WO2021153866A1 - 시트레이트 신타아제의 활성이 약화된 신규한 변이형 폴리펩티드 및 이를 이용한 l-아미노산 생산 방법 - Google Patents
시트레이트 신타아제의 활성이 약화된 신규한 변이형 폴리펩티드 및 이를 이용한 l-아미노산 생산 방법 Download PDFInfo
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- C12Y—ENZYMES
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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
- C12N15/74—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
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- 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.)
- C12N9/1025—Acyltransferases (2.3)
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- 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
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- 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
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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- 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
- C12P13/12—Methionine; Cysteine; Cystine
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/03—Acyl groups converted into alkyl on transfer (2.3.3)
- C12Y203/03001—Citrate (Si)-synthase (2.3.3.1)
Definitions
- the present application relates to a novel mutant polypeptide in which the activity of citrate synthase is weakened, a microorganism comprising the mutant polypeptide, and a method for producing L-amino acids using the microorganism.
- Corynebacterium genus (the genus Corynebacterium )
- Microorganisms particularly Corynebacterium glutamicum (Corynebacterium glutamicum) is a gram-positive microorganism that is widely used for the production of L-amino acids and other useful substances.
- Corynebacterium glutamicum is a gram-positive microorganism that is widely used for the production of L-amino acids and other useful substances.
- various studies for the development of high-efficiency production microorganisms and fermentation process technology are being conducted.
- a target substance-specific approach such as increasing the expression of a gene encoding an enzyme involved in L-lysine biosynthesis or removing a gene unnecessary for biosynthesis is mainly used (US 8048650 B2).
- L-amino acids L-lysine, L-threonine, L-methionine, L-isoleucine, and L-glycine are amino acids derived from aspartate, and the synthesis level of oxaloacetate, a precursor of aspartate, is the same as above. It may affect the level of synthesis of L-amino acids.
- Citrate synthase is an enzyme that produces citrate by polymerizing acetyl-CoA and oxaloacetate produced in the glycolysis of microorganisms, and is also an important enzyme that determines carbon influx into the TCA pathway.
- the present inventors have completed the present invention by confirming that the production of L-amino acids is increased when a novel mutant polypeptide in which citrate synthase activity is attenuated to a specific level is used.
- the present application provides a variant polypeptide having citrate synthase activity in which the amino acid corresponding to the 312th position from the N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.
- the present application provides a polynucleotide encoding the variant polypeptide.
- the present application provides a vector comprising the polynucleotide.
- the present application provides a microorganism for producing L-amino acids, including the variant polypeptide, a polynucleotide encoding the variant polypeptide, or a vector including the polynucleotide.
- the present application provides a method for producing L-amino acids, comprising culturing a microorganism comprising the mutant polypeptide, a polynucleotide encoding the mutant polypeptide, or a vector including the polynucleotide in a medium.
- One aspect of the present application is a variant polypeptide having citrate synthase activity in which the amino acid corresponding to the 312th position from the N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid may be to provide
- the sequence of SEQ ID NO: 1 may be an amino acid sequence having citrate synthase activity.
- the sequence of SEQ ID NO: 1 may be a protein sequence having citrate synthase activity encoded by the gltA gene.
- the amino acid sequence of SEQ ID NO: 1 can be obtained from GenBank of NCBI, a known database. As an example, it may be derived from Corynebacterium glutamicun , but is not limited thereto, and any amino acid sequence of a protein having the same activity as a protein including the amino acid sequence may be included without limitation.
- the protein having citrate synthase activity in the present application may be a protein including the amino acid sequence of SEQ ID NO: 1, insignificant sequence addition before and after the amino acid sequence of SEQ ID NO: 1 or a naturally occurring mutation, or It does not exclude its latent mutation, and if it has the same or corresponding activity as the protein comprising the amino acid sequence of SEQ ID NO: 1, it corresponds to the protein having the citrate synthase activity of the present application.
- the protein having citrate synthase activity of the present application is the amino acid sequence of SEQ ID NO: 1 or 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more thereof.
- It may be a protein composed of an amino acid sequence having homology or identity.
- it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the protein
- a protein having an amino acid sequence in which some sequence is deleted, modified, substituted or added is also included within the scope of the protein subject to mutation of the present application. is self-evident
- citrate synthase is an enzyme that produces citrate by polymerizing acetyl-CoA and oxaloacetate produced in the glycolysis of microorganisms, and is an enzyme that determines carbon inflow into the TCA pathway. It is an important enzyme. Specifically, as a citric acid synthase, it may play a rate-regulating role in the first step of the TCA cycle. The enzyme is also capable of catalyzing the condensation reaction of acetyl coei with a 2-carbon acetate moiety from a molecule of 4-carbon oxaloacetate to form 6-carbon acetate.
- the citrate synthase may be used in combination with citrate synthase, CS, GltA protein or GltA.
- 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 Refers to a polypeptide in which functions or properties are maintained.
- a variant polypeptide differs from the 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.
- variants can generally be identified by modifying one of the polypeptide sequences and evaluating the reactivity of the modified polypeptide.
- 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 means 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.
- positively charged (basic) amino acids include arginine, lysine, and histidine
- negatively charged (acidic) amino acids include glutamic acid and aspartic acid
- Aromatic amino acids include phenylalanine, tryptophan and tyrosine
- hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, proline, glycine and tryptophan.
- conservative substitutions have little or no effect on the activity of the resulting polypeptide.
- variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide.
- the polypeptide can be conjugated with a signal (or leader) sequence at the N-terminus of the protein that is involved in the transfer of the protein either co-translationally or post-translationally.
- the polypeptide may also be conjugated with other sequences or linkers to enable identification, purification, or synthesis of the polypeptide.
- mutant polypeptide having citrate synthase activity refers to a polypeptide having a weakened citrate synthase activity compared to the wild type by substituting a portion of the amino acid sequence of the polypeptide having citrate synthase activity.
- one or more amino acids are mutated in the amino acid sequence of a polypeptide having citrate synthase activity, so that the activity is weakened compared to that of the wild type, and thus it may refer to a mutant polypeptide that efficiently balances carbon flow.
- the variant polypeptide may be a variant polypeptide in which the amino acid corresponding to the 312th position of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid in the various proteins having the above citrate synthase activity.
- the 'other amino acid' refers to an amino acid different from that before the substitution, and is not limited as long as it is an amino acid other than the amino acid before the substitution.
- the variant polypeptide may be a variant polypeptide in which methionine corresponding to position 312 of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid in the various proteins having the above-mentioned citrate synthase activity.
- the methionine is alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine ), isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine (valine) may be substituted with any one or more amino acids selected from the group consisting of, and more specifically, may be a variant sequence substituted with isoleucine, but is not limited thereto.
- the substituted amino acid residue may include not only natural amino acids but also non-natural amino acids.
- the unnatural amino acid can be, for example, a D-amino acid, a homo-amino acid, a beta-homo-amino acid, an N-methyl amino acid, an alpha-methyl amino acid, an unconventional amino acid (such as citrulline or naph thylalanine, etc.), but is not limited thereto.
- a specific amino acid is substituted' in the present application, it is obvious that the amino acid is substituted with an amino acid different from the amino acid before the substitution, even if it is not separately indicated that it is substituted with another amino acid.
- corresponding to refers to an amino acid residue at a position listed in a protein or peptide, or an amino acid residue similar to, identical to, or homologous to a residue listed in a protein or peptide.
- corresponding region generally refers to a similar position in a related protein or reference protein.
- specific numbering may be used for amino acid residue positions in the polypeptides used in this application. For example, by aligning the polypeptide sequence of the present application with the target polypeptide to be compared, it is possible to renumber the position corresponding to the amino acid residue position of the polypeptide of the present application.
- mutant polypeptide having citrate synthase activity provided in the present application, amino acids at specific positions in the aforementioned citrate synthase are substituted, so that the ability to produce L-amino acids can be increased compared to the polypeptide before the mutation.
- the variant polypeptide may have 80% or more and less than 100% sequence homology with the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. Specifically, the variant polypeptide of the present application may be one having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence of SEQ ID NO: 1. It is apparent that proteins having an amino acid sequence in which some sequences are deleted, modified, substituted or added in addition to the amino acid sequence at position 312 are included within the scope of the present application as long as the amino acid sequence has homology and exhibits efficacy corresponding to the protein.
- mutant polypeptide in addition to the specific 312-th mutation that imparts a specific activity, a meaningless sequence before and after the amino acid sequence of the corresponding SEQ ID NO: Addition or naturally occurring mutation, or a mutation thereof It is apparent that latent mutations are not excluded, and that such sequence additions or mutations fall within the scope of the present application.
- the mutant polypeptide in which the amino acid corresponding to the 312th position in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid may include the amino acid sequence of SEQ ID NO: 3. More specifically, the mutant polypeptide in which methionine corresponding to position 312 in the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine may consist of SEQ ID NO: 3. In addition, the variant polypeptide may include the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having 80% or more and less than 100% homology thereto, but is not limited thereto.
- the variant polypeptide of the present application may include SEQ ID NO: 3 and a polypeptide having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 3 there is.
- a protein having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the present application. can be self-evident
- L-amino acid production can be increased through an appropriate balance between the carbon flow into the TCA pathway and the supply of oxaloacetate used as a precursor of L-amino acid biosynthesis through regulation of citrate synthase activity.
- homology refers to the percent identity between two polynucleotide or polypeptide moieties. It refers to the degree of correspondence with a given amino acid sequence or base sequence and may be expressed as a percentage. In the present specification, a homologous sequence having the same or similar activity to a given amino acid sequence or base sequence may be expressed as "% homology". Homology between sequences from one moiety to another can be determined by known art. For example, using standard software that calculates parameters such as score, identity and similarity, specifically BLAST 2.0, or by Southern hybridization experiments under defined stringent conditions. appropriate hybridization conditions is to check, by comparing the definition is within the skill of a method well known to those skilled in the art (e.g., J.
- Another aspect of the present application is that the amino acid corresponding to the 312th position from the N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, a variant having citrate synthase activity
- a polynucleotide encoding a polypeptide may be provided.
- amino acid sequence of SEQ ID NO: 1, citrate synthase and variant polypeptides are the same as described above.
- 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, encoding the variant. polynucleotide fragments.
- the polynucleotide of the present application may be included without limitation as long as it is a polynucleotide sequence encoding a variant polypeptide having citrate synthase activity of the present application.
- the gene encoding the amino acid sequence of citrate synthase is a gltA gene, and the gene may be derived from Corynebacterium glutamicum, but is not limited thereto.
- the gene may be a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, more specifically, may be a sequence including the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.
- the polynucleotide of the present application may contain a variety of coding regions within a range that does not change the amino acid sequence of the polypeptide due to codon degeneracy or considering codons preferred in an organism to express the polypeptide. Deformation can be made. Specifically, any polynucleotide sequence encoding a variant polypeptide in which the amino acid corresponding to position 312 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid may be included without limitation.
- the variant polypeptide of the present application 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 set forth in SEQ ID NO: 4, but is not limited thereto.
- the 312th amino acid in the amino acid sequence of SEQ ID NO: 1 is a different amino acid Any sequence encoding a protein having a substituted citrate synthase activity may be included without limitation.
- the "stringent condition” means a condition that enables specific hybridization between polynucleotides. These conditions are specifically described in the literature (eg, J. Sambrook et al., supra).
- 40% or more, specifically 90% or more, more specifically 95% or more, more specifically 97% or more, particularly specifically 99% or more homology between genes with high homology or identity, or Genes having the same identity are hybridized and genes having homology or identity lower than that are not hybridized, or washing conditions of normal Southern hybridization at 60°C, 1 ⁇ SSC, 0.1% SDS, specifically At a salt concentration and temperature equivalent to 60°C, 0.1 ⁇ SSC, 0.1% SDS, more specifically 68°C, 0.1 ⁇ SSC, 0.1% SDS, conditions for washing once, specifically 2 to 3 times can be listed. there is.
- 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.
- adenosine is complementary to thymine
- cytosine is complementary to guanine.
- 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 above-described conditions.
- 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 depending on the purpose.
- Another aspect of the present application is that the amino acid corresponding to the 312th position from the N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, a variant having citrate synthase activity
- Vectors comprising a polynucleotide encoding a polypeptide can be provided.
- amino acid sequence of SEQ ID NO: 1, citrate synthase, variant polypeptides and polynucleotides are the same as described above.
- the term "vector” refers to a DNA preparation containing the nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable regulatory sequence so that the target polypeptide 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.
- pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used.
- pCR2.1, pDC, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.
- the vector usable in the present application is not particularly limited, and a known expression vector may be used.
- a polynucleotide encoding a target polypeptide can 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 has been inserted, and has a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide. Markers to be given can be used. In an environment treated with a selective agent, only cells expressing a selectable marker survive or exhibit other expression traits, so that transformed cells can be selected.
- the term "transformation” refers 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.
- polynucleotide may include DNA and RNA encoding a target protein.
- 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.
- the transformation method includes any method of introducing a polynucleotide into a cell, and may be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (Ca(H 2 PO 4 ) 2 , CaHPO 4 , or Ca 3 (PO 4 ) 2 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, Polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method, but are not limited thereto.
- electroporation calcium phosphate (Ca(H 2 PO 4 ) 2 , CaHPO 4 , or Ca 3 (PO 4 ) 2 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, Polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method, but are not limited thereto.
- operably linked may mean that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target protein of the present application and the polynucleotide sequence are functionally linked.
- the operable linkage may be prepared using a genetic recombination technique known in the art, and site-specific DNA cleavage and ligation may be made using a cleavage and ligation enzyme in the art, but is not limited thereto.
- Another aspect of the present application is that the amino acid corresponding to the 312th position from the N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, a variant having citrate synthase activity It is possible to provide a microorganism for producing L-amino acids, including a polypeptide, a polynucleotide encoding the variant polypeptide, or a vector including the polynucleotide.
- amino acid sequence of SEQ ID NO: 1, citrate synthase, variant polypeptides, polynucleotides and vectors are as described above.
- the microorganism may include a polynucleotide encoding the variant polypeptide or provide a microorganism transformed with a vector containing a polynucleotide encoding the variant polypeptide, but is not limited thereto.
- the microorganism can improve the production capacity of L-amino acids without inhibiting the growth or sugar consumption rate of the microorganisms compared to the microorganisms containing the wild-type polypeptide, and L-amino acids can be obtained from these microorganisms in high yield. there is.
- the term "microorganism comprising a variant polypeptide” refers to a microorganism in which L-amino acid-producing ability is conferred to a microorganism having a naturally weak L-amino acid-producing ability or a parent strain without L-amino acid-producing ability. .
- the microorganism is a microorganism expressing a variant polypeptide comprising one or more amino acid mutations in a polypeptide having citrate synthase activity, wherein the amino acid mutation is at position 312 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 It may include substitution of the corresponding amino acid with another amino acid.
- the variant polypeptide having a citrate synthase activity expressed from the microorganism may have a weakened activity, but is not limited thereto.
- the microorganism is a cell or microorganism capable of expressing a variant polypeptide by being transformed with a vector containing a polynucleotide encoding a variant polypeptide or a polynucleotide encoding a variant polypeptide, for the purpose of the present application Phase
- the host cell or microorganism may be any microorganism capable of producing L-amino acids including the variant polypeptide.
- the term "microorganism that produces L-amino acids” includes all microorganisms in which genetic modification has occurred, either naturally or artificially, and specific As a microorganism with an enhanced or weakened mechanism, it may be a microorganism in which genetic mutation or activity is weakened for the production of a desired L-amino acid.
- the microorganism producing the L-amino acid refers to a microorganism capable of producing the desired L-amino acid from a carbon source in the medium, including the mutant polypeptide, in excess compared to the wild-type or unmodified microorganism.
- the "microorganism producing L-amino acid” may be used interchangeably with "a microorganism having L-amino acid producing ability" or "a microorganism producing L-amino acid”.
- the L-amino acid produced by the L-amino acid producing microorganism may be any one or more selected from the group consisting of leucine, lysine, valine, isoleucine and O-acetyl homoserine, but is not limited thereto.
- Microorganism producing the L- amino acid is specifically exemplified by, Escherichia (Escherichia) genus, Serratia marcescens (Serratia), An air Winiah (Erwinia) genus, Enterobacter bacteria (Enterobacteria) genus, Salmonella (Salmonella) genus Streptomyces (Streptomyces) genus Pseudomonas (Pseudomonas), a can include microbial strains such as Brevibacterium (Brevibacterium) in or Corynebacterium (Corynebacterium) genus. Specifically, it may be a microorganism of the genus Corynebacterium, and a more specific example may be Corynebacterium glutamicum , but is not limited thereto.
- the microorganism producing L-amino acid is a strain of CJL-8100 having L-leucine production ability by introducing a mutant [leuA_(R558H, G561D)] of isopropyl malate synthase into a microorganism of the genus Corynebacterium, Coryne. Corynebacterium CJ3P (Binder et al.
- Genome Biology 2012, 13 having L-lysine-producing ability by introducing three types of mutations [pyc(P458S), hom(V59A), lysC(T311I)] into the microorganisms of the genus Bacterium :R40), Corynebacterium KCCM11201P (US 8465962 B2), a valine-producing strain, Corynebacterium KCCM11248P (Republic of Korea Patent No.
- an acetyl-homoserine degradation pathway O-acetyl by introducing a mutation (L377K) (US 10662450 B2) for canceling feedback inhibition of L-lysine and L-Threonine in the lysC gene encoding aspartokinase - It may be Corynebacterium glutamicum having a homoserine-producing ability, but is not limited thereto.
- the microorganism producing the L-amino acid may further include the mutant polypeptide, thereby increasing the production ability of the desired
- Corynebacterium genus microorganism is specifically Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum ( Brevibacterium lactofermentum ), Brevibacterium flavum ( Brevibacterium flavum ), Corynebacterium thermoaminogenes ), Corynebacterium efficiens ), etc., but are not necessarily limited thereto.
- the Corynebacterium genus microorganism increases the yield of L-amino acids even if the citrate synthase activity is weakened compared to the unmodified microorganism, and the sugar consumption rate is similar Corynebacterium glutamicum ( Corynebacterium glutamicum ).
- Another aspect of the present application is that the amino acid corresponding to the 312th position from the N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, a variant having citrate synthase activity It is possible to provide a method for producing L-amino acids, comprising culturing a microorganism comprising a polypeptide, a polynucleotide encoding the variant polypeptide, or a vector comprising the polynucleotide in a medium.
- amino acid sequence of SEQ ID NO: 1, citrate synthase, variant polypeptides, polynucleotides, vectors and microorganisms are as described above.
- the microbial culture 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.
- a basic compound eg, sodium hydroxide, potassium hydroxide or ammonia
- an acidic compound eg, phosphoric acid or sulfuric acid
- 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 L-amino acids produced by the culture may be secreted into the medium or remain in the cells.
- the culture medium used is a carbon source that includes sugars and carbohydrates (eg, glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), oils and fats (eg, soybean oil, sunflower seeds). Oil, peanut oil and coconut oil), fatty acids (eg palmitic acid, stearic acid and linoleic acid), alcohols (eg glycerol and ethanol), and organic acids (eg acetic acid) may be used individually or in combination. , but not limited thereto.
- sugars and carbohydrates eg, glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose
- oils and fats eg, soybean oil, sunflower seeds. Oil, peanut oil and coconut oil
- fatty acids eg palmitic acid, stearic acid and linoleic acid
- alcohols eg glycerol and ethanol
- organic acids eg acetic acid
- Nitrogen sources include nitrogen-containing organic compounds (e.g., peptone, yeast extract, broth, malt extract, corn steep liquor, soy meal and urea), or inorganic compounds (e.g., 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 e.g., peptone, yeast extract, broth, malt extract, corn steep liquor, soy meal and urea
- inorganic compounds e.g., 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, but not limited to, other metal salts (eg
- the method may further include the step of recovering L-amino acids from the culture medium or microorganisms after the culturing step, but is not limited thereto.
- the 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 L-amino acid may be recovered from the medium or microorganism using a suitable method known in the art.
- the recovery step may include a purification process, and may be performed using a suitable method known in the art. Accordingly, the recovered L-amino acid may be in a purified form or a microbial fermentation broth containing L-amino acid.
- the L-amino acid may be any one or more selected from the group consisting of leucine, lysine, valine, isoleucine and O-acetyl homoserine, but is not limited thereto.
- compositions for the production of L-amino acids comprising a microorganism of the genus Corynebacterium or a culture thereof comprising the mutant polypeptide having the citrate synthase activity.
- the microorganism may be of the genus Corynebacterium, specifically Corynebacterium glutamicum, but is not limited thereto. This is the same as described above.
- the composition for producing L-amino acid may refer to a composition capable of producing L-amino acid by the mutant polypeptide having citrate synthase activity of the present application.
- the composition may include, without limitation, the variant polypeptide having citrate synthase activity or a composition capable of activating the variant polypeptide having citrate synthase activity.
- the mutant polypeptide having the citrate synthase activity may be in a form included in a vector so that the operably linked gene can be expressed in the introduced host cell.
- the composition may further comprise a cryoprotectant or an excipient.
- the cryoprotectant or excipient may be a non-naturally occurring material or a naturally occurring material, but is not limited thereto.
- the cryoprotectant or excipient may be a material that the microorganism does not naturally contact, or a material that is not naturally included with the microorganism at the same time, but is not limited thereto.
- Another aspect of the present application provides a use for the production of L-amino acids in a microorganism of the genus Corynebacterium comprising a variant polypeptide having the citrate synthase activity.
- a recombinant vector including a part of gltA was first prepared.
- the amino acid sequence and nucleotide sequence of wild-type gltA are the same as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
- Corynebacterium glutamicun Corynebacterium glutamicun
- PCR was performed using the primers of SEQ ID NO: 5 and SEQ ID NO: 6 using the wild line chromosome as a template, and the amplified product was obtained by using the TOPO cloning kit (Invitrogen) for E. coli vector pCR2 cloned into .1 to obtain pCR-gltA.
- a gltA mutation library was prepared based on the vector constructed in Example 1-1.
- the library was constructed using an 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 30 seconds of pre-heating at 94°C 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. .
- the primers used in this Example are shown in Table 1 below.
- the pTOPO-gltA-library prepared in Example 1-2 was transformed into Corynebacterium glutamicun ATCC13032 by electroporation, and then plated on a nutrient medium containing 25 mg/L of kanamycin to transform 10,000 colonies of the strain into which the gene was inserted were secured, and each colony was named from ATCC13032/pTOPO_gltA(mt)1 to ATCC13032/pTOPO_gltA(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.
- 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, 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 having the most improved leucine production capacity compared to the wild-type Corynebacterium glutamicum strain was selected. The leucine concentrations produced in the selected strains are shown in Table 2 below.
- PCR was performed in ATCC13032/pTOPO_gltA(mt)3142 strain using the primers of SEQ ID NO: 7 and SEQ ID NO: 8 to confirm the genetic mutation of the mutant strain and sequencing was performed to compare the gltA gene with the wild-type ATCC13032 and it was confirmed that the above strain contains a mutation in the gltA gene.
- an insertion vector was prepared.
- a site directed mutagenesis method was used to construct a vector for introducing gltA(M312I) mutations.
- PCR was performed using the primer pair of SEQ ID NOs: 9 and 10, and the primer pair of SEQ ID NOs: 11 and 12 using the chromosome of the Corynebacterium glutamicum wild-type as a template. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, followed by polymerization at 72°C for 5 minutes.
- the resultant gene fragment was cloned by linking the homologous sequence of the 15 bases at the terminal between the DNA fragments using a linear pDC vector digested with SmaI restriction enzyme and an In-Fusion enzyme and cloning. Methionine, the 312th amino acid, was replaced with isoleucine.
- a vector pDC-gltA (M312I) was constructed.
- the strain into which the pDC-gltA(M312I) vector prepared in Example 3-1 was transformed into ATCC13032 and the vector was inserted into the chromosome by recombination of the homologous sequence was selected in a medium containing 25 mg/L of kanamycin.
- 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 gltA gene mutation was introduced in the final transformed strain was confirmed that the mutation was introduced in the strain by performing PCR using the primers of SEQ ID NO: 7 and SEQ ID NO: 8 and then analyzing the nucleotide sequence.
- the produced strain was named ATCC13032_gltA_M312I.
- IPMS isopropylmalate synthase
- the strain into which the pDC-leuA (R558H, G561D) vector containing the leuA mutation was transformed into ATCC13032 and the vector was inserted into the chromosome by recombination of the homologous sequence was selected in a medium containing 25 mg/L of kanamycin.
- the selected primary strain was again subjected to a secondary crossover, 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: 13 and SEQ ID NO: 14 and then analyzing the nucleotide sequence.
- the ATCC13032_leuA_(R558H, G561D) strain transformed with the pDC-leuA(R558H, G561D) vector was named CJL-8100.
- the strain CJL-8100 a leucine-producing strain, was transformed with the pDC-gltA (M312I) vector prepared in Example 3-1, and the vector was inserted into the chromosome by recombination of the homologous sequence, containing 25 mg/L of kanamycin. Selected in one medium. The selected primary strain was again subjected to secondary crossover, and a strain into which the mutation of the target gene was introduced was selected. To determine whether the gltA gene mutation was introduced into the final transformed strain, PCR was performed using the primers of SEQ ID NO: 7 and SEQ ID NO: 8, and then the nucleotide sequence was analyzed to confirm that the gltA mutation was introduced into the strain.
- CJL8100_gltA_M312I was named CA13-8104 and deposited with the Korean Culture Center of Microorganisms (KCCM), an international depository under the Budapest Treaty, as of December 20, 2019, and was given a deposit number KCCM12649P.
- KCCM Korean Culture Center of Microorganisms
- the leucine-producing ability of the prepared CA13-8104 strain was evaluated. Flask culture was carried out in the same manner as in Example 2, and leucine production was measured by the method using HPLC after the end of the culture, and the culture results are shown in Table 4 below.
- the leucine-producing ability of the prepared CJL8100/pTOPO_gltA(mt)3142 strain was evaluated. Flask culture was carried out in the same manner as in Example 2, and leucine production was measured by a method using HPLC after completion of the culture, and the culture results are shown in Table 5 below.
- amino acid at position 312 in the amino acid sequence of gltA is an important position for gltA enzymatic activity.
- Example 5 Preparation of CJ3P strain introduced with gltA mutant (M312I) and analysis of lysine production
- each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of a seed medium, and cultured with shaking at 30° C. for 20 hours at 200 rpm. Then, a 250 ml corner-baffle flask containing 24 ml of production medium was inoculated with 1 ml of the seed culture and cultured with shaking at 32° C. for 72 hours at 200 rpm.
- the composition of the species medium and the production medium is as follows, respectively. After completion of the culture, the concentration of L-lysine was measured using HPLC (Waters 2478), and it is shown in Table 6.
- the CJ3P::gltA(M312I) strain introduced with the gltA(M312I) mutation in the Corynebacterium glutamicum CJ3P strain, which is a lysine-producing strain, improved lysine production by 127% compared to the parent strain. did.
- KCCM11201P was transformed with the pDC-gltA(M312I) vector prepared in Example 3-1, and the vector into which the vector was inserted into the chromosome by recombination of the homologous sequence was selected in a medium containing 25 mg/L of kanamycin. The selected primary strain was again subjected to secondary crossover, and a strain into which the mutation of the target gene was introduced was selected. Whether the gltA gene mutation was introduced in the final transformed strain was confirmed that the gltA mutation was introduced in the strain by performing PCR using the primers of SEQ ID NO: 7 and SEQ ID NO: 8 and then analyzing the nucleotide sequence. The produced strain was named KCCM11201P-gltA (M312I).
- valine-producing ability of the KCCM11201P-gltA (M312I) strain prepared above was evaluated. Flask culture was carried out in the same manner as in Example 2-2, and valine production was measured by the method using HPLC after completion of the culture, and the culture results are shown in Table 7 below.
- amino acid at position 312 in the amino acid sequence of gltA is an important position for gltA enzyme activity.
- Example 7 Confirmation of L-isoleucine-producing ability of gltA selection mutation in isoleucine-producing strains
- Example 4 the recombinant plasmid pDC-gltA (M312I) prepared in Example 3-1 was added to Corynebacterium glutamicum KCCM11248P (Korean Patent No. 10-1335789), which is an L-isoleucine-producing strain. Strains introduced by homologous recombination on chromosomes were prepared and named KCCM11248P::gltA(M312I), respectively. The produced strain was cultured in the following manner to compare isoleucine-producing ability.
- Each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the seed medium, and incubated at 30° C. for 20 hours with shaking at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium and cultured with shaking at 30° C. for 48 hours at 200 rpm.
- the composition of the species medium and the production medium is as follows, respectively.
- Glucose 50 g (NH 4 ) 2 SO 4 12.5 g, Soy Protein 2.5 g, Corn Steep Solids 5 g, Urea 3 g, KH 2 PO 4 1 g, MgSO 4 7H 2 O 0.5 g, Biotin 100 ⁇ g, Thiamine Hydrochloride 1000 ⁇ g, Calcium-Pantothenic Acid 2000 ⁇ g, Nicotinamide 3000 ⁇ g, CaCO3 30 g (Based on 1 Liter of Distilled Water)
- PCR was performed using SEQ ID NOs: 15 and 16 or SEQ ID NOs: 17 and 18 primers.
- the sequences of the primers used are shown in Table 9 below.
- PCR conditions were repeated 30 times of denaturation at 95° C. for 5 minutes, then denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 30 seconds, followed by polymerization at 72° C. for 7 minutes.
- a 509 bp DNA fragment in the 5' upper region and a 520 bp DNA fragment in the 3' lower region were obtained based on the mutation of the lysC gene, respectively.
- PCR was performed with primers of SEQ ID NOs: 15 and 18. After denaturation at 95° C. for 5 minutes, denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 60 seconds were repeated 30 times, followed by polymerization at 72° C. for 7 minutes.
- a 1011 bp DNA fragment containing a mutation in the lysC gene encoding an aspartokinase variant in which leucine at position 377 was substituted with lysine was amplified.
- pDZ-lysC L377K
- the pDZ-lysC (L377K) vector obtained above was introduced into the WT strain by an electric pulse method (Appl. Microbiol. Biothcenol. (1999, 52:541-545)) and then transformed in a selective medium containing 25 mg/L of kanamycin. strains were obtained.
- WT::lysC (L377K) a strain in which nucleotide mutations were introduced into the lysC gene by the DNA fragment inserted on the chromosome, was obtained.
- the gene into which the nucleotide mutation was introduced was finally confirmed by comparison with the sequence of the wild-type lysC gene through PCR using the primers of SEQ ID NOs: 15 and 18 and then sequencing.
- PCR was performed using primers of SEQ ID NOs: 19 and 20 and SEQ ID NOs: 21 and 22 using WT genomic DNA as a template.
- the sequences of the primers used are shown in Table 10 below.
- PCR conditions were repeated 30 times of denaturation at 95° C. for 5 minutes, then denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 30 seconds, followed by polymerization at 72° C. for 7 minutes.
- a 220 bp DNA fragment at the 5' upper end and a 220 bp DNA fragment at the 3' lower end were obtained centering on the mutation of the hom gene.
- PCR was performed using the primers of SEQ ID NOs: 5 and 8 using these two PCR products as templates.
- the obtained pDZ-hom (R407H) vector was introduced into the WT::lysC (L377K) strain by an electric pulse method, and then the transformed strain was obtained in a selective medium containing 25 mg/L of kanamycin.
- the recombinant plasmid pDC-gltA(M312I) prepared in Example 3-1 was introduced into the WT::lysC(L377K)-hom(R407H) strain by homologous recombination on the chromosome in the same manner as in the above Example. and WT::lysC(L377K)-hom(R407H)-gltA(M312I).
- a pair of primers for amplifying the 5' upper region around the mutation site and the 3' lower region are amplified
- a pair of primers (SEQ ID NOs: 25 and 26) was designed for In the primers of SEQ ID NOs: 23 and 26, BamHI restriction enzyme sites (indicated by an underline) were inserted at each end, and the primers of SEQ ID NOs: 24 and 25 were designed to cross each other so that nucleotide substitution mutations (indicated by an underline) were located. did.
- the sequences of the primers were shown in Table 11 below.
- PCR was performed using the primers of SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 using the WT chromosome as a template. PCR conditions were repeated 30 times of denaturation at 95° C. for 5 minutes, then denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 30 seconds, followed by polymerization at 72° C. for 7 minutes. As a result, a 627 bp DNA fragment at the 5' upper end and a 608 bp DNA fragment at the 3' lower end were obtained centering on the mutation of the ilvA gene.
- PCR was performed with primers of SEQ ID NO: 23 and SEQ ID NO: 26. After denaturation at 95° C. for 5 minutes, denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 60 seconds were repeated 30 times, followed by polymerization at 72° C. for 7 minutes.
- a 1217 bp DNA fragment containing a mutation in the ilvA gene encoding the IlvA variant in which valine at position 323 was substituted with alanine was amplified.
- the pECCG117 (Republic of Korea Patent No. 10-0057684) vector and a DNA fragment of 1011 bp were treated with a restriction enzyme BamHI, ligated using a DNA conjugation enzyme, and then cloned to obtain a plasmid, which was called pECCG117- ilvA (V323A). named.
- a strain introduced into the pECCG117-ilvA(V323A) vector was prepared in ATCC13032::hom(R407H)-lysC(L377K)-gltA(M312I) of the above example.
- a strain in which only ilvA(V323A) mutation was introduced into ATCC13032::-hom(R407H)-lysC(L377K) as its control was also prepared.
- the strain prepared above was cultured in the same manner as in the flask culture method shown in Example 4-1, and the L-isoleucine concentration in the culture solution was analyzed, and the results are shown in Table 12 below.
- ATCC13032::hom(R407H)-lysC(L377K)/pECCG117-ilvA(V323A) mutation introduced ATCC13032::hom(R407H)-lysC(L377K) compared to wild-type strain ATCC13032::-hom(R407H)-lysC(L377K)/pECCG117-ilvA(V323A) )- In gltA(M312I)/pECCG117-ilvA(V323A), it was confirmed that the concentration of L-isoleucine increased by about 43%.
- Example 8 Production of strains having improved O-acetyl homoserine-producing ability and evaluation of O-acetyl homoserine-producing ability
- metB encoding cystathionine gamma-synthase of the O-acetyl homoserine degradation pathway genes were obtained.
- the nucleotide sequence information of the metB gene (NCBI registration number Ncgl2360, SEQ ID NO: 27) was obtained from the National Institutes of Health (NIH GenBank), and based on this, a primer (sequence) including the N-terminal portion and the linker portion of the metB gene Nos. 28 and 29), primers (SEQ ID NOs: 30 and 31) comprising a C-terminal portion and a linker portion were synthesized. Primer sequences are shown in Table 13 below.
- PCR was performed using the primers of SEQ ID NOs: 28 and 29 and SEQ ID NOs: 30 and 31.
- Polymerase PfuUltra TM high-confidence DNA polymerase (Stratagene) was used, and PCR conditions were 30 seconds of denaturation at 96°C, annealing at 53°C for 30 seconds, and polymerization at 72°C for 1 minute, repeated 30 times.
- a 558 bp amplified gene including the N-terminal portion and linker portion of the metB gene and 527 bp amplified gene including the C-terminal portion and linker portion of the metB gene were obtained, respectively.
- PCR was performed using the two amplified genes obtained above as a template, and PCR conditions were SEQ ID NO: 2 and 10 repetitions of denaturation at 96 ° C. for 60 seconds, annealing at 50 ° C. for 60 seconds, and polymerization at 72 ° C. for 1 minute. 5 was added and the polymerization was repeated 20 more times.
- a 1064bp inactivation cassette including the N-terminal-linker-C-terminus of the metB gene was obtained.
- Restriction enzymes XbaI and SalI contained at the end of the PCR fragment obtained through the PCR were treated, and ligation was performed to pDZ (US 9109242 B2) vector treated with restriction enzymes XbaI and SalI.
- metB inactivation cassette was performed by cloning. A cloned pDZ- ⁇ metB recombinant vector was constructed.
- the prepared pDZ- ⁇ metB vector was transformed into ATCC13032 by an electric pulse method, and through a secondary crossover process, ATCC13032 ⁇ metB in which the metB gene was inactivated was obtained on the chromosome.
- the inactivated metB gene was finally confirmed by comparison with ATCC13032 in which the metB gene was not inactivated after PCR using the primers of SEQ ID NOs: 28 and 31.
- nucleotide sequence information of the metY gene was obtained from the National Institutes of Health's GenBank (NIH GenBank), and based on this, a primer containing the N-terminal portion and the linker portion of the metY gene (sequence) Nos. 33 and 34), primers (SEQ ID NOs: 35 and 36) comprising a C-terminal portion and a linker portion were synthesized. Primer sequences are shown in Table 14 below.
- PCR was performed using the primers of SEQ ID NOs: 33 and 34 and SEQ ID NOs: 35 and 36.
- PfuUltra TM high-confidence DNA polymerase (Stratagene) was used as the polymerase, and PCR conditions were repeated 30 times: denaturation at 96° C. for 30 seconds, annealing at 53° C. for 30 seconds, and polymerization at 72° C. for 1 minute.
- a 548 bp amplified gene including the N-terminal portion and linker portion of the metY gene and 550 bp amplified gene including the C-terminal portion and linker portion of the metY gene were obtained.
- PCR was performed using the two amplified genes obtained above as a template, and PCR conditions were denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds, and polymerization at 72°C for 1 minute repeated 10 times, followed by SEQ ID NOs: 33 and 34 was added and the polymerization reaction was repeated 20 more times.
- an inactivation cassette of 1077 bp including the N-terminal-linker-C-terminus of the metY gene was obtained.
- Restriction enzymes XbaI and SalI contained at the end of the PCR fragment obtained through the PCR were treated, and ligation was performed to pDZ (US 9109242 B2) vector treated with restriction enzymes XbaI and SalI.
- the metY inactivation cassette was performed by cloning. A cloned pDZ- ⁇ metY recombinant vector was constructed.
- the prepared pDZ- ⁇ metY vector was transformed into ATCC13032 ⁇ metB strain by an electric pulse method, and through a secondary crossover process, ATCC13032 ⁇ metB ⁇ metY in which the metY gene was inactivated was obtained on the chromosome.
- the inactivated metY gene was finally confirmed by comparison with ATCC13032 in which the metY gene was not inactivated after PCR using the primers of SEQ ID NOs: 7 and 10.
- L-lysine and L-threonine of the lysC gene to the lysC gene SEQ ID NO: 11
- the pDZ-lysC (L377K) vector prepared in Example 7-2 was transferred to ATCC13032 ⁇ metB ⁇ metY strain Transformed by a pulse method, and through a secondary crossover process, Corynebacterium glutamicum ATCC13032 ⁇ metB ⁇ metY lysC (L377K) in which a nucleotide mutation was introduced into the lysC gene on the chromosome was obtained.
- the gene into which the nucleotide mutation was introduced was finally confirmed by comparison with the sequence of the wild-type lysC gene through PCR
- the pDC-gltA (M312I) vector prepared in Example 3-1 was transformed into ATCC13032 ⁇ metB ⁇ metY lysC (L377K) strain by an electric pulse method, and nucleotide mutations in the gltA gene were detected on the chromosome through a secondary crossover process.
- the introduced Corynebacterium glutamicum ATCC13032 ⁇ metB ⁇ metY lysC (L377K) gltA (M312I) was obtained. Whether or not the gltA gene mutation was introduced in the final transformed strain was confirmed that the mutation was introduced in the strain by performing PCR using the primers of SEQ ID NO: 7 and SEQ ID NO: 8 and then analyzing the nucleotide sequence.
- the strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of the following medium (inoculation loop), and cultured with shaking at 37° C. for 20 hours at 200 rpm.
- the concentration of O-acetyl homoserine was analyzed using HPLC, and the analyzed concentrations were shown in Table 15 below.
- strain name O-AH (g/L) ATCC13032 ⁇ metB ⁇ metY lysC(L377K) 0.70 ATCC13032 ⁇ metB ⁇ metY lysC (L377K) gltA (M312I) 0.92
- the ATCC13032 ⁇ metB ⁇ metY lysC(L377K) gltA(M312I) strain introduced with gltA(M312I) mutation was O-acetyl-L-homo compared to the ATCC13032 ⁇ metB ⁇ metY lysC(L377K) strain. It was confirmed that the serine production capacity was improved by 31%.
- amino acid at position 312 in the amino acid sequence of gltA is an important position for gltA enzyme activity.
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Abstract
Description
서열번호 | 명칭 | 서열(5'→3') |
5 | pCR-gltA F | CTAATCTCGAGGTCACCCATGTTTGAAAGG |
6 | pCR-gltA R | TCGCGAGGAGCGCTAAAACCGGTTGAT |
7 | gltA F | CAATGCTGGCTGCGTACGC |
8 | gltA R | CTCCTCGCGAGGAACCAACT |
9 | gltA M312I Up F | GTGAATTCGAGCTCGGTACCCGCGGGAATCCTGCGTTACCGC |
10 | gltA M312I Up R | TGTAAACGCGGTGTCCGAAGCCGATGAGGCGGACGCCGTCTT |
11 | gltA M312I Down F | AAGACGGCGTCCGCCTCATCGGCTTCGGACACCGCGTTTACA |
12 | gltA M312I Down R | GGTCGACTCTAGAGGATCCCCTTAGCGCTCCTCGCGAGGAAC |
13 | leuA F | ACCGAAATTGGCTTGGGTGCCAGCCCAGCTGATGCCTAC |
14 | leuA R | AAGCTTGCATGCCTGCAGCTTAAAGTCACCTACGTTTTGTAC |
균주명 | 류신 (g/L) |
ATCC13032 | 0.87 |
ATCC13032/pTOPO_gltA(mt)3142 | 1.54 |
균주명 | 류신 (g/L) |
ATCC13032 | 0.87 |
ATCC13032_gltA_M312I | 1.25 |
균주명 | 류신 (g/L) |
ATCC13032 | 0.87 |
ATCC13032_leuA_(R558H, G561D) : CJL-8100 | 2.7 |
CJL8100_gltA_M312I : CA13-8104 | 3.0 |
균주명 | 류신 (g/L) |
CJL8100 | 2.6 |
CJL8100/pTOPO_gltA(mt)3142 | 2.8 |
균주명 | 리신 (g/L) |
CJ3P | 8.8 |
CJ3P::gltA(M312I) | 11.2 |
균주명 | 발린 (g/L) |
KCCM11201P -gltA(M312I) | 2.6 |
KCCM11201P -gltA(M312I) | 2.8 |
균주명 | L-이소류신 (g/L) | ||||
배치 1 | 배치 2 | 배치 3 | 평균 | ||
대조군 | KCCM11248P | 1.2 | 1.6 | 1.4 | 1.4 |
실험군 | KCCM11248P-gltA(M312I) | 2.0 | 1.8 | 1.8 | 1.9 |
서열번호 | 명칭 | 서열(5'→3') |
15 | lysC up F | tcctctagaGCTGCGCAGTGTTGAATACG |
16 | lysC up R | TGGAAATCttTTCGATGTTCACGTTGACAT |
17 | lysC down F | ACATCGAAaaGATTTCCACCTCTGAGATTC |
18 | lysC down R | gactctagaGTTCACCTCAGAGACGATTA |
서열번호 | 명칭 | 서열(5'→3') |
19 | hom up F | tcctctagaCTGGTCGCCTGATGTTCTAC |
20 | hom up R | CACGATCAGATGTGCATCATCAT |
21 | hom down F | ATGATGATGCACATCTGATCGTG |
22 | hom down R | gactctagaTTAGTCCCTTTCGAGGCGGA |
서열번호 | 명칭 | 서열(5'→3') |
23 | ilvA V323A up F | ACGGATCCCAGACTCCAAAGCAAAAGCG |
24 | ilvA V323A up R | ACACCACGgCAGAACCAGGTGCAAAGGACA |
25 | ilvA V323A down F | CTGGTTCTGcCGTGGTGTGCATCATCTCTG |
26 | ilvA V323A down R | ACGGATCCAACCAAACTTGCTCACACTC |
균주명 | L-이소류신 (g/L) | ||||
배치 1 | 배치 2 | 배치 3 | 평균 | ||
대조군 | ATCC13032::-hom(R407H)-lysC(L377K)/pECCG117-ilvA(V323A) | 4.4 | 4.5 | 4.3 | 4.4 |
실험군 | ATCC13032::hom(R407H)-lysC(L377K)- gltA(M312I)/pECCG117-ilvA(V323A) | 6.3 | 6.7 | 5.9 | 6.3 |
서열번호 | 명칭 | 서열(5'→3') |
서열번호 28 | metB_N_del F | TCTAGACGCCCGCATACTGGCTTC |
서열번호 29 | metB_N_del R | CCCATCCACTAAACTTAAACAGATGTGATCGCCCGGC |
서열번호 30 | metB_C_del F | TGTTTAAGTTTAGTGGATGGGGAAGAACCACCCAGGCC |
서열번호 31 | metB_C_del R | GTCGACCAATCGTCCAGAGGGCG |
서열번호 | 명칭 | 서열(5'→3') |
서열번호 33 | metY_N_del F | TCTAGACCATCCTGCACCATTTAG |
서열번호 34 | metY_N_del R | CCCATCCACTAAACTTAAACACGCTCCTGCCAGGTTC |
서열번호 35 | metY_C_del F | TGTTTAAGTTTAGTGGATGGGCTTGGTACGCAACCAAGG |
서열번호 36 | metY_C_del R | GTCGACGATTGCTCCGGCTTCGG |
균주명 | O-AH (g/L) |
ATCC13032 △metB △metY lysC(L377K) | 0.70 |
ATCC13032 △metB △metY lysC(L377K) gltA(M312I) | 0.92 |
Claims (14)
- 서열번호 1의 아미노산 서열로 구성되는 폴리펩티드의 N-말단으로부터 312번째 위치에 상응하는 아미노산이 다른 아미노산으로 치환된, 시트레이트 신타아제(citrate synthase) 활성을 가지는 변이형 폴리펩티드.
- 제1항에 있어서, 상기 312번째 위치에 상응하는 아미노산은 이소류신 (isoleucine)으로 치환된, 변이형 폴리펩티드.
- 제1항에 있어서, 상기 변이형 폴리펩티드는 서열번호 1의 아미노산 서열과 80% 이상, 100% 미만의 서열 상동성을 가지는, 변이형 폴리펩티드.
- 제1항에 있어서, 상기 변이형 폴리펩티드는 서열번호 3의 아미노산 서열로 이루어진 것인, 변이형 폴리펩티드.
- 제1항 내지 제4항 중 어느 한 항의 변이형 폴리펩티드를 코딩하는 폴리뉴클레오티드.
- 제5항의 폴리뉴클레오티드를 포함하는 벡터.
- 제1항 내지 제4항 중 어느 한 항의 변이형 폴리펩티드, 상기 변이형 폴리펩티드를 코딩하는 폴리뉴클레오티드 또는 상기 폴리뉴클레오티드를 포함하는 벡터를 포함하는, L-아미노산을 생산하는 미생물.
- 제7항에 있어서, 상기 L-아미노산은 류신, 리신, 발린, 이소류신 및 O-아세틸 호모세린으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것인, 미생물.
- 제7항에 있어서, 상기 미생물은 코리네박테리움 속(Corynebacterium sp.)인, L-아미노산을 생산하는 미생물.
- 제9항에 있어서, 상기 코리네박테리움 속 미생물은 코리네박테리움 글루타미쿰(Corynebacterium glutamicum)인, L-아미노산을 생산하는 미생물.
- 제1항 내지 제4항 중 어느 한 항의 변이형 폴리펩티드, 상기 변이형 폴리펩티드를 코딩하는 폴리뉴클레오티드 또는 상기 폴리뉴클레오티드를 포함하는 벡터를 포함하는 미생물을 배지에서 배양하는 단계를 포함하는, L-아미노산 생산 방법.
- 제11항에 있어서, 상기 방법은 배양된 배지 또는 미생물에서 L-아미노산을 회수하는 단계를 추가로 포함하는 것인, L-아미노산 생산 방법.
- 제12항에 있어서, 상기 상기 L-아미노산은 류신, 리신, 발린, 이소류신 및 O-아세틸 호모세린으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것인, L-아미노산 생산 방법.
- 서열번호 1의 아미노산 서열로 구성되는 폴리펩티드의 N-말단으로부터 312번째 위치에 상응하는 아미노산이 다른 아미노산으로 치환된, 시트레이트 신타아제 활성을 가지는 변이형 폴리펩티드, 상기 변이형 폴리펩티드를 코딩하는 폴리뉴클레오티드 또는 상기 폴리뉴클레오티드를 포함하는 벡터를 포함하는, L-아미노산을 생산하는 미생물의 L-아미노산의 생산용도.
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US17/779,909 US20230040524A1 (en) | 2020-01-30 | 2020-08-04 | Novel modified polypeptide with attenuated activity of citrate synthase and method for producing l-amino acid using the same |
AU2020426558A AU2020426558A1 (en) | 2020-01-30 | 2020-08-04 | Novel Modified Polypeptide With Attenuated Activity Of Citrate Synthase And Method For Producing L-Amino Acid Using The Same |
CN202080090211.2A CN115175993B (zh) | 2020-01-30 | 2020-08-04 | 柠檬酸合酶活性减弱的新型修饰多肽及使用其生产l-氨基酸的方法 |
BR112022012576A BR112022012576A2 (pt) | 2020-01-30 | 2020-08-04 | Polipeptídeo modificado, polinucleotídeo, vetor, micro-organismo que produz um l-aminoácido, método para produzir um l-aminoácido e uso do dito micro-organismo |
MX2022008211A MX2022008211A (es) | 2020-01-30 | 2020-08-04 | Nuevo polipeptido modificado con actividad atenuada de citrato sintasa y procedimiento de produccion de l-aminoacido mediante el uso del mismo. |
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EP20916402.9A EP4047086A4 (en) | 2020-01-30 | 2020-08-04 | NEW MUTANT POLYPEPTIDE WITH REDUCED ACTIVITY OF CITRATE SYNTHASE AND L-AMINO ACID PRODUCTION PROCESS THEREFORE |
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