WO2022245000A1 - Micro-organisme produisant un acide aminé présentant une activité de protéine agl améliorée, et procédé de production d'acide aminé l'utilisant - Google Patents

Micro-organisme produisant un acide aminé présentant une activité de protéine agl améliorée, et procédé de production d'acide aminé l'utilisant Download PDF

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WO2022245000A1
WO2022245000A1 PCT/KR2022/005843 KR2022005843W WO2022245000A1 WO 2022245000 A1 WO2022245000 A1 WO 2022245000A1 KR 2022005843 W KR2022005843 W KR 2022005843W WO 2022245000 A1 WO2022245000 A1 WO 2022245000A1
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agl
amino acid
microorganism
protein
maltose
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PCT/KR2022/005843
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Korean (ko)
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변효정
배현원
이한형
신용욱
김병수
박상민
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씨제이제일제당 (주)
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Publication of WO2022245000A1 publication Critical patent/WO2022245000A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • 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
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/0102—Alpha-glucosidase (3.2.1.20)

Definitions

  • the present application relates to an amino acid-producing microorganism with enhanced Agl protein activity and an amino acid production method using the same.
  • Amino acids are used in animal feed, pharmaceutical and cosmetic industries, and are mainly produced by fermentation using strains of the genus Corynebacterium or strains of the genus Escherichia. For the production of amino acids, various studies such as development of high-efficiency production strains and fermentation process technology are being conducted.
  • An object of the present application is to provide an amino acid-producing microorganism with enhanced Agl protein activity and an amino acid production method using the same.
  • the present application provides microorganisms of the genus Corynebacterium that produce amino acids with enhanced Agl protein activity.
  • the present application provides a method for producing amino acids, comprising culturing the microorganism in a medium.
  • the present application provides a method for producing the microorganism.
  • the present application provides a composition for amino acid production comprising the microorganism or its culture.
  • the microorganism of the present application can be usefully used for amino acid production.
  • One aspect of the present application provides a microorganism of the genus Corynebacterium that produces amino acids with enhanced Agl protein activity.
  • Al protein in this application refers to a kind of protein having glucosidase activity.
  • glucosidase is an enzyme that catalyzes the cleavage of glycosidic bonds and is involved in breaking down carbohydrates into monomers.
  • the glucosidase may be an enzyme classified as EC 3.2.1, but is not limited thereto.
  • the Agl protein of the present application may have alpha-glucosidase activity.
  • alpha-glucosidase is a type of glucosidase that decomposes sugar into glucose, and means an enzyme capable of decomposing an alpha-glycosidic bond.
  • the alpha-glycosidic bond may be, for example, ⁇ (1 ⁇ 4), ⁇ (1 ⁇ 6), ⁇ (1 ⁇ 2), etc., but is not limited thereto.
  • the alpha-glucosidase of the present application may have an activity of decomposing carbohydrates into glucose. In another embodiment, the alpha-glucosidase of the present application may have an activity of cleaving ⁇ (1 ⁇ 4) and/or ⁇ (1 ⁇ 6) bonds, but is not limited thereto.
  • the Agl protein may include any protein capable of enhancing amino acid production.
  • the Agl protein of the present application may be an enzyme capable of degrading at least one selected from maltose and maltose isomers. Therefore, the microorganism of the present application may use at least one selected from maltose and maltose isomers as a carbon source. The microorganism may thus have increased sugar consumption or sugar utilization of maltose or isomers of maltose.
  • the Agl protein of the present application comprises, has, consists of, or consists essentially of SEQ ID NO: 1 or an amino acid sequence having at least 70% homology or identity thereto, or consisting essentially of the amino acid sequence.
  • the amino acid sequence of the Agl protein of the present application may be a protein sequence having a glucosidase activity encoded by the agl gene.
  • the amino acid sequence may be obtained from various databases such as NCBI's GenBank, which is a known database, but is not limited thereto.
  • the Agl protein of the present application may be derived from Bifidobacterium adolescentis , but is not limited thereto, and a sequence having the same activity as the above amino acid sequence may be included without limitation.
  • the Agl protein of the present application was described as a protein containing SEQ ID NO: 1, meaningless sequence additions to and from the amino acid sequence of SEQ ID NO: 1, mutations that may occur naturally, or silent mutations thereof ), and it is apparent to those skilled in the art that the Agl protein of the present application corresponds to the protein having the same or corresponding activity as the protein containing the amino acid sequence.
  • the Agl protein of the present application comprises the amino acid sequence of SEQ ID NO: 1, or at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89% of the amino acid sequence of SEQ ID NO: 1 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity.
  • any amino acid sequence having the above homology or identity and exhibiting efficacy corresponding to the protein is included within the scope of the present application even if some sequences have amino acid sequences that are deleted, modified, substituted or added.
  • polypeptide or protein comprising the amino acid sequence described in a specific sequence number', 'a polypeptide or protein consisting of the amino acid sequence described in a specific sequence number', or 'a polypeptide or protein having an amino acid sequence described in a specific sequence number'
  • a protein having an amino acid sequence in which some sequence is deleted, modified, substituted, conservatively substituted, or added can also be used in this application. is self-explanatory. For example, it is a case of adding a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation thereof, or a conservative substitution to the N-terminus and/or C-terminus of the amino acid sequence. .
  • the "conservative substitution” refers to the substitution of one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitutions can generally occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the polypeptide.
  • the term 'homology' or 'identity' refers to the degree of identical or similarity between two given amino acid sequences or base sequences and may be expressed as a percentage.
  • the terms homology and identity are often used interchangeably.
  • Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard alignment algorithms, together with default gap penalties established by the program used. Substantially homologous or identical sequences are generally the entire sequence or a portion corresponding to at least about 50%, 60%, 70%, 80% or 90% of the full-length and intermediate or It can hybridize under highly stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing common codons or codons considering codon degeneracy in polynucleotides.
  • GAP program can define the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids).
  • the 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 penalty of 0.10 for each symbol in each gap (or 10 gap opening penalty, 0.5 gap extension penalty); and (3) no penalty for end gaps.
  • the microorganisms in which the activity of the Agl protein of the present application is enhanced include Agl protein; polynucleotides encoding them; And it may be a microorganism containing one or more of the vectors containing the polynucleotide.
  • polynucleotide is a DNA strand of a certain length or longer as a polymer of nucleotides in which nucleotide monomers are connected in a long chain shape by covalent bonds.
  • polynucleotide sequence encoding the Agl protein of the present application may also be referred to as " agl gene” or " aglB gene", which may include the polynucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 1 above.
  • polynucleotide various modifications may be made to the coding region within a range that does not change the amino acid sequence of the polypeptide due to codon degeneracy or in consideration of codons preferred in organisms in which the polypeptide is to be expressed.
  • the polynucleotide may include, consist of, or consist essentially of SEQ ID NO: 2 or a polynucleotide sequence having 70% or more homology or identity thereto, but is not limited thereto.
  • the polynucleotide has 70% or more, 80% or more, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more homology or identity with SEQ ID NO: 2 More specifically, it may consist of a nucleotide sequence of 99% or more, but is not limited thereto.
  • polynucleotide of the present application may include without limitation any sequence capable of hybridizing with a probe, for example, a sequence complementary to all or part of the polynucleotide base sequence under stringent conditions.
  • stringent condition means a condition that allows specific hybridization between polynucleotides. Such conditions are specifically described in the literature (eg, J. Sambrook et al., ibid.).
  • polynucleotides with high homology or identity 40% or more, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, more specifically 99% or more 60 ° C., 1 ⁇ SSC, 0.1 washing conditions for hybridization under conditions in which polynucleotides having the same identity or identity do not hybridize and polynucleotides having less homology or identity do not hybridize, or washing conditions for typical southern hybridization Washing once, specifically 2 to 3 times, at a salt concentration and temperature corresponding to % SDS, specifically 60 ° C, 0.1 ⁇ SSC, 0.1% SDS, more specifically 68 ° C, 0.1 ⁇ SSC, 0.1% SDS may be a condition.
  • Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization.
  • complementary is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine.
  • the polynucleotides of the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to 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 according to the purpose.
  • Appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotide and the degree of complementarity, parameters well known in the art (J. Sambrook et al., supra).
  • vector refers to a DNA preparation for inserting a polynucleotide encoding an Agl protein into a host chromosome or a suitable expression control region (or expression control sequence) so as to express a target polypeptide in a suitable host operably. It may include a DNA preparation containing the nucleotide sequence of the polynucleotide encoding the linked Agl protein.
  • the expression control region may include a promoter capable of initiating transcription, an arbitrary operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating termination of transcription and translation. After transformation into a suitable host cell, the vector can replicate or function independently of the host genome and can integrate into the genome itself.
  • the vector of the present application may be an expression vector for expressing the Agl protein of the present application in a host cell, but is not limited thereto.
  • the vector of the present application may be an insertion vector for inserting the polynucleotide encoding the Agl protein of the present application into a chromosome, but is not limited thereto. 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.
  • a selection marker for determining whether the chromosome is inserted may be further included.
  • the selectable marker is used to select cells transformed with a vector, that is, to determine whether a target nucleic acid molecule has been inserted, and can exhibit selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface polypeptide expression. markers may be used. In an environment treated with a selective agent, only cells expressing the selectable marker survive or exhibit other expression traits, so transformed cells can be selected.
  • the insertion vector may not contain an origin of replication required for replication in the transformed cell.
  • Vectors used in the present application are not particularly limited, and any vectors known in the art may be used.
  • Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages.
  • pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ-based, pBR-based, and pUC-based plasmid vectors , pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, pET-based, etc. can be used.
  • pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.
  • transformation in the present application means introducing a vector containing a polynucleotide encoding the protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell.
  • the transformed polynucleotide may be inserted into and located in the chromosome of the host cell or located outside the chromosome, as long as it can be expressed in the host cell.
  • the polynucleotide includes DNA and/or RNA encoding the protein.
  • the polynucleotide may be introduced in any form as long as it can be introduced into a host cell and expressed.
  • the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a genetic construct containing all elements required 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 necessary for expression in the host cell, but is not limited thereto.
  • operably linked in the present application means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the Agl protein of the present application and the gene sequence are functionally linked.
  • the method of transforming the vector of the present application includes any method of introducing nucleic acid into a cell, and can be performed by selecting an appropriate standard technique as known in the art according to the host cell. For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposomal method, and lithium acetate -DMSO method, etc., but is not limited thereto.
  • microorganism or “strain” includes both wild-type microorganisms and naturally or artificially genetically modified microorganisms, and is derived from causes such as insertion of foreign genes or enhancement or inactivation of endogenous gene activity.
  • a microorganism in which a specific mechanism is weakened or enhanced it may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein or product.
  • the microorganism of the present application may be a microorganism having an amino acid production ability.
  • the microorganism of the present application may be one in which the Agl protein of the present application is introduced into a microorganism naturally having the ability to produce Agl protein or amino acid, or a mother strain having no ability to produce Agl protein or amino acid.
  • the microorganism of the present application is a cell or microorganism in which the activity of the Agl protein is enhanced by being transformed with a polynucleotide encoding the Agl protein of the present application. It may include all microorganisms capable of producing amino acids, including.
  • the microorganism of the present application may be a natural wild-type microorganism or a recombinant strain whose amino acid production ability is increased by enhancing the activity of the Agl protein by introducing a polynucleotide encoding the Agl protein of the present application into a natural wild-type microorganism or a microorganism producing amino acids.
  • the recombinant strain with increased amino acid production ability may be a natural wild-type microorganism or a microorganism with increased amino acid production ability compared to a microorganism in which the activity of the Agl protein of the present application is not enhanced or does not express the Agl protein, but is not limited thereto. not.
  • a microorganism that does not express the Agl protein of the present application which is a target strain for comparing the increase in the amino acid production ability, may be KCCM11016P (Korean Patent No. 10-0159812) or KCCM10770P (US 9109242 B2), Not limited to this.
  • the recombinant strain with increased production capacity may have an amino acid production capacity increased by about 0.001% or more or 0.01% or more compared to the amino acid production capacity of the parent strain or non-modified microorganism before mutation, but the production of the parent strain or non-modified microorganism before mutation As long as it has an increased amount of + value compared to the ability, it is not limited to this.
  • the term “about” includes all ranges of ⁇ 0.5, ⁇ 0.4, ⁇ 0.3, ⁇ 0.2, ⁇ 0.1, etc., and includes all ranges equivalent to or similar to the ranges following the term “about”. Not limited.
  • the term "unmodified microorganism” does not exclude strains containing mutations that may occur naturally in microorganisms, and are wild-type strains or wild-type strains themselves, or are genetically modified by natural or artificial factors. It may mean a strain before change.
  • the unmodified microorganism may refer to a strain without or before the introduction of the Agl protein of the present application.
  • the "unmodified microorganism” may be used interchangeably with "strain before transformation", “microorganism before transformation”, “non-mutated strain”, “unmodified strain", “non-mutated microorganism” or "reference microorganism".
  • microorganism of the present application may be a microorganism of the genus Corynebacterium.
  • "microbes of the genus Corynebacterium” may include all microorganisms of the genus Corynebacterium. Specifically, Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ( Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Corynebacterium efficiens ( Corynebacterium efficiens ) .
  • Corynebacterium striatum Corynebacterium striatum
  • Corynebacterium ammoniagenes Corynebacterium ammoniagenes
  • Corynebacterium pollutisoli Corynebacterium pollutisoli
  • Corynebacterium imitans Corynebacterium imitans
  • Corynebacterium testudino It may be Corynebacterium testudinoris or Corynebacterium flavescens , and more specifically Corynebacterium glutamicum.
  • the term "enhancement" of polypeptide activity means that the activity of the polypeptide is increased relative to the intrinsic activity.
  • the enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase.
  • activation, enhancement, upregulation, overexpression, and increase may include those that exhibit an activity that was not originally possessed, or those that exhibit enhanced activity compared to intrinsic activity or activity before modification.
  • a microorganism expressing the Agl protein or a microorganism into which the Agl protein is introduced can also be referred to as a microorganism in which the activity of the Agl protein is enhanced.
  • the "intrinsic activity” refers to the activity of a specific polypeptide originally possessed by a parent strain or unmodified microorganism before transformation when a character is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activation before transformation”. "Enhancement”, “upregulation”, “overexpression” or “increase” of the activity of a polypeptide compared to the intrinsic activity means that the activity and/or concentration (expression amount) is improved.
  • the enhancement can be achieved by introducing a foreign polypeptide or by enhancing the activity and/or concentration (expression level) of an endogenous polypeptide. Whether or not the activity of the polypeptide is enhanced can be confirmed from an increase in the activity level, expression level, or amount of a product released from the corresponding polypeptide.
  • Enhancement of the activity of the polypeptide can be applied by various methods well known in the art, and is not limited as long as the activity of the target polypeptide can be enhanced compared to the microorganism before transformation. Specifically, it may be using genetic engineering and / or protein engineering, which is well known to those skilled in the art, which is a routine method of molecular biology, but is not limited thereto (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).
  • modification of the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide eg, modification of the polynucleotide sequence of the polypeptide gene to encode the modified polypeptide to enhance the activity of the polypeptide
  • It may be a combination of two or more selected from 1) to 8), but is not particularly limited thereto.
  • amino acid refers to a compound in which an amino group and a carboxy group are bonded to the same carbon atom, and includes proteinaceous amino acids that are the basic building blocks of proteins and non-proteins that are not encoded by the genetic code of organisms. Includes all non-proteinogenic amino acids.
  • an amino acid of the present application may be an L-amino acid.
  • the L-amino acids are, for example, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine Any one selected from the group consisting of L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine may be ideal
  • the L-amino acid may be, as a more specific example, L-lysine, but is not limited thereto.
  • an amino acid of the present application may be a non-proteinaceous amino acid.
  • the non-proteinaceous amino acid may be, for example, one or more selected from the group consisting of citrulline, ornithine, and GABA, but is not limited thereto.
  • Another aspect of the present application provides a method for producing amino acids comprising culturing the microorganism of the present application in a medium.
  • the microorganisms are as described above.
  • any medium and other culture conditions used for culturing the microorganism of the present application may be used without particular limitation as long as it is a medium used for culturing a common microorganism of the genus Corynebacterium.
  • the microorganism of the present application may be used as a suitable carbon source, It can be cultured while controlling temperature, pH, etc. under aerobic or anaerobic conditions in a conventional medium containing a nitrogen source, phosphorus, inorganic compounds, amino acids and/or vitamins.
  • the carbon source includes carbohydrates such as glucose, fructose, sucrose, maltose and isomers thereof; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; Amino acids such as glutamic acid, methionine, lysine, and the like may be included.
  • natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sorghum pomace and corn steep liquor can be used, specifically glucose and sterilized pretreated molasses (i.e. converted to reducing sugar).
  • Carbohydrates such as molasses
  • other carbon sources in an appropriate amount may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.
  • the carbon source that may be included in the culture medium of the present application may include maltose or isomers of maltose. More specifically, it may include one or more selected from glucose, maltose (maltose), and maltose isomers (isomaltose), or a combination of two or more, but is not limited thereto.
  • nitrogen source examples include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, etc., organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or degradation products thereof, defatted soybean cake or degradation products thereof, etc. can be used These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.
  • inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate
  • Amino acids such as glutamic acid, methionine, glutamine, etc.
  • organic nitrogen sources such as peptone, NZ-amine,
  • the number of persons may include monopotassium phosphate, dipotassium phosphate, or a sodium-containing salt corresponding thereto.
  • the inorganic compound sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like may be used.
  • the culture medium may contain metal salts such as magnesium sulfate or iron sulfate necessary for growth.
  • essential growth substances such as amino acids and vitamins may be used in addition to the above substances.
  • precursors suitable for the culture medium may be used. The above raw materials may be added in a batchwise or continuous manner by a method suitable for the culture during the culture process, but is not limited thereto.
  • the pH of the culture may be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the culture in an appropriate manner during the culture of the microorganism.
  • an antifoaming agent such as a fatty acid polyglycol ester.
  • oxygen or oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen or carbon dioxide gas may be injected without gas injection or nitrogen, hydrogen or carbon dioxide gas may be injected to maintain the anaerobic and non-aerobic state, but is limited thereto It doesn't work.
  • the temperature of the culture may be 25 °C to 40 °C, more specifically 28 °C to 37 °C, but is not limited thereto.
  • the culturing period may be continued until a desired production amount of a useful substance is obtained, and specifically may be 1 hour to 100 hours, but is not limited thereto.
  • the amino acid production method of the present application may include recovering amino acids from the microorganism or medium.
  • a desired amino acid may be recovered from the medium using a suitable method known in the art.
  • a suitable method known in the art.
  • centrifugation, filtration, treatment with a precipitating agent for crystallized protein salting out method
  • extraction ultrasonic disruption
  • ultrafiltration dialysis method
  • molecular sieve chromatography gel filtration
  • adsorption chromatography ion exchange chromatography
  • affinity chromatography affinity chromatography
  • the method may include additional purification steps.
  • the purification process may use a suitable method known in the art.
  • Another aspect of the present application provides a method for producing an amino acid-producing microorganism of the genus Corynebacterium, comprising enhancing the activity of the Agl protein in the microorganism of the genus Corynebacterium.
  • the step of enhancing the activity of the Agl protein may be a step of modifying a microorganism of the genus Corynebacterium to express the Agl protein, as described above.
  • composition for amino acid production comprising a microorganism of the genus Corynebacterium or a culture thereof having enhanced Agl protein activity.
  • composition may include maltose and/or maltose isomers, but is not limited thereto.
  • the Agl protein, microorganisms of the genus Corynebacterium whose activity is enhanced, and amino acids are as described above.
  • Another aspect of the present application provides a use of the Agl protein of the present application to increase amino acid production.
  • Another aspect of the present application provides a use of a microorganism of the genus Corynebacterium in which the activity of the Agl protein of the present application is enhanced, for amino acid production.
  • the Agl protein, microorganisms of the genus Corynebacterium whose activity is enhanced, and amino acids are as described above.
  • candidate proteins were selected using the NCBI Protein Database.
  • the protein derived from Bifidobacterium adolescentis has the amino acid sequence of SEQ ID NO: 1.
  • Information on the gene encoding the protein and the surrounding nucleic acid sequence was obtained from NIH GenBank, and the gene has a nucleotide sequence of SEQ ID NO: 2.
  • PCR SolTM Pfu-X DNA polymerase
  • the CJ7 promoter Korean Patent No.
  • PCR was amplified using the genome of Corynebacterium ammoniagenes as a template.
  • the obtained gene fragments were ligated into vector pECCG117 (Korean Patent No. 0057684) digested with XbaI restriction enzyme and subjected to Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix ) method, E. coli DH5 ⁇ was transformed and plated on LB solid medium containing kanamycin (25 mg/L).
  • PCR was performed using primers of SEQ ID NOs: 7 and 8 to select colonies transformed with the vector linked to the gene of interest and pECCG117.
  • a plasmid was obtained from the selected colonies using a commonly known plasmid extraction method, and was named pECCG117-Pcj7-agl (Bad).
  • the protein derived from Bifidobacterium breve has the amino acid sequence of SEQ ID NO: 9.
  • Information on the gene encoding the protein and its surrounding nucleic acid sequence was obtained from NIH GenBank, and the gene has a nucleotide sequence of SEQ ID NO: 10.
  • the gene of SEQ ID NO: 10 was amplified by performing PCR using the primers of SEQ ID NOs: 11 and 12 using the genome of Bifidobacterium breve (KCTC3220) as a template.
  • a plasmid was obtained from the obtained gene fragment by the method described above, and it was named pECCG117-Pcj7-agl(Bbr).
  • the protein derived from Saccharomyces cerevisiae has the amino acid sequence of SEQ ID NO: 13.
  • Information on the gene encoding the protein and the surrounding nucleic acid sequence was obtained from NIH GenBank, and the gene has a nucleotide sequence of SEQ ID NO: 14. Based on the sequence, the gene of SEQ ID NO: 14 was amplified by performing PCR (SolTM Pfu-X DNA polymerase) using the primers of SEQ ID NOs: 15 and 16 using the genome of Saccharomyces cerevisiae as a template. .
  • a plasmid was obtained from the obtained gene fragment using the method described above, and it was named pECCG117-Pcj7-agl(Sce).
  • the protein derived from Corynebacterium glutamicum has the amino acid sequence of SEQ ID NO: 17.
  • Information on the gene encoding the protein and the surrounding nucleic acid sequence was obtained from NIH GenBank, and the gene has a nucleotide sequence of SEQ ID NO: 18.
  • PCR SolTM Pfu-X DNA polymerase
  • the control pECCG117 vector and the above-constructed four vectors (pECCG117-Pcj7-agl (Bad), pECCG117-Pcj7-agl (Bbr), pECCG117-Pcj7-agl (Sce) and pECCG117-Pcj7-agl (cgl)) Transformation of Corynebacterium glutamicum ATCC13032 by electroporation (Van der Rest et al., Appl. Microbiol. Biotecnol. 52:541-545, 1999), kanamycin (25 mg/L) It was smeared on a complex plate medium containing this, and colonies were obtained after incubation at 30 ° C for 48 hours.
  • the composite plate medium composition used above was as follows:
  • Glucose 10 g Peptone 10 g, Beef extract 5 g, Yeast extract 5 g, Brain Heart Infusion 18.5 g, NaCl 2.5 g, Urea 2 g, Sorbitol 91 g, Agar 20 g, Kanamycin 25 mg (based on 1 liter of distilled water)
  • each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of a seed medium containing 25 mg/L of kanamycin, and cultured at 32° C. for 24 hours with shaking at 200 rpm.
  • composition of the selective medium used above is as follows.
  • the 13032::pECCG-Pcj7-agl(Bad) strain into which the Bifidobacterium adolescentis-derived protein was introduced showed the best maltose utilization ability.
  • SEQ ID NO: 21 and PCR were performed using the primers of 22 to amplify the gene of SEQ ID NO: 2.
  • the primers of SEQ ID NOs: 23 and 24 the genome of Corynebacterium ammoniagenes as a template, the genome of Corynebacterium glutamicum using the CJ7 promoter (US 7662943 B2) and the primers of SEQ ID NOs: 25 and 26 Using as a template, the promoter of gapA (NCgl1526) was amplified by PCR.
  • the promoter obtained above, the gene fragment derived from Bifidobacterium adolescentis, and the vector pDZTn (US 8323933 B2) digested with SpeI restriction enzyme were prepared by Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY). 2009, NEBuilder HiFi DNA Assembly Master Mix) method, and then transformed into E. coli DH5 ⁇ and plated on LB solid medium containing kanamycin (25 mg/L). PCR was performed using the primers of SEQ ID NOs: 27 and 28 to select colonies transformed with the vector in which the target gene and pDZTn were ligated.
  • Plasmids were obtained using a commonly known plasmid extraction method using the selected colonies, and these plasmids were named pDZTn-Pcj7-agl and pDZTn-PgapA-agl in that order.
  • Electroporation Appl. Microbiol Biotechnol. PCR and sequencing were performed using primers of SEQ ID NOs: 29 and 30 capable of amplifying adjacent regions including the site where the gene was inserted, and the corresponding genetic manipulation was confirmed.
  • the strains thus obtained were named Corynebacterium glutamicum KCCM11016P::Pcj7-agl and KCCM11016P::PgapA-agl in order.
  • Example 4 In medium containing glucose agl Comparison of L-lysine production ability of transgenic strains
  • KCCM11016P::Pcj7-agl, KCCM11016P::PgapA-agl strains and control KCCM11016P were cultured in the following manner, and cell mass, sugar consumption, and lysine production were compared.
  • each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of seed medium, and cultured at 30° C. for 20 hours with shaking at 200 rpm.
  • a 250 ml corner-baffle flask containing 24 ml of production medium was inoculated with 1 ml of the seed culture and incubated at 37° C. for 42 hours with shaking at 200 rpm. After completion of the culture, the production of L-lysine was measured by HPLC.
  • Glucose 45 g (NH4)2SO4 15 g, soy protein 10 g, molasses containing 50% sugar 10 g, KH2PO4 0.55 g, MgSO4 7H2O 0.6 g, biotin 0.9 mg, thiamine hydrochloride 4.5 mg, calcium-pantothenic acid 4.5 mg, nicotine Amide 30 mg, MnSO4 9 mg, FeSO4 9 mg, ZnSO4 0.45 mg, CuSO4 0.45 mg, CaCO3 30 g (based on 1 liter of distilled water).
  • KCCM11016P Comparison of L-lysine production capacity (KCCM11016P) strain OD 562 Consumed sugar (g/L) Lysine production (g/L) Lysine yield (%) KCCM11016P 33.6 50.0 12.1 24.2 KCCM11016P::Pcj7-agl 33.0 50.0 12.4 24.8 KCCM11016P::PgapA-agl 33.1 50.0 12.3 24.6
  • KCCM11016P::Pcj7-agl and KCCM11016P::PgapA-agl strains with enhanced Agl protein activity of the present application showed increased lysine yield compared to the control strain KCCM11016P.
  • Example 5 In medium containing maltose agl Comparison of L-lysine production ability of transgenic strains
  • KCCM11016P::Pcj7-agl, KCCM11016P::PgapA-agl strains and control KCCM11016P were cultured in the same manner as in Example 4, but 45 g of glucose in the production medium was replaced with maltose by 1 g, 5 g, and 20 g, thereby increasing cell mass and sugar consumption. performance and lysine production ability were compared.
  • Example 6 KCCM10770P strain in maltose containing medium agl Confirmation of gene introduction effect
  • Example 3 Using the method described in Example 3, a strain in which the PgapA-agl gene was inserted at the transposon position on the genome of the Corynebacterium glutamicum KCCM10770P (US 9109242 B2) strain was obtained. The strain thus obtained was named Corynebacterium glutamicum KCCM10770P::PgapA-agl.
  • KCCM10770P::PgapA-agl strain and control KCCM10770P were cultured, and cell mass, sugar consumption, and lysine production were compared.
  • 45 g of glucose in the production medium was replaced with 1 g, 5 g, and 20 g of maltose.
  • the experiment was repeated three times, and the culture results (average values) are shown in Table 10.
  • the KCCM10770P As shown in Table 10, the KCCM10770P :: PgapA-agl strain with enhanced Agl protein activity of the present application showed an increased lysine yield compared to the control strain KCCM10770P in a production medium containing maltose.
  • Example 7 In a medium containing maltose isomers agl Comparison of L-lysine production ability of transgenic strains
  • KCCM11016P::PgapA-agl, KCCM10770P::PgapA-agl strains and the control were cultured in the same manner as in Example 4, but 45 g of glucose in the production medium was replaced with maltose isomers (iso-maltose) by 1 g and 5 g, thereby increasing cell mass and sugar consumption. performance and lysine production ability were compared.
  • KCCM11016P Glucose 44 Isomaltose 1 33.3 49.0 11.9 24.3 KCCM11016P::PgapA-agl Glucose 44 Isomaltose 1 33.1 50.0 12.3 24.6 KCCM10770P Glucose 44 Isomaltose 1 66.5 49.0 7.9 16.1 KCCM10770P::PgapA-agl Glucose 44 Isomaltose 1 67.0 50.0 8.1 16.2 KCCM11016P Glucose 40 Isomaltose 5 31.9 45.0 10.8 24.0 KCCM11016P::PgapA-agl Glucose 40 Isomaltose 5 33.2 50.0 12.4 24.8 KCCM10770P Glucose 40 Isomaltose 5 61.5 45.0 7.2 16.0
  • KCCM11016P::PgapA-agl and KCCM10770P::PgapA-agl strains with enhanced Agl protein activity of the present application were prepared in a production medium containing iso-maltose, the control strain KCCM11016P, Compared to KCCM10770P, maltose isomer availability was shown.
  • a vector was constructed by linking the signal sequences NCgl0801 (SEQ ID NO: 31) and NCgl2101 (SEQ ID NO: 32) between the gapA promoter and the agl gene.
  • Information on the gene encoding the signal sequence and surrounding nucleic acid sequences was obtained from the National Institutes of Health GenBank (NIH GenBank), and the gapA promoter was prepared using SEQ ID NOs: 25 and 37 using the genome of Corynebacterium glutamicum as a template.
  • SEQ ID NO: 31 was amplified using the primers of SEQ ID NOS: 33 and 34
  • SEQ ID NO: 32 was amplified using the primers of SEQ ID NOS: 35 and 36.
  • the prepared plasmids were named pDZTn-PgapA-SP0801-agl and pDZTn-PgapA-SP2101-agl in order.
  • KCCM11016P::PgapA-SP0801-agl strain was named CM03-1572, and on March 31, 2021, it was internationally deposited at the Korea Center for Microorganism Conservation (KCCM), an international depository under the Budapest Treaty, and was given an accession number as KCCM12968P.
  • KCCM Microorganism Conservation
  • Example 10 In a medium containing maltose or maltose isomers agl Comparison of L-lysine production ability of secretory transgenic strains
  • Example 9 The strain prepared in Example 9 was cultured by the method described in Example 4, and the cell mass, sugar consumption capacity, and lysine production capacity were compared (see Example 4, Example 5, and Example 7). The experiment was repeated three times, and the culture results (average values) are shown in Table 13.
  • the KCCM11016P::PgapA-SP0801-agl and KCCM11016P::PgapA-SP2101-agl strains with enhanced Agl protein activity of the present application increased lysine compared to the control strain in a production medium containing maltose. Yield is shown.
  • maltose isomers iso-maltose
  • maltose isomer availability was shown compared to the control strain, KCCM11016P.

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Abstract

La présente invention concerne un micro-organisme produisant un acide aminé présentant une activité de protéine Agl améliorée, et un procédé de production d'acide aminé l'utilisant.
PCT/KR2022/005843 2021-05-18 2022-04-25 Micro-organisme produisant un acide aminé présentant une activité de protéine agl améliorée, et procédé de production d'acide aminé l'utilisant WO2022245000A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
KR20130082124A (ko) * 2012-01-10 2013-07-18 씨제이제일제당 (주) 자일로즈 이용능이 부여된 코리네박테리움 속 미생물 및 이를 이용한 ｌ-라이신의 생산방법
US20140080185A1 (en) * 2008-01-10 2014-03-20 Ajinomoto Co., Inc. Method for Producing a Target Substance by Fermentation
EP3415623A1 (fr) * 2017-06-14 2018-12-19 Evonik Degussa GmbH Procédé de production de produits chimiques fins au moyen d'un corynebacterium sécrétant de l'amylo-?lpha-1,6-glucosidase modifié
KR20200036647A (ko) * 2018-09-28 2020-04-07 씨제이제일제당 (주) 알파-글루코시다제의 활성이 강화된 l-아미노산을 생산하는 미생물 및 이를 이용한 l-아미노산 생산 방법

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
US20140080185A1 (en) * 2008-01-10 2014-03-20 Ajinomoto Co., Inc. Method for Producing a Target Substance by Fermentation
KR20130082124A (ko) * 2012-01-10 2013-07-18 씨제이제일제당 (주) 자일로즈 이용능이 부여된 코리네박테리움 속 미생물 및 이를 이용한 ｌ-라이신의 생산방법
EP3415623A1 (fr) * 2017-06-14 2018-12-19 Evonik Degussa GmbH Procédé de production de produits chimiques fins au moyen d'un corynebacterium sécrétant de l'amylo-?lpha-1,6-glucosidase modifié
KR20200036647A (ko) * 2018-09-28 2020-04-07 씨제이제일제당 (주) 알파-글루코시다제의 활성이 강화된 l-아미노산을 생산하는 미생물 및 이를 이용한 l-아미노산 생산 방법

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DATABASE Nucleotide 10 May 2017 (2017-05-10), ANONYMOUS: "Bifidobacterium adolescentis ATCC 15703 DNA, complete genome", XP055853156, retrieved from NCBI Database accession no. AP009256.1 *

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