WO2017022587A1 - Procédé de production de lipides - Google Patents

Procédé de production de lipides Download PDF

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Publication number
WO2017022587A1
WO2017022587A1 PCT/JP2016/072007 JP2016072007W WO2017022587A1 WO 2017022587 A1 WO2017022587 A1 WO 2017022587A1 JP 2016072007 W JP2016072007 W JP 2016072007W WO 2017022587 A1 WO2017022587 A1 WO 2017022587A1
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protein
gene
amino acid
acid sequence
transformant
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PCT/JP2016/072007
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Japanese (ja)
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慎二 杉原
達郎 尾崎
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花王株式会社
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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats

Definitions

  • the present invention relates to a method for producing lipids.
  • the present invention also relates to ⁇ -ketoacyl-ACP synthase used in the method, a gene encoding the same, and a transformant that promotes the expression of the gene.
  • Fatty acids are one of the main constituents of lipids and constitute lipids (oils and fats) such as triacylglycerol produced by ester bonds with glycerin in vivo. In many animals and plants, fatty acids are also stored and used as energy sources. Fatty acids and lipids stored in animals and plants are widely used for food or industry. For example, derivatives of higher alcohols obtained by reducing higher fatty acids having about 12 to 18 carbon atoms are used as surfactants. Alkyl sulfate esters and alkylbenzene sulfonates are used as anionic surfactants. Polyoxyalkylene alkyl ethers, alkyl polyglycosides, and the like are used as nonionic surfactants.
  • surfactants are used as cleaning agents, disinfectants, and the like.
  • Cationic surfactants such as alkylamine salts and mono- or dialkyl quaternary amine salts, which are derivatives of the same higher alcohol, are routinely used for fiber treatment agents, hair rinse agents, disinfectants, and the like.
  • Benzalkonium-type quaternary ammonium salts are routinely used for bactericides and preservatives.
  • vegetable oils and fats are also used as raw materials for biodiesel fuel. As described above, fatty acids and lipids are widely used, and therefore, attempts have been made to improve the productivity of fatty acids and lipids in vivo in plants and the like.
  • Plant fatty acid synthesis pathway is localized in chloroplasts.
  • acetyl-ACP acyl carrier protein
  • acyl-ACP acyl which is a fatty acid residue
  • a complex comprising a group and an ACP
  • KAS ⁇ -ketoacyl-acyl-carrier-protein synthase
  • KAS III works at the initiation stage of the chain length extension reaction and extends acetyl-ACP (or acetyl-CoA) having 2 carbon atoms to ⁇ -ketoacyl-ACP having 4 carbon atoms.
  • KAS II is mainly involved in the elongation reaction up to 16 carbon atoms palmitoyl-ACP
  • KAS II is mainly involved in the elongation reaction up to 18 carbon atoms stearoyl-ACP.
  • KAS IV is said to be involved in the elongation reaction up to medium chain acyl-ACP having 6 to 14 carbon atoms.
  • a method of accumulating short-chain or medium-chain fatty acids by enhancing or modifying KAS III in plants or Escherichia coli has been proposed (see Patent Document 2, and Non-Patent Documents 2 and 3).
  • algae has attracted attention as being useful for biofuel production.
  • Algae are attracting attention as next-generation biomass resources because they can produce lipids that can be used as biodiesel fuel by photosynthesis and do not compete with food.
  • algae have a higher ability to produce and accumulate lipids than plants.
  • the present invention relates to a method for producing a lipid, in which a transformant in which expression of a gene encoding the following protein (A) or (B) is promoted is cultured to produce a fatty acid or a lipid comprising the same.
  • a protein comprising the amino acid sequence represented by SEQ ID NO: 1.
  • B A protein comprising an amino acid sequence having 67% or more identity with the amino acid sequence of the protein (A) and having ⁇ -ketoacyl-ACP synthase activity (hereinafter also referred to as “KAS activity”).
  • the present invention also relates to the protein (A) or (B).
  • the present invention also relates to a gene encoding the protein (A) or (B). Furthermore, this invention relates to the transformant which promoted the expression of the gene which codes the said protein (A) or (B).
  • the present invention relates to a method for producing a lipid, which improves the productivity of a medium chain fatty acid or a lipid comprising the same.
  • the present invention also relates to a transformant having improved productivity of medium-chain fatty acids or lipids comprising the same.
  • the present inventors newly identified KAS of algae belonging to the genus Nannochloropsis, which is a kind of algae, as an enzyme involved in the synthesis of medium chain fatty acids. As a result of accelerating the expression of KAS in microorganisms, the present inventors have found that the productivity of produced medium chain fatty acids or lipids containing them as a constituent component is significantly improved. The present invention has been completed based on these findings.
  • the productivity of a medium chain fatty acid or a lipid containing this as a constituent can be improved.
  • the transformant of the present invention is excellent in productivity of medium chain fatty acids or lipids containing the same as a constituent component.
  • lipid refers to simple lipids such as neutral fats (such as triacylglycerol), waxes, and ceramides; complex lipids such as phospholipids, glycolipids, and sulfolipids; and fatty acids derived from these lipids And derived lipids such as alcohols and hydrocarbons.
  • Cx: y in the notation of fatty acids and acyl groups constituting fatty acids means that the number of carbon atoms is x and the number of double bonds is y.
  • “Cx” represents a fatty acid or acyl group having x carbon atoms.
  • the identity of a base sequence and an amino acid sequence is calculated by the Lipman-Pearson method (Science, 1985, vol. 227, p. 1435-1441). Specifically, it is calculated by performing an analysis assuming that Unit size to compare (ktup) is 2 using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win.
  • “stringent conditions” include, for example, Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W., et al. Russell., Cold Spring Harbor Laboratory Press].
  • upstream of a gene indicates not a position from the translation start point but a region continuing on the 5 ′ side of the gene or region regarded as a target.
  • downstream of a gene indicates a region continuing 3 ′ side of the gene or region captured as a target.
  • the proteins (A) and (B) are one type of KAS, from acetyl-ACP (or acetyl-CoA) having 2 carbon atoms to ⁇ -ketoacyl- having 4 carbon atoms. It is a protein involved in the elongation reaction to ACP.
  • the protein consisting of the amino acid sequence of SEQ ID NO: 1 is one of KAS derived from Nannochloropsis oculata NIES2145 strain, which is an algae belonging to the genus Nannochloropsis .
  • KAS is an enzyme involved in the control of acyl chain length in the fatty acid synthesis pathway.
  • Plant fatty acid synthesis pathways are generally located in the chloroplast.
  • acetyl-ACP or acetyl-CoA
  • the carbon chain elongation reaction is repeated to finally synthesize acyl-ACP having 16 or 18 carbon atoms.
  • acyl-ACP thioesterase acts to hydrolyze the thioester bond of acyl-ACP to produce a free fatty acid.
  • acetoacetyl ACP is produced by a condensation reaction of acetyl-ACP (or acetyl-CoA) and malonyl ACP. This reaction is catalyzed by KAS. Subsequently, the keto group of acetoacetyl ACP is reduced by ⁇ -ketoacyl-ACP reductase to produce hydroxybutyryl ACP. Subsequently, hydroxybutyryl ACP is dehydrated by ⁇ -hydroxyacyl-ACP dehydrase to produce crotonyl ACP. Finally, crotonyl ACP is reduced by enoyl-ACP reductase to produce butyryl ACP.
  • butyryl ACP in which two carbon chains of the acyl group are extended from acetyl-ACP is generated. Thereafter, by repeating the same reaction, the carbon chain of acyl-ACP is extended, and finally acyl-ACP having 16 or 18 carbon atoms is synthesized.
  • KAS activity means an activity that catalyzes the condensation reaction of acetyl-ACP (or acetyl-CoA) or acyl-ACP with malonyl ACP.
  • the fact that a protein has KAS activity means that, for example, a DNA in which a gene encoding the protein is linked downstream of a promoter that functions in the host cell is introduced into a host cell lacking the fatty acid degradation system, and the introduced gene is expressed.
  • the cells can be cultured under such conditions, and changes in fatty acid composition in the host cells or in the culture medium can be confirmed by a conventional method.
  • a DNA ligated with the gene encoding the protein downstream of a promoter that functions in the host cell into the host cell and culturing the cell under conditions in which the introduced gene is expressed, It can be confirmed by performing a chain length extension reaction using various acyl-ACPs as substrates.
  • KAS is classified as KAS I, KAS II, KAS III, or KAS IV depending on its substrate specificity.
  • KAS III uses acetyl-ACP (or acetyl-CoA) having 2 carbon atoms as a substrate and catalyzes an elongation reaction having 2 to 4 carbon atoms.
  • KAS I mainly catalyzes the elongation reaction of 4 to 16 carbon atoms and synthesizes palmitoyl-ACP having 16 carbon atoms.
  • KAS II mainly synthesizes long-chain acyl-ACPs by catalyzing the elongation reaction to long-chain acyl groups having 18 or more carbon atoms.
  • KAS IV mainly catalyzes the elongation reaction of 6 to 14 carbon atoms to synthesize medium chain acyl-ACP.
  • KAS I to IV are known to have different sensitivities to the inhibitor cerulenin.
  • KAS I and KAS II are sensitive to cerulenin, and KAS III and KAS IV are not sensitive to cerulenin.
  • the proteins (A) and (B) were extended from acetyl-ACP having 2 carbon atoms (or acetyl-CoA) to ⁇ -ketoacyl-ACP having 4 carbon atoms. It is thought to be KAS type III KAS, which is a protein involved in the reaction. As shown in Examples described later, in the transformant in which the expression of the gene encoding the protein (A) is promoted, productivity of medium chain fatty acids having 12 or 14 carbon atoms is improved.
  • the amount of acyl-ACP of each chain length is increased by increasing the amount of short-chain acyl-ACP and increasing the chain-length elongation substrate by the protein (A) which is KASIII type KAS.
  • the protein (A) which is KASIII type KAS.
  • the amount of the substrate increases due to the action of the protein (A)
  • the amount of fatty acid cut out due to the action of TE also increases, so it is considered that the medium chain fatty acid increased.
  • “medium chain” means that the acyl group has 6 to 14 carbon atoms, preferably 8 to 14 carbon atoms, more preferably 10 to 14 carbon atoms, and still more preferably. Means that the number of carbon atoms is 12 or more and 14 or less.
  • a DNA linking a gene encoding a protein downstream of a promoter that functions in the host cell is introduced into a host cell lacking the fatty acid degradation system, It can be confirmed by culturing cells under conditions where the introduced gene is expressed, and analyzing changes in the fatty acid composition in the host cells or culture medium by a conventional method. Moreover, it can confirm by co-expressing TE mentioned later to said system, and comparing with the fatty acid composition at the time of expressing only TE.
  • the identity with the amino acid sequence of the protein (A) is preferably 70% or more, more preferably 74% or more, more preferably 80% or more, more preferably 85% or more. More preferably, 90% or more is more preferable, 92% or more is more preferable, 93% or more is more preferable, 94% or more is more preferable, 95% or more is more preferable, 96% or more is more preferable, and 97% or more is more Preferably, 98% or more is more preferable, and 99% or more is more preferable. Further, as the protein (B), one or more (for example, 1 to 138, preferably 1 to 126, more preferably 1 to 109) amino acid sequences of the protein (A).
  • Examples of the method for introducing a mutation into an amino acid sequence include a method for introducing a mutation into a base sequence encoding an amino acid sequence. Examples of the method for introducing mutation include site-specific mutagenesis.
  • Specific methods for introducing site-specific mutations include a method using SOE-PCR, an ODA method, a Kunkel method, and the like. Also, commercially available kits such as Site-Directed Mutagenesis System Mutan-SuperExpress Km Kit (Takara Bio), Transformer TM Site-Directed Mutagenesis Kit (Clonetech), KOD-Plus-Mutagenesis Kit (Toyobo) may be used. it can. Moreover, after giving a random gene mutation, the target gene can also be obtained by performing enzyme activity evaluation and gene analysis by an appropriate method.
  • the proteins (A) and (B) can be obtained by ordinary chemical techniques, genetic engineering techniques, and the like.
  • a protein derived from a natural product can be obtained by isolation, purification or the like from Nannochloropsis oculata.
  • the proteins (A) and (B) can be obtained by artificial chemical synthesis based on the amino acid sequence information shown in SEQ ID NO: 1.
  • the proteins (A) and (B) may be prepared as recombinant proteins by genetic recombination techniques.
  • the ⁇ -ketoacyl-ACP synthase gene described later can be used.
  • Nannochloropsis oculata can be obtained from a preservation organization such as a private or public laboratory.
  • Nannochloropsis oculata strain NIES-2145 can be obtained from the National Institute for Environmental Studies (NIES).
  • a gene encoding the protein (A) or (B) (hereinafter also referred to as “KAS gene”), a gene comprising the following DNA (a) or (b) (hereinafter also referred to as “NoKASIII gene”).
  • A DNA consisting of the base sequence represented by SEQ ID NO: 2.
  • B DNA encoding the protein (A) or (B) having a base sequence having 62% or more identity with the base sequence of the DNA (a) and having KAS activity.
  • the base sequence of SEQ ID NO: 2 is the base sequence of a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1 (KAS derived from Nannochloropsis oculata strain NIES2145).
  • the identity with the base sequence of the DNA (a) is preferably 70% or more, more preferably 74% or more, more preferably 80% or more, more preferably 85% or more. More preferably, 90% or more is more preferable, 92% or more is more preferable, 93% or more is more preferable, 94% or more is more preferable, 95% or more is more preferable, 96% or more is more preferable, and 97% or more is more Preferably, 98% or more is more preferable, and 99% or more is more preferable.
  • the DNA (b) one or more (for example, 1 or more and 481 or less, preferably 1 or more and 379 or less, more preferably 1 or more and 329 or less) in the base sequence represented by SEQ ID NO: 2, More preferably 1 to 253, more preferably 1 to 189, more preferably 1 to 126, more preferably 1 to 101, more preferably 1 to 88, More preferably 1 or more and 75 or less, more preferably 1 or more and 63 or less, more preferably 1 or more and 50 or less, more preferably 1 or more and 37 or less, more preferably 1 or more and 25 or less, More preferably, DNA encoding a protein having 1 to 12 bases) deleted, substituted, inserted or added, and having KAS activity.
  • the DNA (b) is also preferably a DNA that hybridizes with a DNA comprising a base sequence complementary to the DNA (a) under a stringent condition and encodes a protein having KAS activity.
  • the method for promoting the expression of the KAS gene can be appropriately selected from conventional methods. For example, a method of introducing the KAS gene into a host, a method of modifying an expression regulatory region (promoter, terminator, etc.) of the gene in a host having the KAS gene on the genome, and the like can be mentioned.
  • a method of introducing the KAS gene into a host a method of modifying an expression regulatory region (promoter, terminator, etc.) of the gene in a host having the KAS gene on the genome, and the like can be mentioned.
  • one that promotes the expression of a gene encoding the target protein is also referred to as a “transformant”, and one that does not promote the expression of the gene encoding the target protein is referred to as “host” or “ Also referred to as “wild strain”.
  • the transformant used in the present invention compared to the host itself, is a medium-chain fatty acid or lipid productivity comprising this (the production amount of the medium-chain fatty acid or lipid comprising this component, the total fatty acid produced Or the ratio (ratio) of the medium chain fatty acid in the total lipid or the lipid containing this as a constituent component is significantly improved.
  • the fatty acid composition in the lipid is modified. Therefore, the present invention using the transformant is a lipid having a specific number of carbon atoms, particularly a medium chain fatty acid or a lipid comprising this, preferably a fatty acid having 6 to 14 carbon atoms or a constituent thereof.
  • a fatty acid having 8 to 14 carbon atoms or a lipid comprising this more preferably a fatty acid having 10 to 14 carbon atoms or a lipid comprising this, more preferably Can be suitably used for the production of fatty acids having 12 to 14 carbon atoms or lipids comprising these.
  • the productivity of fatty acids and lipids in the host and transformant can be measured by the method used in the examples.
  • the KAS gene can be obtained by ordinary genetic engineering techniques.
  • the KAS gene can be artificially synthesized based on the amino acid sequence shown in SEQ ID NO: 1 or the base sequence shown in SEQ ID NO: 2.
  • services such as Invitrogen can be used.
  • It can also be obtained by cloning from Nannochloropsis oculata.
  • Nannochloropsis oculata NIES-2145 used in the examples can be obtained from the National Institute for Environmental Studies (NIES).
  • a transformant that can be preferably used in the present invention can be obtained by introducing the KAS gene into the host by a conventional method. Specifically, it can be prepared by preparing a recombinant vector or gene expression cassette capable of expressing the KAS gene in a host cell, introducing it into the host cell, and transforming the host cell.
  • the host for the transformant can be appropriately selected from those usually used.
  • hosts that can be used in the present invention include microorganisms (including algae and microalgae), plants, and animals. From the viewpoint of production efficiency and availability of the obtained lipid, the host is preferably a microorganism or a plant, more preferably a microorganism, and even more preferably a microalgae.
  • the microorganism may be either prokaryotic, eukaryotic, Escherichia (Escherichia) genus microorganisms or Bacillus (Bacillus) genus microorganisms, Synechocystis (Synechocystis) microorganism of the genus, Synechococcus (Synechococcus) genus microorganism of Prokaryotes or eukaryotic microorganisms such as yeast and filamentous fungi can be used.
  • Escherichia coli Escherichia coli
  • Bacillus subtilis Bacillus subtilis
  • red yeast Rhodosporidium toruloides
  • Mortierella sp Mortierella sp.
  • Chlamydomonas Chlamydomonas
  • Chlorella Chlorella
  • Faye Oda Kuti ram Phaeodactylum
  • Nan'nokuroro Algae of the genus Psis are preferred, and algae of the genus Nannochloropsis are more preferred.
  • Nannochloropsis gaditana examples include Nannochloropsis gaditana , Nannochloropsis salina , Nannochloropsis oceanica , Nannochloropsis oceanica , Nannochloropsis oceanica Examples thereof include Nannochloropsis atomus , Nannochloropsis maculata , Nannochloropsis granulata , Nannochloropsis sp.
  • Nannochloropsis oculata or Nannochloropsis gaditana is preferable, and Nannochloropsis oculata is more preferable.
  • Arabidopsis thaliana As the plant body, Arabidopsis thaliana , Brassica napus , Brassica rapa , Cocos nucifera , Palm ( Elaeis guineensis ), caffe, soybean, from the viewpoint of high lipid content in seeds ( Glycine max ), corn ( Zea mays ), rice ( Oryza sativa ), sunflower ( Helianthus annuus ), camphor ( Cinnamomum camphora ), or jatropha ( Jatropha curcas ) are preferable, and Arabidopsis is more preferable.
  • a gene capable of introducing a gene encoding a target protein into a host and expressing the gene in a host cell I just need it.
  • a vector having an expression regulatory region such as a promoter or terminator according to the type of host to be introduced, and a vector having a replication origin or a selection marker can be used.
  • it may be a vector that autonomously grows and replicates outside the chromosome, such as a plasmid, or a vector that is integrated into the chromosome.
  • an expression vector that can be preferably used in the present invention, when a microorganism is used as a host, for example, pBluescript (pBS) II SK (-) (Stratagene), pSTV vector (Takara Bio), pUC vector (Takara Shuzo), pET vector (Takara Bio), pGEX vector (GE Healthcare), pCold vector (Takara Bio), pHY300PLK (Takara Bio), pUB110 ( Mckenzie, T. et al., 1986, Plasmid 15 (2), p.
  • pBR322 (Takara Bio), pRS403 (Stratagene), and pMW218 / 219 (Nippon Gene) It is done.
  • pBluescript II SK ( ⁇ ) or pMW218 / 219 is preferably used.
  • pUC19 manufactured by Takara Bio Inc.
  • P66 Cholamydomonas Center
  • P-322 Cholamydomonas Center
  • pPha-T1 Yangmin Gong, et al., Journal of Basic Microbiology, 2011, vol.
  • pJET1 manufactured by Cosmo Bio
  • the host is an algae belonging to the genus Nannochloropsis
  • pUC19, pPha-T1, or pJET1 is preferably used.
  • pJET1 is an algae belonging to the genus Nannochloropsis, Oliver Kilian, et al., Proceedings of the National Academy of Sciences of the United States of America, 2011, vol.
  • a host can also be transformed with a DNA fragment (gene expression cassette) comprising a target gene, a promoter and a terminator.
  • Examples of the DNA fragment include a DNA fragment amplified by PCR and a DNA fragment cleaved with a restriction enzyme.
  • pRI vectors manufactured by Takara Bio Inc.
  • pBI vectors manufactured by Clontech
  • IN3 vectors manufactured by Implanta Innovations
  • the host is Arabidopsis thaliana
  • pRI vectors or pBI vectors are preferably used.
  • promoter that regulates the expression of the gene encoding the target protein incorporated in the expression vector
  • Promoters that can be preferably used in the present invention can be induced by the addition of lac promoter, trp promoter, tac promoter, trc promoter, T7 promoter, SpoVG promoter, isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG).
  • Promoters related to various derivatives Rubisco operon (rbc), PSI reaction center protein (psaAB), PSII D1 protein (psbA), cauliflower mosil virus 35SRNA promoter, housekeeping gene promoter (eg, tubulin promoter, actin promoter, ubiquitin promoter) etc.), rape or oilseed rape derived Napin gene promoter, a plant-derived Rubisco promoters, Nannochloropsis from the genus violaxanthin / chlorophyll a binding protein gene promoter VCP1 promoter, VCP2 promoter) (Oliver Kilian, et al., Proceedings of the National Academy of Sciences of the United States of America, 2011, vol.
  • Nannochloropsis lipid droplet surface protein gene promoter
  • the promoter of a violaxanthin / chlorophyll a binding protein gene or the promoter of an oleosin-like protein LDSP gene derived from the genus Nannochloropsis can be preferably used.
  • the type of selectable marker for confirming that the gene encoding the target protein has been incorporated can be appropriately selected according to the type of host used.
  • Selectable markers that can be preferably used in the present invention include ampicillin resistance gene, chloramphenicol resistance gene, erythromycin resistance gene, neomycin resistance gene, kanamycin resistance gene, spectinomycin resistance gene, tetracycline resistance gene, blasticidin S Drug resistance genes such as resistance genes, bialaphos resistance genes, zeocin resistance genes, paromomycin resistance genes, and hygromycin resistance genes. Furthermore, a gene deficiency associated with auxotrophy can be used as a selectable marker gene.
  • transformation method can be appropriately selected from conventional methods according to the type of host used. For example, transformation methods using calcium ions, general competent cell transformation methods, protoplast transformation methods, electroporation methods, LP transformation methods, methods using Agrobacterium, particle gun methods, etc. .
  • transformation methods using calcium ions For example, transformation methods using calcium ions, general competent cell transformation methods, protoplast transformation methods, electroporation methods, LP transformation methods, methods using Agrobacterium, particle gun methods, etc.
  • transformation can also be performed using the electroporation method described in Randor Radakovits, et al., Nature Communications, DOI: 10.1038 / ncomms1688, 2012, or the like.
  • ⁇ Selection of transformant introduced with target gene fragment> can be performed by using a selection marker or the like.
  • a drug resistance gene acquired by a transformant as a result of introduction of a drug resistance gene into a host cell together with a target DNA fragment at the time of transformation can be used as an indicator.
  • the introduction of the target DNA fragment can be confirmed by a PCR method using a genome as a template.
  • “Expression regulatory region” refers to a promoter or terminator, and these sequences are generally involved in regulating the expression level (transcription level, translation level) of adjacent genes.
  • the expression regulatory region of the gene is modified to promote the expression of the KAS gene, thereby improving the productivity of medium-chain fatty acids or lipids composed thereof. be able to.
  • Examples of the method for modifying the expression regulatory region include promoter replacement.
  • the expression of the KAS gene can be promoted by replacing the promoter of the gene (hereinafter also referred to as “KAS promoter”) with a promoter having higher transcriptional activity.
  • KAS promoter the promoter of the gene
  • the NoKASIII gene is present immediately below the DNA sequence consisting of the base sequence shown in SEQ ID NO: 50.
  • a promoter region is present in the DNA sequence consisting of the base sequence shown in SEQ ID NO: 50.
  • the promoter used for replacement of the KAS promoter is not particularly limited, and can be appropriately selected from those having higher transcriptional activity than the KAS promoter and suitable for the production of medium chain fatty acids or lipids comprising them.
  • a tubulin promoter a heat shock protein promoter, a promoter of the above-mentioned violaxanthin / chlorophyll a binding protein gene (VCP1 promoter (SEQ ID NO: 22), VCP2 promoter), or Nannochloropsis genus
  • the promoter (SEQ ID NO: 49) of the derived oleosin-like protein LDSP gene can be preferably used.
  • the promoter of the violaxanthin / chlorophyll a-binding protein gene or the promoter of the LDSP gene is more preferable.
  • the above-described promoter modification can be performed according to a conventional method such as homologous recombination. Specifically, a linear DNA fragment containing upstream and downstream regions of the target promoter and containing another promoter instead of the target promoter is constructed and incorporated into the host cell, and the target promoter of the host genome Two homologous recombination occurs at the upstream and downstream side of. As a result, the target promoter on the genome is replaced with another promoter fragment, and the promoter can be modified.
  • a method for modifying a target promoter by homologous recombination is described in, for example, Besher et al., Methods in molecular biology, 1995, vol. 47, p. Reference can be made to documents such as 291-302.
  • the transformant of the present invention preferably promotes expression of a gene encoding TE (hereinafter also referred to as “TE gene”) in addition to the gene encoding the protein (A) or (B).
  • TE is an enzyme that hydrolyzes the thioester bond of acyl-ACP synthesized by a fatty acid synthase such as KAS to produce free fatty acid.
  • the fatty acid synthesis on the ACP is completed by the action of TE, and the cut fatty acid is used for synthesis of polyunsaturated fatty acid, triacylglycerol (hereinafter also referred to as “TAG”) and the like.
  • TAG triacylglycerol
  • the TE that can be used in the present invention may be a protein having acyl-ACP thioesterase activity (hereinafter also referred to as “TE activity”).
  • TE activity refers to an activity of hydrolyzing the thioester bond of acyl-ACP.
  • TE has a plurality of TEs having different reaction specificities depending on the number of carbon atoms and the number of unsaturated bonds of the acyl group (fatty acid residue) constituting the substrate acyl-ACP.
  • TE is considered to be an important factor that determines the fatty acid composition in vivo.
  • it is preferable to promote the expression of the gene encoding TE.
  • the productivity of medium chain fatty acids is improved. Therefore, it is preferable to promote the expression of a gene encoding TE having substrate specificity for medium chain acyl-ACP. By introducing such a gene, the productivity of medium chain fatty acids can be further improved.
  • TE that can be used in the present invention can be appropriately selected from normal TE and functionally equivalent proteins according to the type of host.
  • TE (GenBank ABB71581) from Cuphea calophylla subsp. Mesostemon; TE from Cinnamomum camphora (GenBank AAC49151.1); TE from Myristica fragrans (GenBank AAB71729); TE from Myristica fragrans (GenBank AAB71730); Cuphea lanceolata TE (GenBank CAA54060); TE from Cuphea hookeriana (GenBank Q39513); TE from Ulumus americana (GenBank AAB71731); TE from Sorghum bicolor (GenBank EER87824); TE from Sorghum bicolor (GenBank EER88593); Cocos nucifera TE from CnFatB1 (see Jing et al.
  • the identity with any of the above-described TE amino acid sequences is 50% or more (preferably 70% or more, more preferably 80% or more, and further preferably 90% or more).
  • a protein having the amino acid sequence and having TE activity can also be used.
  • CTE (SEQ ID NO: 7, nucleotide sequence of the gene encoding the same: SEQ ID NO: 8), NoTE (SEQ ID NO: 34, the gene encoding the same) from the viewpoint of substrate specificity for medium chain acyl-ACP Nucleotide sequence: SEQ ID NO: 35), bay-derived TE (SEQ ID NO: 51, nucleotide sequence of the gene encoding the same: SEQ ID NO: 52), TE derived from Nannochloropsis gaditana (SEQ ID NO: 53, the gene encoding the same) Nucleotide sequence: SEQ ID NO: 54), TE derived from Nannochloropsis granulata (SEQ ID NO: 55, nucleotide sequence of the gene encoding this: SEQ ID NO: 56), TE derived from symbiodinium microadriaticum (sequence) No.
  • nucleotide sequence of the gene encoding it SEQ ID NO: 58
  • identity of these TE amino acid sequences of 50% or more preferably Or a protein having a TE activity against medium-chain acyl-ACP (for example, the base sequence shown in SEQ ID NO: 40).
  • a protein encoded by DNA is preferred.
  • sequence information of these TEs and the genes encoding them can be obtained from, for example, the National Center for Biotechnology Information (NCBI).
  • the protein has TE activity, for example, by introducing DNA linked to an acyl-ACP thioesterase gene downstream of a promoter that functions in a host cell such as E. coli into a host cell lacking the fatty acid degradation system, It can be confirmed by culturing under conditions where the gene is expressed, and analyzing changes in the fatty acid composition in the host cell or culture solution using a method such as gas chromatography analysis.
  • Reactions using various acyl-ACPs prepared by the method of Yuan et al. (Yuan L. et al., Proc. Natl. Acad. Sci. USA, 1995, vol. 92 (23), p. 10639-10643) as substrates. By doing so, TE activity can be measured.
  • a transformant that promotes the expression of the TE gene can be prepared by a conventional method.
  • a transformant is obtained by a method of introducing a TE gene into a host, a method of modifying an expression regulatory region of the gene in a host having the TE gene on the genome, etc. Can be produced.
  • KAS I mainly catalyzes an elongation reaction having 4 to 16 carbon atoms to synthesize palmitoyl-ACP having 16 carbon atoms.
  • KAS IV catalyzes an extension reaction mainly having 6 to 14 carbon atoms to synthesize medium chain acyl-ACP. Therefore, by promoting the expression of the gene encoding KAS IV, the productivity of medium chain fatty acids can be further improved.
  • acyltransferase is an enzyme that performs acylation necessary for the biosynthesis of TAG. Therefore, by promoting the expression of a gene encoding a medium chain fatty acid-specific acyltransferase such as a medium chain fatty acid specific diacylglycerol acyltransferase, the productivity of the medium chain fatty acid can be further improved.
  • the KAS and acyltransferase that can be used in the present invention can be appropriately selected from ordinary KAS, acyltransferase, and proteins functionally equivalent to them according to the type of host. Moreover, the transformant which promoted the expression of these genes can be produced by a conventional method.
  • a transformant can be produced by a method of modifying an expression control region of a gene.
  • productivity of medium-chain fatty acids or lipids comprising the same is improved as compared to a host in which expression of the gene encoding the protein (A) or (B) is not promoted. is doing. Therefore, if the transformant of the present invention is cultured under appropriate conditions, and then the medium chain fatty acid or the lipid containing it as a constituent component is recovered from the obtained culture or growth product, the medium chain fatty acid or the constituent component thereof is recovered. Can be produced efficiently.
  • “culture” refers to the culture solution and transformant after culturing
  • “growth” refers to the transformant after growth.
  • the culture conditions of the transformant of the present invention can be appropriately selected depending on the host, and culture conditions usually used for the host can be used. From the viewpoint of fatty acid production efficiency, for example, glycerol, acetic acid, glucose or the like may be added to the medium as a precursor involved in the fatty acid biosynthesis system.
  • the Escherichia coli When Escherichia coli is used as a host, the Escherichia coli can be cultured, for example, in LB medium or Overnight Express Instant TB Medium (Novagen) at 30 to 37 ° C. for 0.5 to 1 day.
  • the medium when E. coli is used as a host, the medium preferably contains cerulenin in order to improve the productivity of medium chain fatty acids. As shown in Examples described later, medium chain fatty acid productivity can be further improved by culturing the transformant in a medium containing cerulenin.
  • the concentration of cerulenin in the medium is preferably a concentration that does not adversely affect the growth of the transformant.
  • the cerulenin concentration is preferably 1 ⁇ M or more, more preferably 10 ⁇ M or more, preferably 50 ⁇ M or less, and more preferably 25 ⁇ M or less.
  • 1 to 50 ⁇ M is preferable, 10 to 50 ⁇ M is more preferable, and 10 to 25 ⁇ M is more preferable.
  • the culture of Arabidopsis thaliana can be performed, for example, at a temperature of 20 to 25 ° C. in soil, continuously irradiated with white light, or under light conditions such as 16 hours of light and 8 hours of dark. Can be cultivated monthly.
  • the culture medium may be based on natural seawater or artificial seawater, or a commercially available culture medium may be used.
  • the medium include f / 2 medium, ESM medium, Daigo IMK medium, L1 medium, and MNK medium.
  • f / 2 medium, ESM medium, or Daigo IMK medium is preferable, f / 2 medium or Daigo IMK medium is more preferable, and f / 2 medium is further included. preferable.
  • nitrogen sources, phosphorus sources, metal salts, vitamins, trace metals, and the like can be appropriately added to the medium.
  • the amount of transformant to be inoculated into the medium can be appropriately selected, and is preferably 1 to 50% (vol / vol), more preferably 1 to 10% (vol / vol) per medium from the viewpoint of growth.
  • the culture temperature is not particularly limited as long as it does not adversely affect the growth of algae, but it is usually in the range of 5 to 40 ° C. From the viewpoint of promoting the growth of algae, improving the productivity of fatty acids, and reducing the production cost, the temperature is preferably 10 to 35 ° C, more preferably 15 to 30 ° C.
  • the algae is preferably cultured under light irradiation so that photosynthesis is possible. The light irradiation may be performed under conditions that allow photosynthesis, and may be artificial light or sunlight.
  • the illuminance during light irradiation is preferably in the range of 100 to 50000 lux, more preferably in the range of 300 to 10000 lux, and still more preferably in the range of 1000 to 6000 lux, from the viewpoint of promoting the growth of algae and improving the productivity of fatty acids. It is. Further, the light irradiation interval is not particularly limited, but from the same viewpoint as described above, it is preferably performed in a light / dark cycle, and the light period in 24 hours is preferably 8 to 24 hours, more preferably 10 to 18 hours, More preferably, it is 12 hours.
  • the culture of algae is preferably performed in the presence of a gas containing carbon dioxide or a medium containing a carbonate such as sodium bicarbonate so that photosynthesis is possible.
  • concentration of carbon dioxide in the gas is not particularly limited, but is preferably 0.03 (similar to atmospheric conditions) to 10%, more preferably 0.05 to 5%, from the viewpoint of promoting growth and improving the productivity of fatty acids. More preferably, it is 0.1 to 3%, and still more preferably 0.3 to 1%.
  • the concentration of the carbonate is not particularly limited.
  • sodium bicarbonate it is preferably 0.01 to 5% by mass, more preferably 0.05 to 2% by mass, from the viewpoint of promoting growth and improving the productivity of fatty acids.
  • the content is 0.1 to 1% by mass.
  • the culture time is not particularly limited, and may be performed for a long period of time (for example, about 150 days) so that algal bodies that accumulate lipids at high concentrations can grow at high concentrations. From the viewpoint of promoting the growth of algae, improving the productivity of fatty acids, and reducing the production cost, the culture period is preferably 3 to 90 days, more preferably 3 to 30 days, and even more preferably 7 to 30 days.
  • the culture may be any of aeration and agitation culture, shaking culture or stationary culture. From the viewpoint of improving aeration, aeration and agitation culture or shaking culture is preferable, and aeration and agitation culture is more preferable.
  • the method for collecting lipid from the culture or growth can be appropriately selected from conventional methods.
  • lipid components can be isolated from the aforementioned culture or growth by filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, chloroform / methanol extraction method, hexane extraction method, ethanol extraction method, or the like. Can be separated and recovered.
  • oil is recovered from the culture or growth by pressing or extraction, and then general purification such as degumming, deoxidation, decolorization, dewaxing, deodorization, etc. is performed to obtain lipids. be able to.
  • a fatty acid can be obtained by hydrolyzing the isolated lipid.
  • Examples of the method for isolating the fatty acid from the lipid component include a method of treating at a high temperature of about 70 ° C. in an alkaline solution, a method of treating with lipase, a method of decomposing using high-pressure hot water, and the like.
  • the lipid produced in the production method of the present invention preferably contains a fatty acid or a fatty acid compound, and more preferably contains a fatty acid or a fatty acid ester compound, from the viewpoint of its availability.
  • the fatty acid or fatty acid ester compound contained in the lipid is preferably a medium chain fatty acid or an ester compound thereof, more preferably a fatty acid having 6 to 14 carbon atoms or an ester compound thereof, from the viewpoint of availability to a surfactant or the like.
  • a fatty acid having 8 to 14 carbon atoms or an ester compound thereof is more preferable, a fatty acid having 10 to 14 carbon atoms or an ester compound thereof is more preferable, and a fatty acid having 12 to 14 carbon atoms or a fatty acid thereof. More preferred are ester compounds.
  • the fatty acid ester compound is preferably a simple lipid or a complex lipid, more preferably a simple lipid, and even more preferably triacylglycerol.
  • Lipids obtained by the production method of the present invention are used as edible, plasticizers, emulsifiers such as cosmetics, detergents such as soaps and detergents, fiber treatment agents, hair rinse agents, or bactericides and preservatives. Can do.
  • the present invention further relates to the embodiments described above, and the following methods for producing lipids, methods for improving lipid productivity, methods for modifying the composition of fatty acids produced, proteins, genes, recombinant vectors, organisms, transformants, and A method for producing a transformant is disclosed.
  • a method for producing a lipid comprising culturing a transformant in which expression of a gene encoding the following protein (A) or (B) is promoted to produce a fatty acid or a lipid comprising the same.
  • a protein comprising the amino acid sequence represented by SEQ ID NO: 1.
  • B) The identity with the amino acid sequence of the protein (A) is 67% or more, preferably 70% or more, more preferably 74% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90%.
  • % Or more more preferably 92% or more, preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more. More preferably, a protein comprising 99% or more amino acid sequence and having KAS activity.
  • ⁇ 2> Promote the expression of the gene encoding the protein (A) or (B), and improve the productivity of medium-chain fatty acids produced in the cells of the transformant or lipids comprising this as a constituent component, Method for improving lipid productivity.
  • ⁇ 3> Promoting the expression of the gene encoding the protein (A) or (B) to improve the productivity of medium-chain fatty acids produced in the cells of the transformant or lipids comprising this A method for modifying the composition of lipids, which modifies the composition of fatty acids or lipids in total fatty acids or total lipids produced.
  • ⁇ 4> The gene according to ⁇ 1> to ⁇ 3>, wherein the gene encoding the protein (A) or (B) is introduced into a host to promote the expression of the gene encoding the protein (A) or (B).
  • ⁇ 5> A method for producing a lipid, comprising culturing a transformant into which a gene encoding the protein (A) or (B) has been introduced, and producing a fatty acid or a lipid comprising the same.
  • Transformants are produced by introducing a gene encoding the protein (A) or (B), and medium-chain fatty acids produced in the cells of the transformants or lipid production comprising them To improve lipid productivity.
  • Transformants are produced by introducing a gene encoding the protein (A) or (B), and medium-chain fatty acids produced in the cells of the transformants or lipid production comprising them A method for modifying the composition of lipids, which improves the properties and modifies the composition of fatty acids or lipids in total fatty acids or total lipids produced.
  • the protein (B) has one or more, preferably 1 or more and 138 or less, more preferably 1 or more and 126 or less, more preferably 1 or more amino acid sequences in the protein (A). 109 or less, more preferably 1 or more and 84 or less, more preferably 1 or more and 63 or less, more preferably 1 or more and 42 or less, more preferably 1 or more and 33 or less, more preferably 1 or more 29 or less, more preferably 1 or more and 25 or less, more preferably 1 or more and 21 or less, more preferably 1 or more and 16 or less, more preferably 1 or more and 12 or less, more preferably 1 or more 8.
  • the protein is a protein in which 8 or less, more preferably 1 to 4 amino acids are deleted, substituted, inserted or added.
  • the gene encoding the protein (A) or (B) is a gene consisting of the following DNA (a) or (b).
  • (B) 62% or more identity with the base sequence of the DNA (a), preferably 70% or more, more preferably 74% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% % Or more, more preferably 92% or more, preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more. More preferably, the DNA encoding the protein (A) or (B) having a nucleotide sequence of 99% or more and having KAS activity.
  • the DNA (b) has one or more, preferably 1 or more and 481 or less, more preferably 1 or more and 379 or less, more preferably 1 or more, in the base sequence of the DNA (a). 329 or less, more preferably 1 or more and 253 or less, more preferably 1 or more and 189 or less, more preferably 1 or more and 126 or less, more preferably 1 or more and 101 or less, more preferably 1 or more 88 or less, more preferably 1 or more and 75 or less, more preferably 1 or more and 63 or less, more preferably 1 or more and 50 or less, more preferably 1 or more and 37 or less, more preferably 1 or more 25 or less, more preferably 1 or more and 12 or less base sequences having a deleted, substituted, inserted, or added base sequence and having the KAS activity (A) or It encodes the protein (A) or (B) that hybridizes under stringent conditions with the DNA encoding (B) or the DNA comprising a base sequence complementary to the DNA (a)
  • the method according to any one of ⁇ 1> to ⁇ 9>, wherein ⁇ 11> The method according to any one of ⁇ 1> to ⁇ 10>, wherein the proteins (A) and (B) are KAS type KAS.
  • ⁇ 12> The method according to any one of ⁇ 1> to ⁇ 11>, wherein expression of a gene encoding TE is promoted in the transformant.
  • ⁇ 13> The method according to ⁇ 12>, wherein the gene encoding TE is introduced into a transformant to promote the expression of the gene encoding TE.
  • ⁇ 14> The method according to ⁇ 12> or ⁇ 13>, wherein the TE is TE having substrate specificity for medium chain acyl-ACP.
  • the protein having the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 34, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, or SEQ ID NO: 57, or the amino acid sequence of the protein has 50 identity % Or more (preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more), and a protein having TE activity against medium chain acyl-ACP.
  • ⁇ 16> The method according to any one of ⁇ 1> to ⁇ 12>, wherein the transformant is a microorganism or a plant.
  • ⁇ 17> The method according to ⁇ 16>, wherein the microorganism is a microalgae.
  • microalgae are algae belonging to the genus Nannochloropsis, preferably Nannochloropsis oculata.
  • micro19> The method according to ⁇ 16>, wherein the microorganism is Escherichia coli.
  • the plant is Arabidopsis thaliana.
  • the lipid is a medium chain fatty acid or an ester compound thereof, preferably a fatty acid having 6 to 14 carbon atoms or an ester compound thereof, more preferably a fatty acid having 8 to 14 carbon atoms or an ester compound thereof, Any of the above ⁇ 1> to ⁇ 20>, more preferably containing a fatty acid having 10 to 14 carbon atoms or an ester compound thereof, more preferably a fatty acid having 12 to 14 carbon atoms or an ester compound thereof.
  • ⁇ 22> The protein (A) or (B) defined in any one of ⁇ 1> to ⁇ 21>.
  • ⁇ 23> A gene encoding the protein according to ⁇ 22>.
  • ⁇ 24> A gene comprising the DNA (a) or (b) defined in any one of ⁇ 1> to ⁇ 21>.
  • ⁇ 25> A recombinant vector containing the gene according to ⁇ 23> or ⁇ 24>.
  • ⁇ 26> A transformant that promotes the expression of the gene according to ⁇ 23> or ⁇ 24>.
  • ⁇ 27> A transformant obtained by introducing the gene according to ⁇ 23> or ⁇ 24> or the recombinant vector according to ⁇ 25> into a host.
  • ⁇ 28> A method for producing a transformant, wherein the gene according to ⁇ 23> or ⁇ 24> or the recombinant vector according to ⁇ 25> is introduced into a host.
  • ⁇ 29> The transformant according to any one of ⁇ 26> to ⁇ 28> or a method for producing the same, wherein expression of a gene encoding TE is promoted.
  • ⁇ 30> The transformant according to ⁇ 29> or a method for producing the same, wherein a gene encoding TE is introduced into a host to promote expression of the gene encoding TE.
  • ⁇ 31> The transformant according to ⁇ 29> or ⁇ 30> or a method for producing the same, wherein the TE is TE having substrate specificity for medium-chain acyl-ACP.
  • the protein having the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 34, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55 or SEQ ID NO: 57, or the amino acid sequence of the protein has 50 identity % Or more (preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more), and a protein having TE activity against medium chain acyl-ACP, ⁇ 29> to ⁇ 31>
  • ⁇ 33> The transformant according to any one of ⁇ 26> to ⁇ 32> or the method for producing the transformant, wherein the transformant or host is a microorganism or a plant.
  • ⁇ 34> The transformant according to ⁇ 33> or the production method thereof, wherein the microorganism is a microalgae.
  • ⁇ 35> The transformant according to ⁇ 34> or the method for producing the same, wherein the microalga is an algae belonging to the genus Nannochloropsis, preferably Nannochloropsis oculata.
  • ⁇ 36> The transformant according to ⁇ 33> or the method for producing the same, wherein the microorganism is Escherichia coli.
  • ⁇ 37> The transformant according to ⁇ 33> or the method for producing the same, wherein the plant is Arabidopsis thaliana.
  • the lipid is a medium chain fatty acid or an ester compound thereof, preferably a fatty acid having 6 to 14 carbon atoms or an ester compound thereof, more preferably a fatty acid having 8 to 14 carbon atoms or an ester compound thereof,
  • Example 1 Production of Transformant Introducing NoKASIII Gene into Escherichia coli and Production of Fatty Acid Using Transformant (1) Construction of Plasmid for Expression of NoKASIII Gene Nannochloropsis Oculata NIES-2145 from National Institute for Environmental Studies (NIES) Stocks were purchased and used.
  • Nannochloropsis oculata strain NIES-2145 is f / 2 liquid medium (NaNO 3 75 mg, NaH 2 PO 4 ⁇ 2H 2 O 6 mg, vitamin B12 0.5 ⁇ g, biotin 0.5 ⁇ g, thiamine 100 ⁇ g, Na 2 SiO 3 ⁇ 9H 2 O 10mg, Na 2 EDTA ⁇ 2H 2 O 4.4mg, FeCl 3 ⁇ 6H 2 O 3.16mg, FeCl 3 ⁇ 6H 2 O 12 ⁇ g, ZnSO 4 ⁇ 7H 2 O 21 ⁇ g, MnCl 2 ⁇ 4H 2 O 180 ⁇ g, CuSO 4 ⁇ 5H 2 O 7 ⁇ g, Na 2 MoO 4 ⁇ 2H 2 O 7 ⁇ g / artificial seawater 1L), inoculate 10% in 50mL f / 2 medium, and at 25 ° C, carbon dioxide 0.3% in an artificial weather machine Cultured for 6 days.
  • RNA was purified using RNeasy Plant Mini Kit (Qiagen). From the obtained total RNA, a cDNA library was prepared using SuperScript III First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen). Using this cDNA as a template, a DNA fragment of NoKASIII gene was obtained by PCR using the primer pair of primer number 3 and primer number 4 shown in Table 1.
  • pSTV28 was amplified by PCR using the plasmid vector pSTV28 (Takara Bio) as a template and the primer pair of primer number 5 and primer number 6 shown in Table 1, and the template was treated by restriction enzyme Dpn I (manufactured by Toyobo). Digestion was performed. These two fragments were purified using the High Pure PCR Product Purification Kit (Roche Applied Science) and then fused using the In-Fusion HD Cloning Kit (Clontech) to construct a NoKASIII gene expression plasmid. did.
  • NoKASIII gene expression plasmid into Escherichia coli
  • E. coli mutant K27 strain (fadD88) (Overath et al, Eur. J. Biochem., 7, p. 559-574, 1969) were transformed by the competent cell transformation method.
  • the transformed K27 strain (pSTV :: NoKASIII) was allowed to stand at 37 ° C overnight, and colonies were obtained from LBCm liquid medium (Bacto Trypton 1%, Yeast Extract 0.5%, NaCl 1%, chloramphenicol) 30 ⁇ g / mL) was inoculated into 1 mL and cultured at 30 ° C. overnight.
  • Nitrogen gas was blown onto the resulting chloroform layer to dry it, 0.7 mL of 0.5N potassium hydroxide / methanol solution was added, and the temperature was kept constant at 80 ° C. for 30 minutes. Subsequently, 1 mL of 14% boron trifluoride-methanol solution (manufactured by SIGMA) was added, and the temperature was kept constant at 80 ° C. for 10 minutes. Thereafter, 1 mL each of hexane and saturated saline was added and stirred vigorously, and allowed to stand at room temperature for 30 minutes. The upper hexane layer was recovered to obtain a fatty acid methyl ester.
  • the fatty acid methyl ester was identified by subjecting the sample to gas chromatograph mass spectrometry analysis under the same conditions.
  • the amount of methyl ester of each fatty acid was quantified from the peak area of the waveform data obtained by gas chromatography analysis. Each peak area was compared with the peak area of 7-pentadecanone, which is an internal standard, to correct between samples, and the amount of each fatty acid per liter of culture solution was calculated. Furthermore, the sum total of each fatty acid amount was made into the total fatty acid amount (FA), and the ratio of each fatty acid amount which occupies for the total fatty acid amount was computed. The results are shown in Table 2.
  • “Fatty acid composition (% TFA)” indicates the ratio (weight percent) of each fatty acid to the total fatty acid.
  • “N” represents an integer of 0 to 5, for example, when “C18: n” is described, C18: 0, C18: 1, C18: 2, C18: 3, C18: 4, and C18: 5 Represents a fatty acid.
  • Example 2 Production of Fatty Acid by Transformant Introduced NoKASIII Gene into Escherichia coli in the Presence of Cerrenin K27 Strain (pSTV :: NoKASIII) Introduced in Example 1 and Introduced with NoKASIII Gene was LBCm Liquid Medium (Bacto Trypton) 1%, Yeast Extract 0.5%, NaCl 1%, chloramphenicol 30 ⁇ g / mL) was inoculated into 1 mL and cultured at 30 ° C. overnight. 20 ⁇ L of the culture solution was inoculated into 2 mL of Overnight Express Instant TB Medium (Novagen) and cultured with shaking at 30 ° C.
  • Example 3 Production of Transformant Introducing NoKASIII Gene and CTE Gene into Escherichia coli, and Production of Fatty Acid Using Transformant
  • the gene to be encoded (base sequence: SEQ ID NO: 8) was introduced into the K27 strain (fadD88) to prepare a transformant (pMW :: CTE).
  • the transformant pMW :: CTE was further transformed by the competent cell transformation method using the NoKASIII gene expression plasmid prepared in Example 1.
  • the CTE gene was cloned into the plasmid vector pMW219 (Nippon Gene).
  • the K27 strain (CTE + pSTV :: KASIII) transformed with the NoKASIII gene and CTE gene was allowed to stand at 37 ° C. overnight, and colonies were obtained from the LBCmKm liquid medium (Bacto Trypton 1%, Yeast Extract 0.5%, NaCl 1%, chloramphenicol 30 ⁇ g / mL, kanamycin sulfate 50 ⁇ g / mL) was inoculated into 1 mL, and cultured at 30 ° C. overnight. 20 ⁇ L of the culture solution was inoculated into 2 mL of Overnight Express Instant TB Medium (Novagen) and cultured with shaking at 30 ° C.
  • Example 4 Production of Fatty Acid by Transformant Introduced NoKASIII Gene and CTE Gene into Escherichia coli in the Presence of Cerrenin K27 Strain (CTE + pSTV :: KASIII) Introduced with NoKASIII Gene and CTE Gene Prepared in Example 3 ) was inoculated into 1 mL of LBCmKm liquid medium (Bacto Trypton 1%, Yeast Extract 0.5%, NaCl 1%, chloramphenicol 30 ⁇ g / mL, kanamycin sulfate 50 ⁇ g / mL) and cultured at 30 ° C. overnight.
  • LBCmKm liquid medium Bostrehalose 1%, Yeast Extract 0.5%, NaCl 1%, chloramphenicol 30 ⁇ g / mL, kanamycin sulfate 50 ⁇ g / mL
  • Example 5 Production of transformant in which NoKASIII gene was introduced into Nannochloropsis oculata, and production of fatty acid by transformant
  • PCR was performed using the primer pair of primer number 11 and primer number 12 and the primer pair of primer number 13 and primer number 14 shown in Table 1, respectively, and the zeocin resistance gene and tubulin promoter sequence Each was amplified. Further, PCR was performed using the genome of Nannochloropsis oculata NIES2145 strain as a template and the primer pair of primer number 15 and primer number 16 shown in Table 1 to amplify the heat shock protein terminator sequence (SEQ ID NO: 17).
  • PCR was carried out using the plasmid vector pUC19 (manufactured by Takara Bio Inc.) as a template and the primer pair of primer number 18 and primer number 19 shown in Table 1 to amplify the plasmid vector pUC19.
  • This expression plasmid consists of an insert sequence linked in the order of a tubulin promoter sequence, a zeocin resistance gene, a heat shock protein terminator sequence, and a pUC19 vector sequence.
  • VCP1 violaxanthin / chlorophyll a binding protein gene of Nannochloropsis sp. W2J3B strain registered in GenBank
  • VCP1 promoter sequence SEQ ID NO: 22
  • VCP1 terminator sequence SEQ ID NO: 23
  • PCR was performed using the primer pair of primer number 24 and primer number 25 shown in Table 1, and the primer pair of primer number 26 and primer number 27, respectively, and the VCP1 promoter sequence and VCP1 terminator sequence were determined. Acquired each.
  • zeocin resistance gene expression plasmid as a template, PCR was performed using the primer pair of primer number 28 and primer number 19 shown in Table 1, and a zeocin resistance gene expression cassette (tubulin promoter sequence, zeocin resistance gene, A fragment consisting of a heat shock protein terminator sequence) and a pUC19 sequence was amplified.
  • This expression plasmid comprises a VUC1 promoter sequence, a NoKASIII gene, a VCP1 terminator sequence, a tubulin promoter sequence, a zeocin resistance gene, and a heat shock protein terminator sequence in that order and a pUC19 vector sequence.
  • NoKASIII gene expression fragment into Nannochloropsis Using the NoKASIII gene expression plasmid as a template, PCR was performed using the primer pair of primer number 16 and primer number 24 shown in Table 1, for NoKASIII gene expression.
  • the fragment (DNA fragment consisting of VCP1 promoter sequence, NoKASIII gene, VCP1 terminator sequence, tubulin promoter sequence, zeocin resistance gene, heat shock protein terminator sequence) was amplified.
  • the amplified DNA fragment was purified using High Pure PCR Product Purification Kit (Roche Applied Science). Note that sterilized water was used for elution during purification, not the elution buffer included in the kit.
  • Nannochloropsis oculata strain NIES2145 obtained from the National Institute for Environmental Studies (NIES)
  • NIES2145 obtained from the National Institute for Environmental Studies (NIES)
  • 384 mM sorbitol solution to completely remove salts, and transformed host cells Used as.
  • About 500 ng of the NoKASIII gene expression fragment amplified above was mixed with host cells, and electroporation was performed under the conditions of 50 ⁇ F, 500 ⁇ , and 2,200 v / 2 mm.
  • N15P5 medium a medium in which the nitrogen concentration of f / 2 medium is increased 15-fold and the phosphorus concentration is increased 5-fold. ° C., under 0.3% CO 2 atmosphere, for 4 weeks shaking culture at 12h / 12h light-dark conditions to the preculture. 10 mL of the preculture was transferred to 40 mL of N15P5 medium, and cultured under shaking in a 12 h / 12 h light / dark condition at 25 ° C. in a 0.3% CO 2 atmosphere. After 3 weeks of culture, the lipid components contained in the culture solution were analyzed by the same method as in Example 1. As a negative control, the same experiment was performed on the Nannochloropsis oculata strain (control) into which only the zeocin resistance gene was introduced. The results are shown in Table 6.
  • Example 6 Production of Transformant Introducing NoKASIII Gene and NoTE Gene into Nannochloropsis Oculata, and Production of Fatty Acid by Transformant
  • Paromomycin Resistance Gene (SEQ ID NO: 29) was artificially synthesized. PCR was performed using the synthesized DNA fragment as a template and the primer pair of primer number 30 and primer number 31 shown in Table 1 to amplify the paromomycin resistance gene. Using the zeocin resistance gene expression plasmid constructed in Example 5 as a template, PCR was performed using the primer pair of primer number 14 and primer number 15 shown in Table 1, tubulin promoter sequence, heat shock protein terminator sequence and pUC19. A gene fragment consisting of a vector was amplified.
  • This expression plasmid consists of an insert sequence in the order of a tubulin promoter sequence, a paromomycin resistance gene, a heat shock protein terminator sequence, and a pUC19 vector sequence.
  • pBluescriptII SK (-) was amplified by PCR using the plasmid vector pBluescriptII SK (-) (Stratagene) as a template and the primer pair of primer number 36 and primer number 37 shown in Table 1, and the restriction enzyme Dpn I
  • the mold was digested by treatment (manufactured by Toyobo Co., Ltd.). These two fragments were purified using High Pure PCR Product Purification Kit (Roche Applied Science) and then fused using In-Fusion HD Cloning Kit (Clontech) to construct NoTE gene plasmid NoTE. .
  • This plasmid NoTE_262 is obtained by removing amino acid residues 1 to 87 from the N-terminal side of the amino acid sequence shown in SEQ ID NO: 34, and upstream of it from the N-terminal side of the LacZ protein derived from the plasmid vector pBluescriptII SK (-) It was constructed to express a protein in which amino acid residues 1 to 29 were fused.
  • “NoTE” is the nucleotide sequence encoding the polypeptide consisting of the amino acid sequence of positions 88 to 287 of SEQ ID NO: 34, and the nucleotide sequence of positions 262 to 864 of SEQ ID NO: 35 is the plasmid.
  • a gene obtained by mutating a part of bases at positions 262 to 864 in the base sequence shown in SEQ ID NO: 35 by PCR using the plasmid NoTE as a template and the primer pair of primer No. 38 and primer No. 39 shown in Table 1 A fragment (SEQ ID NO: 40) was obtained. Using this gene fragment, a NoTE variant expression plasmid NoTE_262 (V204W) was constructed in the same manner as described above.
  • a codon encoding valine at position 204 in the amino acid sequence shown in SEQ ID NO: 34 is replaced with a codon (TGG) encoding tryptophan.
  • PCR was performed using the plasmid NoTE_262 (V204W) as a template and the primer pair of primer number 41 and primer number 42 shown in Table 1 to obtain a NoTE variant gene fragment consisting of the base sequence shown in SEQ ID NO: 40.
  • VCP1 chloroplast transition signal sequence SEQ ID NO: 43
  • complete cds sequence accesion number: JF957601.1
  • VCP1 chloroplast translocation signal sequence Using the DNA fragment of this VCP1 chloroplast translocation signal sequence and the DNA fragment of the VCP1 promoter sequence and VCP1 terminator sequence synthesized in Example 5 as a template, the primer pair of primer number 24 and primer number 25 shown in Table 1, primer number PCR was performed using the primer pair of 44 and primer number 45, and the primer pair of primer number 26 and primer number 27, respectively, and a VCP1 promoter sequence, a VCP1 chloroplast transfer signal sequence, and a VCP1 terminator sequence were obtained.
  • PCR was carried out using the plasmid vector pUC19 (manufactured by Takara Bio Inc.) as a template and the primer pair of primer number 18 and primer number 19 shown in Table 1 to amplify the plasmid vector pUC19.
  • the NoTE variant gene fragment, VCP1 promoter sequence, VCP1 chloroplast translocation signal sequence, and VCP1 terminator sequence obtained above were fused to the plasmid vector pUC19 in the same manner as in Example 5 for expression of the NoTE variant gene.
  • Plasmid NoTE_262 (V204W) _Nanno was constructed. This plasmid consists of a NoTE gene expression sequence linked in the order of a VCP1 promoter sequence, a VCP1 chloroplast transfer signal sequence, a NoTE variant gene fragment, and a VCP1 terminator sequence, and a pUC19 vector sequence.
  • PCR was performed using the plasmid NoTE_262 (V204W) _Nanno as a template and the primer pair of primer number 46 and primer number 28 to obtain a gene fragment consisting of a VCP1 chloroplast transfer signal, a NoTE variant gene, and a VCP1 terminator sequence. . Moreover, PCR was performed using the genome of Nannochloropsis oculata NIES2145 strain as a template and the primer pair of primer number 47 and primer number 48 shown in Table 1 to amplify the LDSP promoter sequence (SEQ ID NO: 49).
  • PCR was performed using the above-mentioned plasmid for paromomycin resistance gene expression as a template and the primer pair of primer number 28 and primer number 19 shown in Table 1, and a cassette for paromomycin resistance gene expression (tubulin promoter sequence, paromomycin resistance gene, A fragment consisting of a heat shock protein terminator sequence) and a pUC19 sequence was amplified.
  • This expression plasmid consists of an LDSP promoter sequence, a VCP1 chloroplast transfer signal sequence, a NoTE variant gene sequence, a VCP1 terminator sequence, a tubulin promoter sequence, a paromomycin resistance gene, and a heat shock protein terminator sequence in that order.
  • PUC19 vector sequence PUC19 vector sequence.
  • NoTE variant gene expression fragment and NoKASIII gene expression fragment were introduced into Nannochloropsis Using the NoTE variant gene expression plasmid as a template, primer pairs of primer numbers 16 and 24 shown in Table 1 were used. PCR was performed to amplify a NoTE variant gene expression fragment (DNA fragment consisting of LDSP promoter sequence, NoTE variant gene, VCP1 terminator sequence, tubulin promoter sequence, paromomycin resistance gene, and heat shock protein terminator sequence). The amplified DNA fragment was purified using High Pure PCR Product Purification Kit (Roche Applied Science). Note that sterilized water was used for elution during purification, not the elution buffer included in the kit.
  • Example 5 the NoTE variant gene expression fragment was introduced into Nannochloropsis oculata strain NIES2145, cultured using paromomycin-containing f / 2 medium, and the resulting colony was transformed into the NoTE variant gene. Selected as an introduced strain (NoTE). Furthermore, using the obtained NoTE variant gene introduction strain (NoTE) as a host, a NoKASIII gene expression fragment was introduced in the same manner as in Example 5, and the obtained colonies were introduced with a NoTE variant gene and a NokASIII gene. Selected as a strain (NoTE + NoKASIII).
  • a transformant with improved productivity of medium chain fatty acids can be produced by promoting the expression of the KAS gene defined in the present invention. By culturing this transformant, the productivity of medium chain fatty acids can be improved.

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Abstract

L'invention concerne un procédé de production de lipides pour la culture d'un transformant ayant une expression accrue d'un gène codant pour la protéine (A) ou (B) ci-dessous et la production d'acides gras ou d'un lipide les contenant à titre de composants structuraux. (A) Protéine comprenant une séquence d'acides aminés représentée par SEQ ID No: 1. (B) Protéine comprenant une séquence d'acides aminés présentant une identité de 67 % ou plus avec la séquence d'acides aminés de la protéine (A) et ayant une activité β-cétoacyl-ACP synthase.
PCT/JP2016/072007 2015-08-06 2016-07-27 Procédé de production de lipides WO2017022587A1 (fr)

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US20030145350A1 (en) * 2000-01-12 2003-07-31 Friedrich Spener Method for increasing the content of fatty acids in plants and micro-organisms
WO2009150435A1 (fr) * 2008-06-13 2009-12-17 University Of Stavanger Vecteurs de transformation du plaste permettant l’excision de gènes marqueurs
JP2011250781A (ja) * 2010-05-06 2011-12-15 Kao Corp チオエステラーゼ及びそれを用いた脂肪酸又は脂質の製造方法
US20130102040A1 (en) * 2011-10-17 2013-04-25 Colorado School Of Mines Use of endogenous promoters in genetic engineering of nannochloropsis gaditana
WO2013082186A2 (fr) * 2011-11-28 2013-06-06 Solazyme, Inc. Souches microbiennes génétiquement modifiées comprenant des gènes de la voie des lipides de prototheca
WO2015005139A1 (fr) * 2013-07-12 2015-01-15 花王株式会社 Acyl-acp thioestérase

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WO2009150435A1 (fr) * 2008-06-13 2009-12-17 University Of Stavanger Vecteurs de transformation du plaste permettant l’excision de gènes marqueurs
JP2011250781A (ja) * 2010-05-06 2011-12-15 Kao Corp チオエステラーゼ及びそれを用いた脂肪酸又は脂質の製造方法
US20130102040A1 (en) * 2011-10-17 2013-04-25 Colorado School Of Mines Use of endogenous promoters in genetic engineering of nannochloropsis gaditana
WO2013082186A2 (fr) * 2011-11-28 2013-06-06 Solazyme, Inc. Souches microbiennes génétiquement modifiées comprenant des gènes de la voie des lipides de prototheca
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