes through several steps from pyruvic acid to ketoisocaproate (2-ketoisocaproate) ) is known to be biosynthesized as a precursor.
- enzymes used for leucine synthesis are equally used in the biosynthesis of branched-chain amino acids, it is difficult to industrially mass-produce one branched-chain amino acid through fermentation.
- bcaT branched-chain amino acid aminotransferase
- An object of the present application is a branched amino acid aminotransferase (branched amino acid aminotransferase) variant in which the 156th valine (V: valine) amino acid residue from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid; a polynucleotide encoding it; a vector containing it; It is to provide a microorganism containing it.
- V valine
- Another object of the present application is to provide a method for producing leucine, comprising the step of culturing the microorganism in a medium.
- the branched-chain amino acid aminotransferase mutant of the present application increases leucine-producing ability compared to the wild-type, it can be widely used for more efficient mass production of leucine.
- One aspect of the present application provides a branched amino acid aminotransferase (branched amino acid aminotransferase) variant in which the 156th valine (V: valine) amino acid residue from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid can do.
- V valine
- the present application may provide a branched-chain amino acid aminotransferase variant comprising one or more substitutions in the amino acid sequence of SEQ ID NO: 1, wherein the amino acid substitution is the 156th amino acid from the N-terminus to an amino acid other than valine. It may include substituted ones.
- the 'other amino acid' is not limited as long as it is an amino acid other than the 156th amino acid of SEQ ID NO: 1, valine.
- the variant may be a protein in which the 156th amino acid is substituted with a nonpolar amino acid in the amino acid sequence of SEQ ID NO: 1, and more specifically, the variant has the 156th valine in the amino acid sequence of SEQ ID NO: 1 alanine It may be a branched chain amino acid aminotransferase variant substituted with, but is not limited thereto.
- branched-chain amino acid aminotransferase (bcaT) is an enzyme involved in the biosynthesis of branched-chain amino acids, and in the present application, the branched-chain amino acid aminotransferase is “bcaT", “trans It may be used interchangeably with “transaminase B", or "ilvE”. In addition, the branched chain amino acid aminotransferase may be one encoded by the ilvE gene, but is not limited thereto.
- leucine is derived from pyruvic acid to acetolactic acid, dihydroxy isovaleric acid, ketoisovaleric acid, 2-isopropylmalic acid, 3-iso It is known to be biosynthesized via isopropylmalic acid and ketoisocaproic acid.
- these biosynthetic processes are acetohydroxy acid synthase, acetohydroxyacid isomeroreductase, dihydroxy acid dehydratase, isopropyl malic acid synthase ( Catalyzed by enzymes such as isopropylmalic acid synthase, isopropylmalic acid dehydratase, isopropylmalic acid dehydrogenase, branched amino acid aminotransferase to be biosynthesized
- the branched-chain amino acid aminotransferase may have its sequence obtained from GenBank of NCBI, a known database, but is not limited thereto, and branched-chain amino acid aminotransferase is secured based on various methods well known in the art. can do. Examples of the method include gene synthesis technology including codon optimization to secure enzymes with high efficiency in microorganisms of the genus Corynebacterium, which are commonly widely used for enzyme expression, or bioinformatics methods based on large-scale genome information of microorganisms. There is a screening method for useful enzyme resources by, but is not limited thereto.
- the branched-chain amino acid aminotransferase to be mutated in the present application may be a branched-chain amino acid aminotransferase derived from Corynebacterium sp., specifically, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 It may be a /protein, but a polypeptide/protein having the same activity may be included without limitation. As an example, it may include the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80% or more homology or identity therewith, but is not limited thereto.
- the branched chain amino acid aminotransferase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to SEQ ID NO: 1. can do.
- a polypeptide/protein having an amino acid sequence in which some sequence is deleted, modified, substituted or added is also a polypeptide/protein having such homology or identity and exhibiting branched-chain amino acid aminotransferase activity, the polypeptide to be mutated in the present application It is obvious that it is included within the scope of /protein.
- a protein or polypeptide having an amino acid sequence described in a specific SEQ ID NO:' or 'a protein or polypeptide having an amino acid sequence described in a specific SEQ ID NO:' in the present application a protein consisting of the amino acid sequence of the corresponding SEQ ID NO: It is apparent that a protein or polypeptide having an amino acid sequence in which some sequences are deleted, modified, substituted or added may also be used in the present application as long as it has the same or corresponding activity as the polypeptide.
- variant means that one or more amino acids differ from the recited sequence in conservative substitution and/or modification, but the function of the protein ( functions) or properties (properties) are maintained.
- a variant differs from an identified sequence by several amino acid substitutions, deletions or additions. Such variants can generally be identified by modifying one of the polypeptide sequences and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the native protein.
- some variants may include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, are removed.
- variants may include variants in which a portion is removed from the N- and/or C-terminus of the mature protein.
- conservative substitution may refer to substituting one amino acid with another amino acid having similar structural and/or chemical properties.
- Such variants may have, for example, one or more conservative substitutions while still retaining one or more biological activities.
- Such amino acid substitutions may generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues.
- conservative substitutions may have little or no effect on the activity of the polypeptide.
- variants may contain deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide.
- the variant, or variant polypeptide is conjugated with a signal (or leader) sequence at the N-terminus of the protein involved in the transfer of the protein co-translationally or post-translationally. can do.
- the variants may also be conjugated with other sequences or linkers to identify, purify, or synthesize the polypeptide.
- variant includes terms such as mutant, modified, mutated protein, mutant polypeptide, mutant, and variant protein (in English, modified, modified protein, modified polypeptide, mutant, mutein, divergent, variant, etc.) may be used, and if the term is used in a mutated sense, it is not limited thereto.
- the variant may have a branched-chain amino acid aminotransferase activity that has been changed to have enhanced leucine-producing ability.
- branched-chain amino acid aminotransferase variant refers to one or more amino acid substitutions in the amino acid sequence of a polypeptide having branched-chain amino acid aminotransferase activity
- branched-chain amino acid aminotransferase variant means It can be used interchangeably with "polypeptide having branched chain amino acid aminotransferase activity”.
- branched chain amino acid aminotransferase variant refers to a variant branched chain amino acid aminotransferase protein, a variant bcaT, a variant bcaT protein, a bcaT variant, a variant transaminase B protein, a variant form.
- Transaminase B, transaminase B mutant, mutant ilvE protein, mutant ilvE, ilvE mutant, etc. may be used interchangeably, but as described above, if the term is used in the mutated sense, it is not limited thereto.
- the 156th valine (V: valine) amino acid residue from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid
- the branched-chain amino acid aminotransferase variant is a branched-chain amino acid aminotransferase activity It is apparent that in the amino acid sequence of a polypeptide having SEQ ID NO: 1, the amino acid corresponding to the 156th position from the N-terminus of SEQ ID NO: 1 may include a protein variant in which another amino acid is substituted.
- the variant may have at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% homology or identity to the sequence of SEQ ID NO: 1 in a sequence of SEQ ID NO: 1 It may include a sequence in which the amino acid at the position corresponding to the 156th position from the N-terminus is substituted with another amino acid.
- the 'other amino acid' may be an amino acid other than the amino acid corresponding to the 156th amino acid of SEQ ID NO: 1.
- the variant may be one in which the amino acid corresponding to the 156th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with a non-polar amino acid having structural and/or chemical properties similar to those of alanine.
- the variant may be one in which the amino acid corresponding to amino acid 156 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, but is not limited thereto.
- corresponding position refers to an amino acid residue at a position listed in a protein or polypeptide, or an amino acid residue similar to, identical to, or homologous to a residue listed in a protein or polypeptide.
- corresponding region generally refers to a similar or corresponding position in a related protein or reference protein.
- specific numbering may be used for amino acid residue positions in proteins used in this application. For example, by aligning the polypeptide sequence of the protein of the present application with the target protein to be compared, it is possible to renumber the position corresponding to the amino acid residue position of the protein of the present application.
- the branched-chain amino acid aminotransferase mutant of the present application may have an activity to increase leucine-producing ability in microorganisms compared to a wild-type or unmutated branched-chain amino acid aminotransferase protein.
- the branched chain amino acid aminotransferase variant has the amino acid sequence of SEQ ID NO: 1 in which the 156th valine (V: valine) amino acid residue from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, more specifically, alanine and 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% or more homology or identity, and more specifically, 90% or more homology or identity with SEQ ID NO: 1 may be to have
- the variant may consist of SEQ ID NO: 3, but is not limited thereto.
- the amino acid corresponding to the 156th position from the N-terminus in SEQ ID NO: 3 is fixed (that is, the amino acid at the position corresponding to the 156th amino acid of SEQ ID NO: 3 is, SEQ ID NO: 3 is alanine identically to the 156th amino acid of SEQ ID NO: 3 and 70% or more, specifically 80%, 85%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% or more homology or identity to an amino acid sequence with or more, but is not limited thereto.
- a protein having an amino acid sequence in which some sequences are deleted, modified, substituted or added in addition to the amino acid sequence at position 156 is also within the scope of the present application. The inclusion is obvious.
- the term 'homology' or 'identity' refers to the degree to which two given amino acid sequences or nucleotide sequences are related to each other and may be expressed as a percentage.
- 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 at least about 50%, 60%, 70%, 80% of the entire or full-length sequence under moderate or high stringent conditions. or more than 90% hybrid. Hybridization is also contemplated for polynucleotides containing degenerate codons instead of codons in the polynucleotides.
- GAP program is 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.
- the term “homology” or “identity” may refer to a relevance between sequences.
- Another aspect of the present application may provide a polynucleotide encoding the branched-chain amino acid aminotransferase variant.
- another aspect of the present application may provide a vector including the polynucleotide.
- polynucleotide refers to a DNA or RNA strand of a certain length or longer as a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds, and more specifically, the branched chain amino acid amino acids. It may be a polynucleotide fragment encoding a transferase variant.
- the polynucleotide encoding the branched-chain amino acid aminotransferase variant of the present application may be included without limitation as long as it is a polynucleotide sequence encoding the branched-chain amino acid aminotransferase variant of the present application.
- the polynucleotide encoding the branched-chain amino acid aminotransferase variant may be included without limitation as long as it is a sequence encoding a variant in which the 156th amino acid is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 1.
- polynucleotide sequence encoding a variant in which the 156th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine may be a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3, but is not limited thereto. More specifically, it may be composed of the polynucleotide sequence of SEQ ID NO: 4, but is not limited thereto.
- polynucleotide various modifications can be made to the coding region within the range that does not change the amino acid sequence of the protein due to codon degeneracy or considering codons preferred in the organism to express the protein. . Accordingly, it is obvious that a polynucleotide that can be translated into a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 or a polypeptide having homology or identity thereto may also be included due to codon degeneracy.
- the 156th amino acid in the amino acid sequence of SEQ ID NO: 1 is changed to another amino acid by hydridation under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the nucleotide sequence. Any sequence encoding a substituted branched-chain amino acid aminotransferase variant may be included without limitation.
- the "stringent condition” may be a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (eg, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989). For example, genes having high homology or identity, 80% or more, 85% or more, specifically 90% or more, more specifically 95% or more, more specifically 97% or more, In particular, the conditions in which genes having 99% or more homology or identity hybridize with each other and genes with lower homology or identity do not hybridize, or at 60° C., which is a washing condition for normal Southern hybridization.
- Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization.
- complementary is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present application may also include isolated nucleic acid fragments that are complementary to substantially similar nucleic acid sequences as well as the entire sequence.
- polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the conditions described above.
- the Tm value may be 60 °C , 63 °C or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.
- the term "vector” refers to a DNA preparation containing the nucleotide sequence of a polynucleotide encoding the variant protein of interest operably linked to a suitable regulatory sequence so that the variant protein of interest can be expressed in a suitable host.
- suitable regulatory sequences 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.
- the vector After transformation into a suitable 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 plasmids, cosmids, viruses and bacteriophages in a natural or recombinant state.
- pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors
- pBR-based, pUC-based, and pBluescriptII-based plasmid vectors may be used as plasmid vectors.
- pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used.
- pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. may be used, but is not limited thereto.
- a polynucleotide encoding a target mutant protein in a chromosome may be replaced with a mutated polynucleotide 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.
- the polynucleotide may further include a selection marker for confirming whether or not the chromosome is inserted.
- Selectable markers are used to select cells transformed with a vector, that is, to confirm whether a target nucleic acid molecule is inserted, and selectable phenotypes such as drug resistance, auxotrophic resistance, resistance to cytotoxic agents, or expression of surface variant proteins. Markers that give ? may 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.
- Another aspect of the present application is the branched chain amino acid aminotransferase variant; a polynucleotide encoding the variant; And it can provide a microorganism comprising any one or more of the vector containing the polynucleotide.
- the microorganism may be a microorganism prepared by transformation with a vector containing a polynucleotide encoding a mutant, but is not limited thereto.
- the term "transformation" may refer to introducing a vector including a polynucleotide encoding a target protein into a host cell so that the protein 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 outside the chromosome, as long as they can be expressed in the host cell.
- the polynucleotide includes DNA and RNA encoding a target protein. The polynucleotide may be introduced into a host cell and expressed in any form, as long as it can be expressed.
- 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 may be one in which the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the branched-chain amino acid aminotransferase of the present application. .
- the microorganism of the present application is the branched chain amino acid aminotransferase variant; a polynucleotide encoding it; And it may be a microorganism having a leucine-producing ability, including any one or more of the vector containing the polynucleotide, but is not limited thereto.
- the microorganism may be a microorganism having a leucine-producing ability naturally, or a microorganism in which a leucine-producing ability is imparted to a parent strain having no leucine-producing ability, but is not limited thereto.
- the microorganism may be any microorganism capable of producing leucine by expressing the branched-chain amino acid aminotransferase variant.
- the term "to be/are/are” a protein may refer to a state in which a target protein is introduced into a microorganism or modified to be expressed in a microorganism.
- the target protein is a protein present in a microorganism, it may mean a state in which its activity is enhanced compared to before intrinsic or modification.
- the "protein of interest” may be the aforementioned branched chain amino acid aminotransferase variant.
- introduction of protein may mean that the microorganism exhibits the activity of a specific protein that it did not originally have, or exhibits improved activity compared to the intrinsic activity or activity before modification of the protein.
- a polynucleotide encoding a specific protein may be introduced into a chromosome in a microorganism, or a vector including a polynucleotide encoding a specific protein may be introduced into the microorganism to exhibit its activity.
- “enhancement of activity” may mean that the activity is improved compared to the intrinsic activity of a specific protein of the microorganism or the activity before modification.
- “Intrinsic activity” may refer to the activity of a specific protein originally possessed by the parent strain before transformation when the trait of a microorganism is changed due to genetic variation caused by natural or artificial factors.
- the enhancement of protein activity may be achieved by enhancing the expression of a gene encoding the protein, but is not limited thereto.
- the enhancement of the activity of the protein variant includes increasing the intracellular copy number of the gene encoding the protein variant, a method of introducing a mutation into the expression control sequence of the gene encoding the protein variant, and the protein variant
- a method of replacing a gene expression control sequence encoding a gene expression control sequence with a strong sequence a method of replacing a gene encoding a native protein having chromosomal branched-chain amino acid aminotransferase activity with a gene encoding the protein variant, the protein variant It may consist of any one or more methods selected from the group consisting of a method of additionally introducing a mutation into the gene encoding the mutant to enhance the activity of the mutant, and a method of introducing a protein mutant into a microorganism, but is not limited thereto.
- modifying the expression control sequence so as to increase the expression of the polynucleotide is not particularly limited thereto, but deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence to further enhance the activity of the expression control sequence, or their It can be carried out by inducing a mutation in the sequence in combination, or by replacing it with a nucleic acid sequence having a stronger activity.
- the expression control sequence is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence for regulating the termination of transcription and translation, and the like.
- a strong promoter may be linked to the upper portion of the polynucleotide expression unit instead of the original promoter, but is not limited thereto.
- Examples of known strong promoters include cj1 to cj7 promoter (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 10584338 B2), O2 promoter (US 10273491 B2), tkt promoter, and yccA promoter, but are not limited thereto.
- the modification of the polynucleotide sequence on the chromosome is not particularly limited thereto, but a mutation in the expression control sequence by deletion, insertion, non-conservative or conservative substitution of a nucleic acid sequence or a combination thereof to further enhance the activity of the polynucleotide sequence. It can be carried out by inducing and replacing with a polynucleotide sequence improved to have stronger activity.
- Incorporation and enhancement of such protein activity is generally performed such that the activity or concentration of the corresponding protein is at least 1%, 10%, 25%, 50%, based on the activity or concentration of the protein in the wild-type or unmodified microbial strain. It may be increased by 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to 1000% or 2000%, but is not limited thereto.
- the microorganism may be any microorganism capable of expressing a mutant by transforming it with a vector containing a protein variant or a polynucleotide encoding a protein variant or a polynucleotide encoding the variant.
- Escherichia sp. Serratia sp., Erwinia sp., Enterobacteria sp., Salmonella sp., Streptomyces
- It may include microorganism strains such as Streptomyces sp., Pseudomonas sp., Brevibacterium sp., or Corynebacterium sp., specifically, Corynebacterium sp.
- It may be a microorganism of the genus Nebacterium, and a more specific example may be Corynebacterium glutamicum, but is not limited thereto.
- the microorganism of the present application may be a microorganism that produces leucine.
- leucine-producing microorganism may be a microorganism in which leucine-producing ability is imparted to a microorganism having a leucine-producing ability naturally or a parent strain having no leucine-producing ability.
- the microorganism producing the leucine is a microorganism genetically modified through any one or more of the branched chain amino acid aminotransferase variant, a polynucleotide encoding the same, and a vector comprising the polynucleotide; a microorganism modified to express the branched chain amino acid aminotransferase variant, or a polynucleotide encoding the same; It may be a recombinant microorganism expressing the branched chain amino acid aminotransferase variant, or a polynucleotide encoding the same, but is not limited thereto.
- the leucine-producing microorganism is a microorganism capable of expressing a mutant by transforming with a vector containing a polynucleotide encoding a protein variant, or a polynucleotide encoding a mutant, and for the purpose of the present application, the microorganism is any microorganism capable of producing leucine, including ilvE mutants.
- Escherichia sp. Serratia sp., Erwinia sp., Enterobacteria sp., Salmonella sp., Streptomyces It may include microorganism strains such as Streptomyces sp., Pseudomonas sp., Brevibacterium sp., or Corynebacterium sp., specifically, Corynebacterium sp. It may be a microorganism of the genus Nebacterium, and a more specific example may be Corynebacterium glutamicum, but is not limited thereto.
- the term "leucine-producing Corynebacterium sp. microorganism” may refer to a microorganism of the genus Corynebacterium having a leucine-producing ability through wild-type or mutation. It was known that the microorganisms of the genus Corynebacterium can produce leucine to some extent, but the leucine-producing ability is remarkably low and it is difficult to industrially produce it.
- a microorganism of the genus Corynebacterium having a leucine-producing ability refers to a native microorganism itself or a gene related to an external leucine production mechanism is inserted, or the activity of an intrinsic gene is strengthened, weakened, or inactivated to improve leucine-producing ability. It may mean a microorganism of the genus Corynebacterium that has been
- the microorganism producing the leucine may include the branched-chain amino acid aminotransferase mutant of the present application, and may be a microorganism characterized in that the leucine-producing ability is increased.
- the microorganism may have an increased leucine-producing ability compared to a microorganism that does not include a natural wild-type strain, the parent strain, or the mutant of the present application.
- strain before modification or "microbe before modification” does not exclude a strain containing a mutation that may occur naturally in a microorganism, it is a wild-type strain or a natural-type strain itself, or caused by natural or artificial factors It may refer to a strain before the trait is changed due to genetic mutation.
- pre-modified strain or “pre-modified microorganism” may be used interchangeably with “unmodified strain”, “unmodified strain”, “unmodified microorganism”, “unmodified microorganism” or “reference microorganism”.
- the microorganism of the genus Corynebacterium may have enhanced expression of a gene in the leucine biosynthetic pathway or attenuated/inactivated expression of a gene involved in a leucine degradation pathway, specifically isopropyl malate
- the synthase (isopropylmalate synthase) activity may be an additionally enhanced, leucine-producing microorganism of the genus Corynebacterium, but is not limited thereto.
- the enhancement of protein activity is as described above.
- weakened/inactivation of protein activity means that the expression of an enzyme or protein is not expressed at all compared to a natural wild-type strain, a parent strain, or a strain in which the protein is unmodified, or has no activity even if it is expressed. It may mean reduced. In this case, the decrease is when the activity of the protein is decreased compared to the activity of the protein possessed by the original microorganism due to mutation of the gene encoding the protein, modification of the expression control sequence, deletion of part or all of the gene, and the gene encoding the same When the overall protein activity level in the cell is lower than that of the native strain or the strain before transformation due to inhibition of expression or translation inhibition, the concept also includes a combination thereof.
- the inactivation/attenuation may be achieved by applying various methods well known in the art.
- the method include: 1) a method of deleting all or part of the gene encoding the protein; 2) modification of the expression control sequence to decrease the expression of the gene encoding the protein, 3) modification of the gene sequence encoding the protein so that the activity of the protein is removed or attenuated, 4) the gene encoding the protein introduction of an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to the transcript of 5)
- an antisense oligonucleotide eg, antisense RNA
- a secondary structure is formed to make attachment of the ribosome impossible Way; 6)
- Another aspect of the present application may provide a leucine production method comprising the step of culturing the microorganism.
- the step of culturing the microorganism is not particularly limited, but may be performed by a known batch culture method, a continuous culture method, a fed-batch culture method, and the like.
- the culture conditions are not particularly limited thereto, but use a basic compound (eg, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg, phosphoric acid or sulfuric acid) to an appropriate pH (eg, pH 5 to 9, specifically can control pH 6 to 8, most specifically pH 6.8) and maintain aerobic conditions by introducing oxygen or an oxygen-containing gas mixture into the culture.
- the culture temperature may be maintained at 20 to 45° C., specifically 25 to 40° C., and may be cultured for about 10 to 160 hours, but is not limited thereto.
- the leucine produced by the culture may be secreted into the medium or remain in the cells.
- the culture medium used is a carbon source, such as sugars and carbohydrates (eg glucose, sucrose, lactose, fructose, maltose, molasse, starch and cellulose), oils and fats (eg soybean oil) , sunflower seed oil, peanut oil and coconut oil), fatty acids (such as palmitic acid, stearic acid and linoleic acid), alcohols (such as glycerol and ethanol) and organic acids (such as acetic acid), either individually or in combination. can be used, but is not limited thereto.
- sugars and carbohydrates eg glucose, sucrose, lactose, fructose, maltose, molasse, starch and cellulose
- oils and fats eg soybean oil
- sunflower seed oil e.g., sunflower seed oil
- peanut oil and coconut oil e.glycerol and ethanol
- organic acids such as acetic acid
- Nitrogen sources include nitrogen-containing organic compounds (such as peptone, yeast extract, broth, malt extract, corn steep liquor, soy flour and urea), or inorganic compounds (such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate) may be used individually or in combination, but is not limited thereto.
- organic compounds such as peptone, yeast extract, broth, malt extract, corn steep liquor, soy flour and urea
- inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate
- potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium-containing salt corresponding thereto, etc. may be used individually or in combination, but is not limited thereto.
- the medium may contain essential growth-promoting substances such as other metal salts (eg, magnesium sulfate or iron sulfate), amino acids and vitamins.
- the production method may further include recovering leucine from the cultured microorganism or culture medium.
- a desired amino acid may be collected from the culture medium using a suitable method known in the art according to the culture method. For example, centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc. may be used, and the desired leucine may be recovered from the medium or microorganism using a suitable method known in the art.
- Another aspect of the present application is a branched amino acid aminotransferase (branched amino acid aminotransferase) variant in which the 156th valine (V: valine) amino acid residue from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid
- a method for increasing leucine-producing ability can be provided, comprising modifying a microorganism to express it.
- Another aspect of the present application may provide a use for increasing leucine production of the protein variant.
- the protein variants are as described above.
- a recombinant vector containing a part of ilvE was first constructed.
- the amino acid sequence and nucleotide sequence of wild-type ilvE are as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
- PCR was performed using the primers of SEQ ID NO: 5 and SEQ ID NO: 6 using the chromosome of Corynebacterium glutamicun ATCC13032 as a template, and the amplified product was E. coli vector pCR2 using TOPO Cloning Kit (Invitrogen) cloned into .1 to obtain pCR-ilvE.
- An ilvE mutation library was prepared based on the vector constructed in Example 1-1.
- the library was prepared using the error-prone PCR kit (clontech Diversify® PCR Random Mutagenesis Kit). Under conditions in which mutations can occur, a PCR reaction was performed using SEQ ID NO: 5 and SEQ ID NO: 6 as primers. Specifically, after pre-heating at 94°C for 30 seconds under the condition that 0 to 3 mutations occur per 1000bp, the process of 30 seconds at 94°C and 1 minute and 30 seconds at 68°C was repeated 25 times.
- Example 1-2 a leucine-producing strain derived from ATCC13032 was prepared, and the library vector prepared in Example 1-2 was introduced to select variants.
- IPMS isopropylmalate synthase
- the variant includes a mutation in which G, which is the 1673th nucleotide of the leuA gene encoding isopropylmalate synthase, is substituted with A, and arginine, the 558th amino acid of the IPMS protein, is substituted with histidine, and 1682th and 1683th nucleotides It contains a mutation in which GC is substituted with AT, and glycine, which is the 561th amino acid, is substituted with aspartic acid (US 2020-0032305 A1).
- the pDC-leuA (R558H, G561D) vector containing the leuA mutation was transformed into wild-type ATCC13032, and the strain into which the vector was inserted into the chromosome by recombination of the homologous sequence was transformed into a medium containing 25 mg/L of kanamycin. was selected from
- the selected primary strain was again subjected to a secondary cross-over, and a strain into which a mutation of the leuA gene was introduced was selected. Whether or not the mutation was introduced into the final transformed strain was confirmed that the mutation was introduced by performing PCR using the primers of SEQ ID NO: 7 and SEQ ID NO: 8 and then analyzing the nucleotide sequence.
- the ATCC13032_leuA_(R558H, G561D) strain transformed with the pDC-leuA(R558H, G561D) vector was named CA13-8100.
- the pTOPO-ilvE-library prepared in Example 1-2 was transformed into the leucine-producing strain CA13-8100 prepared in Example 2-1 by electroporation, and then spread on a nutrient medium containing 25 mg/L of kanamycin. Thus, 10,000 colonies of strains into which the mutant gene was inserted were secured, and each colony was named from CA13-8100/pTOPO_ilvE(mt)1 to CA13-8100/pTOPO_ilvE(mt)10000.
- Fermentation potency was evaluated in the following manner for each colony in order to confirm the colonies with increased leucine production among the secured 10,000 colonies.
- - Nutrient medium glucose 10g, broth 5g, polypeptone 10g, sodium chloride 2.5g, yeast extract 5g, agar 20g, urea 2g (based on 1 liter of distilled water)
- Glucose 50 g Ammonium Sulfate 20 g, Corn Steep Solids 20 g, Potassium Dibasic 1 g, Magnesium Sulfate 0.5 g, Biotin 100 ⁇ g, Thiamine-HCl 1 mg, Calcium Carbonate 15 g (based on 1 liter of distilled water), pH 7.0
- Each colony was inoculated into a 250 ml corner-barpool flask containing 25 ug/ml of kanamycin in 25 ml of autoclaved production medium using platinum ear, and then cultured with shaking at 30° C. for 60 hours at 200 rpm. After completion of the culture, leucine production was measured by a method using high-performance liquid chromatography (HPLC, SHIMAZDU LC20A), and one strain with the most improved leucine production capacity compared to the CA13-8100 strain was selected. The leucine concentrations produced in the selected strains are shown in Table 1 below.
- PCR was performed in the CA13-8100/pTOPO_ilvE(mt)3012 strain using the primers of SEQ ID NO: 9 and SEQ ID NO: 10 and sequencing was performed, the ilvE gene was compared with ilvE of wild-type ATCC13032, and it was confirmed that the strain contains a mutation in the ilvE gene.
- an insertion vector was prepared.
- a site directed mutagenesis method was used to construct a vector for introducing ilvE (V156A) mutations.
- Corynebacterium glutamicum PCR was performed using the primers of SEQ ID NOs: 11 and 12 and primer pairs of SEQ ID NOs: 13 and 14 using the ATCC13032 chromosome as a template. PCR was performed 30 times of denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute and 30 seconds, followed by polymerization at 72°C for 5 minutes.
- the resulting gene fragment was cloned by fusion of the homologous sequence of the terminal 15 base between the DNA fragments using the pDC vector on the line cut with PstI and XbaI restriction enzymes and the In-Fusion enzyme, and the 156th amino acid, valine (Val), was replaced with alanine ( Ala), a vector pDC-ilvE (V156A) was constructed.
- the strain CA13-8100 a leucine-producing strain, was transformed with the pDC-ilvE (V156A) vector prepared in Example 3-1, and the vector was inserted into the chromosome by recombination of the homologous sequence, 25 mg of kanamycin. Selected on medium containing /L. The selected primary strain was again subjected to secondary cross-over, and a strain into which the mutation of the target gene was introduced was selected. Whether or not the ilvE gene mutation was introduced into the final transformed strain was confirmed that the ilvE mutation was introduced into the strain by performing PCR using the primers of SEQ ID NO: 9 and SEQ ID NO: 10 and then analyzing the nucleotide sequence.
- the manufactured CA13-8100_ilvE_V156A was named CA13-8107.
- wild-type Corynebacterium glutamicum ATCC13032 was transformed with the pDC-ilvE (V156A) vector, and the prepared strain ATCC13032_ilvE_V156A was named CA13-8106.
- Flask culture was carried out in the same manner as in Example 2-2, and leucine production was measured by the method using HPLC after the culture was completed, and the culture results are shown in Table 2 below.
- the CA13-8107 strain was deposited with the Korean Culture Center of Microorganisms (KCCM), an international depository under the Budapest Treaty, on November 15, 2019 and was given an accession number KCCM12630P.
- KCCM Korean Culture Center of Microorganisms
- the recombinant vector pDC-ilvE (V156A) prepared in Example 3-1 was transferred to leucine-producing strains Corynebacterium glutamicum KCCM11661P (US 10351859 B2), KCCM11662P (US 10351859 B2) by homologous recombination on chromosomes. transformed.
- the above two strains were wild-type Corynebacterium glutamicum ATCC 14067 and Corynebacterium glutamicum ATCC 13869 with N-methyl-N'-nitro-N-nitrosoguanidine (N-Methyl-N'-nitro -Nnitrosoguanidine, NTG) is a mutant strain that has the ability to produce leucine.
- the recombinant strains were named Corynebacterium glutamicum KCCM11661P_ilvE_V156A and KCCM11662P_ilvE_V156A.
- flask culture was carried out in the same manner as in Example 2-2. After the culture was completed, the leucine production was measured by using HPLC, and the measured leucine concentrations are shown in Table 3 below. .
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Description
균주명 | 류신 (g/L) |
CA13-8100 | 3.4 |
CA13-8100/pTOPO_ilvE(mt)3012 | 3.97 |
균주명 | 류신 (g/L) |
ATCC13032 | 0.14 |
ATCC13032_ilvE_V156A : CA13-8106 | 0.19 |
ATCC13032_leuA_(R558H, G561D) : CA13-8100 | 3.4 |
CA13-8100_ilvE_V156A : CA13-8107 | 4.1 |
균주명 | 류신 (g/L) |
KCCM11661P | 2.7 |
KCCM11661P_ilvE_V156A | 3.3 |
KCCM11662P | 3.0 |
KCCM11662P_ilvE_V156A | 3.6 |
Claims (11)
- 서열번호 1의 아미노산 서열의 N-말단으로부터 156번째 발린(V: valine) 아미노산이 다른 아미노산으로 치환된, 분지쇄 아미노산 아미노트랜스퍼라제(branched amino acid aminotransferase) 변이체.
- 제1항에 있어서, 상기 156번째 발린은 알라닌(Alanine)으로 치환되는 것인, 분지쇄 아미노산 아미노트랜스퍼라제 변이체.
- 제1항에 있어서, 상기 변이체는 서열번호 1의 아미노산 서열과 90% 이상의 상동성 또는 동일성을 갖는 것인, 분지쇄 아미노산 아미노트랜스퍼라제 변이체.
- 제1항에 있어서, 상기 변이체는 서열번호 3의 아미노산 서열로 이루어진 것인, 분지쇄 아미노산 아미노트랜스퍼라제 변이체.
- 제1항 내지 제4항 중 어느 한 항의 변이체를 코딩하는 폴리뉴클레오티드.
- 제5항의 폴리뉴클레오티드를 포함하는 벡터.
- 제1항 내지 제4항 중 어느 한 항의 변이체; 상기 변이체를 코딩하는 폴리뉴클레오티드; 및 상기 폴리뉴클레오티드를 포함하는 벡터 중 어느 하나 이상을 포함하는, 코리네박테리움(Corynebacterium sp.) 속 미생물.
- 제7항에 있어서, 상기 코리네박테리움 속 미생물은 류신(leucine)을 생산하는, 코리네박테리움(Corynebacterium sp.) 속 미생물.
- 제7항에 있어서, 상기 코리네박테리움 속 미생물은 코리네박테리움 글루타미쿰(Corynebacterium glutamicum)인, 코리네박테리움 속 미생물.
- 제7항의 미생물을 배지에서 배양하는 단계를 포함하는, 류신 생산방법.
- 제10항에 있어서, 상기 배양된 미생물 또는 배지로부터 류신을 분리 또는 회수하는 단계를 더 포함하는, 류신 생산방법.
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
BR112022011021A BR112022011021A2 (pt) | 2019-12-06 | 2020-11-24 | Variante de aminotransferase de aminoácido de cadeia ramificada, polinucleotídeo, vetor, microrganismo e método para a produção de leucina |
MX2022006827A MX2022006827A (es) | 2019-12-06 | 2020-11-24 | Nueva variante de aminotransferasa de aminoacido de cadena ramificada y procedimiento para producir leucina usando la misma. |
EP20897508.6A EP4047085A4 (en) | 2019-12-06 | 2020-11-24 | NOVEL MUTANT BRANCHED CHAIN AMINO ACID AMINOTRANSFERASE AND METHOD FOR PRODUCING LEUCINE USING THE SAME |
US17/756,627 US20220411832A1 (en) | 2019-12-06 | 2020-11-24 | Novel branched-chain amino acid aminotransferase variant and method for producing leucine using the same |
AU2020398425A AU2020398425B2 (en) | 2019-12-06 | 2020-11-24 | Novel branched-chain amino acid aminotransferase variant and method for producing leucine using the same |
CN202080083985.2A CN115038787B (zh) | 2019-12-06 | 2020-11-24 | 新型支链氨基酸氨基转移酶变体和使用其的亮氨酸生产方法 |
JP2022529090A JP7378621B2 (ja) | 2019-12-06 | 2020-11-24 | 新規な分枝鎖アミノ酸アミノトランスフェラーゼ変異体及びそれを用いたロイシン生産方法 |
ZA2022/06109A ZA202206109B (en) | 2019-12-06 | 2022-06-01 | Novel branched-chain amino acid aminotransferase variant and method for producing leucine using the same |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020190161716A KR102143964B1 (ko) | 2019-12-06 | 2019-12-06 | 신규한 분지쇄 아미노산 아미노트랜스퍼라제 변이체 및 이를 이용한 류신 생산방법 |
KR10-2019-0161716 | 2019-12-06 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2021112469A1 true WO2021112469A1 (ko) | 2021-06-10 |
Family
ID=72038972
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/KR2020/016675 WO2021112469A1 (ko) | 2019-12-06 | 2020-11-24 | 신규한 분지쇄 아미노산 아미노트랜스퍼라제 변이체 및 이를 이용한 류신 생산방법 |
Country Status (11)
Country | Link |
---|---|
US (1) | US20220411832A1 (ko) |
EP (1) | EP4047085A4 (ko) |
JP (1) | JP7378621B2 (ko) |
KR (1) | KR102143964B1 (ko) |
CN (1) | CN115038787B (ko) |
AR (1) | AR120644A1 (ko) |
AU (1) | AU2020398425B2 (ko) |
BR (1) | BR112022011021A2 (ko) |
MX (1) | MX2022006827A (ko) |
WO (1) | WO2021112469A1 (ko) |
ZA (1) | ZA202206109B (ko) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022186487A1 (ko) | 2021-03-05 | 2022-09-09 | 씨제이제일제당 (주) | 이소프로필말레이트 신타제 변이체 및 이를 이용한 l-류신의 생산 방법 |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR102143964B1 (ko) * | 2019-12-06 | 2020-08-12 | 씨제이제일제당 주식회사 | 신규한 분지쇄 아미노산 아미노트랜스퍼라제 변이체 및 이를 이용한 류신 생산방법 |
KR102470602B1 (ko) * | 2020-12-11 | 2022-11-25 | 씨제이제일제당 주식회사 | 신규한 분지 연쇄 아미노산 아미노트렌스퍼라아제 변이체 및 이를 이용한 이소류신 생산 방법 |
KR102495918B1 (ko) | 2021-01-26 | 2023-02-06 | 씨제이제일제당 주식회사 | aroG 알돌라아제 (Phospho-2-dehydro-3-deoxyheptonate aldolase) 변이체 및 이를 이용한 분지쇄 아미노산 생산 방법 |
KR102527096B1 (ko) | 2021-02-01 | 2023-04-28 | 씨제이제일제당 주식회사 | 프리페네이트 탈수 효소 (Prephenate dehydratase) 변이체 및 이를 이용한 분지쇄 아미노산 생산 방법 |
Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5120654A (en) * | 1986-10-29 | 1992-06-09 | Hoechst Aktiengesellschaft | Cloning and use of the transaminase gene ilve |
US20030100054A1 (en) * | 2000-12-20 | 2003-05-29 | Brigitte Bathe | Nucleotide sequences which code for the ilvE gene |
US7220572B2 (en) * | 2002-06-25 | 2007-05-22 | Ajinomoto Co., Inc. | Method for producing L-leucine |
US20080261278A1 (en) * | 2001-02-13 | 2008-10-23 | Tabolina Ekaterina Aleksandrov | Method for producing l-amino acid using bacteria belonging to the genus escherichia |
US7662943B2 (en) | 2004-12-16 | 2010-02-16 | Cj Cheiljedang Corporation | Promoter sequences from Corynebacterium ammoniagenes |
KR20120098235A (ko) * | 2011-02-28 | 2012-09-05 | 이화여자대학교 산학협력단 | 비천연 아미노산 생산 균주 및 이를 이용한 비천연 아미노산의 제조 방법 |
US10273491B2 (en) | 2015-01-29 | 2019-04-30 | Cj Cheiljedang Corporation | Promoter and uses thereof |
US10351859B2 (en) | 2015-08-25 | 2019-07-16 | Cj Cheiljedang Corporation | Microorganism producing L-leucine and method for producing L-leucine using the same |
US20200032305A1 (en) | 2016-12-28 | 2020-01-30 | Cj Cheiljedang Corporation | A novel isopropylmalate synthase variant and a method of producing l-leucine using the same |
US10584338B2 (en) | 2016-08-31 | 2020-03-10 | Cj Cheiljedang Corporation | Promoter and use thereof |
KR102143964B1 (ko) * | 2019-12-06 | 2020-08-12 | 씨제이제일제당 주식회사 | 신규한 분지쇄 아미노산 아미노트랜스퍼라제 변이체 및 이를 이용한 류신 생산방법 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102286505B (zh) * | 2011-05-26 | 2013-04-03 | 江南大学 | 用于发酵生产l-缬氨酸的重组dna、菌株及方法 |
CN109294966A (zh) * | 2018-10-26 | 2019-02-01 | 江南大学 | 一种高产l-亮氨酸的谷氨酸棒杆菌重组菌及其构建方法 |
CN109402034A (zh) * | 2018-10-26 | 2019-03-01 | 江南大学 | 只产一种支链氨基酸的重组菌及其应用 |
-
2019
- 2019-12-06 KR KR1020190161716A patent/KR102143964B1/ko active IP Right Grant
-
2020
- 2020-11-24 MX MX2022006827A patent/MX2022006827A/es unknown
- 2020-11-24 WO PCT/KR2020/016675 patent/WO2021112469A1/ko unknown
- 2020-11-24 AU AU2020398425A patent/AU2020398425B2/en active Active
- 2020-11-24 EP EP20897508.6A patent/EP4047085A4/en active Pending
- 2020-11-24 JP JP2022529090A patent/JP7378621B2/ja active Active
- 2020-11-24 BR BR112022011021A patent/BR112022011021A2/pt unknown
- 2020-11-24 CN CN202080083985.2A patent/CN115038787B/zh active Active
- 2020-11-24 US US17/756,627 patent/US20220411832A1/en active Pending
- 2020-12-01 AR ARP200103331A patent/AR120644A1/es unknown
-
2022
- 2022-06-01 ZA ZA2022/06109A patent/ZA202206109B/en unknown
Patent Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5120654A (en) * | 1986-10-29 | 1992-06-09 | Hoechst Aktiengesellschaft | Cloning and use of the transaminase gene ilve |
US20030100054A1 (en) * | 2000-12-20 | 2003-05-29 | Brigitte Bathe | Nucleotide sequences which code for the ilvE gene |
US20080261278A1 (en) * | 2001-02-13 | 2008-10-23 | Tabolina Ekaterina Aleksandrov | Method for producing l-amino acid using bacteria belonging to the genus escherichia |
US7220572B2 (en) * | 2002-06-25 | 2007-05-22 | Ajinomoto Co., Inc. | Method for producing L-leucine |
US7662943B2 (en) | 2004-12-16 | 2010-02-16 | Cj Cheiljedang Corporation | Promoter sequences from Corynebacterium ammoniagenes |
KR20120098235A (ko) * | 2011-02-28 | 2012-09-05 | 이화여자대학교 산학협력단 | 비천연 아미노산 생산 균주 및 이를 이용한 비천연 아미노산의 제조 방법 |
US10273491B2 (en) | 2015-01-29 | 2019-04-30 | Cj Cheiljedang Corporation | Promoter and uses thereof |
US10351859B2 (en) | 2015-08-25 | 2019-07-16 | Cj Cheiljedang Corporation | Microorganism producing L-leucine and method for producing L-leucine using the same |
US10584338B2 (en) | 2016-08-31 | 2020-03-10 | Cj Cheiljedang Corporation | Promoter and use thereof |
US20200032305A1 (en) | 2016-12-28 | 2020-01-30 | Cj Cheiljedang Corporation | A novel isopropylmalate synthase variant and a method of producing l-leucine using the same |
KR102143964B1 (ko) * | 2019-12-06 | 2020-08-12 | 씨제이제일제당 주식회사 | 신규한 분지쇄 아미노산 아미노트랜스퍼라제 변이체 및 이를 이용한 류신 생산방법 |
Non-Patent Citations (12)
Title |
---|
"Atlas Of Protein Sequence And Structure", 1979, NATIONAL BIOMEDICAL RESEARCH FOUNDATION, pages: 353 - 358 |
"Guide to Huge Computers", 1994, ACADEMIC PRESS |
"Needleman-Wunsch algorithm", J. MOL. BIOL., vol. 48, 1970, pages 443 - 453 |
ATSCHUL, S., J MOLEC BIOL, vol. 215, 1990, pages 403 |
CARILLO, SIAM J APPLIED MATH, vol. 48, 1988, pages 1073 |
DEVEREUX, J. ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, 1984, pages 387 |
GRIBSKOV ET AL., NUCL. ACIDS RES., vol. 14, 1986, pages 6745 |
J SAMBROOK ET AL.: "Molecular Cloning, A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS |
NEEDLEMAN ET AL., J MOL BIOL., vol. 48, 1970, pages 443 |
PEARSON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 2444 |
RICE ET AL., TRENDS GENET, vol. 16, 2000, pages 276 - 277 |
SMITHWATERMAN, ADV. APPL. MATH, vol. 2, 1981, pages 482 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022186487A1 (ko) | 2021-03-05 | 2022-09-09 | 씨제이제일제당 (주) | 이소프로필말레이트 신타제 변이체 및 이를 이용한 l-류신의 생산 방법 |
Also Published As
Publication number | Publication date |
---|---|
EP4047085A1 (en) | 2022-08-24 |
JP2023503077A (ja) | 2023-01-26 |
US20220411832A1 (en) | 2022-12-29 |
CN115038787A (zh) | 2022-09-09 |
AU2020398425A1 (en) | 2022-06-09 |
AR120644A1 (es) | 2022-03-09 |
CN115038787B (zh) | 2024-04-12 |
KR102143964B1 (ko) | 2020-08-12 |
JP7378621B2 (ja) | 2023-11-13 |
AU2020398425B2 (en) | 2024-07-11 |
EP4047085A4 (en) | 2022-12-28 |
ZA202206109B (en) | 2023-04-26 |
MX2022006827A (es) | 2022-07-12 |
BR112022011021A2 (pt) | 2022-08-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2019160301A1 (ko) | 시트레이트 신타아제의 활성이 약화된 변이형 폴리펩타이드 및 이를 이용한 l-아미노산 생산방법 | |
WO2021112469A1 (ko) | 신규한 분지쇄 아미노산 아미노트랜스퍼라제 변이체 및 이를 이용한 류신 생산방법 | |
WO2019117398A1 (ko) | 5'-이노신산을 생산하는 미생물 및 이를 이용한 5'-이노신산의 생산 방법 | |
WO2015064917A1 (ko) | L-라이신 생산능이 향상된 코리네박테리움 속 미생물 및 이를 이용한 l-라이신 생산방법 | |
WO2022231369A1 (ko) | 신규한 포르메이트 의존성 포스포리보실글리신아미드 포밀 전이효소 변이체 및 이를 이용한 imp 생산 방법 | |
WO2022225322A1 (ko) | 신규한 f0f1 atp 합성효소 서브유닛 알파 변이체 및 이를 이용한 xmp 또는 gmp 생산 방법 | |
WO2022163917A1 (ko) | 신규한 단백질 변이체 및 이를 이용한 l-발린 생산 방법 | |
WO2022154190A1 (ko) | 신규한 포스포노아세테이트 하이드롤라제 변이체 및 이를 이용한 xmp 또는 gmp 생산 방법 | |
WO2022154191A1 (ko) | 신규한 2,5-다이케토-d-글루콘산 리덕타제 변이체 및 이를 이용한 xmp 또는 gmp 생산 방법 | |
WO2022154177A1 (ko) | 신규한 3d-(3,5/4)-트리하이드록시사이클로헥세인-1,2-다이온 아실하이드롤라아제 변이체 및 이를 이용한 imp 생산 방법 | |
WO2022154181A1 (ko) | 신규한 1,4-알파-글루칸-분지 효소 변이체 및 이를 이용한 imp 생산 방법 | |
WO2022225319A1 (ko) | 신규한 l-세린 암모니아 분해 효소 변이체 및 이를 이용한 xmp 또는 gmp 생산 방법 | |
WO2022225320A1 (ko) | 신규한 포스포글리세린산 디하이드로게나제 변이체 및 이를 이용한 xmp 또는 gmp 생산 방법 | |
WO2022163922A1 (ko) | 신규한 아스파라긴 신타제 변이체 및 이를 이용한 l-발린 생산 방법 | |
WO2022163935A1 (ko) | 신규한 글루코사민-6-포스페이트 디아미나제 변이체 및 이를 이용한 l-글루탐산 생산 방법 | |
WO2022154178A1 (ko) | 신규한 혐기성 코프로포르피리노겐 iii 옥시다제 변이체 및 이를 이용한 imp 생산 방법 | |
WO2022163920A1 (ko) | 신규한 시스테인 설피네이트 디설피나제 변이체 및 이를 이용한 l-발린 생산 방법 | |
WO2022225321A1 (ko) | 신규한 f0f1 atp 합성효소 서브유닛 감마 변이체 및 이를 이용한 xmp 또는 gmp 생산 방법 | |
WO2022231371A1 (ko) | 신규한 5-(카르복시아미노)이미다졸리보뉴클레오티드합성효소 변이체 및 이를 이용한 imp 생산 방법 | |
WO2022158646A1 (ko) | 신규한 쿠퍼익스포팅 p-type 에이티피에이즈 a 변이체 및 이를 이용한 l-트립토판 생산 방법 | |
WO2022231067A1 (ko) | 신규한 이중기능성 pyr 오페론 전사조절자/우라실 포스포리보실 전달 효소 변이체 및 이를 이용한 imp 생산 방법 | |
WO2022225100A1 (ko) | 신규한 이중기능성 메틸렌테트라히드로폴레이트 탈수소효소/메테닐테트라하이드로폴레이트 사이클로하이드롤라아제 변이체 및 이를 이용한 xmp 또는 gmp 생산 방법 | |
WO2022163916A1 (ko) | 신규한 dahp 신타아제 변이체 및 이를 이용한 l-라이신 생산 방법 | |
WO2022215800A1 (ko) | 신규한 분지쇄아미노산 투과효소 변이체 및 이를 이용한 l-발린 생산 방법 | |
WO2022158652A1 (ko) | 신규한 사이토신 퍼미에이즈 변이체 및 이를 이용한 l-트립토판 생산 방법 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 20897508 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2022529090 Country of ref document: JP Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2020897508 Country of ref document: EP Effective date: 20220516 |
|
ENP | Entry into the national phase |
Ref document number: 2020398425 Country of ref document: AU Date of ref document: 20201124 Kind code of ref document: A |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112022011021 Country of ref document: BR |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 112022011021 Country of ref document: BR Kind code of ref document: A2 Effective date: 20220606 |