WO2016190239A1 - Lipid production method - Google Patents

Abstract

A lipid production method comprising culturing a transformant that is produced by introducing a gene encoding any one of proteins (a) to (f) as mentioned below into a host, thereby producing a fatty acid or a lipid containing the fatty acid as a constituent. (a) A protein comprising the amino acid sequence represented by SEQ ID NO: 1; (b) a protein which comprises an amino acid sequence having 85% or more identity to the amino acid sequence for the protein (a) and has a β-ketoacyl-ACP synthase activity; (c) a protein comprising the amino acid sequence represented by SEQ ID NO: 3; (d) a protein which comprises an amino acid sequence having 85% or more identity to the amino acid sequence for the protein (c) and has a β-ketoacyl-ACP synthase activity; (e) a protein comprising the amino acid sequence represented by SEQ ID NO: 5; and (f) a protein which comprises an amino acid sequence having 85% or more identity to the amino acid sequence for the protein (e) and has a β-ketoacyl-ACP synthase activity.

Description

Method for producing lipid

The present invention relates to a method for producing a fatty acid or a lipid comprising the same, and a transformant and β-ketoacyl-ACP synthase used therefor.

Fatty acids are one of the major constituents of lipids, and constitute lipids 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 (oils and fats) 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 or disinfectants. Similarly, cationic surfactants such as alkylamine salts and mono- or dialkyl quaternary ammonium salts are routinely used as fiber treatment agents, hair rinse agents, or bactericides as higher alcohol derivatives. Benzalkonium-type quaternary ammonium salts are routinely used as bactericides and preservatives. Furthermore, vegetable oils and fats are also used as raw materials for biodiesel fuel.

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 having 16 or 18 carbon atoms (acyl group and acyl which are fatty acid residues) A complex consisting of a carrier protein) 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 extension reaction, and extends acetyl-ACP having 2 carbon atoms to acyl-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 C16 palmitoyl-ACP, and KAS II is mainly involved in the elongation reaction up to C18 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. As for KAS IV, not much knowledge has been obtained in plants, and KAS IV is regarded as a KAS unique to plants that accumulate medium chain fatty acids such as cuphea (see Patent Document 1 and Non-Patent Document 1).

International Publication No. 98/46776 Pamphlet

In the present invention, a transformant obtained by introducing a gene encoding any of the following proteins (a) to (f) into a host is cultured to produce a fatty acid or a lipid comprising this as a constituent. It relates to a manufacturing method.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (a), and β-ketoacyl-ACP synthase activity (C) a protein having the amino acid sequence represented by SEQ ID NO: 3 (d) a protein having the identity of 85% or more with the protein (c) (E) a protein having the KAS activity (e) a protein comprising the amino acid sequence represented by SEQ ID NO: 5 (f) an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (e), and a KAS activity Protein with

The present invention also relates to a transformant obtained by introducing a gene encoding any of the proteins (a) to (f) into a host.
The present invention also relates to the proteins (a) to (f).
Furthermore, the present invention relates to a gene encoding any one of the proteins (a) to (f).

The present invention relates to a method for producing a lipid, which improves the productivity of a fatty acid having a specific carbon number, or a lipid comprising this fatty acid.
Moreover, this invention relates to the transformant which improved the productivity of the fatty acid of a specific carbon number, or the lipid which uses this as a structural component.

As a result of intensive studies, the present inventors have identified new plant-derived KAS from California Bay ( Umbellularia californica ) and camphor ( Cinnamomum camphora ). And when the host was transformed using the gene which codes these, it discovered that productivity of the fatty acid of a specific carbon number improved in a transformant. The present invention has been completed based on these findings.

According to the method for producing a lipid of the present invention, it is possible to improve the productivity of a fatty acid having a specific carbon number or a lipid comprising this as a constituent.
Moreover, the transformant of the present invention is excellent in the productivity of a fatty acid having a specific carbon number, or a lipid comprising this as a constituent.
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, waxes, and ceramides; complex lipids such as phospholipids, glycolipids, and sulfolipids; and fatty acids, alcohols, and hydrocarbons derived from these lipids And other derived lipids.
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.

The transformant of the present invention is transformed with a gene encoding any of the following proteins (a) to (f) (hereinafter also referred to as “KAS gene”).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) a protein comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (a) and having KAS activity (the protein ( a) Functionally equivalent protein)
(C) a protein comprising the amino acid sequence represented by SEQ ID NO: 3 (d) a protein comprising the amino acid sequence having 85% or more identity with the protein (c) and having KAS activity (the protein ( c) Functionally equivalent protein)
(E) a protein comprising the amino acid sequence represented by SEQ ID NO: 5 (f) a protein comprising the amino acid sequence having 85% or more identity with the amino acid sequence of the protein (e) and having KAS activity (the protein ( e) Functionally equivalent protein)

The protein comprising the amino acid sequence of SEQ ID NO: 1 is KAS derived from California Bay seeds (hereinafter also referred to as “BKAS431”) obtained from Sierra Seed Supply, USA. The protein consisting of the amino acid sequence of SEQ ID NO: 3 is KAS derived from California Bay seeds (hereinafter also referred to as “BKAS1082”) obtained from Sierra Seed Supply, USA. The protein consisting of the amino acid sequence of SEQ ID NO: 5 is KAS (hereinafter also referred to as “KKAS250”) derived from camphor seeds collected from the Kao Wakayama Factory.
KAS is an enzyme involved in the control of acyl chain length in the fatty acid synthesis pathway. Plant fatty acid synthesis pathway is localized in chloroplasts. In the chloroplast, acetyl-ACP 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. Subsequently, acyl-ACP thioester bond is hydrolyzed by the action of acyl-ACP thioesterase (hereinafter also referred to as “TE”) to produce free fatty acid.
In the first step of fatty acid synthesis, acetoacetyl ACP is produced by the condensation reaction of acetyl-ACP 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.

As used herein, “KAS activity” means an activity of catalyzing the condensation reaction of acetyl-ACP or acyl-ACP with malonyl ACP.
The fact that a protein has KAS activity means that, for example, a fusion gene 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 It can be confirmed by culturing the cells under the expressing condition and analyzing the change of the fatty acid composition in the host cell or in the culture solution by a conventional method. Alternatively, a fusion gene in which a gene encoding the protein is linked downstream of a promoter that functions in the host cell is introduced into the host cell, and the cell is cultured under conditions where the introduced gene is expressed. On the other hand, it can be confirmed by performing a chain 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 having 2 carbons as a substrate and catalyzes an elongation reaction having 2 to 4 carbons. KAS I mainly catalyzes an elongation reaction having 4 to 16 carbon atoms to synthesize palmitoyl-ACP having 16 carbon atoms. KAS II mainly catalyzes an elongation reaction having 16 to 18 carbon atoms to synthesize stearoyl ACP having 18 carbon atoms. KAS IV catalyzes the elongation reaction of 6 to 14 carbon atoms to synthesize medium chain acyl-ACP.
As shown in Examples described later, the protein (a) (BKAS431) has substrate specificity for fatty acids having 16 carbon atoms and selectively synthesizes them. Therefore, it is considered that the proteins (a) and (b) are KAS I.
In addition, the protein (c) (BKAS1082) and the protein (e) (KKAS250) have substrate specificity for medium chain acyl-ACP as shown in the Examples described later. Therefore, the proteins (c) to (f) are considered to be KAS IV. Here, the amino acid sequence of the protein (c) has 98.7% identity with the amino acid sequence of the protein (e). In the present specification, “substrate specificity for medium chain acyl-ACP” means that KAS mainly uses an acyl ACP having 4 to 12 carbon atoms as a substrate and an elongation reaction of synthesis of medium chain acyl ACP up to 14 carbon atoms. It means to catalyze. In this specification, “medium chain” means that the acyl group has 6 to 14 carbon atoms.
Regarding the substrate specificity of KAS, for example, a fusion gene in which a gene encoding a 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. It can be confirmed by culturing the cells under the conditions and analyzing the 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 TE alone. In addition, a fusion gene in which a gene encoding a protein is linked downstream of a promoter that functions in the host cell is introduced into the host cell, and the cell is cultured under conditions where the introduced gene is expressed. This can be confirmed by performing a chain length 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 90% or more, more preferably 95% or more, and further preferably 98% or more. In addition, as the protein (b), one or several (for example, 1 to 73, preferably 1 to 49, more preferably 1 to 25) amino acid sequences of the protein (a) And, more preferably, a protein in which 1 to 10 amino acids are deleted, substituted, inserted or added.
In the protein (d), from the viewpoint of KAS activity, the identity with the amino acid sequence of the protein (c) is preferably 90% or more, more preferably 95% or more, and further preferably 98% or more. In addition, as the protein (d), one or several (for example, 1 to 86, preferably 1 to 57, more preferably 1 to 29) amino acid sequences of the protein (c) And, more preferably, a protein in which 1 to 12 amino acids are deleted, substituted, inserted or added.
In the protein (f), from the viewpoint of KAS activity, the identity with the amino acid sequence of the protein (e) is preferably 90% or more, more preferably 95% or more, and further preferably 98% or more. Further, as the protein (f), one or several (for example, 1 to 86, preferably 1 to 57, more preferably 1 to 29) amino acid sequences of the protein (e) And, more preferably, a protein in which 1 to 12 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 Splicing overlap extension (SOE) -PCR reaction, ODA method, Kunkel method and the like. Site-Directed Mutagenesis System Mutan-SuperExpress Km kit (trade name, Takara Bio), Transformer TM Site-Directed Mutagenesis kit (trade name, Clonetech), KOD-Plus-Mutagenesis Kit (trade name, Toyobo) A commercially available kit can also be used. 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.

An example of the KAS gene is a gene consisting of any of the following DNA (g) to (l).

(G) DNA comprising the base sequence represented by SEQ ID NO: 2
(H) DNA comprising a base sequence having 85% or more identity with the base sequence of DNA (g) and encoding a protein having KAS activity
(I) DNA comprising the base sequence represented by SEQ ID NO: 4
(J) DNA comprising a base sequence having 85% or more identity with the base sequence of DNA (i) and encoding a protein having KAS activity
(K) DNA comprising the base sequence represented by SEQ ID NO: 6
(L) DNA comprising a base sequence having 85% or more identity with the base sequence of DNA (k) and encoding a protein having KAS activity

The base sequence of SEQ ID NO: 2 is a base sequence of a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1. The base sequence of SEQ ID NO: 4 is a base sequence of a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 3. The base sequence of SEQ ID NO: 6 is a base sequence of a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 5.

In the DNA (h), from the viewpoint of KAS activity, the identity with the base sequence of the DNA (g) is preferably 90% or more, and more preferably 95% or more. In addition, the DNA (h) is one or several in the nucleotide sequence represented by SEQ ID NO: 2 (for example, 1 to 218, preferably 1 to 145, more preferably 1 to 73) Also preferred is a DNA encoding a protein having the above-mentioned base deleted, substituted, inserted or added and having KAS activity. Furthermore, as the DNA (h), it encodes the protein (a) or (b) which hybridizes with a DNA having a base sequence complementary to the DNA (g) under stringent conditions and has KAS activity. DNA is also preferred.
In the DNA (j), from the viewpoint of KAS activity, the identity with the base sequence of the DNA (i) is preferably 90% or more, and more preferably 95% or more. In addition, the DNA (j) is one or several (for example, 1 to 256, preferably 1 to 171 and more preferably 1 to 86) in the base sequence represented by SEQ ID NO: 4. Also preferred is a DNA encoding a protein having the above-mentioned base deleted, substituted, inserted or added and having KAS activity. Furthermore, the DNA (j) encodes the protein (c) or (d) which hybridizes with a DNA having a base sequence complementary to the DNA (i) under stringent conditions and has KAS activity. DNA is also preferred.
In the DNA (l), from the viewpoint of KAS activity, the identity with the base sequence of the DNA (k) is preferably 90% or more, and more preferably 95% or more. In addition, the DNA (l) is one or several in the nucleotide sequence represented by SEQ ID NO: 6 (for example, 1 to 256, preferably 1 to 171 and more preferably 1 to 86) Also preferred is a DNA encoding a protein having the above-mentioned base deleted, substituted, inserted or added and having KAS activity. Furthermore, as the DNA (l), the protein (e) or (f) that hybridizes under stringent conditions with DNA comprising a base sequence complementary to the DNA (k) and has KAS activity is encoded. DNA is also preferred.

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, 3 or 5 or the base sequence shown in SEQ ID NO: 2, 4 or 6. For the synthesis of the KAS gene, for example, services such as Invitrogen can be used. It can also be obtained by cloning from California Bay or camphor genomes. For example, Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press (2001)] can be used.

The transformant of the present invention is preferably one obtained by introducing a gene encoding TE (hereinafter also referred to as “TE gene”) into the host in addition to the KAS gene.
TE is an enzyme that hydrolyzes the acyl-ACP thioester bond synthesized by a fatty acid synthase such as KAS to produce a free fatty acid. The fatty acid synthesis on the ACP is terminated by the action of TE, and the cut fatty acid is used for synthesis of triacylglycerol and the like. Therefore, by co-introducing the KAS gene and the TE gene into the host, the lipid productivity of the transformant, particularly the fatty acid productivity, can be further improved.
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, TE is considered to be an important factor that determines the fatty acid composition in vivo. When a host that originally does not have a gene encoding TE is used for transformation, co-introduction of a gene encoding TE, preferably a gene encoding TE having substrate specificity for medium chain acyl-ACP, may be performed. It is effective. By co-introducing such a gene, the productivity of 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.
Specifically, Umbellularia californica TE (GenBank AAA34215.1); Cuphea calophylla subsp. mesostemon TE (GenBank ABB71581); Cinnamomum camphora TE (GenBank AAC49151.1); Myristica fragrans TE (GenBank AAB71729); Myristica fragrans TE (GenBank AAB71730); Cuphea lanceolata TE (GenBank CAA54060); Cuphea hookeriana TE (GenBank Q39513); Ulumus americana TE (GenBank AAB71731); Sorghum bicolor TE (GenBank EER87824); Sorghum bicolor TE (GenBank EER88593); Cocos nucifera TE (CnFatB1: Jing et al. BMC Biochemistry 2011, 12:44 Cocos nucifera TE (CnFatB2: Jing et al. BMC Biochemistry 2011, 12:44); Cuphea viscosissima TE (CvFatB1: Jing et al. BMC Biochemistry 2011, 12:44); Cuphea viscosissima TE ( CvFatB2: Jing et al. BMC Biochemistry 2011, 12:44); Cuphea viscosissima TE (CvFatB3: Jing et al. BMC Biochemistry 2011, 12:44); Elaeis guineensis TE (GenBank AAD42220); Desulfovibrio vulgaris TE (GenBank ACL08376); Bacteriodes fragilis TE (GenBank CAH09236); Parabacteriodes distasonis TE (GenBank ABR43801); Bacteroides thetaiotaomicron TE (GenBank AAO77182); Clostridium asparagiforme TE (GenBank EEG55387); Bryanthella formatexigens TE (GenBank EET61113); Geobacillus sp. ; Streptococcus dysgalactiae of TE (GenBank BAH81730); Lactobacillus brevis of TE (GenBank ABJ63754); Lactobacillus plantarum in TE (GenBank CAD63310); Anaerococcus tetradius of TE (GenBank EEI82564); Bdellovibrio bacteriovorus of TE (GenBank CAE80300); Clostridium thermocellum of TE (GenBank ABN54268); Cocos nucifera TE (CnFatB3: Jing et al. BMC Biochemistry 2011, 12:44, SEQ ID NO: 7, nucleotide sequence of the gene encoding it: SEQ ID NO: 8); TE of Nannochloropsis oculata (SEQ ID NO: 9, nucleotide sequence of the gene encoding it: SEQ ID NO: 10); Nannochloropsis gaditana TE (SEQ ID NO: 11, nucleotide sequence of the gene encoding it: SEQ ID NO: 12); Nannochloropsis granulata TE (SEQ ID NO: 13, nucleotide sequence of the gene encoding it: SEQ ID NO: 14); Symbiodinium microadriaticum TE (SEQ ID NO: 15, nucleotide sequence of gene encoding the same: SEQ ID NO: 16), and the like. Moreover, as a protein functionally equivalent to these, it has 50% or more (preferably 70% or more, more preferably 80% or more, further preferably 90% or more) identity with any of the TE amino acid sequences described above. A protein having an amino acid sequence having TE activity can also be used. Alternatively, one or several (for example, 1 or more and 147 or less, preferably 1 or more and 119 or less, more preferably 1 or more and 59 or less, more preferably 1 or more) in any of the above-described amino acid sequences of TE Proteins having 30 or less amino acids deleted, substituted, inserted or added and having TE activity can also be used.
Among these TEs, Umbellularia californica TE, Cocos nucifera TE, Cinnamonum camphorum TE, Elaeis guineensis TE, Cuphea TE, Nannochloropsis oculata TE, Nannochloropsis gaditana TE, Nannochloropsis granulata TE, Symbiodinium microadriatic , A protein comprising an amino acid sequence having 50% or more identity with these TE amino acid sequences (preferably 70% or more, more preferably 80% or more, more preferably 90% or more) and having TE activity, or these 1 or several (for example, 1 or more and 147 or less, preferably 1 or more and 119 or less, more preferably 1 or more and 59 or less, and further preferably 1 or more and 30 or less) amino acid sequences of TE Are preferably deleted, substituted, inserted or added and have TE activity.
The sequence information of these TEs and the genes encoding them can be obtained from, for example, the National Center for Biotechnology Information (NCBI).

TE has specificity for the fatty acid chain length and the degree of unsaturation of acyl-ACP as a substrate. Therefore, by changing the type of TE to be introduced, it is possible to cause cyanobacteria to produce free fatty acids having a desired chain length and degree of unsaturation.
For example, TE derived from Umbellularia californica has substrate specificity for an acyl group having 12 carbon atoms, and the generated free fatty acids are mainly free fatty acids having 12 carbon atoms such as lauric acid (C12: 0). Moreover, TE of Cinnamonum camphorum and Cocos nucifera has substrate specificity for an acyl group having 14 carbon atoms, and the free fatty acids to be produced are mainly 14 free fatty acids such as myristic acid (C14: 0). Moreover, Escherichia coli K-12 strain TE has substrate specificity for an acyl group having 16 or 18 carbon atoms, and the free fatty acids produced are mainly palmitic acid (C16: 0) and palmitoleic acid (C16: 1). , Free fatty acids having 16 or 18 carbon atoms such as stearic acid (C18: 0), oleic acid (C18: 1), linoleic acid (C18: 2), and linolenic acid (C18: 3).

In the present invention, the TE activity can be achieved, for example, by introducing a fusion gene in which a TE gene is linked downstream of a promoter that functions in the host cell into a host cell that lacks the fatty acid degradation system, and under conditions where the introduced TE gene is expressed. It can be confirmed by culturing the cells and analyzing changes in the fatty acid composition in the host cells or culture medium by conventional methods. Alternatively, a fusion gene in which a TE gene is linked downstream of a promoter that functions in the host cell is introduced into the host cell, and the cell is cultured under conditions in which the introduced TE gene is expressed. By carrying out reactions using various acyl-ACPs as substrates according to these methods (Yuan L. et al., Proc. Natl. Acad. Sci. USA, 1995, vol. 92 (23), p. 10639-10643). I can confirm.

The transformant of the present invention can be obtained by introducing the KAS gene into a host. The transformant significantly improves the productivity of a fatty acid having a specific carbon number and a lipid containing this fatty acid as a constituent component, as compared with the host itself. In the transformant, the fatty acid composition in the lipid is modified as compared with the host. The productivity of fatty acids and lipids in the host and transformant can be measured by the method used in the examples.

The transformant of 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 an expression vector capable of expressing the KAS gene in a host cell, introducing the vector into the host cell, and transforming the host cell.
A transformant in which the TE gene is introduced in addition to the KAS gene can also be prepared by a conventional method.

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, algae, 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 plant.
The microorganism may be either a prokaryote or a eukaryote, and a prokaryote such as a microorganism belonging to the genus Escherichia or a genus Bacillus, or a eukaryotic microorganism such as a yeast or filamentous fungus is used. it can. Of these, Escherichia coli , Bacillus subtilis , red yeast ( Rhodosporidium toruloides ), or Mortierella sp. Is preferred, and Escherichia coli is more preferred from the viewpoint of fatty acid productivity.
From the viewpoint of establishing a genetic recombination technique, the microalgae include algae of the genus Chlamydomonas , algae of the genus Chlorella , algae of the genus Phaeodactylum , or Nannochloropsis ( Algae of the genus Nannochloropsis ) are preferred, and algae of the genus Nannochloropsis are more preferred.
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.

The vector (plasmid) serving as the parent of the plasmid vector for gene expression may be any vector as long as it can introduce a gene encoding the target protein into the host and can express the gene in the host cell. 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.
Expression vectors that can be preferably used in the present invention include pUC vectors (Takara Bio), pBluescript (pBS) II SK (-) (Stratagene), pSTV vectors (Takara Bio), 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), pMW218 / 219 (Nippon Gene), pRI vector (Takara Bio), pBI vectors (Clontech), IN3 vectors (Implanta Innovations), pUC19 (Takara Bio), P66 (Chlamydomonas Center), P-322 (Chlamydomonas Center), pPha-T1 (Yangmin Gong, Xiaojing) Guo, Xia Wan, Zhuo Liang, Mulan Jiang "Characterization of a novel thioesterase (PtTE) from Phaeodactylum tricornutum", Journal of Basic Microbiology, 2011 December, Volume 51, p.666-672. Reference), and pJET1 (manufactured by Cosmo Bio Co., Ltd.). In the present invention, when Arabidopsis thaliana is used as a host, a pRI vector or a pBI vector is 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, and violaxanthin / chlorophyll a binding protein gene from Nannochloropsis genus promoter Chromatography, and the like. In the present invention, when Arabidopsis thaliana is used as a host, Western rape or rape-derived Napin gene promoter 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. Transformation methods include calcium ion transformation method, general competent cell transformation method, protoplast transformation method, electroporation method, LP transformation method, method using Agrobacterium, particle gun method Etc.

In the transformant of the present invention, the productivity of fatty acids having a specific carbon number and lipids comprising the same is improved as compared with the host. Therefore, the lipid can be efficiently produced by culturing the transformant of the present invention under appropriate conditions, and then recovering the lipid from the obtained culture or growth. 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 according to the host of the transformant, and culture conditions usually used for the host can be used. From the viewpoint of fatty acid production efficiency, for example, glycerol, acetic acid, or malonic acid 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 transformant 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.
When Arabidopsis thaliana is used as a host, the transformant can be cultured, for example, in soil at a temperature of 20 to 25 ° C. under continuous irradiation with white light or light conditions such as 16 hours of light and 8 hours of dark. Can be cultivated for 2 months.
When using algae as a host, a medium based on natural seawater or artificial seawater may be used, or a commercially available culture medium may be used. 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 inoculated into the medium can be appropriately selected, and is preferably 1 to 50% (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. The algae is preferably cultured under light irradiation so that photosynthesis is possible. 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 transformant may be cultured by aeration and agitation culture, shaking culture or stationary culture, and shaking culture is preferred from the viewpoint of improving aeration.

The method for recovering the lipid produced by the transformant can be appropriately selected from conventional methods. For example, by filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, chloroform / methanol extraction method, hexane extraction method, ethanol extraction method, etc. from the aforementioned culture, growth product or transformant The lipid component can be isolated and recovered. In the case of larger-scale culture, oil is recovered from the culture, growth, or transformant by pressing or extraction, and then general purification such as degumming, deoxidation, decolorization, dewaxing, deodorization, etc. is performed. , Lipids can be obtained. 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 obtained by the production method of the present invention preferably contains one or more selected from simple lipids and derived lipids, more preferably contains derived lipids, fatty acids or It is more preferable that it contains the ester, and it is still more preferable that it is a fatty acid or its ester.

The lipid obtained by the production method of the present invention can be used as an edible material, as an emulsifier for cosmetics and the like, a detergent such as soap and detergent, a fiber treatment agent, a hair rinse agent, or a bactericide and preservative.

Regarding the above-described embodiments, the present invention further discloses the following lipid production method, transformant, transformant production method, protein, gene, and lipid productivity improvement method.

<1> Manufacture of lipids by culturing a transformant obtained by introducing a gene encoding any of the following proteins (a) to (f) into a host to produce fatty acids or lipids comprising the same Method.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more with the amino acid sequence of the protein (a) (C) a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 and (d) an amino acid sequence of the protein (c) of 85% or more, preferably A protein comprising an amino acid sequence having 90% or more, more preferably 95% or more, and even more preferably 98% or more identity, and having KAS activity (e) a protein comprising an amino acid sequence represented by SEQ ID NO: 5 ( f) 85% or more, preferably 90% or more, more preferably 95% or more, with the amino acid sequence of the protein (e) Preferably of an amino acid sequence having 98% or more, identity and protein having KAS activity

<2> The protein (b) has one or several, preferably 1 to 73, more preferably 1 to 49, still more preferably 1 or more amino acid sequences in the protein (a). The method according to <1> above, wherein the protein is a protein in which 25 or less, more preferably 1 or more and 10 or less amino acids are deleted, substituted, inserted or added.
<3> The method according to <1> or <2> above, wherein the gene encoding the protein (a) or (b) is a gene consisting of the following DNA (g) or (h).
(G) DNA comprising the base sequence represented by SEQ ID NO: 2
(H) a DNA comprising a base sequence having 85% or more, preferably 90% or more, more preferably 95% or more identity with the DNA (g) base sequence, and encoding a protein having KAS activity
<4> The DNA (h) has one or more, preferably 1 or more and 218 or less, more preferably 1 or more and 145 or less, more preferably 1 or more, in the base sequence of the DNA (g). A DNA encoding the protein (a) or (b) having a KAS activity consisting of a base sequence in which 73 or fewer bases are deleted, substituted, inserted or added, or complementary to the DNA (g) <3> The method according to <3>, wherein the DNA is a DNA that hybridizes with a DNA comprising a basic sequence under stringent conditions and encodes the protein (a) or (b) having KAS activity.
<5> The method according to any one of <1> to <4>, wherein the proteins (a) and (b) are KAS I.
<6> The protein (d) has one or several, preferably 1 to 86, more preferably 1 to 57, and still more preferably 1 or more amino acid sequences in the protein (c). 29. The method according to <1> above, wherein the protein is a protein in which 29 or less, more preferably 1 or more and 12 or less amino acids are deleted, substituted, inserted or added.
<7> The method according to <1> or <6> above, wherein the gene encoding the protein (c) or (d) is a gene consisting of the following DNA (i) or (j).
(I) DNA comprising the base sequence represented by SEQ ID NO: 4
(J) a DNA comprising a base sequence having 85% or more, preferably 90% or more, more preferably 95% or more identity with the DNA (i) base sequence, and encoding a protein having KAS activity
<8> The DNA (j) has one or more, preferably 1 to 256, more preferably 1 to 171 and even more preferably 1 or more nucleotide sequences in the DNA (i). A DNA encoding a protein (c) or (d) having a KAS activity, comprising a nucleotide sequence with 86 or fewer bases deleted, substituted, inserted, or added, or complementary to the DNA (i) The method according to <7> above, which is a DNA that hybridizes under stringent conditions with a DNA comprising a basic nucleotide sequence and encodes the protein (c) or (d) having KAS activity.
<9> The method according to any one of <1> and <6> to <8>, wherein the proteins (c) and (d) are KAS IV.
<10> The protein (f) has one or several, preferably 1 to 86, more preferably 1 to 57, and still more preferably 1 or more amino acid sequences in the protein (e). 29. The method according to <1> above, wherein the protein is a protein in which 29 or less, more preferably 1 or more and 12 or less amino acids are deleted, substituted, inserted or added.
<11> The method according to <1> or <10> above, wherein the gene encoding the protein (e) or (f) is a gene consisting of the following DNA (k) or (l).
(K) DNA comprising the base sequence represented by SEQ ID NO: 6
(L) a DNA encoding a protein having a KAS activity and comprising a base sequence having 85% or more, preferably 90% or more, more preferably 95% or more identity with the base sequence of DNA (i)
<12> The DNA (l) has one or more, preferably 1 to 256, more preferably 1 to 171 and still more preferably 1 or more nucleotide sequences in the DNA (k). A DNA encoding a protein (e) or (f) having a KAS activity consisting of a base sequence in which 86 or fewer bases are deleted, substituted, inserted, or added, or complementary to the DNA (k) <11> The method according to <11> above, which is a DNA that hybridizes under stringent conditions with a DNA comprising a basic nucleotide sequence and encodes the protein (e) or (f) having KAS activity.
<13> The method according to any one of <1> and <10> to <12>, wherein the proteins (e) and (f) are KAS IV.
<14> The method according to any one of <1> to <13>, wherein the host is a microorganism or a plant.
<15> The method according to <14>, wherein the plant is Arabidopsis thaliana.
<16> The method according to <15>, wherein the transformant is produced by introducing a gene encoding any of the proteins (a) to (f) into Arabidopsis as a host using Agrobacterium. .
<17> The method according to <15> or <16> above, wherein the fatty acid produced by the transformant or a lipid comprising the fatty acid is collected from Arabidopsis seeds.
<18> Any one of <1> to <17>, wherein a gene encoding TE, preferably a gene encoding TE having substrate specificity for medium chain acyl-ACP, has been introduced into the host. The method described.
<19> the TE is, Umbellularia californica the TE, Cocos nucifera of TE, TE of Cinnamonum camphorum, TE of Elaeis guineensis, Cuphea species TE, Nannochloropsis oculata of TE, TE of Nannochloropsis gaditana, Nannochloropsis granulata of TE, and Symbiodinium Microadriaticum <18> The method according to <18>, wherein the method is at least one TE selected from the group consisting of:

<20> A transformant obtained by introducing a gene encoding any of the proteins (a) to (f) into a host.
<21> A method for producing a transformant, wherein a gene encoding any of the proteins (a) to (f) is introduced into a host.
<22> A method for improving the lipid productivity of a host, wherein a gene encoding any of the proteins (a) to (f) is introduced into a host, and the lipid productivity of the obtained transformant is improved.
<23> A gene encoding any one of the proteins (a) to (f) is introduced into a host, the fatty acid composition in the lipid produced by the obtained transformant is modified, and in the lipid produced by the host A method of modifying the fatty acid composition.

<24> The protein (b) has one or several, preferably 1 to 73, more preferably 1 to 49, and still more preferably 1 or more amino acid sequences of the protein (a). The transformant according to any one of the above <20> to <23>, which is a protein in which 25 or less, more preferably 1 or more and 10 or less amino acids are deleted, substituted, inserted or added Method.
<25> The transformation according to any one of <20> to <24>, wherein the gene encoding the protein (a) or (b) is a gene comprising the DNA (g) or (h) Body or method.
<26> The DNA (h) has one or more, preferably 1 or more and 218 or less, more preferably 1 or more and 145 or less, more preferably 1 or more, in the base sequence of the DNA (g). A DNA encoding the protein (a) or (b) having a KAS activity consisting of a base sequence in which 73 or fewer bases are deleted, substituted, inserted or added, or complementary to the DNA (g) The transformant according to <25>, wherein the transformant is a DNA that hybridizes with a DNA comprising a basic nucleotide sequence under stringent conditions and encodes the protein (a) or (b) having KAS activity. Or method.
<27> The transformant or method according to any one of <20> to <26>, wherein the proteins (a) and (b) are KAS I.
<28> The protein (d) has one or several, preferably 1 to 86, more preferably 1 to 57, more preferably 1 or more amino acid sequences in the protein (c). The transformant according to any one of the above <20> to <23>, which is a protein having 29 or less, more preferably 1 to 12 amino acids deleted, substituted, inserted or added, or Method.
<29> Any one of the above <20> to <23> and <28>, wherein the gene encoding the protein (c) or (d) is the gene consisting of the DNA (i) or (j) The transformant or method as described.
<30> The DNA (j) has one or more, preferably 1 to 256, more preferably 1 to 171 and even more preferably 1 or more nucleotide sequences in the DNA (i). A DNA encoding a protein (c) or (d) having a KAS activity, comprising a nucleotide sequence with 86 or fewer bases deleted, substituted, inserted, or added, or complementary to the DNA (i) <29> The transformant according to <29>, wherein the transformant is a DNA that hybridizes with a DNA having a basic nucleotide sequence under stringent conditions and encodes the protein (c) or (d) having KAS activity. Or method.
<31> The transformant or method according to any one of <20> to <23> and <28> to <30>, wherein the proteins (c) and (d) are KAS IV.
<32> The protein (f) has one or several, preferably 1 to 86, more preferably 1 to 57, still more preferably 1 or more amino acid sequences of the protein (e). The transformant according to any one of the above <20> to <23>, which is a protein having 29 or less, more preferably 1 to 12 amino acids deleted, substituted, inserted or added, or Method.
<33> Any one of the above <20> to <23> and <32>, wherein the gene encoding the protein (e) or (f) is the gene consisting of the DNA (k) or (l) The transformant or method as described.
<34> One or more, preferably 1 to 256, more preferably 1 to 171 and even more preferably 1 or more of the DNA (l) in the base sequence of the DNA (k) A DNA encoding a protein (e) or (f) having a KAS activity consisting of a base sequence in which 86 or fewer bases are deleted, substituted, inserted, or added, or complementary to the DNA (k) The transformant according to <33>, wherein the transformant is a DNA that hybridizes with a DNA comprising a basic sequence under stringent conditions and encodes the protein (e) or (f) having KAS activity. Or method.
<35> The transformant or method according to any one of <20> to <23> and <32> to <34>, wherein the proteins (e) and (f) are KAS IV.
<36> The transformant or method according to any one of <20> to <35>, wherein the host is a microorganism or a plant.
<37> The transformant or method according to <36>, wherein the plant is Arabidopsis thaliana.
<38> The gene according to <37>, wherein the gene encoding any one of the proteins (a) to (f) is introduced into Arabidopsis thaliana as a host using Agrobacterium, and the transformant is produced. Converter or method.
<39> Any one of <20> to <38>, wherein a gene encoding TE, preferably a gene encoding TE having substrate specificity for medium chain acyl-ACP, has been introduced into the host. The transformant or method as described.
<40> the TE is, Umbellularia californica the TE, TE of Cocos nucifera, TE of Cinnamonum camphorum, TE of Elaeis guineensis, Cuphea species TE, TE of Nannochloropsis oculata, Nannochloropsis gaditana of TE, Nannochloropsis granulata of TE, and Symbiodinium Microadriaticum The transformant or method according to <39>, wherein the transformant is the at least one TE selected from the group consisting of TEs.

<41> The protein (a) to (f).
<42> A gene encoding any one of the proteins according to <41>.
<43> A gene comprising any one of the DNAs (g) to (l).

EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.
Here, the base sequences of the primers used in this example are shown in Table 1.

(Preparation Example 1) Preparation of coconut cDNA After coconut solid endosperm was frozen in liquid nitrogen, it was crushed using a multi-bead shocker (manufactured by Yasui Kikai). To the crushed solid endosperm, phenol / chloroform and 50 mM Tris-HCl (pH 9) were added and mixed, and centrifuged at 7500 rpm for 10 minutes, and the supernatant was collected. The recovered supernatant was again subjected to the same phenol / chloroform treatment. The upper layer was collected, ethanol precipitation operation was performed on this, and the nucleic acid component contained therein was purified.
For purification of RNA components, RNeasy Plant Mini Kit (Qiagen, Valencia, California) was used. The nucleic acid pellet after ethanol precipitation was vortexed by adding RLT Buffer supplemented with 1/100 volume of 1M DTT, and applied to a QIA shredder spin column. Thereafter, the operation was performed according to the manual attached to the kit, and finally the total RNA derived from coconut was eluted with deionized water (dH ₂ O).
DNase I (manufactured by Thermo Scientific) was added to the obtained RNA solution together with a buffer, and the mixture was treated at 37 ° C. for 1 hour. Thereafter, phenol / chloroform treatment and ethanol precipitation treatment were carried out to prepare a coconut endosperm-derived RNA solution.
Subsequently, coconut cDNA was prepared from the obtained RNA using PrimeScript II 1st strand cDNA Synthesis Kit (trade name, manufactured by Takara Bio Inc.).

Preparation Example 2 Preparation of California Bay cDNA California Bay seeds obtained from Sierra Seed Supply, USA were frozen in liquid nitrogen and then crushed using a mortar and breast. RNA was extracted from the crushed tissue using Fruit Mate (trade name, manufactured by Takara Bio Inc.) and NucleoSpin RNA plant (trade name, manufactured by Takara Bio Inc.).
Subsequently, a California Bay cDNA was prepared from the obtained RNA using PrimeScript II 1st strand cDNA Synthesis Kit (trade name, manufactured by Takara Bio Inc.).

(Preparation Example 3) Preparation of camphor tree cDNA The camphor seeds collected from the Kao Wakayama Factory were frozen in liquid nitrogen and then crushed using a mortar and breast. RNA was extracted from the crushed tissue using Fruit Mate (trade name, manufactured by Takara Bio Inc.) and NucleoSpin RNA plant (trade name, manufactured by Takara Bio Inc.).
Subsequently, camphor tree cDNA was prepared from the obtained RNA using PrimeScript II 1st strand cDNA Synthesis Kit (trade name, manufactured by Takara Bio Inc.).

(Preparation Example 4) Construction of TE gene expression plasmid derived from coconut palm Using the PowerPlant DNA Isolation Kit (trade name, manufactured by MO BIO Laboratories, USA), collected in oilseed rape and Mashiko-machi, Tochigi Genomic DNA was extracted from each rape.
Using the rape genomic DNA collected in Itako, Ibaraki Prefecture as a template, DNA polymerase PrimeSTAR (trade name, manufactured by Takara Bio Inc.) and primer Nos. Shown in Table 1 were used. 1 (SEQ ID NO: 22) and primer no. PCR was performed using 2 (SEQ ID NO: 23) to amplify the Napin gene promoter.
Moreover, using the rape genomic DNA collected in Mashiko-machi, Tochigi Prefecture as a template, DNA polymerase PrimeSTAR (trade name, manufactured by Takara Bio Inc.) and primer Nos. Shown in Table 1 were used. 3 (SEQ ID NO: 24) and primer no. PCR was performed using 4 (SEQ ID NO: 25) to amplify the terminator of the Napin gene.
The PCR product confirmed to be amplified was used as a template, and the promoter of Napin gene was the primer No. shown in Table 1. 5 (SEQ ID NO: 26) and primer no. 6 (SEQ ID NO: 27), the terminator of the Napin gene is the primer No. shown in Table 1. 3 (SEQ ID NO: 24) and primer no. PCR was performed again using 7 (SEQ ID NO: 28). In this manner, a Napin gene promoter sequence fragment (SEQ ID NO: 17) and a Napin gene terminator sequence fragment (SEQ ID NO: 18) were prepared, respectively.
These amplified gene fragments were each treated with Mighty TA-cloning Kit (trade name, manufactured by Takara Bio Inc.) and then inserted into a pMD20-T vector (trade name, manufactured by Takara Bio Inc.) by a ligation reaction. Thus, plasmid pPNapin1 into which the Napin gene promoter region was introduced and pTNapin1 into which the Napin gene terminator region was introduced were constructed.

Using the plasmid pPNapin1 as a template, PrimeSTAR and the primer Nos. Shown in Table 1 were used. 8 (SEQ ID NO: 29) and primer no. PCR was performed using 9 (SEQ ID NO: 30) to amplify a promoter sequence having restriction enzyme recognition sequences added to both ends. Further, using the plasmid pTNapin1 as a template, PrimeSTAR and the primer Nos. Shown in Table 1 were used. 10 (SEQ ID NO: 31) and primer no. A PCR reaction was performed using 11 (SEQ ID NO: 32) to amplify the terminator sequence.
The amplified PCR product was treated with Mighty TA-cloning Kit (trade name, manufactured by Takara Bio Inc.) and then inserted into the pMD20-T vector by ligation reaction to construct plasmid pPNapin2 and plasmid pTNapin2. Plasmid pPNapin2 was treated with restriction enzymes Sal I and Not I, plasmid pTNapin2 was treated with restriction enzymes Sma I and Not I, respectively, and ligated to pRI909 vector (Takara Bio) treated with Sal I and Sma I, Plasmid p909PTnapin was constructed.

Next, a gene (SEQ ID NO: 19) encoding a chloroplast translocation signal peptide derived from California Bay (hereinafter also abbreviated as BTE) gene is provided by a contract synthesis service provided by Invitrogen (Carlsbad, California). Acquired using. Using the obtained plasmid containing the gene sequence as a template, PrimeSTAR and the primer Nos. Shown in Table 1 were used. 12 (SEQ ID NO: 33) and primer no. PCR was performed using 13 (SEQ ID NO: 34) to amplify a gene fragment encoding a signal peptide. Using Mighty TA-cloning Kit (trade name, manufactured by Takara Bio Inc.), deoxyadenine (dA) was added to both ends of the amplified gene fragment, and then ligated to pMD20-T vector (Takara Bio Inc.). The plasmid pSignal was constructed by insertion. The constructed plasmid pSignal was treated with the restriction enzyme Not I and ligated to the Not I site of the plasmid p909PTnapin by a ligation reaction to obtain a plasmid p909PTnapin-S.

Using the cDNA derived from coconut endosperm prepared in Preparation Example 1 as a template, restriction enzyme PrimeSTAR MAX (trade name, manufactured by Takara Bio Inc.), and primer No. 1 shown in Table 1. 14 (SEQ ID NO: 35) and primer no. PCR was performed using 15 (SEQ ID NO: 36) to amplify a gene fragment (SEQ ID NO: 8) encoding coconut palm-derived TE (hereinafter also abbreviated as CTE).
In addition, using the above-mentioned p909PTnapin-S as a template, the primer numbers shown in Table 1 were used. 16 (SEQ ID NO: 37) and primer no. 17 (SEQ ID NO: 38) was used to amplify a linear fragment of p909PTnapin-S.

The CTE gene fragment and the linear p909PTnapin-S fragment were ligated by In-fusion reaction using In-Fusion Advantage PCR Cloning Kit (trade name, manufactured by Clontech) to construct plasmid p909CTE. The plasmid p909CTE is designed such that the expression of the CTE gene is controlled by the promoter of the Napin gene derived from rape, and the expressed CTE gene is transferred to the chloroplast by the chloroplast transfer signal peptide derived from the BTE gene.

(Preparation Example 5) Construction of plasmid for KAS gene expression Encoding phosphinothricin acetyltransferase derived from Streptomyces hygroscopicus with reference to the sequence of transformation vector pYW310 (ACCESSION NO. DQ469641) disclosed in NCBI Gene Bank The bialaphos resistance gene (Bar gene, SEQ ID NO: 20) was obtained using a commissioned synthesis service provided by Gene Script.
Using the synthesized Bar gene as a template, PrimeSTAR and primer Nos. Shown in Table 1 were used. 18 (SEQ ID NO: 39) and primer no. PCR was performed using 19 (SEQ ID NO: 40) to amplify the Bar gene. On the other hand, using pRI909 as a template, PrimeSTAR and the primer numbers shown in Table 1 were used. 20 (SEQ ID NO: 41) and primer no. A PCR reaction was performed using 21 (SEQ ID NO: 42) to amplify a region excluding the kanamycin resistance gene from the pRI909 vector. These gene fragments fragment was treated with Nde I and Spe I, and ligated in ligation reaction, the kanamycin resistance gene vector pRI909 held originally to construct a plasmid pRI909Bar substituted on Bar gene.

Reference to the sequence of Brassica napus napin Promoter (ACCESSION NO. EU416279) disclosed in NCBI's Gene Bank, using the contract synthesis service provided by Gene Script for the Napin promoter (SEQ ID NO: 21) expressed in the seeds of rape And acquired.
Using the synthesized Napin promoter as a template, primer Nos. Shown in Table 1 were used. 22 (SEQ ID NO: 43) and primer no. PCR was performed using 23 (SEQ ID NO: 44) to amplify the Napin promoter. In addition, using the plasmid pRI909Bar as a template, the primer Nos. Shown in Table 1 were used. 24 (SEQ ID NO: 45) and primer no. PCR was performed using 25 (SEQ ID NO: 46) to amplify a linear fragment of pRI909Bar. Furthermore, using the plasmid p909CTE as a template, the primer numbers shown in Table 1 were used. 26 (SEQ ID NO: 47) and primer no. PCR was performed using 27 (SEQ ID NO: 48) to amplify the CTE-Tnapin sequence. These gene fragments were subjected to In-fusion reaction in the same manner as in Preparation Example 4 to construct plasmid p909Pnapus-CTE-Tnapin.

Using the plasmid p909Pnapus-CTE-Tnapin as a template, the primer numbers shown in Table 1 were used. 28 (SEQ ID NO: 49) and primer no. PCR was performed using 29 (SEQ ID NO: 50) to amplify the gene fragment. In addition, using primer derived from California Bay seed as a template, primer Nos. Shown in Table 1 were used. 30 (SEQ ID NO: 51) and primer no. PCR was performed using 31 (SEQ ID NO: 52) to amplify a gene fragment (SEQ ID NO: 2) encoding BKAS431. These gene fragments were ligated by In-fusion reaction to construct p909Pnapus-BKAS431-Tnapin.
Similarly, primer Nos. Shown in Table 1 were used. 32 (SEQ ID NO: 53) and primer no. 33 (SEQ ID NO: 54) was used to amplify a gene fragment (SEQ ID NO: 4) encoding BKAS1082, and the BKAS431 gene of p909Pnapus-BKAS431-Tnapin was replaced with the BKAS1082 gene, and p909Pnapus-BKAS1082-Tnapin was subjected to In-fusion reaction. It was constructed.
Furthermore, using the camphor seed-derived cDNA as a template, primer Nos. Shown in Table 1 were used. 34 (SEQ ID NO: 55) and primer no. PCR was performed using 35 (SEQ ID NO: 56) to amplify a gene fragment (SEQ ID NO: 6) encoding KKAS250. Then, p909Pnapus-KKAS250-Tnapin was constructed by replacing the BKAS431 gene of p909Pnapus-BKAS431-Tnapin with the KKAS250 gene by In-fusion reaction.

(Preparation Example 6) Preparation of transformant Using the plasmid p909CTE, a transformant of Arabidopsis thaliana ( Arabidopsis thaliana , Colombia strain) into which the CTE gene had been introduced was obtained using the Arabidopsis transformation contract service by Implanta Innovations. . The obtained Arabidopsis thaliana transformants (WT :: CTE) and wild-type Arabidopsis thaliana (WT) were grown at 22 ° C. under fluorescent lighting for 24 hours (about 4000 lux). After about 2 months of cultivation, the seeds were harvested.

Using the Arabidopsis thaliana WT :: CTE introduced with p909CTE thus obtained as a parent strain, the following transformation experiments were performed.
The Agrobacterium tumefaciens GV3101 strain into which the plasmids p909Pnapus-BKAS431-Tnapin, p909Pnapus-BKAS1082-Tnapin, and p909Pnapus-KKAS250-Tnapin were introduced were transformed into Arabidopsis thaliana WT :: CTE introduced with p909CTE. A conversion operation was performed. The inflorescences of Arabidopsis grown about 1.5 months after sowing were grown for 6-7 days after excision and infected with Agrobacterium into which each plasmid was introduced.
Seeds grown for about 1-2 months after treatment with Agrobacterium were sown on MS agar medium (containing 100 μg / mL kraforan and 7 μg / mL bialaphos) and transformed (WT :: CTE: : BKAS431, WT :: CTE :: BKAS1082, WT :: CTE :: KKAS250) were selected. The selected transformant was grown at 22 ° C. under fluorescent light illumination under the condition of a light period of 24 hours, and seeds were harvested after cultivation for about 2 months.

(Test Example 1) Extraction of lipids from Arabidopsis seeds and methyl esterification of the lipids Each Arabidopsis seed obtained was pulverized with a multi-bead shocker (manufactured by Yasui Kikai Co., Ltd.). To this pulverized product, 20 μL of 7-pentadecanone (0.5 mg / mL methanol) (internal standard) and 0.25 mL of chloroform added with 20 μL of acetic acid and 0.5 mL of methanol were added, and the mixture was sufficiently stirred and allowed to stand for 15 minutes. Further, 0.25 mL of 1.5% KCl and 0.25 mL of chloroform were added and stirred sufficiently, and allowed to stand for 15 minutes. After centrifugation at room temperature and 1500 rpm for 5 minutes, the lower layer portion was collected and dried with nitrogen gas.
Triacylglycerol was hydrolyzed by adding 100 μL of 0.5N potassium hydroxide-methanol solution to the dried sample and incubating at 70 ° C. for 30 minutes. Furthermore, 0.3 mL of 3-boron fluoride methanol complex solution was added to dissolve the dried product, and the mixture was incubated at 80 ° C. for 10 minutes to carry out methyl esterification of fatty acid. Thereafter, 0.2 mL of saturated saline and 0.3 mL of hexane were added, and the mixture was sufficiently stirred and allowed to stand for 30 minutes. A hexane layer (upper layer portion) containing a fatty acid methyl ester was collected and subjected to gas chromatography (GC) analysis under the following conditions.

<Gas chromatography (GC) conditions>
Column: DB1-MS (J & W Scientific, Folsom, California)
Analyzer: 6890 (Agilent technology, Santa Clara, California)
Column oven temperature: 150 ° C. holding 0.5 minutes → 150 to 320 ° C. (20 ° C./minute temperature increase) → 320 ° C. holding 2 minutes
Injection method: split mode (split ratio = 75: 1)
Sample injection volume: 5 μL
Column flow rate: 0.3 ml / min Constant detector: FID
Carrier gas: Hydrogen makeup gas: Helium

From the peak area of the waveform data obtained by GC analysis, the amount of methyl ester of each fatty acid was quantified. The GC peak corresponding to each lipid in the seeds was identified by the retention time of the standard methyl ester of each fatty acid. In addition, each peak area was compared with the peak area of 7-pentadecanone, which is an internal standard, to correct between samples, and the ratio of each fatty acid amount to the total fatty acid amount contained in all seeds subjected to analysis was calculated. . The results are shown in Table 2. In Table 2, the value of one line was shown for the wild type and the average value of three independent lines for the other.

As shown in Table 2, the C12: 0 fatty acid content was about 3% and the C14: 0 fatty acid content was about 13% in the seed of the transformant WT :: CTE into which only the CTE gene was introduced, compared to the wild type seed. The amount of C16: 0 fatty acid increased by about 15%.
On the other hand, in the transformant WT :: CTE :: BKAS431 into which the CTE gene and the BKAS431 gene were introduced, the amount of C16: 0 fatty acid and the amount of C18: n fatty acid increased compared to the transformant WT :: CTE. However, the amount of C12: 0 fatty acid and the amount of C14: 0 fatty acid decreased.
Further, in the transformant WT :: CTE :: BKAS1082 introduced with the CTE gene and the BKAS1082 gene, and the transformant WT :: CTE :: KKAS250 introduced with the CTE gene and the KKAS250 gene, the transformant WT :: CTE and In comparison, the amount of C12: 0 fatty acid increased and the amount of C16: 0 fatty acid decreased.

As described above, by introducing the KAS gene defined in the present invention into a host, a transformant with improved productivity of a fatty acid having a specific carbon number can be produced. And the productivity of a specific fatty acid can be improved by culturing this transformant.

(Test Example 2) Identification of BKAS431, BKAS1082 and KKAS250 The BKAS431, BKAS1082 and KKAS250 were identified using a homology search by the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). It was.
As a result, BKAS431 was annotated as KAS I. As mentioned above, KAS I extends acyl-ACP to 16 carbon atoms. This is consistent with the result shown in Table 2 in which the amount of C16: 0 fatty acid was increased by introducing the BKAS431 gene.
On the other hand, BKAS1082 and KKAS250 were annotated as KAS II. As described above, KAS II is an enzyme that catalyzes the reaction of converting acyl-ACP having 16 carbon atoms into acyl-ACP having 18 carbon atoms. However, the result of this annotation does not agree with the result shown in Table 2 above, in which the amount of medium chain fatty acids such as C12: 0 is increased by introducing the BKAS1082 gene or the KKAS250 gene. These results are thought to be because the conventional knowledge about plant KAS is insufficient and the KAS IV gene involved in the production of medium chain fatty acids has hardly been identified.

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. 2015-104992 filed in Japan on May 22, 2015, which is hereby incorporated herein by reference. Capture as part.

Claims (43)

1. A method for producing a lipid, comprising culturing a transformant obtained by introducing a gene encoding any one of the following proteins (a) to (f) into a host to produce a fatty acid or a lipid comprising this as a constituent.
   (A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (a) and having β-ketoacyl-ACP synthase activity Protein (c) a protein comprising the amino acid sequence represented by SEQ ID NO: 3 (d) comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (c), and having β-ketoacyl-ACP synthase activity (E) a protein comprising the amino acid sequence represented by SEQ ID NO: 5 (f) an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (e), and β-ketoacyl-ACP synthase activity Protein with
2. The method according to claim 1, wherein the protein (b) is a protein in which 1 to 73 amino acids are deleted, substituted, inserted or added to the amino acid sequence of the protein (a).
3. The method according to claim 1 or 2, wherein the gene encoding the protein (a) or (b) is a gene consisting of the following DNA (g) or (h).
   (G) DNA comprising the base sequence represented by SEQ ID NO: 2
   (H) a DNA encoding a protein comprising a base sequence having 85% or more identity with the base sequence of DNA (g) and having β-ketoacyl-ACP synthase activity
4. The DNA (h) has a base sequence in which 1 to 218 bases are deleted, substituted, inserted or added to the base sequence of the DNA (g), and has a β-ketoacyl-ACP synthase activity Which hybridizes under stringent conditions with DNA encoding the protein (a) or (b) having the above or a DNA comprising a base sequence complementary to the DNA (g), and β-ketoacyl-ACP synthase activity The method according to claim 3, wherein the DNA encodes the protein (a) or (b).
5. The method according to any one of claims 1 to 4, wherein the proteins (a) and (b) are β-ketoacyl-ACP synthase I.
6. The method according to claim 1, wherein the protein (d) is a protein in which 1 to 86 amino acids are deleted, substituted, inserted or added to the amino acid sequence of the protein (c).
7. The method according to claim 1 or 6, wherein the gene encoding the protein (c) or (d) is a gene comprising the following DNA (i) or (j).
   (I) DNA comprising the base sequence represented by SEQ ID NO: 4
   (J) DNA encoding a protein comprising a base sequence having 85% or more identity with the base sequence of DNA (i) and having β-ketoacyl-ACP synthase activity
8. The DNA (j) has a base sequence in which 1 to 256 bases are deleted, substituted, inserted or added to the base sequence of the DNA (i), and has a β-ketoacyl-ACP synthase activity A β-ketoacyl-ACP synthase activity that hybridizes under stringent conditions with a DNA encoding the protein (c) or (d) having the above or a DNA comprising a base sequence complementary to the DNA (i) The method according to claim 7, wherein the protein encodes the protein (c) or (d).
9. The method according to any one of claims 1 and 6 to 8, wherein the proteins (c) and (d) are β-ketoacyl-ACP synthase IV.
10. The method according to claim 1, wherein the protein (f) is a protein in which 1 to 86 amino acids are deleted, substituted, inserted or added to the amino acid sequence of the protein (e).
11. The method according to claim 1 or 10, wherein the gene encoding the protein (e) or (f) is a gene consisting of the following DNA (k) or (l).
    (K) DNA comprising the base sequence represented by SEQ ID NO: 6
    (L) a DNA encoding a protein comprising a base sequence having 85% or more identity with the base sequence of DNA (k) and having β-ketoacyl-ACP synthase activity
12. The DNA (l) has a base sequence in which 1 to 256 bases are deleted, substituted, inserted or added to the base sequence of the DNA (k), and has a β-ketoacyl-ACP synthase activity A β-ketoacyl-ACP synthase activity that hybridizes under stringent conditions with a DNA encoding the protein (e) or (f) having the above or a DNA comprising a base sequence complementary to the DNA (k) The method according to claim 11, wherein the DNA encodes the protein (e) or (f).
13. The method according to any one of claims 1 and 10 to 12, wherein the proteins (e) and (f) are β-ketoacyl-ACP synthase IV.
14. The method according to any one of claims 1 to 13, wherein the host is a microorganism or a plant.
15. The method according to claim 14, wherein the plant is Arabidopsis thaliana.
16. The method according to claim 15, wherein the transformant is produced by introducing a gene encoding any of the proteins (a) to (f) into Arabidopsis as a host using Agrobacterium.
17. The method according to claim 15 or 16, wherein the fatty acid produced by the transformant or a lipid comprising the fatty acid is collected from Arabidopsis seeds.
18. The method according to any one of claims 1 to 17, wherein a gene encoding acyl-ACP thioesterase is introduced into the host.
19. The acyl-ACP thioesterase comprises Umbellularia californica acyl-ACP thioesterase, Cocos nucifera acyl-ACP thioesterase, Cinnamonum camphorum acyl-ACP thioesterase, Elaeis guineensis acyl-ACP thioesterase, Cuphea species acyl-ACP At least one acyl selected from the group consisting of thioesterase, acyl-ACP thioesterase of Nannochloropsis oculata , acyl-ACP thioesterase of Nannochloropsis gaditana , acyl-ACP thioesterase of Nannochloropsis granulata , and acyl-ACP thioesterase of Symbiodinium microadriaticum The method of claim 18, which is an ACP thioesterase.
20. A transformant obtained by introducing a gene encoding any of the following proteins (a) to (f) into a host.
    (A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (a) and having β-ketoacyl-ACP synthase activity Protein (c) a protein comprising the amino acid sequence represented by SEQ ID NO: 3 (d) comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (c), and having β-ketoacyl-ACP synthase activity (E) a protein comprising the amino acid sequence represented by SEQ ID NO: 5 (f) an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (e), and β-ketoacyl-ACP synthase activity Protein with
21. A method for producing a transformant, wherein a gene encoding any of the following proteins (a) to (f) is introduced into a host.
    (A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (a) and having β-ketoacyl-ACP synthase activity Protein (c) a protein comprising the amino acid sequence represented by SEQ ID NO: 3 (d) comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (c), and having β-ketoacyl-ACP synthase activity (E) a protein comprising the amino acid sequence represented by SEQ ID NO: 5 (f) an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (e), and β-ketoacyl-ACP synthase activity Protein with
22. A method for improving lipid productivity of a host, wherein a gene encoding any one of the following proteins (a) to (f) is introduced into a host, and the lipid productivity of the obtained transformant is improved.
    (A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (a) and having β-ketoacyl-ACP synthase activity Protein (c) a protein comprising the amino acid sequence represented by SEQ ID NO: 3 (d) comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (c), and having β-ketoacyl-ACP synthase activity (E) a protein comprising the amino acid sequence represented by SEQ ID NO: 5 (f) an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (e), and β-ketoacyl-ACP synthase activity Protein with
23. A gene encoding any of the following proteins (a) to (f) is introduced into a host, and the fatty acid composition in the lipid produced by the host is modified by modifying the fatty acid composition in the lipid produced by the obtained transformant. How to modify.
    (A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (a) and having β-ketoacyl-ACP synthase activity Protein (c) a protein comprising the amino acid sequence represented by SEQ ID NO: 3 (d) comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (c), and having β-ketoacyl-ACP synthase activity (E) a protein comprising the amino acid sequence represented by SEQ ID NO: 5 (f) an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (e), and β-ketoacyl-ACP synthase activity Protein with
24. The protein (b) is a protein in which 1 to 73 amino acids are deleted, substituted, inserted or added to the amino acid sequence of the protein (a). The transformant or method as described.
25. The transformant or method according to any one of claims 20 to 24, wherein the gene encoding the protein (a) or (b) is a gene comprising the following DNA (g) or (h).
    (G) DNA comprising the base sequence represented by SEQ ID NO: 2
    (H) a DNA encoding a protein comprising a base sequence having 85% or more identity with the base sequence of DNA (g) and having β-ketoacyl-ACP synthase activity
26. The DNA (h) has a base sequence in which 1 to 218 bases are deleted, substituted, inserted or added to the base sequence of the DNA (g), and has a β-ketoacyl-ACP synthase activity Which hybridizes under stringent conditions with DNA encoding the protein (a) or (b) having the above or a DNA comprising a base sequence complementary to the DNA (g), and β-ketoacyl-ACP synthase activity The transformant or method according to claim 25, which is a DNA encoding the protein (a) or (b).
27. The transformant or method according to any one of claims 20 to 26, wherein the proteins (a) and (b) are β-ketoacyl-ACP synthase I.
28. The protein (d) is a protein in which 1 to 86 amino acids are deleted, substituted, inserted or added to the amino acid sequence of the protein (c). The transformant or method as described.
29. The transformant or method according to any one of claims 20 to 23 and 28, wherein the gene encoding the protein (c) or (d) is a gene comprising the following DNA (i) or (j).
    (I) DNA comprising the base sequence represented by SEQ ID NO: 4
    (J) DNA encoding a protein comprising a base sequence having 85% or more identity with the base sequence of DNA (i) and having β-ketoacyl-ACP synthase activity
30. The DNA (j) has a base sequence in which 1 to 256 bases are deleted, substituted, inserted or added to the base sequence of the DNA (i), and has a β-ketoacyl-ACP synthase activity A β-ketoacyl-ACP synthase activity that hybridizes under stringent conditions with a DNA encoding the protein (c) or (d) having the above or a DNA comprising a base sequence complementary to the DNA (i) 30. The transformant or method according to claim 29, which is a DNA encoding the protein (c) or (d).
31. The transformant or method according to any one of claims 20 to 23 and 28 to 30, wherein the proteins (c) and (d) are β-ketoacyl-ACP synthase IV.
32. The protein (f) is a protein in which 1 to 86 amino acids are deleted, substituted, inserted or added to the amino acid sequence of the protein (e). The transformant or method as described.
33. The transformant or method according to any one of claims 20 to 23 and 32, wherein the gene encoding the protein (e) or (f) is a gene comprising the following DNA (k) or (l).
    (K) DNA comprising the base sequence represented by SEQ ID NO: 6
    (L) a DNA encoding a protein comprising a base sequence having 85% or more identity with the base sequence of DNA (k) and having β-ketoacyl-ACP synthase activity
34. The DNA (l) has a base sequence in which 1 to 256 bases are deleted, substituted, inserted or added to the base sequence of the DNA (k), and has a β-ketoacyl-ACP synthase activity A β-ketoacyl-ACP synthase activity that hybridizes under stringent conditions with a DNA encoding the protein (e) or (f) having the above or a DNA comprising a base sequence complementary to the DNA (k) 34. The transformant or method according to claim 33, which is a DNA encoding the protein (e) or (f).
35. The transformant or method according to any one of claims 20 to 23 and 32-34, wherein the proteins (e) and (f) are β-ketoacyl-ACP synthase IV.
36. The transformant or method according to any one of claims 20 to 35, wherein the host is a microorganism or a plant.
37. The transformant or method according to claim 36, wherein the plant is Arabidopsis thaliana.
38. The transformant or method according to claim 37, wherein a gene encoding any of the proteins (a) to (f) is introduced into Arabidopsis thaliana as a host using Agrobacterium to produce the transformant.
39. The transformant or method according to any one of claims 20 to 38, wherein a gene encoding acyl-ACP thioesterase is introduced into the host.
40. The acyl-ACP thioesterase comprises Umbellularia californica acyl-ACP thioesterase, Cocos nucifera acyl-ACP thioesterase, Cinnamonum camphorum acyl-ACP thioesterase, Elaeis guineensis acyl-ACP thioesterase, Cuphea species acyl-ACP At least one acyl selected from the group consisting of thioesterase, acyl-ACP thioesterase of Nannochloropsis oculata , acyl-ACP thioesterase of Nannochloropsis gaditana , acyl-ACP thioesterase of Nannochloropsis granulata , and acyl-ACP thioesterase of Symbiodinium microadriaticum 40. The transformant or method of claim 39, which is an ACP thioesterase.
41. The following proteins (a) to (f).
    (A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (a) and having β-ketoacyl-ACP synthase activity Protein (c) a protein comprising the amino acid sequence represented by SEQ ID NO: 3 (d) comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (c), and having β-ketoacyl-ACP synthase activity (E) a protein comprising the amino acid sequence represented by SEQ ID NO: 5 (f) an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (e), and β-ketoacyl-ACP synthase activity Protein with
42. A gene encoding any one of the proteins according to claim 41.
43. A gene comprising any one of the following DNA (g) to (l).
    (G) DNA comprising the base sequence represented by SEQ ID NO: 2
    (H) a DNA encoding a protein comprising a base sequence having 85% or more identity with the base sequence of DNA (g) and having β-ketoacyl-ACP synthase activity
    (I) DNA comprising the base sequence represented by SEQ ID NO: 4
    (J) DNA encoding a protein comprising a base sequence having 85% or more identity with the base sequence of DNA (i) and having β-ketoacyl-ACP synthase activity
    (K) DNA comprising the base sequence represented by SEQ ID NO: 6
    (L) a DNA encoding a protein comprising a base sequence having 85% or more identity with the base sequence of DNA (k) and having β-ketoacyl-ACP synthase activity