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WO2018030526A1 - Transformant et procédé de production d'acide 3-hydroxypropionique - Google Patents

Transformant et procédé de production d'acide 3-hydroxypropionique Download PDF

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WO2018030526A1
WO2018030526A1 PCT/JP2017/029169 JP2017029169W WO2018030526A1 WO 2018030526 A1 WO2018030526 A1 WO 2018030526A1 JP 2017029169 W JP2017029169 W JP 2017029169W WO 2018030526 A1 WO2018030526 A1 WO 2018030526A1
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gene
gene encoding
transformant
mcr
pombe
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Japanese (ja)
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正浩 漆原
薫 竹川
明子 陶山
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旭硝子株式会社
国立大学法人九州大学
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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
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/52—Propionic acid; Butyric acids

Definitions

  • Schizosaccharomyces pombe (hereinafter also referred to as “S. pombe”), which is suitable for the production of 3-hydroxypropionic acid (hereinafter also referred to as “3-HP”), is used as a host.
  • S. pombe which is suitable for the production of 3-hydroxypropionic acid
  • the present invention relates to a transformant incorporating a foreign gene or the like.
  • 3-HP is a very useful compound as a raw material for various chemical products such as acrylic esters.
  • 3-HP can be produced through a fermentation process with microorganisms. For example, in a microorganism into which a gene encoding malonyl CoA reductase (MCR) has been introduced, 3-HP is produced from malonyl CoA. In addition, in a microorganism into which a gene encoding glycerol dehydratase, aldehyde dehydrogenase or the like has been introduced, 3-HP is produced from glycerin via 3-hydroxypropionaldehyde (3-HPA).
  • MCR malonyl CoA reductase
  • 3-HP is produced from glycerin via 3-hydroxypropionaldehyde (3-HPA).
  • heterologous protein production system using eukaryotic microorganisms such as yeast
  • eukaryotic microorganisms such as yeast
  • part or all of the genome part of the host unnecessary or harmful to heterologous protein production is removed.
  • improved hosts that have been deleted or inactivated.
  • Patent Document 3 S.A. In Pombe, it is described that the production efficiency of heterologous proteins can be improved by using an improved host in which at least one gene selected from a gene encoding a specific protease (protease gene) is deleted or inactivated. Yes.
  • S. cerevisiae can produce 3-HP with high productivity without requiring neutralization with alkali. It is an object of the present invention to provide a transformant of pombe and a method for producing 3-HP by culturing the transformant.
  • the transformant according to the present invention is S. cerevisiae.
  • a transformant using Pombe as a host, and a gene encoding MCR, a gene encoding ACC, a gene encoding pyruvate oxidase, and a gene encoding acetyl CoA synthetase (ACS) are expressed in the cytoplasm.
  • Genes that have been introduced so as to be expressed or that can form a gene encoding MCR, a gene encoding ACC, and a pyruvate dehydrogenase complex hereinafter also referred to as “PDH”).
  • the pyruvate oxidase is an enzyme that catalyzes a reaction for generating acetic acid from pyruvate and water.
  • the introduced ACC-encoding gene is preferably a gene encoding ACC originally possessed by the host, and the introduced ACS-encoding gene is originally introduced by the host. It is preferably a gene encoding ACS.
  • the gene encoding pyruvate oxidase is preferably poxB, and the introduced gene encoding MCR is derived from Chloroflexus aurantiacus. It is preferably a gene encoding MCR.
  • the PDH is a complex composed of pyruvate dehydrogenase / decarboxylase (E1), dihydrolipoylacetyltransferase (E2), and dehydrolipoamide dehydrogenase (E3).
  • the gene group capable of forming PDH is a prokaryotic cell-derived gene group.
  • a gene encoding at least one protease inherent in the host is deleted or inactivated, and the gene encoding the protease is a metalloprotease.
  • a gene selected from the group consisting of a gene encoding, a gene encoding serine protease, a gene encoding cysteine protease, and a gene encoding aspartic protease is preferable.
  • the method for producing 3-HP according to the present invention is characterized in that the transformant is cultured in a liquid medium, and 3-hydroxypropionic acid is obtained from the liquid medium.
  • the liquid medium preferably contains glucose or sucrose, and the total concentration thereof is 1 to 50% by mass.
  • 3-HP can be efficiently produced without requiring neutralization treatment.
  • FIG. 4 is a schematic diagram of the structure of the pJK148-hsp9p-cut6-LPIT vector.
  • FIG. 4 is a schematic diagram of the structure of the pML7a-hsp9p-Camcr-inv1t vector. It is a schematic diagram of the structure of the pSM-u4pAde vector. It is a schematic diagram of the structure of the pSN-u4pAde vector.
  • 1 is a schematic diagram of the structure of a pSM-u4pAde-poxB-acs1 vector.
  • Example 1 it is the figure which showed the result of having measured the OD 600 value of each culture solution of mcr / cut6-2 introduction strain (broken line: control) and each culture solution of mcr / cut6 / poxB / acs1 introduction strain (solid line) with time. is there.
  • the 3-HP concentration (g / L) of each culture solution of mcr / cut6-2 introduced strain (broken line: control) and mcr / cut6 / poxB / acs1 introduced strain (solid line) was measured over time. It is the figure which showed the result.
  • 1 is a schematic diagram of the structure of a pSN-u4pAde-PDH vector.
  • Example 2 the OD 600 value of each culture solution of mcr / cut6-3 introduced strain (broken line: control) and mcr / cut6 / aceE / aceF / lpd introduced strain (solid line) was measured over time.
  • FIG. 4 is a graph showing the results of measuring the 3-HP concentration (g / L) of each culture solution of mcr / cut6 / aceE / aceF / lpd-introduced strain (solid black triangle) over time.
  • a “foreign” gene is not a structural gene originally possessed by a host (a structural gene contained in a natural host prior to transformation), but by a transformation operation or the like. It means the introduced structural gene.
  • “introduced” gene means a structural gene introduced by a transformation operation or the like, and it is the same gene as the gene originally possessed by the host, as well as genes derived from organisms other than the host and artificial genes. It may be.
  • the gene (mcr) encoding MCR and the gene (poxB) encoding pyruvate oxidase (PoxB), which is an enzyme that catalyzes the reaction of generating acetic acid from pyruvate and water, are foreign genes.
  • the gene encoding ACC, the gene encoding ACS, and the gene capable of forming PDH may be foreign genes or genes originally possessed by the host.
  • a gene encoding a protein such as an enzyme may be referred to as “protein name + gene”.
  • a gene encoding MCR is also referred to as “MCR gene”.
  • the transformant according to the present invention is S. cerevisiae.
  • a transformant having a gene encoding MCR (MCR gene) and a gene encoding ACC (ACC gene) with Pombe as a host and having increased the production of acetyl CoA from pyruvate in the cytoplasm is there.
  • Wild type S. cerevisiae In Pombe 3-HP cannot be produced from acetyl CoA, but in a transformant introduced with MCR gene and ACC gene, malonyl CoA is produced from acetyl CoA by ACC, and 3-HP from malonyl CoA by MCR. Produced. That is, S. cerevisiae into which MCR gene and ACC gene were introduced.
  • acetyl CoA is a precursor of 3-HP, and increasing the amount of acetyl CoA in the cytoplasm can increase the amount of 3-HP produced.
  • the transformant according to the present invention further has a gene encoding PoxB (PoxB gene) and a gene encoding ACS (ACS gene), or a gene group capable of forming PDH (PDH gene group).
  • the PDH gene group refers to a plurality of genes that respectively encode a plurality of enzymes that form PDH.
  • the transformant according to the present invention is specifically S. cerevisiae.
  • a transformant using Pombe as a host, and in addition to MCR and ACC, PoxB and ACS or PDH can be expressed in the cytoplasm.
  • acetic acid is produced from pyruvate by PoxB in the cytoplasm, and this acetic acid is converted to acetyl CoA by ACS.
  • acetyl CoA is produced from pyruvate by PDH in the cytoplasm.
  • the transformant when the transformant is cultured in a liquid medium containing acetic acid, a step of separating 3-HP secreted from the transformant into the liquid medium from acetic acid is required.
  • the transformant according to the present invention can produce acetyl CoA from pyruvic acid originally present in the cytoplasm, it is not necessary to contain acetic acid in the culture medium used for 3-HP production. It is not necessary to separate and purify 3-HP from acetic acid.
  • S. host is a yeast belonging to the genus Schizosaccharomyces (fission yeast), and is a microorganism particularly excellent in acid resistance compared to other yeasts.
  • S. Pombe is superior to other yeasts such as Saccharomyces cerevisiae to produce 3-HP under high concentrations of glucose, and is also suitable for high-density culture (culture using a large amount of yeast). I understood it. Therefore, S. By using a Pombe transformant, 3-HP can be produced with extremely high productivity.
  • PoxB is an enzyme that catalyzes the reaction of generating acetic acid from pyruvic acid and water. S. In the pombe cytoplasm, PoxB is not originally expressed. For this reason, among the transformants according to the present invention, transformants in which PoxB is expressed in the cytoplasm are obtained from the S. It is obtained by introducing it into the pombe. Examples of the PoxB gene introduced into the transformant according to the present invention include S. cerevisiae.
  • Any structural gene that can express a protein that exhibits PoxB activity (enzyme activity that catalyzes the reaction of generating acetic acid from pyruvic acid and water) when introduced into pombe can be a PoxB gene derived from any species. May be. Further, the PoxB gene possessed by the transformant may be one type or two or more types.
  • PoxB encoded by the PoxB gene introduced as a foreign gene examples include PoxB derived from Escherichia coli (Escherichia coli) or Corynebacterium glutamicum. Moreover, the modified body of these PoxB may be sufficient.
  • the variant of PoxB include a polypeptide having an amino acid sequence in which one or several amino acids of these PoxB amino acid sequences are substituted, added, or deleted, and having PoxB activity. The number of amino acids to be substituted is not particularly limited as long as it does not impair the PoxB activity, but is preferably 20 or less, more preferably 10 or less, and even more preferably 5 or less.
  • the modified variant of PoxB comprises an amino acid sequence having a sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more with these amino acid sequences of PoxB, and PoxB Also included are polypeptides having activity.
  • poxB derived from E. coli is preferable, and the sequence of poxB derived from E. coli optimized for expressing PoxB (SEQ ID NO: 1) is preferable.
  • transformants in which PoxB is expressed in the cytoplasm can also express ACS in the cytoplasm.
  • the transformant according to the present invention has the introduced ACS gene and can express a sufficient amount of ACS in the cytoplasm. This increases the amount of acetyl CoA converted from acetic acid, resulting in an increase in 3-HP production.
  • ACS encoded by the ACS gene to be introduced examples include S. Examples include ACS derived from Pombe and Saccharomyces cerevisiae. These ACS variants may also be used. ACS variants include, for example, polypeptides having an ACS activity consisting of an amino acid sequence in which one or several amino acids of these ACS amino acid sequences are substituted, added, or deleted. The number of amino acids to be substituted is not particularly limited as long as it does not impair the ACS activity, but is preferably 20 or less, more preferably 10 or less, and even more preferably 5 or less.
  • ACS variants include amino acid sequences having sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more of these ACS amino acid sequences, and ACS. Also included are polypeptides having activity.
  • ACS genes possessed by the transformant according to the present invention include ACS2 (SEQ ID NO: 2) or S. cerevisiae derived from Saccharomyces cerevisiae. Pombe derived acs1 (SEQ ID NO: 3) is preferred.
  • PDH is a complex enzyme having an enzyme activity that catalyzes a reaction (pyruvate decarboxylation reaction) for converting pyruvate to acetyl CoA.
  • PDH may be expressed in the cytoplasm instead of PoxB and ACS.
  • PDH is S.D. In Pombe, it is localized in the mitochondrial matrix. Therefore, a group of genes encoding each enzyme capable of forming PDH is obtained by genetic engineering methods. By introducing into pombe, a transformant capable of expressing PDH in the cytoplasm can be obtained.
  • PDH for example, a complex composed of pyruvate dehydrogenase / decarboxylase (E1), dihydrolipoylacetyltransferase (E2), and dehydrolipoamide dehydrogenase (E3) can be used.
  • the PDH composed of E1, E2, and E3 is preferably PDH derived from prokaryotic cells such as Escherichia coli because it can be stably expressed in the cytoplasm.
  • E. coli-derived PDH consists of three enzymes: AceE (corresponding to E1), AceF (corresponding to E2), and Lpd (corresponding to E3).
  • aceE (SEQ ID NO: 4) encoding AceE, aceF (SEQ ID NO: 5) encoding AceF, and lpd (SEQ ID NO: 6) encoding Lpd are genetically engineered by S. cerevisiae.
  • S. cerevisiae By introducing into a pombe, a transformant capable of expressing the AceE / AceF / Lpd complex enzyme in the cytoplasm is obtained.
  • E1, E2 and E3 to be expressed in the cytoplasm may be proteins inherent in prokaryotic cells, or may be modified forms thereof.
  • the variant include a polypeptide having an amino acid sequence in which one or several amino acids of the amino acid sequence of E1 originally possessed by prokaryotic cells are substituted, added, or deleted, and having E1 enzyme activity.
  • the number of amino acids to be substituted is not particularly limited as long as it does not impair the E1 enzyme activity, but is preferably 20 or less, more preferably 10 or less, and even more preferably 5 or less.
  • an amino acid having a sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, and further preferably 95% or more with the amino acid sequence of E1 originally possessed by prokaryotic cells Polypeptides consisting of sequences and having E1 enzyme activity are also included. The same applies to E2 and E3.
  • the transformant according to the present invention has the MCR gene.
  • S. Pombe does not naturally have the MCR gene. Therefore, S.
  • the MCR gene derived from organisms other than Pombe can be obtained by genetic engineering.
  • a transformant is obtained by introducing into pombe. Examples of the MCR gene introduced into the transformant include S. cerevisiae. It may be a structural gene that can express a protein that exhibits MCR activity when introduced into pombe, and may be an MCR gene derived from any species. Further, the MCR gene possessed by the transformant may be one type or two or more types.
  • Examples of the MCR encoded by the introduced MCR gene include, for example, Chloroflexus aurantiacus, Chloroflexus aggregans, Roseiflexus castenholzii, and Roseiflexus SP. Roseiflexus sp.), Roseiflexus sp. Strain S-1 (Roseiflexus sp. Strain S-1), Erythrobacter sp., Erythrobacter sp strain NAP1 (Erythrobactersp.
  • Strain SNAP1 Gamma Proteobacteria Umm (gamma proteobacterium), Metallosphaera sedula, Sulfolobus tokodaii, Sulfolobus metallicus, Sulfolobus metallicus, Sulfolobus metallicus (Su lfolobus sp.), Acidianus brierleyi, Acidianus infernos, Acidianus ambivalens, Stygiolobus azomarii (Stygiolobus azoricus) MCR and the like. Moreover, the modified body of these MCR may be sufficient.
  • MCR variants examples include polypeptides having an MCR activity consisting of an amino acid sequence in which one or several amino acids of these MCR amino acid sequences are substituted, added, or deleted.
  • mcr derived from Chloroflexus aurantiacus hereinafter also referred to as “Camcr”
  • the sequence of Camcr optimized for expressing MCR More preferred is SEQ ID NO: 7).
  • the transformant according to the present invention has the introduced ACC gene.
  • the ACC gene introduced into the transformant include S. cerevisiae. It may be a structural gene that can express a protein that exhibits ACC activity when introduced into pombe, and may be an ACC gene derived from any species. Further, the ACC gene possessed by the transformant may be one type or two or more types.
  • Examples of ACC encoded by the introduced ACC gene include S. Pombe, Saccharomyces cerevisiae, Bacillus cereus, Bacillus subtilis, Bos taurus, Brevibacterium thiogenitalis, Lactobacillus plantarum, plant Lactobacillus Candida lipolytica, Candida catenulata, Candida gropengiesseri, Candida rugosa, Candida sonorensis, Candida sonorensis (Candida andsonorensis) methanosorbosa, Candida curvata, Kluyveromyces marxianus, Kluyveromyces thermotolerans, I Ittchenkia orientalis, Rhodotorula sglutinis, Pichia haplophila, Aspergillus clavatus, Aspergillus ⁇ ⁇ sperm, sperm Aspergillus terreus, Aspergillus oryzae, Aspergillus ochraceus, Aspergillus niger, Crypto
  • the modified body of these ACC may be sufficient.
  • the modified ACC include a polypeptide having an ACC activity, which is composed of an amino acid sequence in which one or several amino acids of these ACC amino acid sequences are substituted, added, or deleted.
  • the ACC gene possessed by the transformant according to the present invention is preferably cut6 (SEQ ID NO: 8). Note that cut6 is S.I. It is a gene encoding ACC that Pombe originally has.
  • protease protease gene
  • protease gene protease gene
  • the transformant according to the present invention includes a gene encoding serine protease (serine protease gene), a gene encoding aminopeptidase (aminopeptidase gene), a gene encoding carboxypeptidase (carboxypeptidase gene) and a dipeptidase. It is preferable that one or more genes selected from the group consisting of genes to be deleted (dipeptidase genes) have been deleted or inactivated.
  • protease gene only one type of protease gene may be deleted, or two or more types of protease genes may be deleted. Among them, it is selected from the group consisting of a gene encoding a metalloprotease (metalloprotease gene), a serine protease gene, a gene encoding cysteine protease (cysteine protease gene), and a gene encoding aspartic protease (aspartic protease gene).
  • metalloprotease metalloprotease gene
  • serine protease gene a gene encoding cysteine protease
  • cysteine protease gene cysteine protease
  • aspartic protease gene aspartic protease gene
  • a transformant from which at least one gene is deleted is preferred, and the group is composed of at least one gene selected from the group consisting of a metalloprotease gene and a serine protease gene, a cysteine protease gene and an aspartic protease gene
  • a transformant from which at least one gene selected from is deleted is also preferred.
  • protease genes of Pombe include the following. Metalloprotease genes: cdb4 (SPAC23H4.09), mas2 (SPBC18E5.12c), pgp1 (SPCC1259.10), ppp20 (SPAC4F10.02), ppp22 (SPBC14C8.03), ppp51 (SPAC22G7.01c), ppp52 (SPBC18A7. 01), ppp53 (SPAP14E8.04).
  • Serine protease genes isp6 (SPAC4A8.04), ppp16 (SPBC1711.12), psp3 (SPAC1006.01), sxa2 (SPAC1296.03c).
  • Cysteine protease genes ppp80 (SPAC19B12.08), pca1 (SPCC1840.04), cut1 (SPCC5E4.04), gpi8 (SPCC11E10.02c). Aspartic protease genes: sxa1 (SPAC26A3.01), yps1 (SPCC1795.09), ppp81 (SPAC25B8.17).
  • the metalloprotease gene deleted in the transformant according to the present invention is preferably at least one of cdb4, pgp1, ppp20, ppp22, ppp52, or ppp53, and more preferably at least one of cdb4, ppp22, or ppp53 preferable.
  • the serine protease gene deleted in the transformant according to the present invention is preferably at least one of isp6, ppp16, psp3, or sxa2.
  • the cysteine protease gene deleted in the transformant according to the present invention is preferably ppp80.
  • the transformant according to the present invention includes a total of one or more genes selected from the group consisting of cdb4, ppp22, and ppp53 and two or more genes selected from the group consisting of isp6, ppp16, psp3, and sxa2.
  • genes selected from the group consisting of cdb4, ppp22, and ppp53 and two or more genes selected from the group consisting of isp6, ppp16, psp3, and sxa2.
  • 3 or more types of genes are preferred, and those in which a total of 3 or more types of genes including at least one gene selected from the group consisting of ppp53 and cdb4, and isp6 and psp3 have been deleted, etc. More preferred.
  • the transformant according to the present invention is preferably one in which four or more genes including ppp53, isp6, psp3, and ppp16 have been deleted, and includes ppp53, isp6, psp3, ppp16, and ppp22.
  • genes of more than one species are more preferable
  • those in which 6 or more genes including ppp53, isp6, psp3, ppp16, ppp22, and sxa2 are deleted are more preferable
  • psp3, isp6, ppp53 , Ppp16, ppp22, sxa2, ppp80, and ppp20 are more preferable.
  • transformants capable of expressing PoxB and ACS in the cytoplasm are S. cerevisiae serving as hosts. It can be produced by introducing the PoxB gene, ACS gene, MCR gene, and ACC gene into pombe by a genetic engineering method.
  • transformants capable of expressing PDH in the cytoplasm are S. cerevisiae serving as hosts. It can be produced by introducing the E1, E2, E3, MCR, and ACC genes into pombe by a genetic engineering method.
  • the pombe may be a wild type or a mutant type in which a specific gene is deleted or inactivated depending on the use.
  • a method for deleting or inactivating a specific gene a known method can be used. Specifically, the gene can be deleted by using the Latour method (described in Nucleic Acids Res, 2006, 34, e11, International Publication No. 2007/063919).
  • mutation isolation methods using mutants Yeast Molecular Genetics Experimental Method, 1996, Society Press Center
  • random mutation methods using PCR PCR Methods Application, Vol. 2, pages 28-33, 1992
  • the part where a specific gene is deleted or inactivated may be an ORF (open reading frame) part or an expression regulatory sequence part.
  • a particularly preferred method is a method of deletion or inactivation by PCR-mediated homologous recombination method (Yeast, Vol. 14, pages 943-951, 1998) in which the ORF portion of the structural gene is replaced with a marker gene.
  • yeast hosts of the genus Schizosaccharomyces from which a specific gene has been deleted or inactivated are described in, for example, International Publication No. 2002/101038, International Publication No. 2007/015470, International Publication No. 2013/137277, etc. .
  • the host used in the present invention include S. cerevisiae.
  • at least one protease gene originally possessed by Pombe is deleted or inactivated, and at least one gene selected from the group consisting of a serine protease gene, an aminopeptidase gene, a carboxypeptidase gene, and a dipeptidase gene is present.
  • At least one gene selected from the group consisting of metalloprotease gene, serine protease gene, cysteine protease gene, and aspartic protease gene is deleted or inactivated. More preferred are those in which psp3, isp6, ppp53, ppp16, ppp22, sxa2, ppp80, and ppp20 are deleted or inactivated.
  • S. is used as a host. It is preferable to use a pombe having a marker for selecting a transformant. For example, it is preferable to use a host in which a specific nutritional component is essential for growth because a certain gene is missing. When a transformant is produced by transforming with a vector containing the target gene sequence, by incorporating this missing gene (an auxotrophic complementary marker) into the vector, the transformant is required for host nutrition. Sex disappears. Due to the difference in auxotrophy between the host and the transformant, a transformant can be obtained by distinguishing the two.
  • ura4 orotidine phosphate decarboxylase gene
  • a transformant in which the vector is incorporated can be obtained by selecting those that have lost uracil requirement.
  • the gene that becomes auxotrophic due to deletion in the host is not limited to ura4 as long as it is used for selection of transformants, and may be an isopropylmalate dehydrogenase gene (leu1) or the like.
  • Each gene to be introduced is S. cerevisiae. It is preferably introduced into the pombe chromosome. By introducing a gene into the chromosome, a transformant having excellent passage stability can be obtained. A plurality of genes can be introduced into a chromosome.
  • a known method can be used as a method for introducing a gene into a host by a genetic engineering method.
  • the gene As a method for introduction into the pombe chromosome, a method of introduction by homologous recombination using a vector having an expression cassette having a gene and a recombination site is preferred.
  • An expression cassette is a combination of DNAs necessary for expressing a target protein, and includes a structural gene encoding the target protein, a promoter that functions in the host, and a terminator.
  • the expression cassette used in the production of the transformant according to the present invention comprises a structural gene to be introduced and S. cerevisiae. A promoter that functions in Pombe Including a terminator that functions within the pombe.
  • the expression cassette may contain any one or more of 5′-untranslated region and 3′-untranslated region. Furthermore, the auxotrophic complementary marker may be included. Multiple expression genes may exist in one expression cassette.
  • the number of structural genes in one expression cassette is preferably 1-8, and more preferably 1-5.
  • the MCR gene and the ACC gene may be introduced into the host by separate expression cassettes, or both genes may be introduced into the host by one expression cassette.
  • the E1, E2, and E3 genes may be introduced into the host by separate expression cassettes, or each gene may be introduced into the host as one expression cassette.
  • the gene encoded by the wild type may be used as it is, but S.
  • the wild type gene sequence was transformed into S. cerevisiae. It may be modified to a codon frequently used in pombe.
  • promoters that function in pombe include S. cerevisiae. Pombe's inherent promoter (preferably having high transcription initiation activity) or S.
  • a promoter eg, a virus-derived promoter
  • Two or more promoters may be present in the vector.
  • the promoter and terminator of the expression cassette introduced into the transformant according to the present invention even if 3-HP accumulates and becomes acidic (below pH 6), it functions in the transformant and is a structural gene. Those capable of maintaining the expression of the protein encoded by are preferred.
  • promoters inherent to Pombe include alcohol dehydrogenase gene promoter, nmt1 gene promoter involved in thiamine metabolism, fructose-1, 6-bisphosphatase gene promoter involved in glucose metabolism, and invertase gene involved in catabolite repression. Examples include promoters (see International Publication No. 99/23223), heat shock protein gene promoters (see International Publication No. 2007/26617, International Publication No. 2014/030644), and the like. S.
  • promoters that Pombe does not originally include are promoters derived from animal cell viruses described in JP-A-5-15380, JP-A-7-163373, and JP-A-10-234375, and hCMV A promoter, SV40 promoter is preferred.
  • S. Pombe's inherent terminator and S.P. A terminator that Pombe does not have can be used. Two or more terminators may be present in the vector. Examples of the terminator include human terminators described in JP-A-5-15380, JP-A-7-163373, and JP-A-10-234375, and human lipocortin I terminator is preferable. .
  • the transformant according to the present invention has an expression cassette containing the structural gene to be introduced in the chromosome or as an extrachromosomal gene.
  • Having an expression cassette in the chromosome means that the expression cassette is incorporated at one or more positions in the chromosome of the host cell, and having as an extrachromosomal gene means having a plasmid containing the expression cassette in the cell. That is.
  • a transformant containing each expression cassette is used as a host using a vector containing each expression cassette. It is obtained by transforming pombe.
  • the vector can be produced by incorporating the expression cassette into a vector having a circular DNA structure or a linear DNA structure.
  • the vector may contain a sequence for replication in the host cell, that is, an autonomously replicating sequence (AutonomouslynomReplicating). Sequence: ARS) is preferred.
  • ARS autonomously replicating sequence
  • the vector is assumed to have a linear DNA structure and no ARS. It is preferable to be introduced into.
  • the vector may be a vector composed of linear DNA, or a vector having a circular DNA structure provided with a restriction enzyme recognition sequence for cleavage into linear DNA upon introduction into a host.
  • the vector is a plasmid having ARS, it can be introduced into the host after forming a linear DNA structure by deleting the ARS part or a linear DNA structure in which the function of ARS is inactivated by cleaving the ARS part.
  • the vector preferably has a marker for selecting a transformant.
  • the marker include ura4 and leu1.
  • the vector was transformed into S. cerevisiae.
  • the recombination site of the vector is S. cerevisiae. This is a site having a base sequence that allows homologous recombination to be performed on a target site for homologous recombination in the pombe chromosome.
  • the target site is S. pneumoniae. This is a target site for integrating the expression cassette in the pombe chromosome.
  • the target site can be freely set by setting the recombination site of the vector to a base sequence that allows homologous recombination to be performed on the target site.
  • the homology between the base sequence of the recombination site and the base sequence of the target site needs to be 70% or more. Further, the homology between the base sequence of the recombination site and the base sequence of the target site is preferably 90% or more, and more preferably 95% or more from the viewpoint that homologous recombination is likely to occur.
  • the expression cassette is incorporated into the target site by homologous recombination.
  • the length (number of bases) of the recombination site is preferably 20 to 2000 bp. If the length of the recombination site is 20 bp or more, homologous recombination is likely to occur.
  • the length of the recombination site is 2000 bp or less, it is easy to prevent the vector from becoming too long and causing homologous recombination to hardly occur.
  • the length of the recombination site is more preferably 100 bp or more, and further preferably 200 bp or more. Further, the length of the recombination site is more preferably 800 bp or less, and further preferably 400 bp or less.
  • the vector may have other DNA regions in addition to the expression cassette and the recombination site.
  • a replication initiation region called “ori” necessary for replication in E. coli and an antibiotic resistance gene (neomycin resistance gene, etc.) can be mentioned. These are genes usually required when constructing a vector using Escherichia coli.
  • the replication initiation region is preferably removed when the vector is integrated into the host chromosome as described later.
  • the vector When integrating a gene into a chromosome, the vector is S. cerevisiae. When introducing into a pombe cell, it is preferable to introduce it in a linear DNA structure. That is, in the case of a vector having a circular DNA structure such as a commonly used plasmid DNA, S. It is preferable to introduce into pombe cells. In this case, the position for opening the vector having a circular DNA structure is within the recombination site. As a result, the recombination sites partially exist at both ends of the opened vector, and the entire vector is integrated into the target site of the chromosome by homologous recombination.
  • the vector may be constructed by a method other than the method of cutting a vector having a circular DNA structure, as long as it can have a linear DNA structure in which a part of the recombination site exists at each end.
  • the vector for example, plasmids derived from E. coli such as pBR322, pBR325, pUC118, pUC119, pUC18, and pUC19 are preferably used.
  • the plasmid vector used for homologous recombination preferably has a replication initiation region called “ori” that is necessary for replication in E. coli.
  • ori replication initiation region
  • the method for constructing the vector from which the replication initiation region has been removed is not particularly limited, but the method described in JP-A-2000-262284 is preferably used.
  • a method is preferred in which a precursor vector in which a replication initiation region is inserted at the cleavage site in the recombination site is constructed so that the replication initiation region is excised at the same time as the linear DNA structure as described above. Thereby, a vector from which the replication initiation region has been easily removed can be obtained.
  • expression vectors described in JP-A-5-15380, JP-A-7-163373, International Publication No. 96/23890, JP-A-10-234375, and the construction method thereof are applied for expression.
  • a method may be used in which a precursor vector having a cassette and a recombination site is constructed, and a vector used for homologous recombination is obtained by further removing the replication initiation region from the precursor vector by an ordinary genetic engineering technique.
  • Target sites that incorporate the vector are S. cerevisiae. It may be present only at one location in the pombe chromosome, or may be present at two or more locations. When two or more target sites exist, S.P.
  • the vector can be integrated at two or more positions on the pombe chromosome.
  • a plurality of genes when a plurality of genes are included in one expression cassette, a plurality of genes can be incorporated into one target site.
  • an expression cassette can be incorporated into two or more target sites using two or more vectors having recombination sites corresponding to the respective target sites.
  • Transformation method Any known transformation method may be used as the transformation method.
  • the transformation method include conventionally known methods such as lithium acetate method, electroporation method, spheroplast method, glass bead method, and the method described in JP-A-2005-198612.
  • a commercially available yeast transformation kit may also be used.
  • the obtained transformant is usually selected after homologous recombination.
  • the selection method include the following methods. Screening is performed with a medium capable of selecting transformants using the auxotrophic marker, and a plurality of colonies obtained are selected. Next, after separately culturing them in liquid culture, the expression level of the heterologous protein in each liquid medium is examined, and a transformant with a higher expression level of the heterologous protein is selected.
  • the number of vectors integrated into the chromosome and the number of expression cassettes can be examined. The number of vectors integrated into the chromosome can be adjusted to some extent by adjusting the integration conditions. Depending on the size (number of bases) and structure of the vector, the integration efficiency and the number of integrations may change.
  • the method for producing 3-HP according to the present invention is a method for culturing the transformant according to the present invention in a liquid medium and obtaining 3-HP from the liquid medium.
  • 3-HP is produced in the cytoplasm of the transformant. Since the produced 3-HP is secreted into the liquid medium, 3-HP can be recovered from the liquid medium after culturing the transformant.
  • liquid medium used for the production of 3-HP a known yeast culture medium containing sugar can be used.
  • the liquid medium include S.I. Contains a nitrogen source, inorganic salts, etc. that can be utilized by Pombe; Those capable of efficiently cultivating the pombe are preferred.
  • the liquid medium a natural medium or a synthetic medium may be used.
  • sugars such as glucose, fructose, sucrose, and maltose.
  • nitrogen source include inorganic acids such as ammonia, ammonium chloride, and ammonium acetate, or ammonium salts of inorganic acids, peptone, casamino acid, yeast extract, and the like.
  • inorganic salts include magnesium phosphate, magnesium sulfate, sodium chloride and the like.
  • a fermentation promoting factor such as proteolipid can be included.
  • a liquid medium containing glucose or sucrose as sugar.
  • the concentration of glucose or sucrose in the liquid medium (100% by mass) at the initial stage of culture is preferably 1% by mass or more, more preferably 1 to 50% by mass, and further preferably 2 to 20% by mass. It is preferable to continue the culture by adding glucose as necessary, because the glucose concentration is lowered by the culture.
  • the glucose concentration at the end of the culture may be 1% by mass or less.
  • the productivity of 3-HP is further improved.
  • the production efficiency of 3-HP is further improved by setting the glucose in the liquid medium to 20% by mass or less.
  • the initial bacterial cell concentration of the transformant in the liquid medium is preferably 0.1 to 5 g / L in terms of dry cell weight. More preferably, the initial cell concentration of the transformant in the liquid medium is 0.2 to 2 g / L in terms of dry cell weight.
  • High productivity can be achieved in a short time by increasing the initial cell concentration.
  • the initial bacterial cell concentration is too high, problems such as bacterial cell aggregation and a reduction in purification efficiency may occur.
  • cell concentration indicated in the Examples etc. described later is Shimadzu UV-visible spectrophotometer UVmini-1240 light absorbance at a wavelength of 600nm as measured by (OD 600) values.
  • a known yeast culture method can be used for the culture, for example, shaking culture, stirring culture, or the like.
  • the culture temperature is preferably 23 to 37 ° C.
  • the culture time can be determined as appropriate.
  • the culture may be batch culture or continuous culture.
  • the cells can be separated from the liquid medium to obtain a liquid medium containing 3-HP.
  • the continuous culture method for example, a part of the liquid medium is extracted from the culture tank being cultured, and 3-HP is separated from the extracted liquid medium, and the culture supernatant is recovered, and glucose and A method of continuously culturing by repeatedly adding a new liquid medium or the like and returning it to the culture tank can be mentioned. By performing continuous culture, the productivity of 3-HP is further improved.
  • 3-HP can be produced without neutralization even if the pH is lowered due to the accumulation of 3-HP (pH of about 2 to 4). Therefore, even after the pH of the liquid medium becomes 3.5 or less, 3-HP can be produced by continuous culture that continues the culture.
  • the pH at the end of culture and the pH in continuous culture are preferably 3.5 or less, and particularly preferably 2.3 to 3.5. In order to increase the productivity of 3-HP, it is preferable to continue the cultivation after the pH of the liquid medium becomes 3.5 or lower. Since the transformant according to the present invention has excellent acid resistance, the culture can be continued without neutralizing 3-HP in the liquid medium produced by the transformant.
  • 3-HP For obtaining 3-HP from the liquid medium, a known method is used. In particular, it is preferable to obtain 3-HP by separating the liquid medium and 3-HP without neutralizing 3-HP in the liquid medium. For example, by separating the bacterial cells from the liquid medium after completion of the culture by centrifugation, extracting with diethyl ether or ethyl acetate after adjusting the pH to 1 or less, a method of leaching after adsorbing to an ion exchange resin and washing, activated carbon And a method of removing impurities using, a method of distilling after reacting with an alcohol in the presence of an acid catalyst, and a method of separating using a separation membrane.
  • 3-HP can be obtained by neutralizing 3-HP in the liquid medium and then separating the liquid medium and the 3-HP salt.
  • 3-HP can be obtained by converting 3-HP in a liquid medium into a calcium salt or a lithium salt and crystallizing the neutralized salt.
  • Example 1 S.
  • the MGF438 strain in which psp3, isp6, ppp53, ppp16, ppp22, sxa2, ppp80, and ppp20 have been deleted (genotype: h - leu1-32 ura4-D18 psp3-D13 isp6-D14 oma1-D10 ppp16- D20 fma2-D13 sxa2-D15 aap1-D17 ppp80-D11) to which adenine requirement was obtained by disrupting ade7, mcr (Camcr) derived from Chloroflexus aurantiacus and S.
  • the amount of 3-HP produced by the transformants into which pombe-derived acs1 was introduced was compared.
  • the transformant was transformed with pML7a-hsp9p-Camcr-inv1t cleaved with the restriction enzyme NotI, and the vector 5′-Tf2 (a) -SV40 promoter-URA3-hsp9 promoter-mcr-inv1 terminator- A transformant in which the 3′-TF2 (b) gene was introduced into the Tf2 locus of the chromosome was selected.
  • the transformant was designated as mcr / cut6-1 introduced strain (No. 395).
  • a strain in which ade7 of MGF438 strain was disrupted to impart adenine requirement was transformed with the amplified gene, and a transformant in which the amplified gene was introduced downstream of the Gpm1 locus of the chromosome was selected.
  • the transformant was designated as mcr / cut6-2 introduced strain (No. 408).
  • PCR using the primer Eno101 up Fw (25mer, SEQ ID NO: 14) and the primer Eno101 dw Rv (33mer, SEQ ID NO: 15) was carried out using the pSN-u4pAde vector (6246 bp, FIG. 4, SEQ ID NO: 16) Eno101 up-
  • the loxP-ura4 promoter-ade7-ura4 terminator-loxP-Eno101 Dw gene was amplified.
  • a strain in which ade7 of the MGF438 strain was disrupted to impart adenine requirement was transformed with the amplified gene, and a transformant in which the amplified gene was introduced downstream of the Eno101 locus of the chromosome was selected.
  • the transformant was designated as mcr / cut6-3 introduced strain (No. 409).
  • the Gpm1 up-loxP-ura4 promoter of the pSM-u4pAde-poxB-acs1 vector-ade7-ura4 terminator-loxP-LPI terminator-acs1-vCM1-hV1 promoter The terminator-poxB-hsp9 promoter-Gpm1 Dw gene was amplified.
  • the mcr / cut6-1-introduced strain was transformed with the amplified gene, and a transformant in which the amplified gene was introduced downstream of the Gpm1 locus of the chromosome was selected.
  • the transformant was designated as mcr / cut6 / poxB / acs1 introduced strain.
  • the mcr / cut6-2 introduced strain was inoculated into a 20% glucose concentration MM-leu-ura-adeneine medium (a medium in which leucine, uracil and adenine were removed from the MM medium) at a temperature of 30 ° C.
  • the culture was performed for 48 hours under the condition of shaking speed of 200 rpm.
  • the 3-HP concentration (g / L) of the culture solution was measured every 24 hours from the start of the culture and after the end of the culture under the HPLC conditions shown below.
  • the growth of the mcr / cut6-2 introduced strain and the mcr / cut6 / poxB / acs1 introduced strain were compared. Specifically, the mcr / cut6-2 introduced strain and the mcr / cut6 / poxB / acs1 introduced strain were respectively inoculated in a MM-leu-ura-adeneine medium having a glucose concentration of 20%, and shaken at a temperature of 30 ° C. Culturing was performed for 48 hours under the condition of a speed of 200 rpm.
  • MM-leu-ura-adeneine medium having a glucose concentration of 20%
  • the OD 600 value of the culture broth was measured every 24 hours from the start of the culture and after the end of the culture with an absorptiometer.
  • Example 2 A strain obtained by disrupting ade7 of the MGF438 strain and imparting adenine requirement to mcr and S. cerevisiae derived from Chloroflexus aurantiacus. A transformant into which pombe-derived cut6 was introduced; The amount of 3-HP produced by transformants into which pombe-derived cut6, E. coli-derived aceE, E. coli-derived aceF, and E. coli-derived lpd were introduced was compared.
  • PCR using the primer Eno101 up Fw and the primer Eno101 dw Rv was carried out by using the pSN-u4pAde-PDH vector Eno101 up-loxP-ura4 promoter-ade7-ura4 terminator-loxP-LPI terminator-aceF-hCMV promoter-invCM1
  • the aceE-hsp9 promoter-adh1 promoter-lpd-adh1 terminator-Eno101 Dw gene was amplified.
  • the mcr / cut6-1 introduced strain was transformed with the amplified gene, and a transformant in which the amplified gene was introduced downstream of the Eno101 locus of the chromosome was selected.
  • the transformant was designated as mcr / cut6 / aceE / aceF / lpd-introduced strain.
  • the 3-HP production amount of the mcr / cut6-3 introduced strain and the mcr / cut6 / aceE / aceF / lpd introduced strain was compared.
  • the culture of the mcr / cut6-3 introduced strain and the mcr / cut6 / aceE / aceF / lpd introduced strain and the measurement of the 3-HP concentration (g / L) of the culture solution were carried out with the mcr / cut6-2 introduced strain in Example 1. The same was done.
  • the measurement results of the growth (OD 600 value) of each culture solution are shown in FIG. As a result, both strains reached a stationary phase in 70 hours from the start of culture.
  • the OD 600 value of mcr / cut6-3 introduced strain (broken line) in 70 hours
  • OD 600 value of mcr / cut6 / aceE / aceF / lpd introduced strain (solid line) in 7.1,70 hours was 6.9 .
  • Example 3 A strain obtained by disrupting ade7 of the MGF438 strain and imparting adenine requirement to mcr and S. cerevisiae derived from Chloroflexus aurantiacus.
  • the 3-HP concentration (g / L) of the culture solution was measured by HPLC every hour from the start of the culture.
  • the 3-HP concentration of the culture solution was measured in the same manner as in Example 1.
  • the measurement results of the 3-HP concentration (g / L) of each culture solution are shown in FIG.
  • the amount of 3-HP produced by the mcr / cut6-2-introduced strain (dashed circle) is 1.41 g after completion of the culture, and the amount of 3-HP produced by the mcr / cut6 / poxB / acs1-introduced strain (solid black circle).
  • the amount of 3-HP produced by the mcr / cut6 / poxB / acs1 introduced strain was 7.7 times higher than that of the mcr / cut6-2 introduced strain.
  • the 3-HP production amount of the mcr / cut6-3 introduced strain (broken triangle) is 1.34 g after completion of the culture, and the 3-HP of the mcr / cut6 / aceE / aceF / lpd introduced strain (solid black triangle).
  • the production amount was 9.82 g / L, and the 3-HP production amount of the mcr / cut6 / aceE / aceF / lpd-introduced strain was improved by 7.3 times that of the mcr / cut6-3-introduced strain. From these results, S. cerevisiae having the MCR gene and cut6 introduced therein was introduced.

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Abstract

L'invention concerne un transformant de Schizosaccharomyces pombe (S.pombe), lequel permet de produire avec un grand rendement et sans nécessiter de neutralisation alcaline, de l'acide 3-hydroxypropionique (3-HP). L'invention concerne également un procédé de production de 3-HP par mise en culture du transformant susmentionné.　Ce transformant met en oeuvre Schizosaccharomyces pombe en tant qu'hôte et se caractérise en ce que sont introduits: un gène codant pour une malonyl-CoA réductase, un gène codant pour une acétyl-CoA carboxylase, un gène codant pour une pyruvate oxydase et un gène codant pour une acétyl-CoA synthétase, ou en ce que sont introduits: un gène codant pour une malonyl-CoA réductase, un gène codant pour une acétyl-CoA carboxylase, et un ensemble de gènes pouvant former un complexe pyruvate déshydrogénase, la pyruvate oxydase susmentionnée étant une enzyme catalyseur d'une réaction produisant de l'acide acétique à partir d'acide pyruvique et d'eau.
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