WO2017022587A1 - Lipid production method - Google Patents

Abstract

A lipid production method for cultivating a transformant having enhanced expression of a gene encoding protein (A) or (B) below and producing fatty acids or a lipid having the same as structural components. (A) Protein comprising an amino acid sequence represented by SEQ ID NO: 1. (B) Protein comprising an amino acid sequence having 67% or greater identity with the amino acid sequence of protein (A) and having β-ketoacyl-ACP synthase activity.

Description

Method for producing lipid

The present invention relates to a method for producing lipids. The present invention also relates to β-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. All of these 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. Furthermore, 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. Furthermore, since the use and usefulness of fatty acids depend on the number of carbon atoms, attempts have been made to control the number of carbon atoms of fatty acids, that is, the chain length. For example, a method of accumulating fatty acid having 12 carbon atoms by introduction of acyl-ACP (acyl-carrier-protein) thioesterase derived from bay ( Umbellularia californica (California bay)) (Patent Document 1, Non-Patent Document 1), etc. Proposed.

Plant fatty acid synthesis pathway is localized in chloroplasts. In chloroplasts, acetyl-ACP (acyl carrier protein) is used as a starting material, and the carbon chain elongation reaction is repeated. Finally, acyl-ACP (acyl which is a fatty acid residue) having 16 or 18 carbon atoms is used. A complex comprising a group and an ACP) is synthesized. Among enzymes involved in this fatty acid synthesis pathway, β-ketoacyl-acyl-carrier-protein synthase (hereinafter also referred to as “KAS”) is an enzyme involved in the control of the acyl group chain length. In plants, it is known that there are four types of KAS having different functions of KAS I, KAS II, KAS III, and KAS IV. Among these, 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 β-ketoacyl-ACP having 4 carbon atoms. Subsequent elongation reactions involve KAS I, KAS II, and KAS IV. KAS I is mainly involved in the elongation reaction up to 16 carbon atoms palmitoyl-ACP, and KAS II is mainly involved in the elongation reaction up to 18 carbon atoms stearoyl-ACP. On the other hand, 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).

In recent years, 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. There are also reports that algae have a higher ability to produce and accumulate lipids than plants. Although research has begun on the algae lipid synthesis mechanism and production technology using it, there are many unexplained parts.

International Publication No. 92/20236 International Publication No. 2001/051647

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) 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 β-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.

According to the method for producing a lipid of the present invention, the productivity of a medium chain fatty acid or a lipid containing this as a constituent can be improved.
Moreover, the transformant of the present invention is excellent in productivity of medium chain fatty acids or lipids containing the same as a constituent component.
These and other features and advantages of the present invention will become more apparent from the following description.

As used herein, “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.
In this specification, “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.
Furthermore, in this specification, 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.
In this specification, “stringent conditions” include, for example, Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W., et al. Russell., Cold Spring Harbor Laboratory Press]. For example, in a solution containing 6 × SSC (composition of 1 × SSC: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhart and 100 mg / mL herring sperm DNA Examples of the conditions include hybridization with the probe at 65 ° C. for 8 to 16 hours.
Furthermore, in the specification, “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. On the other hand, the “downstream” of a gene indicates a region continuing 3 ′ side of the gene or region captured as a target.

The proteins (A) and (B) (hereinafter, also referred to as “NoKASIII”) are one type of KAS, from acetyl-ACP (or acetyl-CoA) having 2 carbon atoms to β-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. In the chloroplast, acetyl-ACP (or acetyl-CoA) is used as a starting material, and the carbon chain elongation reaction is repeated to finally synthesize acyl-ACP having 16 or 18 carbon atoms. Next, acyl-ACP thioesterase (hereinafter, also simply referred to as “TE”) acts to hydrolyze the thioester bond of acyl-ACP to produce a free fatty acid.
In the first step of fatty acid synthesis, 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 β-ketoacyl-ACP reductase to produce hydroxybutyryl ACP. Subsequently, hydroxybutyryl ACP is dehydrated by β-hydroxyacyl-ACP dehydrase to produce crotonyl ACP. Finally, crotonyl ACP is reduced by enoyl-ACP reductase to produce butyryl ACP. By a series of these reactions, 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.

Both the proteins (A) and (B) have β-ketoacyl-ACP synthase activity (hereinafter also referred to as “KAS activity”). As used herein, “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. Alternatively, after introducing 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.

From the results of Blast of the amino acid sequence and the base sequence, the proteins (A) and (B) were extended from acetyl-ACP having 2 carbon atoms (or acetyl-CoA) to β-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. It is considered that 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. In addition, since 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.
In the present specification, “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.

Regarding the KAS activity of the proteins (A) and (B), for example, 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. In addition, after introducing a DNA linking a gene encoding a 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, This can be confirmed by performing a chain extension reaction.

In the protein (B), from the viewpoint of KAS activity, 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). , More preferably 1 to 84, more preferably 1 to 63, more preferably 1 to 42, more preferably 1 to 33, and more preferably 1 to 29 1 or more, 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 and 8 or less. And, more preferably, a protein in which 1 to 4 amino acids are deleted, substituted, inserted or added.
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. For example, a protein derived from a natural product can be obtained by isolation, purification or the like from Nannochloropsis oculata. Further, 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. Alternatively, the proteins (A) and (B) may be prepared as recombinant proteins by genetic recombination techniques. When producing a recombinant protein, the β-ketoacyl-ACP synthase gene described later can be used.
In addition, algae such as Nannochloropsis oculata can be obtained from a preservation organization such as a private or public laboratory. For example, Nannochloropsis oculata strain NIES-2145 can be obtained from the National Institute for Environmental Studies (NIES).

As an example of 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”). Can be mentioned.

(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).

In the DNA (b), from the viewpoint of KAS activity, 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.
In addition, as 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.
Furthermore, 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.
Hereinafter, in the present specification, 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. As a result, in the transformant, 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. More preferably, 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.

A method for introducing the KAS gene into a host to promote the expression of the gene will be described.
The KAS gene can be obtained by ordinary genetic engineering techniques. For example, 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. For the synthesis of the KAS gene, for example, services such as Invitrogen can be used. It can also be obtained by cloning from Nannochloropsis oculata. For example, Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press (2001)]. In addition, 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. Examples of 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. Among these, Escherichia coli , Bacillus subtilis , red yeast ( Rhodosporidium toruloides ), or Mortierella sp. Is preferred, and Escherichia coli is more preferred from the viewpoint of lipid productivity.
As the algae and micro-algae, from the viewpoint of gene recombination techniques has been established, Chlamydomonas (Chlamydomonas) genus algae, Chlorella (Chlorella) genus algae, Faye Oda Kuti ram (Phaeodactylum) genus algae, or Nan'nokuroro Algae of the genus Psis are preferred, and algae of the genus Nannochloropsis are more preferred. Specific examples of the algae of the genus Nannochloropsis include Nannochloropsis gaditana , Nannochloropsis salina , Nannochloropsis oceanica , Nannochloropsis oceanica , Nannochloropsis oceanica Examples thereof include Nannochloropsis atomus , Nannochloropsis maculata , Nannochloropsis granulata , Nannochloropsis sp. Among these, from the viewpoint of lipid productivity, Nannochloropsis oculata or Nannochloropsis gaditana is preferable, and Nannochloropsis oculata is more preferable.
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.

As a plasmid vector for gene expression or a vector (plasmid) serving as a base of a gene expression cassette, 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. For example, 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. Further, 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.
As 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. 93-103), pBR322 (Takara Bio), pRS403 (Stratagene), and pMW218 / 219 (Nippon Gene) It is done. In particular, when the host is Escherichia coli, pBluescript II SK (−) or pMW218 / 219 is preferably used.
When using algae or microalgae as a host, for example, pUC19 (manufactured by Takara Bio Inc.), P66 (Chlamydomonas Center), P-322 (Chlamydomonas Center), pPha-T1 (Yangmin Gong, et al., Journal of Basic Microbiology, 2011, vol. 51, p.666-672), and pJET1 (manufactured by Cosmo Bio). In particular, when the host is an algae belonging to the genus Nannochloropsis, pUC19, pPha-T1, or pJET1 is preferably used. When the host 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. With reference to the method described in 108 (52), 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.
When plant cells are used as hosts, for example, pRI vectors (manufactured by Takara Bio Inc.), pBI vectors (manufactured by Clontech), and IN3 vectors (manufactured by Implanta Innovations) can be mentioned. In particular, when the host is Arabidopsis thaliana, pRI vectors or pBI vectors are preferably used.

In addition, the type of promoter that regulates the expression of the gene encoding the target protein incorporated in the expression vector can be appropriately selected according to the type of host used. 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 β-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. 108 (52)), and an oleosin-like protein LDSP from the genus Nannochloropsis ( lipid droplet surface protein) gene promoter (Astrid Vieler, et al., PLOS Genetics, 2012; 8 (11): e1003064. doi: 10.1371). When Nannochloropsis is used as a host in the present invention, 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.
In addition, 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.

Introduction of a gene encoding the target protein into the vector can be performed by a conventional method such as restriction enzyme treatment or ligation.
The 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. . When an algae belonging to the genus Nannochloropsis is used as a host, 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. For example, 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. In addition, the introduction of the target DNA fragment can be confirmed by a PCR method using a genome as a template.

A method for promoting expression of the gene by modifying the expression regulatory region of the gene in a host having the KAS gene on the genome will be described.
“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. In a host having the KAS gene on the genome, 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. In a host having the KAS gene on the genome, 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. For example, in Nannochloropsis oculata strain NIES-2145, which is one of the hosts having the KAS gene on the genome, 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. By replacing part or all of the DNA sequence consisting of the base sequence shown in SEQ ID NO: 50 with a promoter having higher transcriptional activity, the expression of the KAS gene can be promoted.

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.
When Nannochloropsis is used as a host, 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. From the viewpoint of improving the productivity of medium-chain fatty acids or lipids containing these as constituents, 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.
Such 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. In particular, when the host 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 specific region in the genome can be modified by homologous recombination with reference to literature such as 108 (52).

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). .
As described above, 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. Therefore, by promoting the expression of the TE gene in addition to the KAS gene, it is possible to further improve the lipid productivity, particularly the fatty acid productivity, of the transformant used for lipid production. Furthermore, as shown also in the below-mentioned Example, the sum total of each fatty acid amount (total fatty acid amount) can also be improved by promoting the expression of TE gene in addition to KAS gene.
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”). Here, “TE activity” refers to an activity of hydrolyzing the thioester bond of acyl-ACP.

It is known that 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. Thus, like KAS, TE is considered to be an important factor that determines the fatty acid composition in vivo. In addition, when a host that originally does not have a gene encoding TE is used, it is preferable to promote the expression of the gene encoding TE. Moreover, by promoting the expression of the KAS gene, 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.
For example, 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. BMC Biochemistry 2011, 12:44); TE from Cocos nucifera (see CnFatB2: Jing et al., BMC Biochemistry, 2011, 12:44); TE from Cuphea viscosissima (CvFatB1 : Jing et al., BMC Biochemistry, 2011, 12:44); TE from Cuphea viscosissima (see CvFatB2: Jing et al., BMC Biochemistry 2011, 12:44); TE from Cuphea viscosissima (CvFatB3: Jing et al., BMC Biochemistry, 2011, 12:44); El TE from aeis guineensis (GenBank AAD42220); TE from Desulfovibrio vulgaris (GenBank ACL08376); TE from Bacteriodes fragilis (GenBank CAH09236); TE from Parabacteriodes distasonis (GenBank ABR43801); O from Bacteroides thetaiotaomicron ; TE derived from Clostridium asparagiforme (GenBank EEG55387); TE derived from Bryanthella formatexigens (GenBank EET61113); Geobacillus sp. Derived TE (GenBank EDV77528); Streptococcus dysgalactiae derived TE (GenBank BAH81730); Lactobacillus brevis derived TE (GenBank ABJ63754); Lactobacillus plantarum derived TE (GenBank CAD63310); Anaerococcus tetradius derived TE (GenBank EEI82564); Bdellovibrio bacteriovorus TE derived from (GenBank CAE80300); TE derived from Clostridium thermocellum (GenBank ABN54268); TE derived from coconut (hereinafter also referred to as “CTE”) (SEQ ID NO: 7, nucleotide sequence of the gene encoding it: SEQ ID NO: 8); TE derived from Nannochloropsis oculata (hereinafter also referred to as “NoTE”) (SEQ ID NO: 34, nucleotide sequence of gene encoding the same: SEQ ID NO: 35); TE derived from bay (GenBank AAA34215.1, SEQ ID NO: 51, Nucleotide sequence of the gene encoding this: SEQ ID NO: 52); TE derived from Nannochloropsis gaditana (SEQ ID NO: 53, encoding this) Gene base sequence: SEQ ID NO: 54); Nannochloropsis-Guranyurata derived TE (SEQ ID NO: 55, the gene of the nucleotide sequence encoding it: SEQ ID NO: 56); Simbionix Oddi iodonium Micro Adria Chi cam (Symbiodinium microadriaticum ) Derived TE (SEQ ID NO: 57, base sequence of gene encoding the same: SEQ ID NO: 58), and the like.
In addition, as functionally equivalent proteins, 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.

Among the above-mentioned TEs, 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. 57, nucleotide sequence of the gene encoding it: SEQ ID NO: 58), or 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.
The 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.
In addition, after introducing the DNA linked to the TE gene downstream of a promoter that functions in a host cell such as E. coli into the host cell and culturing the cell under conditions where the introduced TE gene is expressed, 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. For example, in the same manner as the method for promoting the expression of the KAS gene described above, 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.

Furthermore, in the transformant of the present invention, in addition to the gene encoding the protein (A) or (B), the expression of a gene encoding KAS, a gene encoding acyltransferase, etc. is also promoted. Is preferred.
As described above, 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.
Furthermore, 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. For example, in the same manner as the method for promoting the expression of the KAS gene described above, a method for introducing a gene encoding KAS or acyltransferase into the host, or a method for the host having a gene encoding KAS or acyltransferase on the genome. A transformant can be produced by a method of modifying an expression control region of a gene.

In the transformant of the present invention, 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. Here, “culture” refers to the culture solution and transformant after culturing, and “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.

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.
In addition, 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. Specifically, the cerulenin concentration is preferably 1 μM or more, more preferably 10 μM or more, preferably 50 μM or less, and more preferably 25 μM or less. Alternatively, 1 to 50 μM is preferable, 10 to 50 μM is more preferable, and 10 to 25 μM is more preferable.

When Arabidopsis thaliana is used as a host, 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.

When using algae as a host, the culture medium may be based on natural seawater or artificial seawater, or a commercially available culture medium may be used. Specific examples of the medium include f / 2 medium, ESM medium, Daigo IMK medium, L1 medium, and MNK medium. Among these, from the viewpoint of improving lipid productivity and nutrient concentration, 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. In order to promote the growth of algae and improve the productivity of fatty acids, 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.
In addition, 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. The 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. For example, when sodium bicarbonate is used, 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. More preferably, 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. For example, 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. When larger-scale cultivation is performed, 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. Thus, after isolating a lipid component, 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. Preferably, 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.
From the viewpoint of productivity, 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.

<1> 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) 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). The method of any one of Claims.

<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.
<6> 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.
<7> 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.

<8> 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 method according to any one of <1> to <7>, wherein the protein is a protein in which 8 or less, more preferably 1 to 4 amino acids are deleted, substituted, inserted or added.
<9> The method according to any one of <1> to <8>, wherein the gene encoding the protein (A) or (B) is a gene consisting of the following DNA (a) or (b).
(A) DNA consisting of the base sequence represented by SEQ ID NO: 2.
(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.
<10> 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) and having KAS activity 10. 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.
<15> 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. The method according to any one of <14>.
<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.
<18> The method according to <17>, wherein the microalgae are algae belonging to the genus Nannochloropsis, preferably Nannochloropsis oculata.
<19> The method according to <16>, wherein the microorganism is Escherichia coli.
<20> The method according to <16>, wherein the plant is Arabidopsis thaliana.
<21> 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. The method according to claim 1.

<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.
<32> 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> The transformant according to any one of the above or a method for producing the transformant.
<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.

<38> A protein, gene, vector, transformant, or a transformant obtained by the method for producing a transformant according to any one of <26> to <37> for producing a lipid use.
<39> 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, The use according to <38> above, more preferably comprising 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.

Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited thereto. Here, the base sequences of the primers used in this example are shown in Table 1.

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 μg, biotin 0.5 μg, thiamine 100 μg, 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 12μg, ZnSO 4 · 7H 2 O 21μg, MnCl 2 · 4H 2 O 180μg, CuSO 4 · 5H 2 O 7μg, Na ₂ MoO ₄ · 2H ₂ O 7μg / 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. The sample collected after the culture was crushed with a multi-bead shocker, and 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.
Also, 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.

(2) Introduction of NoKASIII gene expression plasmid into Escherichia coli Using the constructed NoKASIII gene expression plasmid, 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 μg / mL) was inoculated into 1 mL and cultured at 30 ° C. overnight. 20 μL of the culture solution was inoculated into 2 mL of Overnight Express Instant TB Medium (Novagen) and cultured with shaking at 30 ° C. After 24 hours of culture, lipid components contained in the culture solution were analyzed by the following method.
As a negative control, E. coli K27 strain (control) transformed with plasmid vector pSTV28 was also tested in the same manner.

(3) Extraction of lipids and analysis of constituent fatty acids After adding 50 μL of 1 mg / mL 7-pentadecanone as an internal standard to 1 mL of culture solution, add 0.5 mL of chloroform, 1 mL of methanol, and 10 μL of 2N hydrochloric acid to the culture solution, and vigorously Stir and let stand for 30 minutes. Thereafter, 0.5 mL of chloroform and 0.5 mL of 1.5% KCl were further added and stirred. Centrifugation was performed at 3,000 rpm for 15 minutes, and a chloroform layer (lower layer) was recovered with a Pasteur pipette.
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 obtained fatty acid methyl ester was subjected to gas chromatography analysis under the measurement conditions shown below.
<Gas chromatography conditions>
Capillary column: DB-1 MS (30m × 200μm × 0.25μm, manufactured by J & W Scientific)
Moving bed: High purity helium column flow rate: 1.0 mL / min
Temperature rising program: 100 ° C (1 minute) → 10 ° C / min → 300 ° C (5 minutes)
Equilibration time: 1 minute Inlet: Split injection (split ratio: 100: 1), pressure 14.49psi, 104mL / min
Injection volume: 1μL
Washing vial: Methanol / chloroform Detector temperature: 300 ° C

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. In the following table, “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.

As shown in Table 2, introduction of the NoKASIII gene significantly increased the content of C14: 0 fatty acids and the total amount of fatty acids.

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 μg / mL) was inoculated into 1 mL and cultured at 30 ° C. overnight. 20 μL of the culture solution was inoculated into 2 mL of Overnight Express Instant TB Medium (Novagen) and cultured with shaking at 30 ° C. At this time, cerulenin (manufactured by Wako Pure Chemical Industries, Ltd.) was added to Overnight Express Instant TB Medium to a final concentration of 10 μM. After 24 hours of culture, lipid components contained in the culture solution were analyzed by the same method as in Example 1.
As a negative control, E. coli K27 strain (control) transformed with plasmid vector pSTV28 was also tested in the same manner.
The results are shown in Table 3.

As shown in Table 3, even in the system containing cerulenin in the medium, introduction of the NoKASIII gene increased the content of C14: 0 fatty acids and the total amount of fatty acids.

Example 3 Production of Transformant Introducing NoKASIII Gene and CTE Gene into Escherichia coli, and Production of Fatty Acid Using Transformant According to the method described in JP2011-250781CTE (amino acid sequence: SEQ ID NO: 7) The gene to be encoded (base sequence: SEQ ID NO: 8) was introduced into the K27 strain (fadD88) to prepare a transformant (pMW :: CTE). Then, 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 μg / mL, kanamycin sulfate 50 μg / mL) was inoculated into 1 mL, and cultured at 30 ° C. overnight. 20 μL of the culture solution was inoculated into 2 mL of Overnight Express Instant TB Medium (Novagen) and cultured with shaking at 30 ° C. After 24 hours of culture, lipid components contained in the culture solution were analyzed by the same method as in Example 1.
As a negative control, an experiment was similarly performed on a strain transformed with the plasmid vector pSTV28 (pMW :: CTE + pSTV28).
The results are shown in Table 4.

As shown in Table 4, the content of C12: 0 fatty acid and C14: 1 fatty acid and the total fatty acid amount were significantly increased by further introducing the NoKASIII gene into the strain into which the CTE gene was introduced.

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 μg / mL, kanamycin sulfate 50 μg / mL) and cultured at 30 ° C. overnight. 20 μL of the culture solution was inoculated into 2 mL of Overnight Express Instant TB Medium (Novagen) and cultured with shaking at 30 ° C. At this time, cerulenin (manufactured by Wako Pure Chemical Industries, Ltd.) was added to Overnight Express Instant TB Medium to a final concentration of 10 μM. After 24 hours of culture, lipid components contained in the culture solution were analyzed by the same method as in Example 1.
As a negative control, an experiment was similarly performed on a strain transformed with the plasmid vector pSTV28 (pMW :: CTE + pSTV28).
The results are shown in Table 5.

As shown in Table 5, in the system containing cerulenin in the culture medium, the content of C12: 0 fatty acid and the total amount of fatty acids were significantly increased by further introducing the NoKASIII gene into the strain into which the CTE gene was introduced.

Example 5 Production of transformant in which NoKASIII gene was introduced into Nannochloropsis oculata, and production of fatty acid by transformant (1) Construction of plasmid for expression of zeocin resistance gene Zeocin resistance gene (SEQ ID NO: 9), and literature (Randor Radakovits, et al., Nature Communications, DOI: 10.1038 / ncomms1688, 2012), a tubulin promoter sequence (SEQ ID NO: 10) derived from Nannochloropsis gaditana strain CCMP526 was artificially synthesized.
Using the synthesized DNA fragment as a template, 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).
Furthermore, 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.

These four amplified fragments were each treated with a restriction enzyme Dpn I (manufactured by Toyobo Co., Ltd.) and purified using a High Pure PCR Product Purification Kit (manufactured by Roche Applied Science). Thereafter, the four fragments obtained were fused using an In-Fusion HD Cloning Kit (Clontech) to construct a plasmid for expression of a zeocin resistant gene.
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.

(2) Acquisition of NoKASIII gene and construction of NoKASIII gene expression plasmid Total RNA of Nannochloropsis oculata NIES2145 strain (obtained from National Institute for Environmental Studies (NIES)) was extracted, and SuperScript ™ III First -Strand Synthesis cDNA was obtained by reverse transcription using SuperMix for qRT-PCR (manufactured by Invitrogen). Using this cDNA as a template, a NoKASIII gene fragment was obtained by PCR using the primer pair of primer number 20 and primer number 21 shown in Table 1.
Furthermore, from the complete cds sequence (Accession number: JF957601.1) of the VCP1 (violaxanthin / chlorophyll a binding protein) gene of Nannochloropsis sp. W2J3B strain registered in GenBank, the VCP1 promoter sequence ( SEQ ID NO: 22) and VCP1 terminator sequence (SEQ ID NO: 23) were artificially synthesized. Using the synthesized DNA fragment as a template, 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.
Further, using the above-mentioned 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.

These four amplified fragments were fused in the same manner as described above to construct a NoKASIII gene expression plasmid.
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.

(3) Introduction of 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.

Approximately 1 × 10 ⁹ cells of Nannochloropsis oculata strain NIES2145 (obtained from the National Institute for Environmental Studies (NIES)) were washed with a 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 μF, 500Ω, and 2,200 v / 2 mm.
f / 2 liquid medium (NaNO ₃ 75 mg, NaH ₂ PO ₄ · 2H ₂ O 6 mg, vitamin B12 0.5 μg, biotin 0.5 μg, thiamine 100 μg, Na ₂ SiO ₃ · 9H ₂ O 10 mg, Na ₂ EDTA · 2H ₂ O 4.4 _{mg, FeCl 3 · 6H 2 O} 3.16mg, FeCl 3 · 6H 2 O 12μg, ZnSO 4 · 7H 2 O 21μg, MnCl 2 · 4H 2 O 180μg, CuSO 4 · 5H 2 O 7μg, Na 2 MoO 4 · 2H 2 O 7 μg / artificial seawater 1 L) for 1 day. Thereafter, the mixture was applied to a 2 μg / mL zeocin-containing f / 2 agar medium, and cultured at 25 ° C. in a 0.3% CO ₂ atmosphere under 12 h / 12 h light / dark conditions for 2 to 3 weeks. From the obtained colonies, a Nannochloropsis oculata strain (NoKASIII) containing a NoKASIII gene expression fragment was selected by PCR.

(4) Production of fatty acid by transformant The selected strain is seeded in 50 mL of a medium (hereinafter referred to as “N15P5 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.

As shown in Table 6, the C14: 0 fatty acid content was significantly increased by introducing the NoKASIII gene.

Example 6 Production of Transformant Introducing NoKASIII Gene and NoTE Gene into Nannochloropsis Oculata, and Production of Fatty Acid by Transformant (1) Construction of Plasmid for Expression of Paromomycin Resistance Gene 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.

These two gene fragments were fused in the same manner as in Example 5 to construct a paromomycin resistance gene expression plasmid.
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.

(2) Acquisition of NoTE gene and construction of NoTE gene expression plasmid Using the Nannochloropsis oculata strain NIES2145 prepared in Example 1 as a template, the primer pairs of primer numbers 32 and 33 shown in Table 1 were used. A gene fragment consisting of the nucleotide sequence from position 262 to position 864 of SEQ ID NO: 35 was obtained by PCR used.
Further, 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.
In the following plasmid notation, “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. Means that

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. Here, in the base sequence shown in SEQ ID NO: 40, 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.

An artificially synthesized VCP1 chloroplast transition signal sequence (SEQ ID NO: 43) from the complete cds sequence (Accession number: JF957601.1) of the VCP1 gene of Nannochloropsis sp. W2J3B strain registered in GenBank did. 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.
Furthermore, 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).
Furthermore, 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.

These three gene fragments were fused in the same manner as in Example 5 to construct a NoTE variant gene expression plasmid.
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.

(3) Introduction of NoTE variant gene expression fragment and NoKASIII gene expression fragment 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.

In the same manner as in 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).

(4) Production of fatty acid by transformant Each selected strain was cultured by the same method as in Example 5, and lipid components contained in the culture solution were analyzed. The results are shown in Table 7.

As shown in Table 7, the content of C12: 0 fatty acid and C14: 0 fatty acid was significantly increased by further introducing the NoKASIII gene into the strain into which the NoTE gene was introduced.

As described above, 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.

While this invention has been described in conjunction with its embodiments, we do not intend to limit our invention in any detail of the description unless otherwise specified and are contrary to the spirit and scope of the invention as set forth in the appended claims. I think it should be interpreted widely.

This application claims priority based on Japanese Patent Application No. 2015-155865 filed in Japan on August 6, 2015, which is hereby incorporated herein by reference. Capture as part.

Claims (23)

1. Culturing a transformant in which expression of the gene encoding the following protein (A) or (B) and the gene encoding acyl-ACP thioesterase is promoted to produce a fatty acid or a lipid comprising the same. A method for producing lipids.
   (A) 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 β-ketoacyl-ACP synthase activity.
2. Medium chain fatty acids produced in the cells of the transformant, or components thereof, that promote the expression of the genes encoding the following proteins (A) or (B) and the genes encoding acyl-ACP thioesterase A method for improving lipid productivity, which improves lipid productivity.
   (A) 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 β-ketoacyl-ACP synthase activity.
3. The medium chain fatty acid produced in the cells of the transformant by promoting the expression of the gene encoding the following protein (A) or (B) and the gene encoding acyl-ACP thioesterase The lipid composition modification method for improving the productivity of lipids to be produced and modifying the composition of fatty acids or lipids in total fatty acids or total lipids produced.
   (A) 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 β-ketoacyl-ACP synthase activity.
4. The gene according to any one of claims 1 to 3, wherein a gene encoding the protein (A) or (B) and a gene encoding an acyl-ACP thioesterase are introduced into a host to promote expression of the gene. Method.
5. Production of lipid by culturing a transformant introduced with a gene encoding the following protein (A) or (B) and a gene encoding acyl-ACP thioesterase to produce fatty acid or a lipid comprising the same. Method.
   (A) 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 β-ketoacyl-ACP synthase activity.
6. A medium chain fatty acid produced in the transformant cell by introducing a gene encoding the following protein (A) or (B) and a gene encoding acyl-ACP thioesterase, or this A method for improving lipid productivity, which improves the productivity of lipids comprising a component.
   (A) 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 β-ketoacyl-ACP synthase activity.
7. A medium chain fatty acid produced in the transformant cell by introducing a gene encoding the following protein (A) or (B) and a gene encoding acyl-ACP thioesterase, or this A method for modifying a lipid composition, which improves the productivity of lipids comprising a component and modifies the total fatty acids produced or the composition of fatty acids or lipids in the total lipids.
   (A) 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 β-ketoacyl-ACP synthase activity.
8. The method according to any one of claims 1 to 7, wherein the gene encoding the protein (A) or (B) is a gene comprising the following DNA (a) or (b).
   (A) DNA consisting of the base sequence represented by SEQ ID NO: 2.
   (B) A DNA encoding the protein (A) or (B), which has a base sequence having 62% or more identity with the base sequence of the DNA (a) and has β-ketoacyl-ACP synthase activity.
9. The method according to any one of claims 1 to 8, wherein the transformant is a microorganism or a plant.
10. The method according to claim 9, wherein the microorganism is a microalgae.
11. The method according to claim 10, wherein the microalgae are algae belonging to the genus Nannochloropsis .
12. The method according to claim 9, wherein the microorganism is Escherichia coli.
13. The method according to any one of claims 1 to 12, wherein the lipid comprises a medium chain fatty acid or an ester compound thereof.
14. The following protein (A) or (B).
    (A) A protein comprising the amino acid sequence represented by SEQ ID NO: 1.
    (B) A protein comprising an amino acid sequence having 92% or more identity with the amino acid sequence of the protein (A) and having β-ketoacyl-ACP synthase activity.
15. A gene encoding the protein according to claim 14.
16. A gene comprising the following DNA (a) or (b).
    (A) DNA consisting of the base sequence represented by SEQ ID NO: 2.
    (B) A DNA encoding the following protein (A) or (B) having a base sequence having an identity of 74% or more with the base sequence of the DNA (a) and having β-ketoacyl-ACP synthase activity.
    (A) A protein comprising the amino acid sequence represented by SEQ ID NO: 1.
    (B) A protein comprising an amino acid sequence having 92% or more identity with the amino acid sequence of the protein (A) and having β-ketoacyl-ACP synthase activity.
17. A recombinant vector containing the gene according to claim 15 or 16.
18. A transformant in which expression of the gene according to claim 15 or 16 and the gene encoding acyl-ACP thioesterase is promoted.
19. 19. The transformant according to claim 18, wherein the gene according to claim 15 or 16 and the gene encoding acyl-ACP thioesterase are introduced into a host.
20. 20. The transformant according to claim 18 or 19, wherein the transformant is a microorganism or a plant.
21. The transformant according to claim 20, wherein the microorganism is a microalgae.
22. The transformant according to claim 21, wherein the microalgae are algae belonging to the genus Nannochloropsis .
23. The transformant according to claim 20, wherein the microorganism is Escherichia coli.