JP2018099107A - Lipid production methods - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a lipid production method for enhancing the productivity of medium chain fatty acid or lipid having the fatty acid as a constituent thereof, as well as a transformant having enhanced productivity of medium chain fatty acid or lipid having the fatty acid as a constituent thereof.SOLUTION: The invention provides a lipid production method for producing a medium chain fatty acid or a lipid having the fatty acid as a constituent thereof by culturing a transformant transduced with a gene encoding a β-ketoacyl-ACP synthase derived from a plant that does not contain medium chain fatty acids as a main depot lipid, and a gene encoding an acyl-ACP thioesterase having a substrate specificity to a medium chain acyl ACP. The invention also provides a transformant comprising a gene encoding a β-ketoacyl-ACP synthase derived from a plant that does not contain medium chain fatty acids as a main depot lipid, and a gene encoding an acyl-ACP thioesterase having a substrate specificity to a medium chain acyl ACP.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing a fatty acid or a lipid comprising the same, and a transformant 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 stored in animals and plants are widely used for food or industry.
For example, higher alcohol derivatives 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, plant-derived fats and oils are used as raw materials for biodiesel fuel.
Thus, the use of fatty acids and lipids is diverse. For this reason, 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.

In general, plant fatty acid synthesis pathways are localized in the chloroplast. In chloroplasts, an acetyl-acyl carrier protein (hereinafter also referred to as “ACP”) is used as a starting material, and the carbon chain elongation reaction is repeated. Finally, acyl-ACP (fatty acid) having 16 or 18 carbon atoms is obtained. A complex composed of an acyl group as a residue and ACP, where the number of carbon atoms indicates the number of carbon atoms in the acyl group, and may be expressed in the same manner below). Subsequently, the acyl-ACP thioester bond is hydrolyzed by the action of acyl-ACP thioesterase (hereinafter also simply referred to as “TE”) to produce free fatty acids.
Among enzymes involved in this fatty acid synthesis pathway, β-ketoacyl-ACP synthase (hereinafter also referred to as “KAS”) is an enzyme involved in the control of acyl 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. Of these, KAS III works at the beginning 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 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.

Regarding the KAS IV involved in the elongation reaction up to medium-chain acyl-ACP, little knowledge has been obtained even in plants, and it is a KAS unique to plants that accumulate medium-chain fatty acids such as cuphea ( Cuphea ) ( (See Patent Document 1 and Non-Patent Documents 1 and 2).
In contrast, in plants that accumulate long-chain fatty acids and do not contain medium-chain fatty acids as the main accumulated lipid, a method for improving the productivity of medium-chain fatty acids, and the synthesis of medium-chain fatty acids derived from such plants No knowledge has been obtained about KAS involved in.

International Publication No. 98/46776

The Plant Journal, 1998, vol. 15 (3), p. 383-390 The Plant Journal, 1998, vol. 13 (5), p. 621-628

This invention makes it a subject to provide the manufacturing method of lipid which improves productivity of a medium chain fatty acid or lipid which uses this as a structural component.
Another object of the present invention is to provide a transformant with improved productivity of medium-chain fatty acids or lipids comprising the same.

The present inventor has intensively studied in view of the above problems. As a result, information on a protein annotated as KAS II involved in the elongation reaction up to stearoyl-ACP having 18 carbon atoms was obtained from plants not containing medium-chain fatty acids as the main accumulated lipid. It was also found that when the expression of a gene encoding this and a gene encoding TE having substrate specificity for medium chain acyl-ACP is promoted, productivity of medium chain fatty acids is improved in the transformant.
The present invention has been completed based on these findings.

The present invention provides a transformant that promotes the expression of a plant-derived KAS gene that does not contain medium-chain fatty acids as the main accumulated lipid, and a gene that encodes TE having substrate specificity for medium-chain acyl-ACP. The present invention relates to a method for producing a lipid, which is cultured to produce a fatty acid or a lipid containing the fatty acid or a constituent thereof.
The present invention also relates to a transformant comprising a gene encoding a plant-derived KAS that does not contain medium-chain fatty acids as the main accumulated lipid, and a gene encoding TE having substrate specificity for medium-chain acyl-ACP. .

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.

As used herein, “lipid” refers to neutral lipids (such as triacylglycerol), simple lipids such as wax and ceramide; complex lipids such as phospholipids, glycolipids, and sulfolipids; and fatty acids derived from these lipids. (Free fatty acids), derived lipids such as alcohols and hydrocarbons are included.
Fatty acids generally classified as derived lipids refer to the fatty acids themselves, meaning “free fatty acids”. In the present invention, a fatty acid moiety or an acyl group moiety in simple lipid and complex lipid molecules is referred to as “fatty acid residue”. Unless otherwise specified, “fatty acid” is used as a general term for “free fatty acid” and “fatty acid residue”.
In the present specification, “fatty acid or lipid containing this as a constituent” is used generically as “free fatty acid” and “lipid having the fatty acid residue”. Further, in the present specification, the “fatty acid composition” means the weight ratio of each fatty acid to the total fatty acid (total fatty acid) weighted by regarding the free fatty acid and the fatty acid residue as a fatty acid. The weight (production amount) and fatty acid composition of the fatty acid can be measured by the methods used in the examples.

In the present specification, “Cx: y” in the notation of fatty acids and acyl groups constituting fatty acid residues 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, examples of “stringent conditions” include the method described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. 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 A condition for hybridization with a probe at 65 ° C. for 8 to 16 hours is mentioned.
Furthermore, in the present specification, “upstream” of a gene refers to a region continuing on the 5 ′ side of a gene or region regarded as a target, not a position from the translation start point. 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 lipid production method of the present invention promotes the expression of a plant-derived KAS gene that does not contain medium-chain fatty acids as the main accumulated lipid and a gene that encodes TE having substrate specificity for medium-chain acyl-ACP. Transformants are used.
As described above, KAS involved in the elongation reaction up to medium chain acyl-ACP was considered to be a KAS specific to plants that accumulate medium chain fatty acids (Patent Document 1, and Non-Patent Documents 1 and 2). reference). A transformant prepared by introducing a gene encoding KAS specific to plants that accumulate medium chain fatty acids and a gene encoding TE having substrate specificity for medium chain acyl-ACP is It is also known that productivity is improved (see Patent Document 1 and Non-Patent Document 2).
In contrast, the present inventors have found that KAS found from plants that do not contain medium-chain fatty acids as the main accumulating lipid is used in combination with TE having substrate specificity for medium-chain acyl-ACP. It has been found that the productivity of chain fatty acids is improved.

In the present specification, the “medium chain” means 6 to 14 carbon atoms, preferably 8 to 14 carbon atoms, more preferably 10 to 14 carbon atoms, and still more preferably carbon atoms. Refers to a 10, 12, or 14 chain hydrocarbon group. Here, in the case of a fatty acid or a fatty acid residue, the number of carbon atoms of the chain hydrocarbon group includes carbonyl carbon. The “long chain” refers to a chain hydrocarbon group having 15 or more carbon atoms, preferably 16 or more, more preferably 16 or more and 24 or less. Here, also in the case of a fatty acid or a fatty acid residue, the number of carbon atoms of the chain hydrocarbon group includes carbonyl carbon.
By the way, as described above, in the present specification, fatty acid is a general term for “free fatty acid” and “fatty acid residue”. In addition, the “medium chain” is defined in advance, but in the present specification, “a plant that does not contain a medium chain fatty acid as a main accumulated lipid” means a free fatty acid having 8 to 14 carbon atoms and a fatty acid residue. 10% by mass of the total amount of fatty acids contained in the seed or pulp lipids, that is, the total amount of fatty acids of all free fatty acids and fatty acids regarded from all fatty acid residues. Hereinafter, the plant is preferably 5% by mass or less, more preferably 1% by mass or less. Examples of the main compound of medium chain fatty acid include lauric acid (C12: 0) and myristic acid (C14: 0).
Furthermore, “a plant that does not contain medium-chain fatty acids as the main accumulated lipid” in the present specification may be defined by the ratio of the amount of long-chain fatty acids. That is, a plant that accumulates a certain amount of long-chain fatty acids in the lipids of seeds or pulp, specifically, fatty acids regarded as free fatty acids having 16 to 22 carbon atoms and fatty acid residues among the lipids in seeds or pulps The plant of which the sum total of the ratio of the amount of fatty acids is 60 mass% or more with respect to the total amount of all the fatty acids, Preferably it is 70 mass% or more, More preferably, it is 80 mass% or more. Examples of plants that accumulate a certain amount of long-chain fatty acids in seed or pulp lipids include general oil plants (for example, “http://pci.kaneda.co.jp/contents/introduce/ see composition / list01.html).
The “plant that does not contain medium-chain fatty acids as the main accumulated lipid” according to the present invention can be explained by either the above-mentioned definition of the amount of medium-chain fatty acids or the definition of the amount of long-chain fatty acids.

The “plant that does not contain medium-chain fatty acids as the main accumulated lipid” as defined in the present specification can be appropriately selected from ordinary oil plants that do not contain medium-chain fatty acids as the main accumulated lipid. For example, Euphorbiaceae plant, Brassicaceae plant, Fabaceae plant, Poaceae plant, Vitaceae plant, Asteraceae plant, Examples include at least one plant selected from the group consisting of plants of the family Linaceae, plants of the family Malvaceae, plants of the Pedaliaceae family, and plants of the family Oleaceae. Preferable examples include Jatropha curcas , Ricinus communis , Arabidopsis thaliana , Brassica rapa , Soybean ( Glycine max ), Corn ( Zea mays ), European grape ( Vitis vinifera ), Camerina ( Camelina sativa ), sunflower ( Helianthus annuus ), flax ( Linum usitatissimum ), safflower ( Carthamus tinctorius ), groundnut ( Arachis hypogaea ), cotton genus ( Gossypium spp.), Sesame ( Sesamum indicum ), rice ( Ory ), sativa ( Olea europaea ).
Table 1 below shows the fatty acid composition in the fats and oils contained in the seeds or pulp of the aforementioned oil plants. For reference, the fatty acid composition in the coconut oil is also shown in Table 1. As shown in Table 1, long-chain fatty acids having 16 to 18 carbon atoms are mainly accumulated in oil plants other than coconut palm. However, accumulation of medium chain fatty acids having 10 to 14 carbon atoms has not been observed in oil plants other than coconut palm.

The KAS used in the present invention is preferably a plant-derived KAS II that does not contain medium-chain fatty acids as the main accumulated lipid.
As described above, KAS II is mainly involved in the elongation reaction up to stearoyl-ACP having 18 carbon atoms. Therefore, normally, KAS II is not used for productivity of medium chain fatty acids. As shown in the examples described later, even when the expression of only the gene encoding KAS II is promoted, the productivity of medium chain fatty acids is not improved. However, when KAS II is used in combination with TE described later, the productivity of medium chain fatty acids in the transformant is improved.

The KAS used in the present invention can be appropriately selected from plant-derived KAS that does not contain the aforementioned medium-chain fatty acids as the main accumulated lipid. Specific examples include the following proteins (A) to (R). The following proteins (A) to (R) are annotated as KAS II from the homology search by the BLAST program and the registered name in the database.

(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 60.
(B) A protein comprising 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 (hereinafter also referred to as “KAS activity”).
(C) A protein consisting of the amino acid sequence represented by SEQ ID NO: 61.
(D) A protein comprising an amino acid sequence having an identity of 85% or more with the amino acid sequence of the protein (C) and having KAS activity.
(E) A protein consisting of the amino acid sequence represented by SEQ ID NO: 62.
(F) A protein comprising an amino acid sequence having an identity of 85% or more with the amino acid sequence of the protein (E) and having KAS activity.
(G) A protein consisting of the amino acid sequence represented by SEQ ID NO: 63.
(H) A protein comprising an amino acid sequence having an identity of 85% or more with the amino acid sequence of the protein (G) and having KAS activity.
(I) A protein comprising the amino acid sequence represented by SEQ ID NO: 64.
(J) A protein comprising an amino acid sequence having an identity of 85% or more with the amino acid sequence of the protein (I) and having KAS activity.
(K) A protein consisting of the amino acid sequence represented by SEQ ID NO: 65.
(L) A protein comprising an amino acid sequence having an identity of 85% or more with the amino acid sequence of the protein (K) and having KAS activity.
(M) A protein consisting of the amino acid sequence represented by SEQ ID NO: 66.
(N) A protein consisting of an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (M) and having KAS activity.
(O) A protein consisting of the amino acid sequence represented by SEQ ID NO: 67.
(P) A protein comprising an amino acid sequence having an identity of 85% or more with the amino acid sequence of the protein (O) and having KAS activity.
(Q) A protein consisting of the amino acid sequence represented by SEQ ID NO: 68.
(R) A protein consisting of an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (Q) and having KAS activity.

The protein (A) having the amino acid sequence represented by SEQ ID NO: 60 is KAS II derived from Jatropha (see Jatropha Genome Database (http://www.kazusa.or.jp/jatropha/), Jcr4S04655.30). . Jatropha is a deciduous shrub belonging to the genus Jatropha , and is used as a raw material for biodiesel. In the present specification, the proteins (A) and (B) are collectively referred to as “JcKASII”.
The protein (C) consisting of the amino acid sequence represented by SEQ ID NO: 61 is castor-derived KAS II (see Genbank, XP_002516228). The castor is a perennial plant belonging to the genus Ricinus , and its seed is used as a raw material for vegetable oil. In the present specification, the proteins (C) and (D) are collectively referred to as “RiKASII”.
The protein (E) consisting of the amino acid sequence represented by SEQ ID NO: 62 is KAS II derived from Arabidopsis thaliana (see TAIR (www.arabidopsis.org), AT1G74960). Arabidopsis is an annual plant of the genus Arabidopsis and is widely used as a model organism. In the present specification, the proteins (E) and (F) are collectively referred to as “AtKASII”.
The protein (G) consisting of the amino acid sequence represented by SEQ ID NO: 63 is rape-derived KAS II (see Genbank, XP — 009104733). Brassica is a biennial plant belonging to the genus Brassica , and is used as a raw material for vegetable oil. In the present specification, the proteins (G) and (H) are collectively referred to as “BrKASII”.
Protein (I) consisting of the amino acid sequence represented by SEQ ID NO: 64 is KAS II derived from soybean (see Genbank, AAW88763). Soybean is an annual plant of the genus Soybean ( Glycine ), and its seeds are edible. In the present specification, the proteins (I) and (J) are collectively referred to as “GmKASII”.
The protein (K) consisting of the amino acid sequence represented by SEQ ID NO: 65 is KAS II derived from corn (see Genbank, NM_001148515). Corn is an annual plant of the genus Zea , and is used as a food for human food and livestock as a cereal. In the present specification, the proteins (K) and (L) are collectively referred to as “ZmKASII”.
The protein (M) consisting of the amino acid sequence represented by SEQ ID NO: 66 is KAS II derived from European grapes (see Genbank, XP_002276214). European grapes are plants of the genus Vitis that are used as raw materials for processed foods such as wine and raisins in addition to raw food. In the present specification, the proteins (M) and (N) are collectively referred to as “VvKASII”.
The protein (O) consisting of the amino acid sequence represented by SEQ ID NO: 67 is Camerina-derived KAS II (see Genbank, XP_010498338). Camerina is an annual plant of the genus Camelina and is widely cultivated as a crop rotation of wheat. In the present specification, the proteins (O) and (P) are collectively referred to as “CsKASII”.
The protein (Q) consisting of the amino acid sequence represented by SEQ ID NO: 68 is KAS II derived from sunflower (see Genbank, DQ835562). Sunflower is an annual plant belonging to the genus Helianthus and is cultivated for food, vegetable oil, and ornamental use. In the present specification, the proteins (Q) and (R) are collectively referred to as “HaKASII”.

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. Subsequently, the acyl-ACP thioester bond is hydrolyzed by the action of acyl-ACP thioesterase (hereinafter also simply referred to as “TE”) to produce free fatty acids.
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.

All of the proteins (A) to (R) have KAS activity. As used herein, “KAS activity” means an activity of catalyzing 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 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 elongation reactions with 4 to 16 carbon atoms to synthesize palmitoyl-ACP with 16 carbon atoms. KAS II mainly synthesizes a long-chain acyl-ACP by catalyzing an extension reaction to a long-chain acyl group 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.
From the results of Blast of the amino acid sequence and the base sequence, the proteins (A) to (R) are considered to be KAS II. However, as shown in the Examples described later, the gene encoding any one of the proteins (A) to (R), although KAS II has substrate specificity for long-chain acyl-ACP, In the transformant in which expression of a gene encoding TE described later is promoted, productivity of medium chain fatty acids having 10 to 14 carbon atoms is improved.

  Regarding the substrate specificity of KAS, for example, DNA 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 deficient in the fatty acid degradation system, and the introduced gene is expressed. The cells can be cultured under the condition, and the change in the fatty acid composition in the host cell or culture medium can be confirmed 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, 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 90% or more, more preferably 93% or more, more preferably 95% or more, and more than 97%. 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 60, preferably 1 to 40, more preferably 1 to 28) amino acid sequences of the protein (A). More preferably 1 to 20 amino acids, more preferably 1 to 12 amino acids, more preferably 1 to 8 amino acids, more preferably 1 to 4 amino acids). Or the added protein is mentioned.
The protein (B) may be a protein comprising an amino acid sequence in which a signal peptide involved in protein transport or secretion is added to the amino acid sequence of the protein (A) or (B).

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 93% or more, more preferably 95% or more, and more than 97%. More preferably, 98% or more is more preferable, and 99% or more is more preferable. Further, as the protein (D), one or more (for example, 1 to 84, preferably 1 to 56, more preferably 1 to 39) amino acid sequences of the protein (C) More preferably 1 or more and 28 or less, more preferably 1 or more and 17 or less, more preferably 1 or more and 12 or less, more preferably 1 or more and 6 or less) amino acids are deleted, substituted or inserted Or the added protein is mentioned.
The protein (D) may be a protein comprising an amino acid sequence in which a signal peptide involved in protein transport or secretion is added to the amino acid sequence of the protein (C) or (D).

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 93% or more, more preferably 95% or more, and more than 97%. More preferably, 98% or more is more preferable, and 99% or more is more preferable. In addition, as the protein (F), one or more (for example, 1 or more and 82 or less, preferably 1 or more and 55 or less, more preferably 1 or more and 38 or less) in the amino acid sequence of the protein (E) More preferably 1 to 28, more preferably 1 to 17, more preferably 1 to 11, and more preferably 1 to 6 amino acids). Or the added protein is mentioned.
The protein (F) may also be a protein comprising an amino acid sequence in which a signal peptide involved in protein transport or secretion is added to the amino acid sequence of the protein (E) or (F).

In the protein (H), from the viewpoint of KAS activity, the identity with the amino acid sequence of the protein (G) is preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more. More preferably, 98% or more is more preferable, and 99% or more is more preferable. Further, as the protein (H), one or more (for example, 1 to 82, preferably 1 to 55, more preferably 1 to 39) amino acid sequences of the protein (G) More preferably 1 to 28, more preferably 1 to 17, more preferably 1 to 11, and more preferably 1 to 6 amino acids). Or the added protein is mentioned.
The protein (H) may be a protein comprising an amino acid sequence in which a signal peptide involved in protein transport or secretion is added to the amino acid sequence of the protein (G) or (H).

In the protein (J), from the viewpoint of KAS activity, the identity with the amino acid sequence of the protein (I) is preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more. More preferably, 98% or more is more preferable, and 99% or more is more preferable. In addition, as the protein (J), one or more (for example, 1 to 74, preferably 1 to 49, more preferably 1 to 35) amino acid sequences of the protein (I) More preferably 1 to 25, more preferably 1 to 15 and more preferably 1 to 10 and more preferably 1 to 5 amino acids). Or the added protein is mentioned.
The protein (J) may be a protein comprising an amino acid sequence in which a signal peptide involved in protein transport or secretion is added to the amino acid sequence of the protein (I) or (J).

In the protein (L), from the viewpoint of KAS activity, the identity with the amino acid sequence of the protein (K) is preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more. More preferably, 98% or more is more preferable, and 99% or more is more preferable. Further, as the protein (L), one or more (for example, 1 to 70, preferably 1 to 47, more preferably 1 to 33) amino acid sequences of the protein (K). More preferably 1 to 24, more preferably 1 to 14 and more preferably 1 to 10 and more preferably 1 to 5 amino acids). Or the added protein is mentioned.
The protein (L) may be a protein comprising an amino acid sequence in which a signal peptide involved in protein transport or secretion is added to the amino acid sequence of the protein (K) or (L).

In the protein (N), from the viewpoint of KAS activity, the identity with the amino acid sequence of the protein (M) is preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more. More preferably, 98% or more is more preferable, and 99% or more is more preferable. In addition, as the protein (N), one or more (for example, 1 to 84, preferably 1 to 56, more preferably 1 to 39) amino acid sequences of the protein (M) More preferably 1 or more and 28 or less, more preferably 1 or more and 17 or less, more preferably 1 or more and 12 or less, more preferably 1 or more and 6 or less) amino acids are deleted, substituted or inserted Or the added protein is mentioned.
The protein (N) may be a protein comprising an amino acid sequence in which a signal peptide involved in protein transport or secretion is added to the amino acid sequence of the protein (M) or (N).

In the protein (P), from the viewpoint of KAS activity, the identity with the amino acid sequence of the protein (O) is preferably 90% or more, more preferably 93% or more, more preferably 95% or more, and more than 97%. More preferably, 98% or more is more preferable, and 99% or more is more preferable. In addition, as the protein (P), one or more (for example, 1 to 80, preferably 1 to 54, more preferably 1 to 38) amino acid sequences of the protein (O). More preferably 1 to 27, more preferably 1 to 16, more preferably 1 to 11, and more preferably 1 to 6 amino acids). Or the added protein is mentioned.
The protein (P) may also be a protein comprising an amino acid sequence in which a signal peptide involved in protein transport or secretion is added to the amino acid sequence of the protein (O) or (P).

In the protein (R), from the viewpoint of KAS activity, the identity with the amino acid sequence of the protein (Q) is preferably 90% or more, more preferably 93% or more, more preferably 95% or more, and more than 97%. More preferably, 98% or more is more preferable, and 99% or more is more preferable. Further, as the protein (R), one or more (for example, 1 to 74, preferably 1 to 50, more preferably 1 to 35) amino acid sequences of the protein (Q). More preferably 1 to 25, more preferably 1 to 15 and more preferably 1 to 10 and more preferably 1 to 5 amino acids). Or the added protein is mentioned.
The protein (R) may also be a protein comprising an amino acid sequence in which a signal peptide involved in protein transport or secretion is added to the amino acid sequence of the protein (Q) or (R).

  In general, an amino acid sequence encoding an enzyme protein does not necessarily indicate enzyme activity unless the sequence of the entire region is conserved, and there are regions that do not affect enzyme activity even if the amino acid sequence changes. It is known to exist. In such a region that is not essential for enzyme activity, the original activity of the enzyme can be maintained even if a mutation such as amino acid deletion, substitution, insertion or addition is introduced. Also in the present invention, a protein that retains KAS activity and is partially mutated in amino acid sequence can be used.

  Table 2 summarizes the amino acid sequence identities between the aforementioned KAS II derived from various plants listed as KAS that can be used in the present invention.

  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) to (R) can be obtained by ordinary chemical methods, genetic engineering methods, and the like. For example, a protein derived from a natural product can be obtained by isolation, purification, or the like from a plant that does not contain medium-chain fatty acids as the main accumulated lipid. Moreover, the said protein (A)-(R) can be obtained by artificially chemically synthesizing based on the amino acid sequence information represented by any one of sequence number 60-68. Alternatively, the proteins (A) to (R) may be produced as recombinant proteins by gene recombination techniques. When producing a recombinant protein, the KAS gene described below can be used.
One kind of KAS used in the present invention may be used, or two or more kinds of KAS may be used in combination.

As an example of a gene (hereinafter also referred to as “KAS gene” or “KASII gene”) that encodes a plant-derived KAS that does not contain medium-chain fatty acids as the main accumulated lipid, preferably the proteins (A) to (R), A gene comprising any one of the following DNAs (a) to (r) can be mentioned.

(A) DNA consisting of the base sequence represented by SEQ ID NO: 51.
(B) A DNA encoding a protein having a KAS activity and having a base sequence having an identity of 80% or more with the base sequence of the DNA (a).
(C) DNA consisting of the base sequence represented by SEQ ID NO: 52.
(D) DNA encoding a protein comprising a base sequence having 80% or more identity with the base sequence of DNA (c) and having KAS activity.
(E) DNA consisting of the base sequence represented by SEQ ID NO: 53.
(F) DNA encoding a protein comprising a base sequence having an identity of 80% or more with the base sequence of DNA (e) and having KAS activity.
(G) DNA consisting of the base sequence represented by SEQ ID NO: 54.
(H) A DNA encoding a protein comprising a base sequence having an identity of 80% or more with the base sequence of the DNA (g) and having KAS activity.
(I) DNA consisting of the base sequence represented by SEQ ID NO: 55.
(J) DNA encoding a protein comprising a base sequence having an identity of 80% or more with the base sequence of DNA (i) and having KAS activity.
(K) DNA consisting of the base sequence represented by SEQ ID NO: 56.
(L) A DNA encoding a protein comprising a base sequence having an identity of 80% or more with the base sequence of DNA (k) and having KAS activity.
(M) DNA consisting of the base sequence represented by SEQ ID NO: 57.
(N) A DNA encoding a protein comprising a base sequence having an identity of 80% or more with the base sequence of the DNA (m) and having KAS activity.
(O) DNA consisting of the base sequence represented by SEQ ID NO: 58.
(P) A DNA encoding a protein comprising a base sequence having 80% or more identity with the base sequence of the DNA (o) and having KAS activity.
(Q) DNA consisting of the base sequence represented by SEQ ID NO: 59.
(R) DNA encoding a protein comprising a base sequence having an identity of 80% or more with the base sequence of DNA (q) and having KAS activity.

The base sequence of SEQ ID NO: 51 in the DNA (a) and (b) is the base sequence of the gene encoding the protein (A) consisting of the amino acid sequence represented by SEQ ID NO: 60. In the present specification, the gene consisting of the DNA (a) or (b) is also referred to as “JcKASII gene”.
The base sequence of SEQ ID NO: 52 in the DNAs (c) and (d) is the base sequence of the gene (GenBank accession number: XP002516228) encoding the protein (C) consisting of the amino acid sequence represented by SEQ ID NO: 61. . In the present specification, the gene consisting of the DNA (c) or (d) is also referred to as “RiKASII gene”.
The base sequence of SEQ ID NO: 53 in the DNAs (e) and (f) is the base sequence of the gene encoding the protein (E) consisting of the amino acid sequence represented by SEQ ID NO: 62. In the present specification, the gene consisting of the DNA (e) or (f) is also referred to as “AtKASII gene”.
The base sequence of SEQ ID NO: 54 in the DNAs (g) and (h) is the base sequence of the gene encoding the protein (G) consisting of the amino acid sequence represented by SEQ ID NO: 63. In the present specification, the gene consisting of the DNA (g) or (h) is also referred to as “BrKASII gene”.
The base sequence of SEQ ID NO: 55 in the DNA (i) and (j) is the base sequence of the gene encoding the protein (I) consisting of the amino acid sequence represented by SEQ ID NO: 64. In the present specification, the gene consisting of DNA (i) or (j) is also referred to as “GmKASII gene”.
The base sequence of SEQ ID NO: 56 in the DNAs (k) and (l) is the base sequence of the gene encoding the protein (K) consisting of the amino acid sequence represented by SEQ ID NO: 65. The DNA consisting of the base sequence of SEQ ID NO: 56 is optimized for a part of the DNA sequence of ZmKASII (NM_001148515) using the GeneArt artificial gene synthesis service of Thermo Fisher Scientific according to the host used in the examples described later. It has become. In the present specification, the gene consisting of the DNA (k) or (l) is also referred to as “ZmKASII gene”.
The base sequence of SEQ ID NO: 57 in the DNA (m) and (n) is the base sequence (GenBank accession number: XP002276214) of the gene encoding the protein (M) consisting of the amino acid sequence represented by SEQ ID NO: 66 . In the present specification, the gene consisting of the DNA (m) or (n) is also referred to as “VvKASII gene”.
The base sequence of SEQ ID NO: 58 in the DNAs (o) and (p) is the base sequence (GenBank accession number: XP010498338) encoding the protein (O) consisting of the amino acid sequence represented by SEQ ID NO: 67. . In the present specification, the gene consisting of the DNAs (o) and (p) is also referred to as “CsKASII gene”.
The base sequence of SEQ ID NO: 59 in the DNA (q) and (r) is the base sequence (GenBank accession number: DQ835562) of the gene encoding the protein (Q) consisting of the amino acid sequence represented by SEQ ID NO: 68 . In the present specification, the gene consisting of the DNAs (q) and (r) is also referred to as “HaKASII gene”.

In the DNA (b), from the viewpoint of KAS activity, the identity with the base sequence of the DNA (a) is preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more. More preferably, 97% or more is more preferable, 98% or more is more preferable, and 99% or more is more preferable. In addition, as the DNA (b), one or several (for example, 1 or more and 250 or less, preferably 1 or more and 181 or less, more preferably 1 or more and 121 or less) in the base sequence represented by SEQ ID NO: 51, Preferably 1 or more and 85 or less, more preferably 1 or more and 61 or less, more preferably 1 or more and 37 or less, more preferably 1 or more and 25 or less, more preferably 1 or more and 13 or less. A DNA encoding the protein (A) or (B) having a base deleted, substituted, inserted, or added and having KAS activity is also preferred. Furthermore, the DNA (b) encodes the protein (A) or (B) which hybridizes with a DNA having a base sequence complementary to the DNA (a) under stringent conditions and has KAS activity. DNA is also preferred.
The DNA (b) may be a DNA comprising a base sequence in which a base sequence encoding a signal peptide involved in protein transport or secretion is added to the base sequence of the DNA (a) or (b). Good.

In the DNA (d), from the viewpoint of KAS activity, the identity with the base sequence of the DNA (c) is preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more. More preferably, 97% or more is more preferable, 98% or more is more preferable, and 99% or more is more preferable. Further, as the DNA (d), one or several (for example, 1 or more and 333 or less, preferably 1 or more and 250 or less, more preferably 1 or more and 167 or less) in the base sequence represented by SEQ ID NO: 52, Preferably 1 or more and 117 or less, more preferably 1 or more and 84 or less, more preferably 1 or more and 50 or less, more preferably 1 or more and 34 or less, more preferably 1 or more and 17 or less. A DNA encoding the protein (C) or (D) having a base deleted, substituted, inserted or added and having KAS activity is also preferred. Furthermore, the DNA (d) encodes the protein (C) or (D) that hybridizes under stringent conditions with DNA comprising a base sequence complementary to the DNA (c) and has KAS activity. DNA is also preferred.
The DNA (d) may be a DNA comprising a base sequence in which a base sequence encoding a signal peptide involved in protein transport or secretion is added to the base sequence of the DNA (c) or (d). Good.

In the DNA (f), from the viewpoint of KAS activity, the identity with the base sequence of the DNA (e) is preferably 85% or more, more preferably 90% or more, more preferably 93% or more, and more than 95%. More preferably, 97% or more is more preferable, 98% or more is more preferable, and 99% or more is more preferable. Further, as the DNA (f), one or several (for example, 1 or more and 326 or less, preferably 1 or more and 244 or less, more preferably 1 or more and 163 or less) in the base sequence represented by SEQ ID NO: 53, Preferably 1 or more and 114 or less, more preferably 1 or more and 82 or less, more preferably 1 or more and 49 or less, more preferably 1 or more and 33 or less, and more preferably 1 or more and 17 or less A DNA encoding the protein (E) or (F) having a base deleted, substituted, inserted, or added and having KAS activity is also preferred. Furthermore, the DNA (f) encodes the protein (E) or (F) that hybridizes with a DNA having a base sequence complementary to the DNA (e) under stringent conditions and has KAS activity. DNA is also preferred.
Further, the DNA (f) may be a DNA comprising a base sequence in which a base sequence encoding a signal peptide involved in protein transport or secretion is added to the base sequence of the DNA (e) or (f). Good.

In the DNA (h), from the viewpoint of KAS activity, the identity with the base sequence of the DNA (g) is preferably 85% or more, more preferably 90% or more, more preferably 93% or more, and more than 95%. More preferably, 97% or more is more preferable, 98% or more is more preferable, and 99% or more is more preferable. Further, as the DNA (h), one or several (for example, 1 or more and 329 or less, preferably 1 or more and 247 or less, more preferably 1 or more and 165 or less) in the base sequence represented by SEQ ID NO: 54, Preferably 1 or more and 115 or less, more preferably 1 or more and 83 or less, more preferably 1 or more and 50 or less, more preferably 1 or more and 33 or less, more preferably 1 or more and 17 or less) A DNA encoding the protein (G) or (H) having a base deleted, substituted, inserted, or added and having KAS activity is also preferred. Furthermore, as the DNA (h), it encodes the protein (G) or (H) 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.
The DNA (h) may be a DNA comprising a base sequence in which a base sequence encoding a signal peptide involved in protein transport or secretion is added to the base sequence of the DNA (g) or (h). Good.

In the DNA (j), from the viewpoint of KAS activity, the identity with the base sequence of the DNA (i) is preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more. More preferably, 97% or more is more preferable, 98% or more is more preferable, and 99% or more is more preferable. Further, as the DNA (j), one or several (for example, 1 or more and 294 or less, preferably 1 or more and 221 or less, more preferably 1 or more and 147 or less) in the base sequence represented by SEQ ID NO: 55, Preferably 1 or more and 103 or less, more preferably 1 or more and 74 or less, more preferably 1 or more and 45 or less, more preferably 1 or more and 30 or less, more preferably 1 or more and 15 or less. A DNA encoding the protein (I) or (J) having a base deleted, substituted, inserted, or added and having KAS activity is also preferred. Further, the DNA (j) encodes the protein (I) or (J) that hybridizes with a DNA having a base sequence complementary to the DNA (i) under stringent conditions and has KAS activity. DNA is also preferred.
The DNA (j) may be a DNA comprising a base sequence in which a base sequence encoding a signal peptide involved in protein transport or secretion is added to the base sequence of the DNA (i) or (j). Good.

In the DNA (l), from the viewpoint of KAS activity, the identity with the base sequence of the DNA (k) is preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more. More preferably, 97% or more is more preferable, 98% or more is more preferable, and 99% or more is more preferable. Further, as the DNA (l), one or several (for example, 1 or more and 278 or less, preferably 1 or more and 208 or less, more preferably 1 or more and 139 or less) in the base sequence represented by SEQ ID NO: 56, Preferably 1 or more and 98 or less, more preferably 1 or more and 70 or less, more preferably 1 or more and 42 or less, more preferably 1 or more and 28 or less, more preferably 1 or more and 14 or less) A DNA encoding the protein (K) or (L) having a base deleted, substituted, inserted, or added and having KAS activity is also preferred. Furthermore, as the DNA (l), the protein (K) or (L) that hybridizes with a DNA comprising a base sequence complementary to the DNA (k) under stringent conditions and has KAS activity is encoded. DNA is also preferred.
Further, the DNA (l) may be a DNA having a base sequence in which a base sequence encoding a signal peptide involved in protein transport or secretion is added to the base sequence of the DNA (k) or (l). Good.

In the DNA (n), from the viewpoint of KAS activity, the identity with the base sequence of the DNA (m) is preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more. More preferably, 97% or more is more preferable, 98% or more is more preferable, and 99% or more is more preferable. Further, as the DNA (n), one or several (for example, 1 or more and 334 or less, preferably 1 or more and 251 or less, more preferably 1 or more and 167 or less) in the base sequence represented by SEQ ID NO: 57, Preferably 1 or more and 117 or less, more preferably 1 or more and 84 or less, more preferably 1 or more and 51 or less, more preferably 1 or more and 34 or less, more preferably 1 or more and 17 or less) A DNA encoding the protein (M) or (N) having a base deleted, substituted, inserted, or added and having KAS activity is also preferred. Furthermore, as the DNA (n), it encodes the protein (M) or (N) which hybridizes with a DNA having a base sequence complementary to the DNA (m) under stringent conditions and has KAS activity. DNA is also preferred.
The DNA (n) may be a DNA comprising a base sequence in which a base sequence encoding a signal peptide involved in protein transport or secretion is added to the base sequence of the DNA (m) or (n). Good.

In the DNA (p), from the viewpoint of KAS activity, the identity with the base sequence of the DNA (o) is preferably 85% or more, more preferably 90% or more, more preferably 93% or more, and more than 95%. More preferably, 97% or more is more preferable, 98% or more is more preferable, and 99% or more is more preferable. In addition, as the DNA (p), one or several (for example, 1 or more and 320 or less, preferably 1 or more and 240 or less, more preferably 1 or more and 160 or less) in the base sequence represented by SEQ ID NO: 58, Preferably 1 or more and 112 or less, more preferably 1 or more and 80 or less, more preferably 1 or more and 48 or less, more preferably 1 or more and 32 or less, more preferably 1 or more and 16 or less A DNA encoding the protein (O) or (P) having a base deleted, substituted, inserted, or added and having KAS activity is also preferred. Further, the DNA (p) encodes the protein (O) or (P) which hybridizes with a DNA having a base sequence complementary to the DNA (o) under stringent conditions and has KAS activity. DNA is also preferred.
In addition, the DNA (p) may be a DNA having a base sequence in which a base sequence encoding a signal peptide involved in protein transport or secretion is added to the base sequence of the DNA (o) or (p). Good.

In the DNA (r), from the viewpoint of KAS activity, the identity with the base sequence of the DNA (q) is preferably 85% or more, more preferably 90% or more, more preferably 93% or more, and more than 95%. More preferably, 97% or more is more preferable, 98% or more is more preferable, and 99% or more is more preferable. In addition, as the DNA (r), one or several (for example, 1 or more and 296 or less, preferably 1 or more and 222 or less, more preferably 1 or more and 148 or less) in the base sequence represented by SEQ ID NO: 59, Preferably 1 or more and 104 or less, more preferably 1 or more and 74 or less, more preferably 1 or more and 45 or less, more preferably 1 or more and 30 or less, more preferably 1 or more and 15 or less) A DNA encoding the protein (Q) or (R) having a base deleted, substituted, inserted or added and having KAS activity is also preferred. Furthermore, as the DNA (r), it encodes the protein (Q) or (R) which hybridizes with a DNA having a base sequence complementary to the DNA (q) under stringent conditions and has KAS activity. DNA is also preferred.
The DNA (r) may be a DNA comprising a base sequence in which a base sequence encoding a signal peptide involved in protein transport or secretion is added to the base sequence of the DNA (q) or (r). Good.

  Table 3 summarizes the identity of the base sequences between the KAS II genes derived from the various plants described above as KAS genes that can be used in the present invention.

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 represented by any one of SEQ ID NOs: 60 to 68 or the base sequence represented by any one of SEQ ID NOs: 51 to 59. For the synthesis of the KAS gene, for example, services such as Invitrogen can be used. It can also be obtained by cloning from the genome of a plant that does not contain medium-chain fatty acids as the main accumulated lipid. For example, it can be performed by the method described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press (2001)]. Moreover, you may optimize a part of base sequence represented by any one of sequence number 51-59 according to the kind of host to be used. For example, the GeneArt artificial gene synthesis service of Thermo Fisher Scientific can be used.
Further, the KAS gene used in the present invention may be one kind or a combination of two or more kinds of KAS genes.

The transformant of the present invention comprises a plant-derived KAS gene that does not accumulate medium-chain fatty acids, a gene encoding TE having substrate specificity for medium-chain acyl-ACP (hereinafter also referred to as “TE gene”). Expression is also promoted.
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 terminated by the action of TE, and the cut fatty acid is used for synthesis of triacylglycerol and the like.
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.

  TE is known to have a plurality of TEs having different reaction specificities depending on the number of carbon atoms and the number of unsaturated bonds in 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. In addition, when a host that does not originally have a gene encoding TE is used, it is necessary to promote the expression of the gene encoding TE. In addition, by promoting the expression of a TE gene having substrate specificity for medium chain acyl-ACP, the productivity of medium chain fatty acids is improved.

The TE having substrate specificity for medium-chain acyl-ACP used in the present invention can be appropriately selected from normal TE and proteins functionally equivalent to them 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: BMC Biochemistry 2011, 12:44; TE from Cocos nucifera (see CnFatB2: BMC Biochemistry, 2011, 12:44); TE from Cuphea viscosissima (CvFatB1: BMC Biochemistry, 2011, 12:44) TE from Cuphea viscosissima (see CvFatB2: BMC Biochemistry 2011, 12:44); TE from Cuphea viscosissima (see CvFatB3: BMC Biochemistry 2011, 12:44); TE from Elaeis guineensis (GenBank AAD42220); Desulfovibrio vulg aris derived TE (GenBank ACL08376); Bacteriodes fragilis derived TE (GenBank CAH09236); Parabacteriodes distasonis derived TE (GenBank ABR43801); Bacteroides thetaiotaomicron derived TE (GenBank AAO77182); Clostridium asparagiforme derived TE (GenBank EEG55387); Bryanthella formatexigens- derived TE (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 Cocos nucifera (hereinafter also referred to as “CTE”) (SEQ ID NO: 69, nucleotide sequence of gene encoding the same: sequence No. 70); TE derived from Nannochloropsis oculata (hereinafter also referred to as “NoTE”) (SEQ ID NO: 71, nucleotide sequence of the gene encoding this: SEQ ID NO: 72); California Bay ( Umbellularia californica ) -Derived TE (hereinafter also referred to as “BTE”) (SEQ ID NO: 73, nucleotide sequence of the gene encoding this: SEQ ID NO: 74); Nannochloropsis-Gaditana (Nannochloropsis Gaditana) derived from TE (hereinafter, also referred to as "NgaTE") (SEQ ID NO: 75, the gene of the nucleotide sequence encoding it: SEQ ID NO: 76); Nannochloropsis-Guranyurata ( TE derived from Nannochloropsis granulata (hereinafter also referred to as “NgrTE”) (SEQ ID NO: 77, nucleotide sequence of the gene encoding this: SEQ ID NO: 78); TE derived from Symbiodinium microadriaticum ( Symbiodinium microadriaticum ) (Hereinafter also referred to as “SmTE”) (SEQ ID NO: 79, nucleotide sequence of the gene encoding the same: SEQ ID NO: 80), and the like.
Further, as a protein functionally equivalent to these, the identity with any one of the above-mentioned TE amino acid sequences is 50% or more (preferably 60% or more, more preferably 65% or more, more preferably 70% or more, More preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more), and TE activity against medium chain acyl-ACP Proteins having the above can also be used. Alternatively, one or several (for example, 1 or more and 149 or less, preferably 1 or more and 119 or less, more preferably 1 or more and 104 or less, more preferably 1) in any one of the above-described amino acid sequences of TE 90 or more, more preferably 1 or more and 75 or less, more preferably 1 or more and 60 or less, more preferably 1 or more and 45 or less, more preferably 1 or more and 30 or less, more preferably 1 A protein having at least 15 and no more than 15 amino acids deleted, substituted, inserted or added and having TE activity against medium chain acyl-ACP can also be used.

  Among the above-mentioned TEs, CTE (SEQ ID NO: 69), NoTE (SEQ ID NO: 71), BTE (SEQ ID NO: 73), NgaTE (SEQ ID NO: 75), NgrTE (sequence) from the viewpoint of substrate specificity for medium chain acyl-ACP No. 77), or SmTE (SEQ ID NO: 79), and the identity of any one amino acid sequence of these TEs is 50% or more (preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more Preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more) and has TE activity against medium chain acyl-ACP. 1 or several (for example, 1 or more and 149 or less, preferably 1 or more and 119 or less, more preferably 1 or less) in the amino acid sequence of any one of these proteins or TE 104 or less, more preferably 1 or more and 90 or less, more preferably 1 or more and 75 or less, more preferably 1 or more and 60 or less, more preferably 1 or more and 45 or less, more preferably 1 A protein having 30 or less, more preferably 1 or more and 15 or less) amino acids deleted, substituted, inserted or added and having TE activity against medium chain acyl-ACP is preferred. When a plant cell is used as a host, CTE (SEQ ID NO: 69) or BTE (SEQ ID NO: 73), and the identity of any one of these TEs is 50% or more (preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more) 1 or several (for example, 1 to 149, preferably 1 to 119, more preferably, a protein having TE activity against medium chain acyl-ACP, or any one amino acid sequence of these TEs. Is 1 or more and 104 or less, more preferably 1 or more and 90 or less, more preferably 1 or more and 75 or less, more preferably 1 or more and 60 or less, More preferably 1 to 45, more preferably 1 to 30 and more preferably 1 to 15 amino acids are deleted, substituted, inserted or added, and against medium chain acyl-ACP A protein having TE activity is more preferred.

  The TE gene used in the present invention includes CTE gene (SEQ ID NO: 70), NoTE gene (SEQ ID NO: 72), BTE gene (SEQ ID NO: 74), NgaTE gene (sequence) from the viewpoint of substrate specificity for medium chain acyl-ACP. No. 76), NgrTE gene (SEQ ID NO: 78), or SmTE gene (SEQ ID NO: 80), the identity of any one of these TE genes is 50% or more (preferably 60% or more, more preferably 65%) Or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more) And a gene comprising a DNA encoding a protein having TE activity against medium-chain acyl-ACP, one or several (for example, one of these TE genes) 1 to 447, preferably 1 to 358, more preferably 1 to 313, more preferably 1 to 268, more preferably 1 to 224, more preferably 1 to 179, more preferably 1 to 134, more preferably 1 to 90, more preferably 1 to 45) nucleotide sequences are deleted, substituted, inserted or added. And hybridizing under stringent conditions with a gene comprising a DNA encoding a protein having TE activity against medium-chain acyl-ACP, or a DNA comprising a base sequence complementary to any one of these TE genes And a gene comprising a DNA encoding a protein having TE activity against medium chain acyl-ACP is preferred. When a plant cell is used as a host, the CTE gene (SEQ ID NO: 70) or BTE gene (SEQ ID NO: 74) has an identity of 50% or more (preferably 60% or more, preferably one of these TE genes) More preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more) One or several genes (for example, 1 to 447, preferably 1 to 447, preferably a gene consisting of DNA encoding a protein encoding a protein having TE activity against medium chain acyl-ACP) Is from 1 to 358, more preferably from 1 to 313, more preferably from 1 to 268, more preferably from 1 to 224 Or less, preferably 1 or more and 179 or less, more preferably 1 or more and 134 or less, more preferably 1 or more and 90 or less, more preferably 1 or more and 45 or less) A gene comprising a DNA encoding a protein inserted or added and having a TE activity against medium-chain acyl-ACP, or a DNA comprising a base sequence complementary to any one of these TE genes and stringent More preferred is a gene consisting of DNA that hybridizes under conditions and encodes a protein having TE activity against medium chain acyl-ACP.

  The sequence information of the TE and the gene encoding it can be obtained from, for example, the National Center for Biotechnology Information (NCBI).

The fact that a protein has TE activity means that, for example, a DNA in which a TE gene is linked downstream of a promoter that functions in a host cell such as E. coli is introduced into a host cell lacking the fatty acid degradation system, and the introduced TE gene is expressed. It can be confirmed by culturing under conditions and analyzing the change in 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, By carrying out reactions using various acyl-ACPs prepared by the method of Yuan et al. (Proc. Natl. Acad. Sci. USA, 1995, vol. 92 (23), p. 10639-10643), TE activity was obtained. Can be measured.

The method for promoting the expression of the KAS gene and TE gene can be appropriately selected from conventional methods. Examples thereof include a method of introducing the KAS gene and TE gene into a host, and a method of modifying the expression regulatory region (promoter, terminator, etc.) of the gene in a host having the KAS gene and TE gene on the genome. . Of these, a method of introducing the KAS gene and TE gene into a host and promoting the expression of the KAS gene and TE gene is preferable.
Hereinafter, in the present specification, a substance obtained by promoting the expression of genes (KAS gene and TE gene) encoding target proteins (KAS and TE) by performing the above-mentioned treatment on the host is also referred to as “transformant”. Those not promoting the expression of the gene encoding the protein of interest are also referred to as “host” or “wild strain”.
The transformant of the present invention is more productive of medium chain fatty acids or lipids comprising them as a constituent, compared to the host or the wild strain itself, in particular, the total fatty acids produced or the medium chain fatty acids occupying the total lipids or the same. The proportion of lipid as a 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 as a constituent, more preferably a fatty acid having 10 to 14 carbon atoms or a lipid comprising this, more preferably Fatty acids having 10, 12, or 14 carbon atoms or lipids comprising the same, more preferably saturated fatty acids (capric acid, lauric acid, myristic acid) having 10, 12, or 14 carbon atoms or Lipids as constituents, more preferably saturated fatty acids having 12 or 14 carbon atoms (lauric acid, myristic acid) or fats containing them as constituents , More preferably it can be suitably used for the production of lipids, to lauric acid or which constituents.
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 and TE 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 gene in a host cell, introducing the gene into a 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), plant cells, and animal cells. From the viewpoint of production efficiency and availability of the obtained lipid, the host is preferably a microorganism or a plant cell, and more preferably a plant cell.
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 Nannochloropsis are preferred, and algae of the genus Nannochloropsis are more preferred.
From the viewpoint of high lipid content in the seeds, the plant body includes Arabidopsis, Brassica napus , oilseed rape, coconut palm, palm ( Elaeis guineensis ), caffe, soybean, corn, rice, sunflower, camphor ( Cinnamomum camphora ) Or Jatropha is preferable, and Arabidopsis is more preferable.

As a vector (plasmid) serving as a base for a plasmid for gene expression or a cassette for gene expression, a vector capable of introducing a gene encoding a target protein into a host and expressing the target gene in a host cell If it is. For example, a vector having an expression control region such as a promoter or terminator according to the type of host to be used, and a vector having a replication origin, a selection marker, or the like can be used. Further, it may be a vector that grows and replicates autonomously outside the chromosome, such as a plasmid, or may be 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 ( 1986, Plasmid 15 (2), p. 93-103), pBR322 (Takara Bio), pRS403 (Stratagene), and pMW218 / 219 (Nippon Gene). 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 (Journal of Basic Microbiology, 2011, vol. 51) , p. 666-672), or 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. If the host is an algae belonging to the genus Nannochloropsis, refer to the method described in Proceedings of the National Academy of Sciences of the United States of America, 2011, vol. 108 (52). A host can also be transformed with a DNA fragment (gene expression cassette) comprising a promoter and a terminator.
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 a plant cell such as Arabidopsis thaliana, 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), c-phycocyanin β subunit (cpcB), cauliflower mozil virus 35SRNA promoter, housekeeping gene promoter (eg , tubulin promoter, actin promoter, ubiquitin promoter), rape or oilseed rape derived Napin gene promoter, a plant-derived Rubisco promoters, Nannochloropsis from the genus violaxanthin / chlorophyll a binding Tan Promoters of protein genes (VCP1 promoter, VCP2 promoter) (Proceedings of the National Academy of Sciences of the United States of America, 2011, vol. 108 (52)) and oleosin-like protein LDSP (lipid) from the genus Nannochloropsis droplet surface protein) gene promoter (PLOS Genetics, 2012; 8 (11): e1003064. doi: 10. 1371). When plant cells such as Arabidopsis thaliana are used as the host in the present invention, 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, gentamicin 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. .

  Selection of the transformant introduced with the target gene fragment can be performed by utilizing 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 the expression of the gene by modifying the expression regulatory region of the gene in a host having the KAS gene and the TE 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 or TE gene on the genome, the expression control region of the gene is modified to promote the expression of each of the KAS gene and TE gene, so that the medium chain fatty acid or this is used as a constituent component Lipid productivity can be improved.

Examples of the method for modifying the expression regulatory region include exchanging promoters. In a host having the KAS gene and TE gene on the genome, the expression of the KAS gene and TE gene can be promoted by replacing the promoter of the gene with a promoter having higher transcriptional activity.
The promoter used for the replacement of the promoter is not particularly limited, and may be appropriately selected from those having higher transcriptional activity than promoters of the KAS gene and TE gene and suitable for the production of medium-chain fatty acids or lipids comprising them. it can.

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 can be performed with reference to documents such as Methods in molecular biology, 1995, vol. 47, p. 291-302. In particular, when the host is an algae belonging to the genus Nannochloropsis, the genome is obtained by homologous recombination with reference to documents such as Proceedings of the National Academy of Sciences of the United States of America, 2011, vol. 108 (52). Specific regions within can be modified.

In addition to the KAS gene and TE gene, the transformant of the present invention includes acyl-CoA synthetase (hereinafter also simply referred to as “ACS”), ACP, acyltransferase (hereinafter also simply referred to as “AT”), and the like. It is preferable that the expression of the gene encoding the protein related to fatty acid synthesis is also promoted.
Among fatty acid synthesis-related proteins, ACS is a protein that participates in the production of acyl-CoA by adding CoA to biosynthesized fatty acids (free fatty acids). ACP functions as a scaffold (carrier) for fatty acid biosynthesis (fatty acid elongation reaction). The acyl group of the fatty acid forms a thioester bond with the phosphopantethein group bonded to the serine residue of ACP. In this state, the fatty acid is elongated. AT is a protein that catalyzes acylation of glycerol compounds such as glycerol triphosphate, lysophosphatidic acid, and diacylglycerol. Fatty acyl CoA or acyl ACP in which CoA is bonded to free fatty acid is taken into the glycerol skeleton by various ATs, and is accumulated as triacylglycerol formed by esterifying three fatty acid molecules to one glycerol molecule.
Therefore, in addition to the KAS gene and TE gene, the expression of genes encoding fatty acid synthesis-related proteins such as ACP, AT, ACS (preferably ACP, AT, ACS having specificity for medium chain fatty acids) is promoted. Thereby, the lipid productivity of the transformant used for the production of lipid, particularly the productivity of fatty acid can be further improved.

The amino acid sequence information of the aforementioned fatty acid synthesis-related proteins, the sequence information of the genes encoding them, and the like can be obtained from, for example, the National Center for Biotechnology Information (NCBI).
Moreover, the transformant which promoted the expression of the gene which codes a fatty acid synthesis related protein can be produced by a conventional method. For example, as in the method for promoting the expression of the KAS gene and TE gene described above, a method for introducing the various genes into a host, a method for modifying the expression regulatory region of the gene in a host having the various genes on the genome, etc. Thus, a transformant can be prepared.

In the transformant of the present invention, the productivity of medium-chain fatty acids or lipids comprising them is improved as compared with the host in which the expression of the KAS gene and TE gene is not promoted. Therefore, if the transformant of the present invention is cultured or cultivated under appropriate conditions, and then the medium chain fatty acid or the lipid comprising this as a constituent component is recovered from the obtained culture or growth product, the medium chain fatty acid or this Lipids as constituent components 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 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 using Escherichia coli 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 is used as a host, the transformant is cultured, for example, in soil at a temperature of 20 to 25 ° C., continuously irradiated with white light, or under 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 the transformant inoculated on the medium can be appropriately selected, and is preferably 1 to 50% (vol / vol) per medium from the viewpoint of growth. Although culture | cultivation temperature will not be restrict | limited especially if it is a range which does not have a bad influence on the growth of algae, Usually, it is the range of 5-40 degreeC. 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 culture of the transformant may be any of aeration and agitation culture, shaking culture or stationary culture, and 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, 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 produced in the production method of the present invention preferably contains a free fatty acid, a simple lipid having a fatty acid residue, or a complex lipid from the viewpoint of its availability, and is mediated by a free fatty acid or an ester bond. More preferably, it contains simple lipids or complex lipids having fatty acid residues (hereinafter sometimes referred to as fatty acid ester compounds).
The free fatty acid or fatty acid ester compound contained in the lipid is preferably a free medium chain fatty acid or a fatty acid ester compound thereof from the viewpoint of availability to a surfactant or the like, and a free fatty acid having 6 to 14 carbon atoms or a fatty acid thereof Fatty acid ester compounds are more preferred, free fatty acids having 8 to 14 carbon atoms or fatty acid ester compounds thereof are more preferred, free fatty acids having 10 to 14 carbon atoms or fatty acid ester compounds thereof are more preferred, and carbon atoms. Is more preferably a free fatty acid having 10, 12, or 14 or a fatty acid ester compound thereof, more preferably a free saturated fatty acid having 10, 12, or 14 carbon atoms (capric acid, lauric acid, myristic acid) or a fatty acid ester compound thereof. Preferably, a free saturated fatty acid having 12 or 14 carbon atoms (La Phosphoric acid, myristic acid) or a fatty acid ester compounds are more preferred, lauric acid or a fatty acid ester compounds are more preferred.
From the viewpoint of productivity, the fatty acid ester compound is 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 discloses the following methods for producing lipids, methods for modifying the composition of fatty acids produced, transformants, methods for producing transformants, proteins, genes, and recombinant vectors with respect to the above-described embodiments. .

<1> A plant-derived KAS gene that does not contain medium-chain fatty acid as a main accumulated lipid and a transformant that promotes the expression of a TE gene having substrate specificity for medium-chain acyl-ACP are cultured, and the fatty acid or this A method for producing a lipid, wherein a lipid as a constituent is produced.
<2> Promoting the expression of plant-derived KAS genes that do not contain medium-chain fatty acids as the main accumulated lipid and TE genes having substrate specificity for medium-chain acyl-ACP, and are produced in the cells of transformants A method for producing a lipid, which improves the productivity of a chain fatty acid or a lipid comprising the same.
<3> Plant-derived KAS gene that does not contain medium-chain fatty acids as the main accumulated lipid and TE gene having substrate specificity for medium-chain acyl-ACP are promoted to be produced in the cells of the transformant. A method for modifying the fatty acid composition, which increases the proportion of medium chain fatty acids in the total fatty acids.
<4> The method according to any one of <1> to <3>, wherein the KAS gene and the TE gene are introduced into a host to promote expression of the gene.

<5> Plant-derived KAS gene not containing medium-chain fatty acid as the main accumulated lipid and a transformant introduced with TE gene having substrate specificity for medium-chain acyl-ACP are cultured, and fatty acid or this as a constituent A method for producing lipid, wherein the lipid is produced.
<6> Plant-derived KAS gene not containing medium-chain fatty acid as a main accumulated lipid and a transformant introduced with a TE gene having substrate specificity for medium-chain acyl-ACP are cultured, and produced medium-chain fatty acid or A method for producing lipids, which improves the productivity of lipids comprising this as a constituent.
<7> Plant-derived KAS gene that does not contain medium-chain fatty acid as the main accumulated lipid and a transformant introduced with TE gene having substrate specificity for medium-chain acyl-ACP are cultured and accounted for the total fatty acids produced A method of modifying the fatty acid composition, increasing the proportion of medium chain fatty acids.

<8> The total amount of fatty acids in the plant is a fatty acid that is regarded as a free fatty acid having 8 to 14 carbon atoms and a fatty acid residue is 10% by mass of the total amount of fatty acids contained in the seed or pulp lipid. The method according to <1> to <7> above, wherein the plant is preferably 5% by mass or less, more preferably 1% by mass or less.
<9> 60 mass of the total amount of fatty acids contained in seeds or pulp lipids in which the plant is a fatty acid having 16 to 24 carbon atoms and a fatty acid amount of fatty acids regarded from the fatty acid residues. % Or more, preferably 70% by mass or more, more preferably 80% by mass or more, the method according to <1> to <8> above.
<10> The plant is a Euphorbiaceae plant, Brassicaceae plant, Legume plant, Gramineae plant, Grapeaceous plant, Asteraceae plant, Flaxaceae plant, Mallow family plant, Sesameaceae plant At least one plant selected from the group consisting of plants and asteraceae plants, preferably Jatropha, castor, Arabidopsis, rape, soybean, corn, European grape, camelina, sunflower, flax, safflower, groundnut, cotton genus, sesame At least one plant selected from the group consisting of rice, rice and olives, more preferably at least one plant selected from the group consisting of jatropha, castor, Arabidopsis, rape, soybean, corn, European grape, camelina, and sunflower The method according to <1> to <9> above, wherein

<11> The method according to any one of <1> to <10>, wherein the KAS is KAS II.
<12> The method according to any one of <1> to <11>, wherein the KAS is any one of the following proteins (A) to (R).
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 60.
(B) 85% or more identity with the amino acid sequence of the protein (A), preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98 % Or more, more preferably 99% or more of an amino acid sequence, and a protein having KAS activity.
(C) A protein consisting of the amino acid sequence represented by SEQ ID NO: 61.
(D) 85% or more identity with the amino acid sequence of the protein (C), preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98 % Or more, more preferably 99% or more of an amino acid sequence, and a protein having KAS activity.
(E) A protein consisting of the amino acid sequence represented by SEQ ID NO: 62.
(F) 85% or more identity with the amino acid sequence of the protein (E), preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98 % Or more, more preferably 99% or more of an amino acid sequence, and a protein having KAS activity.
(G) A protein consisting of the amino acid sequence represented by SEQ ID NO: 63.
(H) 85% or more identity with the amino acid sequence of the protein (G), preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98 % Or more, more preferably 99% or more of an amino acid sequence, and a protein having KAS activity.
(I) A protein comprising the amino acid sequence represented by SEQ ID NO: 64.
(J) 85% or more identity with the amino acid sequence of the protein (I), preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98 % Or more, more preferably 99% or more of an amino acid sequence, and a protein having KAS activity.
(K) A protein consisting of the amino acid sequence represented by SEQ ID NO: 65.
(L) 85% or more identity with the amino acid sequence of the protein (K), preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98 % Or more, more preferably 99% or more of an amino acid sequence, and a protein having KAS activity.
(M) A protein consisting of the amino acid sequence represented by SEQ ID NO: 66.
(N) 85% or more identity with the amino acid sequence of the protein (M), preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98 % Or more, more preferably 99% or more of an amino acid sequence, and a protein having KAS activity.
(O) A protein consisting of the amino acid sequence represented by SEQ ID NO: 67.
(P) 85% or more identity with the amino acid sequence of the protein (O), preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98 % Or more, more preferably 99% or more of an amino acid sequence, and a protein having KAS activity.
(Q) A protein consisting of the amino acid sequence represented by SEQ ID NO: 68.
(R) 85% or more identity with the amino acid sequence of the protein (Q), preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98 % Or more, more preferably 99% or more of an amino acid sequence, and a protein having KAS activity.

<13> The protein (B) has one or more, preferably 1 to 60, more preferably 1 to 40, more preferably 1 or more amino acid sequences in the protein (A). 28 or less, more preferably 1 or more and 20 or less, more preferably 1 or more and 12 or less, more preferably 1 or more and 8 or less, more preferably 1 or more and 4 or less amino acids, The method according to <12> above, wherein the protein is a substituted, inserted, or added protein.
<14> The protein (D) has one or more, preferably 1 to 84, more preferably 1 to 56, more preferably 1 or more amino acid sequences in the protein (C). 39 or less, more preferably 1 or more and 28 or less, more preferably 1 or more and 17 or less, more preferably 1 or more and 12 or less, more preferably 1 or more and 6 or less amino acids, The method according to <12> above, wherein the protein is a substituted, inserted, or added protein.
<15> The protein (F) has one or more, preferably 1 to 82, more preferably 1 to 55, more preferably 1 or more amino acid sequences of the protein (E). 38 or less, more preferably 1 or more and 28 or less, more preferably 1 or more and 17 or less, more preferably 1 or more and 11 or less, more preferably 1 or more and 6 or less amino acids, The method according to <12> above, wherein the protein is a substituted, inserted, or added protein.
<16> The protein (H) has one or more, preferably 1 to 82, more preferably 1 to 55, more preferably 1 or more amino acid sequences in the protein (G). 39 or less, more preferably 1 or more and 28 or less, more preferably 1 or more and 17 or less, more preferably 1 or more and 11 or less, more preferably 1 or more and 6 or less amino acids, The method according to <12> above, wherein the protein is a substituted, inserted, or added protein.
<17> The protein (J) has one or more, preferably 1 to 74, more preferably 1 to 49, more preferably 1 or more amino acid sequences in the protein (I). 35 or less, more preferably 1 or more and 25 or less, more preferably 1 or more and 15 or less, more preferably 1 or more and 10 or less, more preferably 1 or more and 5 or less amino acids, The method according to <12> above, wherein the protein is a substituted, inserted, or added protein.
<18> The protein (L) has one or more, preferably 1 to 70, more preferably 1 to 47, more preferably 1 or more amino acid sequences in the protein (K). 33 or less, more preferably 1 or more and 24 or less, more preferably 1 or more and 14 or less, more preferably 1 or more and 10 or less, more preferably 1 or more and 5 or less amino acids, The method according to <12> above, wherein the protein is a substituted, inserted, or added protein.
<19> The protein (N) has one or more, preferably 1 to 84, more preferably 1 to 56, more preferably 1 or more amino acid sequences in the protein (M). 39 or less, more preferably 1 or more and 28 or less, more preferably 1 or more and 17 or less, more preferably 1 or more and 12 or less, more preferably 1 or more and 6 or less amino acids, The method according to <12> above, wherein the protein is a substituted, inserted, or added protein.
<20> The protein (P) has one or more, preferably 1 to 80, more preferably 1 to 54, more preferably 1 or more amino acid sequences in the protein (O). 38 or less, more preferably 1 or more and 27 or less, more preferably 1 or more and 16 or less, more preferably 1 or more and 11 or less, more preferably 1 or more and 6 or less amino acids, The method according to <12> above, wherein the protein is a substituted, inserted, or added protein.
<21> The protein (R) has one or more, preferably 1 to 74, more preferably 1 to 50, more preferably 1 or more amino acid sequences in the protein (Q). 35 or less, more preferably 1 or more and 25 or less, more preferably 1 or more and 15 or less, more preferably 1 or more and 10 or less, more preferably 1 or more and 5 or less amino acids, The method according to <12> above, wherein the protein is a substituted, inserted, or added protein.

<22> The gene encoding the KAS, preferably any one of the proteins (A) to (R), is a gene consisting of any one of the following DNA (a) to (r): The method according to any one of <1> to <21>.
(A) DNA consisting of the base sequence represented by SEQ ID NO: 51.
(B) 80% or more of identity with the base sequence of DNA (a), preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97 % Or more, more preferably 98% or more, more preferably 99% or more, and a DNA encoding a protein having KAS activity.
(C) DNA consisting of the base sequence represented by SEQ ID NO: 52.
(D) The identity with the base sequence of the DNA (c) is 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97 % Or more, more preferably 98% or more, more preferably 99% or more, and a DNA encoding a protein having KAS activity.
(E) DNA consisting of the base sequence represented by SEQ ID NO: 53.
(F) 80% or more of identity with the base sequence of DNA (e), preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97 % Or more, more preferably 98% or more, more preferably 99% or more, and a DNA encoding a protein having KAS activity.
(G) DNA consisting of the base sequence represented by SEQ ID NO: 54.
(H) The identity with the base sequence of the DNA (g) is 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97 % Or more, more preferably 98% or more, more preferably 99% or more, and a DNA encoding a protein having KAS activity.
(I) DNA consisting of the base sequence represented by SEQ ID NO: 55.
(J) 80% or more of identity with the base sequence of DNA (i), preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97 % Or more, more preferably 98% or more, more preferably 99% or more, and a DNA encoding a protein having KAS activity.
(K) DNA consisting of the base sequence represented by SEQ ID NO: 56.
(L) 80% or more of identity with the base sequence of DNA (k), preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97 % Or more, more preferably 98% or more, more preferably 99% or more, and a DNA encoding a protein having KAS activity.
(M) DNA consisting of the base sequence represented by SEQ ID NO: 57.
(N) The identity with the base sequence of the DNA (m) is 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97 % Or more, more preferably 98% or more, more preferably 99% or more, and a DNA encoding a protein having KAS activity.
(O) DNA consisting of the base sequence represented by SEQ ID NO: 58.
(P) The identity with the base sequence of the DNA (o) is 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97 % Or more, more preferably 98% or more, more preferably 99% or more, and a DNA encoding a protein having KAS activity.
(Q) DNA consisting of the base sequence represented by SEQ ID NO: 59.
(R) The identity with the base sequence of DNA (q) is 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97 %, More preferably 98% or more, more preferably 99% or more of a base sequence, and a DNA encoding a protein having KAS activity.

<23> The DNA (b) has one or more, preferably 1 to 250, more preferably 1 to 181 and more preferably 1 or more nucleotide sequences in the DNA (a). 121 or less, preferably 1 or more and 85 or less, more preferably 1 or more and 61 or less, more preferably 1 or more and 37 or less, more preferably 1 or more and 25 or less, more preferably 1 or more and 13 DNA encoding the protein (A) or (B) having a KAS activity consisting of a base sequence in which not more than one base is deleted, substituted, inserted or added, or complementary to the DNA (a) <2 which is a DNA encoding the protein (A) or (B) having a KAS activity and hybridizing under stringent conditions with a DNA comprising a simple nucleotide sequence. The method of> Item described.
<24> The DNA (d) has one or more, preferably 1 to 333, more preferably 1 to 250, more preferably 1 or more nucleotide sequences in the DNA (c). 167 or less, preferably 1 or more and 117 or less, more preferably 1 or more and 84 or less, more preferably 1 or more and 50 or less, more preferably 1 or more and 34 or less, more preferably 1 or more and 17 A DNA encoding the protein (C) or (D) having a KAS activity consisting of a base sequence in which not more than one base is deleted, substituted, inserted, or added, or complementary to the DNA (c) A DNA encoding the protein (C) or (D) having a KAS activity and hybridizing with a DNA comprising a simple nucleotide sequence under stringent conditions, The method of 2> above, wherein.
<25> The DNA (f) has one or more, preferably 1 to 326, more preferably 1 to 244, more preferably 1 or more nucleotide sequences in the DNA (e). 163 or less, preferably 1 or more and 114 or less, more preferably 1 or more and 82 or less, more preferably 1 or more and 49 or less, more preferably 1 or more and 33 or less, more preferably 1 or more and 17 A DNA encoding a protein (E) or (F) having a KAS activity consisting of a base sequence in which not more than one base is deleted, substituted, inserted or added, or complementary to the DNA (e) A DNA encoding the protein (E) or (F) having a KAS activity and hybridizing with a DNA comprising a simple nucleotide sequence under stringent conditions, The method of 2> above, wherein.
<26> The DNA (h) has one or more, preferably 1 or more and 329 or less, more preferably 1 or more and 247 or less, more preferably 1 or more, in the base sequence of the DNA (g). 165 or less, preferably 1 or more and 115 or less, more preferably 1 or more and 83 or less, more preferably 1 or more and 50 or less, more preferably 1 or more and 33 or less, more preferably 1 or more and 17 A DNA encoding a protein (G) or (H) having a KAS activity consisting of a base sequence in which not more than one base is deleted, substituted, inserted or added, or complementary to the DNA (g) A DNA encoding the protein (G) or (H) having a KAS activity and hybridizing with a DNA comprising a simple nucleotide sequence under stringent conditions, The method of 2> above, wherein.
<27> The DNA (j) has one or more, preferably 1 or more and 294 or less, more preferably 1 or more and 221 or less, more preferably 1 or more, in the base sequence of the DNA (i). 147 or less, preferably 1 or more and 103 or less, more preferably 1 or more and 74 or less, more preferably 1 or more and 45 or less, more preferably 1 or more and 30 or less, more preferably 1 or more and 15 A DNA encoding the protein (I) or (J) having a KAS activity consisting of a base sequence in which not more than one base is deleted, substituted, inserted or added, or complementary to the DNA (i) A DNA encoding the protein (I) or (J) having a KAS activity and hybridizing with a DNA comprising a simple nucleotide sequence under stringent conditions, The method of 2> above, wherein.
<28> The DNA (l) has one or more, preferably 1 or more and 278 or less, more preferably 1 or more and 208 or less, more preferably 1 or more, in the base sequence of the DNA (k). 139 or less, preferably 1 or more and 98 or less, more preferably 1 or more and 70 or less, more preferably 1 or more and 42 or less, more preferably 1 or more and 28 or less, more preferably 1 or more and 14 DNA encoding the protein (K) or (L) having a KAS activity, which is composed of a base sequence in which not more than one base is deleted, substituted, inserted or added, or complementary to the DNA (k) <2 which is a DNA that hybridizes with a DNA comprising a simple nucleotide sequence under stringent conditions and encodes the protein (K) or (L) having KAS activity. The method of> Item described.
<29> The DNA (n) has one or more, preferably 1 to 334, more preferably 1 to 251 and more preferably 1 or more nucleotide sequences in the DNA (m). 167 or less, preferably 1 or more and 117 or less, more preferably 1 or more and 84 or less, more preferably 1 or more and 51 or less, more preferably 1 or more and 34 or less, more preferably 1 or more and 17 DNA encoding the protein (M) or (N) having a KAS activity consisting of a base sequence in which not more than one base is deleted, substituted, inserted or added, or complementary to the DNA (m) A DNA encoding the protein (M) or (N) having a KAS activity and hybridizing with a DNA comprising a simple nucleotide sequence under stringent conditions, The method of 2> above, wherein.
<30> The DNA (p) has one or more, preferably 1 to 320, more preferably 1 to 240, more preferably 1 or more nucleotide sequences in the DNA (o). 160 or less, preferably 1 or more and 112 or less, more preferably 1 or more and 80 or less, more preferably 1 or more and 48 or less, more preferably 1 or more and 32 or less, more preferably 1 or more and 16 or more DNA encoding the protein (O) or (P) having a KAS activity, which is composed of a base sequence in which not more than one base is deleted, substituted, inserted or added, or complementary to the DNA (o) A DNA that hybridizes with a DNA consisting of a simple nucleotide sequence under stringent conditions and encodes the protein (O) or (P) having KAS activity, The method of 2> above, wherein.
<31> The DNA (r) has one or more, preferably 1 to 296, more preferably 1 to 222, more preferably 1 or more nucleotide sequences in the DNA (q). 148 or less, preferably 1 or more and 104 or less, more preferably 1 or more and 74 or less, more preferably 1 or more and 45 or less, more preferably 1 or more and 30 or less, more preferably 1 or more and 15 DNA encoding the protein (Q) or (R) having a KAS activity consisting of a base sequence in which not more than one base is deleted, substituted, inserted or added, or complementary to the DNA (q) A DNA that hybridizes with a DNA comprising a simple nucleotide sequence under stringent conditions and encodes the protein (Q) or (R) having KAS activity, The method of 2> above, wherein.

<32> The method according to any one of <1> to <31>, wherein the TE is TE having substrate specificity for medium chain acyl-ACP.
<33> The TE consists of an amino acid sequence represented by SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77 or SEQ ID NO: 79 (preferably SEQ ID NO: 69 or SEQ ID NO: 73). 50% or more of the identity of the protein and the amino acid sequence of the protein (preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, More preferably 85% or more, more preferably 90% or more, more preferably 95% or more) and has a TE activity against medium chain acyl-ACP, or 1 or 1 in the amino acid sequence of the protein Several (preferably 1 or more and 149 or less, more preferably 1 or more and 119 or less, more preferably 1 or less 104 or less, more preferably 1 or more and 90 or less, more preferably 1 or more and 75 or less, more preferably 1 or more and 60 or less, more preferably 1 or more and 45 or less, more preferably 1 or more <1> to <32, which is a protein having 30 or less, more preferably 1 to 15) amino acids deleted, substituted, inserted or added, and having TE activity against medium chain acyl-ACP The method of any one of>.
<34> A base encoding the TE-encoding gene represented by SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78 or SEQ ID NO: 80 (preferably SEQ ID NO: 70 or SEQ ID NO: 74) DNA comprising a sequence and identity to any one base sequence of the DNA is 50% or more (preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more DNA which preferably comprises 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more) and a protein encoding a protein having TE activity against medium chain acyl-ACP, One or several (preferably 1 to 447, more preferably 1 to 358) of any one base sequence of the DNA Lower, more preferably 1 to 313, more preferably 1 to 268, more preferably 1 to 224, more preferably 1 to 179, more preferably 1 to 134 Or less, and more preferably 1 to 90, more preferably 1 to 45) is deleted, substituted, inserted or added, and encodes a protein having TE activity against medium chain acyl-ACP DNA that encodes a protein that hybridizes under stringent conditions with a DNA that comprises a base sequence complementary to any one of the DNA sequences, and has a TE activity against medium-chain acyl-ACP, The method according to any one of <1> to <33>, wherein the gene is a gene comprising:

<35> The method according to any one of <1> to <34>, wherein the transformant is a microorganism or a plant transformant, preferably a plant transformant.
<36> The method according to the above <35>, wherein the transformed plant is Arabidopsis thaliana, Western rape, rape, coconut palm, palm, coffea, soybean, corn, rice, sunflower, camphor, or Jatropha, preferably Arabidopsis. .
<37> The method according to <36> above, wherein the transformant is produced by introducing the gene encoding KAS into Arabidopsis thaliana as a host using Agrobacterium.
<38> The method according to <36> or <37>, wherein the fatty acid produced by the transformant or a lipid comprising the fatty acid is collected from the seed of the plant.
<39> The lipid is a free medium chain fatty acid or a fatty acid ester compound thereof, preferably a free fatty acid having 6 to 14 carbon atoms or a fatty acid ester compound thereof, more preferably a free fatty acid having 8 to 14 carbon atoms. Or a fatty acid ester compound thereof, more preferably a free fatty acid having 10 to 14 carbon atoms or a fatty acid ester compound thereof, more preferably a free fatty acid having 10, 12, or 14 carbon atoms or a fatty acid ester compound thereof, more preferably Is a free saturated fatty acid having 10, 12 or 14 carbon atoms (capric acid, lauric acid, myristic acid) or a fatty acid ester compound thereof, more preferably a free saturated fatty acid having 12 or 14 carbon atoms (lauric acid, myristic acid) Acid) or fatty acid ester compounds thereof, more preferably Gaulin acid or fatty acid ester compounds, including, the <1> to any one method according to <38>.

<40> The proteins (A) to (R).
<41> A gene encoding the protein according to <40>.
<42> A gene comprising any one of the DNAs (a) to (r).
<43> A recombinant vector or DNA cassette containing the gene according to <41> or <42>.

<44> A transformant comprising a plant-derived KAS gene containing no medium chain fatty acid as a main accumulated lipid and a TE gene having substrate specificity for medium chain acyl-ACP.
<45> A transformant in which expression of a plant-derived KAS gene containing no medium chain fatty acid as a main accumulated lipid and a TE gene having substrate specificity for medium chain acyl-ACP is promoted.
<46> A method for producing a transformant, wherein a plant-derived KAS gene containing no medium chain fatty acid as a main accumulated lipid and a TE gene having substrate specificity for medium chain acyl-ACP are introduced.
<47> The total amount of fatty acids of the fatty acid in which the plant is a free fatty acid having 8 or more and 14 or less carbon atoms and a fatty acid residue is 10% by mass of the total amount of fatty acids contained in the seed or pulp lipid The transformant according to any one of the above <44> to <46> or a method for producing the same, which is a plant which is preferably 5% by mass or less, more preferably 1% by mass or less.
<48> 60 mass of the total fatty acid amount contained in the seed or pulp lipid in which the plant is a fatty acid having 16 or more and 24 or less carbon atoms and the fatty acid amount of the fatty acid regarded as its fatty acid residue. % Or more, preferably 70% by mass or more, more preferably 80% by mass or more, The transformant according to any one of <44> to <47> or a method for producing the same.
<49> said plant is Euphorbiaceae plant, cruciferous plant, legume plant, gramineous plant, grape plant, asteraceae plant, flaxaceae plant, mallow family plant, sesameaceae plant At least one plant selected from the group consisting of plants and asteraceae plants, preferably Jatropha, castor, Arabidopsis, rape, soybean, corn, European grape, camelina, sunflower, flax, safflower, groundnut, cotton genus, sesame At least one plant selected from the group consisting of rice, rice and olives, more preferably at least one plant selected from the group consisting of jatropha, castor, Arabidopsis, rape, soybean, corn, European grape, camelina, and sunflower The transformation according to any one of <44> to <48>, wherein Or a method for manufacturing the same.
<50> The transformant according to any one of <44> to <49> or the method for producing the same, wherein the KAS is KAS II.
<51> The transformant according to any one of <44> to <50> or the method for producing the same, wherein the KAS is any one of the proteins (A) to (R).
<52> The gene encoding the KAS, preferably any one of the proteins (A) to (R), is the gene consisting of any one of the DNAs (a) to (r). The transformant of any one of <44>-<51> or its production method.
<53> The transformant according to any one of <44> to <52> or a method for producing the same, wherein the TE is a TE having a substrate specificity for medium-chain acyl-ACP.
<54> The transformant according to any one of <44> to <53> or a method for producing the same, wherein the TE is a protein defined in the item <33>.
<55> The transformant according to any one of <44> to <54> or a method for producing the same, wherein the gene encoding TE is a gene defined in <34>.
<56> The transformant according to any one of <44> to <55> or a method for producing the transformant, wherein the transformant is a microorganism or a plant transformant, preferably a plant transformant.
<57> The trait according to the above <56>, wherein the transformed plant is Arabidopsis thaliana, western rape, rape, coconut palm, palm, coffea, soybean, corn, rice, sunflower, camphor, or jatropha, preferably Arabidopsis thaliana A converter or a production method thereof.

<58> Produced by the method for producing a protein, gene, recombinant vector or DNA cassette, transformant, or transformant according to any one of <40> to <57> for producing a lipid Use of transformants.
<59> The lipid is a free medium chain fatty acid or a fatty acid ester compound thereof, preferably a free fatty acid having 6 to 14 carbon atoms or a fatty acid ester compound thereof, more preferably a free fatty acid having 8 to 14 carbon atoms. Or a fatty acid ester compound thereof, more preferably a free fatty acid having 10 to 14 carbon atoms or a fatty acid ester compound thereof, more preferably a free fatty acid having 10, 12, or 14 carbon atoms or a fatty acid ester compound thereof, more preferably Is a free saturated fatty acid having 10, 12 or 14 carbon atoms (capric acid, lauric acid, myristic acid) or a fatty acid ester compound thereof, more preferably a free saturated fatty acid having 12 or 14 carbon atoms (lauric acid, myristic acid) Acid) or fatty acid ester compounds thereof, more preferably Gaulin acid or fatty acid ester compound, comprising the use of the <58> above, wherein.

EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.
Here, Table 4 shows the base sequences of the primers used in this example.

(Preparation Example 1) Extraction of RNA from plant tissue and synthesis of cDNA Each plant tissue of Arabidopsis thaliana and rape was frozen in liquid nitrogen and crushed using a mortar and pestle. RNA was purified by applying Fruit Mate (Takara Bio) and RNAiso plus (Takara Bio) to the crushed tissue. CDNA was prepared from the obtained RNA using PrimeScript II 1st strand cDNA Synthesis Kit (manufactured by Takara Bio Inc.).

Preparation Example 2 Construction of Coco Gene-Derived TE Gene Expression Plasmid A rape-derived Napin gene promoter (SEQ ID NO: 46) was obtained from a wild rape-like plant collected in Itako, Ibaraki Prefecture. Furthermore, the rape-derived Napin gene terminator (SEQ ID NO: 47) was obtained from a wild rape-like plant collected from Mashiko-machi, Tochigi Prefecture.
Genomic DNA was extracted from wild rape-like plants using the PowerPlant DNA Isolation Kit (trade name, MO BIO Laboratories, USA). Using the extracted genomic DNA as a template, PCR was performed using PrimeSTAR (trade name, manufactured by Takara Bio Inc.) to amplify the Napin gene promoter and terminator, respectively. Specifically, PCR was performed using primer number 1 and primer number 2 shown in Table 1 to amplify the Napin gene promoter. In addition, PCR was performed using the primer numbers 3 and 4 shown in Table 1 to amplify the terminator of the Napin gene.
Using the PCR product confirmed to be amplified as a template, the Napin gene promoter was subjected to PCR again using primer number 5 and primer number 6 shown in Table 1, and the Napin gene terminator was used as primer number 3 and primer number 7 shown in Table 1. went. In this manner, a Napin gene promoter sequence fragment (Pnapin, SEQ ID NO: 46) and a Napin gene terminator sequence fragment (Tnapin, SEQ ID NO: 47) were prepared.
The amplified gene fragments (Pnapin, Tnapin) were each treated with Mighty TA-cloning Kit (trade name, manufactured by Takara Bio Inc.), and then inserted into pMD20-T vector (Takara Bio Inc.) by a ligation reaction. In this way, plasmid pPNapin1 containing the Napin gene promoter and plasmid pTNapin1 containing the Napin gene terminator were constructed, respectively.

Using the plasmid pPNapin1 as a template, PCR was performed using PrimeSTAR and primer numbers 8 and 9 shown in Table 1 to amplify a gene fragment having restriction enzyme recognition sequences added to both ends. Further, PCR was performed using the plasmid pTNapin1 as a template and PrimeSTAR, and primer numbers 10 and 11 shown in Table 1 to amplify a gene fragment having restriction enzyme recognition sequences added to both ends.
The amplified gene fragment was treated with Mighty TA-cloning Kit (trade name, manufactured by Takara Bio Inc.) and then inserted into pMD20-T vector by ligation reaction to construct plasmids pPNapin2 and pTNapin2, respectively. Plasmid pPNapin2 with restriction enzymes Sal I and Not I, treated respectively pTNapin2 with restriction enzymes Sma I and Not I, and ligated in ligation reaction pRI909 vector treated with Sal I and Sma I (manufactured by Takara Bio Inc.), the plasmid p909PTnapin was constructed.

Commissioned synthesis provided by Invitrogen (Carlsbad, California) for a gene (positions 1 to 255 of SEQ ID NO: 74) encoding a chloroplast transit signal peptide (positions 1 to 85 of SEQ ID NO: 73) of BTE (SEQ ID NO: 73) Obtained using the service. Using the plasmid containing the obtained sequence as a template, PCR was performed using PrimeSTAR and primer number 12 and primer number 13 shown in Table 1 to amplify the gene fragment encoding the signal peptide. Deoxyadenine (dA) is added to both ends of the gene fragment amplified using Mighty TA-cloning Kit (trade name, manufactured by Takara Bio Inc.), and then inserted into pMD20-T vector (Takara Bio Inc.) by ligation reaction Plasmid pSignal was constructed. The constructed pSignal was treated with the restriction enzyme Not I and ligated with the Not I site of the plasmid p909PTnapin by a ligation reaction to obtain the plasmid p909PTnapin-S.

Using a cDNA prepared from RNA derived from coconut endosperm as a template, PCR was performed using PrimeSTAR MAX (trade name, manufactured by Takara Bio Inc.) and the primer numbers 14 and 15 shown in Table 1 to amplify the CTE gene fragment. .
Further, PCR was performed using the above-mentioned p909PTnapin-S as a template and using primer number 16 and primer number 17 shown in Table 1, and p909PTnapin-S was amplified as a linear fragment.

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 expression of this plasmid is controlled by the Napin gene promoter derived from rape, and the base sequence of the gene encoding CTE (hereinafter referred to as “BTEsignal-CTE”) that is transferred to the chloroplast by the chloroplast transfer signal peptide derived from BTE. Abbreviated SEQ ID NO: 48) has been introduced into the plant introduction plasmid p909CTE.

(Preparation Example 3) Construction of various KAS gene expression plasmids
With reference to the sequence of the transformation vector pYW310 (ACCESSION NO. DQ469641) disclosed at NCBI's Gene Bank, the bialaphos resistance gene encoding the phosphinothricin acetyltransferase derived from Streptomyces hygroscopicus SEQ ID NO: 49), also referred to as “Bar gene”, was obtained using a commissioned synthesis service provided by Gene Script.
Using the synthesized Bar gene as a template, PCR was performed using PrimeSTAR and primer number 18 and primer number 19 shown in Table 1 to amplify the Bar gene fragment. On the other hand, PCR was performed using pRI909 as a template, PrimeSTAR, and primer number 20 and primer number 21 shown in Table 1 to amplify a region excluding the kanamycin resistance gene from the pRI909 vector.
The amplified these gene fragments were 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.

With reference to the sequence of the promoter of rape Napin gene (ACCESSION NO. EU416279) disclosed in NCBI's Gene Bank, a strong promoter expressed in the seeds of rape (hereinafter referred to as "Napin Promoter of rape") SEQ ID NO: 50) The gene was obtained using a commissioned synthesis service provided by Gene Script. Using the synthesized rape Napin Promoter gene as a template, PCR was performed using the primer number 22 and primer number 23 shown in Table 1 to amplify the rape Napin Promoter.
Moreover, pRI909Bar was used as a template, PCR was performed using primer number 24 and primer number 25 shown in Table 1, and the pRI909Bar fragment was amplified.
Furthermore, PCR was performed using the plasmid p909CTE as a template and the primer numbers 26 and 27 shown in Table 1 to amplify the CTE-Tnapin sequence.
These were ligated by In-fusion reaction to construct plasmid p909Pnapus-CTE-Tnapin.

Using the plasmid p909Pnapus-CTE-Tnapin as a template, PCR was performed using primer number 28 and primer number 29 shown in Table 1 to amplify the gene fragment.
Using the amplified gene fragment, a plasmid in which PCR fragments of KAS derived from various plants were ligated by an in-fusion reaction was prepared according to the method shown below.

(1) Jatropha-derived KAS (JcKASII)
The DNA sequence of the Jatropha-derived KAS gene was obtained using a commissioned synthesis service provided by Operon Biotechnology or Thermo Fisher Scientific. Using the obtained DNA sequence as a template, PCR was performed using primer number 81 and primer number 82 shown in Table 1, and the JcKASII gene fragment (SEQ ID NO: 51) was amplified. Plasmid p909Pnapus-JcKASII-Tnapin was constructed using the obtained amplified fragment.

(2) Castor-derived KAS (RiKASII)
The DNA sequence of the castor-derived KAS gene was obtained using a commissioned synthesis service provided by Operon Biotechnology or Thermo Fisher Scientific. Using the obtained DNA sequence as a template, PCR was performed using the primer numbers 44 and 45 shown in Table 1 to amplify the RiKASII gene fragment (SEQ ID NO: 52). Plasmid p909Pnapus-RiKASII-Tnapin was constructed using the obtained amplified fragment.

(3) Arabidopsis derived KAS (AtKASII)
Using the Arabidopsis thaliana-derived cDNA prepared in Preparation Example 1 as a template, PCR was performed using primer number 30 and primer number 31 shown in Table 1, to amplify the AtKASII gene fragment (SEQ ID NO: 53). Plasmid p909Pnapus-AtKASSII-Tnapin was constructed using the obtained amplified fragment.

(4) Brassica-derived KAS (BrKASII)
Using the rape-derived cDNA prepared in Preparation Example 1 as a template, PCR was performed using the primer numbers 32 and 33 shown in Table 1 to amplify the BrKASII gene fragment (SEQ ID NO: 54). Plasmid p909Pnapus-BrKASII-Tnapin was constructed using the obtained amplified fragment.

(5) KAS derived from soybean (GmKASII)
The DNA sequence of the soybean-derived KAS gene was obtained using a commissioned synthesis service provided by Operon Biotechnology or Thermo Fisher Scientific. Using the obtained DNA sequence as a template, PCR was performed using primer number 34 and primer number 35 shown in Table 1 to amplify the GmKASII gene fragment (SEQ ID NO: 55). Plasmid p909Pnapus-GmKASII-Tnapin was constructed using the obtained amplified fragment.

(6) Corn-derived KAS (ZmKASII)
Using the commissioned synthesis service provided by Operon Biotechnology or Thermo Fisher Scientific, the maize-derived KAS gene DNA sequence was obtained. Using the obtained DNA sequence as a template, PCR was performed using primer number 36 and primer number 37 shown in Table 1 to amplify the ZmKASII gene fragment (SEQ ID NO: 56). Plasmid p909Pnapus-ZmKASII-Tnapin was constructed using the obtained amplified fragment.

(7) European grape-derived KAS (VvKASII)
The DNA sequence of the European grape-derived KAS gene was obtained using a commissioned synthesis service provided by Operon Biotechnology or Thermo Fisher Scientific. Using the obtained DNA sequence as a template, PCR was performed using primer number 38 and primer number 39 shown in Table 1 to amplify the VvKASII gene fragment (SEQ ID NO: 57). Plasmid p909Pnapus-VvKASII-Tnapin was constructed using the obtained amplified fragment.

(8) Camerina-derived KAS (CsKASII)
The DNA sequence of the camelina-derived KAS gene was obtained using a commissioned synthesis service provided by Operon Biotechnology or Thermo Fisher Scientific. Using the obtained DNA sequence as a template, PCR was performed using primer number 40 and primer number 41 shown in Table 1 to amplify the CsKASII gene fragment (SEQ ID NO: 58). Plasmid p909Pnapus-CsKASII-Tnapin was constructed using the obtained amplified fragment.

(9) Sunflower-derived KAS (HaKASII)
The DNA sequence of the sunflower-derived KAS gene was obtained using a contracted synthesis service provided by Operon Biotechnology or Thermo Fisher Scientific. Using the obtained DNA sequence as a template, PCR was performed using the primer numbers 42 and 43 shown in Table 1 to amplify the HaKASII gene fragment (SEQ ID NO: 59). Plasmid p909Pnapus-HaKASII-Tnapin was constructed using the obtained amplified fragment.

(Preparation Example 4) Preparation of transformants Using the plasmid p909CTE, a transformant of Arabidopsis thaliana (Colombia strain) into which the CTE gene had been introduced was obtained using the Arabidopsis transformation service provided by Implanta Innovations.
The obtained Arabidopsis thaliana transformants (WT :: CTE) and wild-type Arabidopsis thaliana (WT) were grown at room temperature of 22 ° C. under fluorescent light illumination for 24 hours (about 4,000 lux). After about 2 months of cultivation, the seeds were harvested.

Using the Arabidopsis transformant (WT :: CTE) into which the plasmid p909CTE thus obtained was introduced or the wild-type Arabidopsis thaliana (WT) as a parent strain, the following transformation was performed.
The plasmids p909Pnapus-RiKASII-Tnapin, p909Pnapus-AtKASSII-Tnapin, p909Pnapus-BrKASII-Tnapin, p909Pnapus-GmKASII-Tnapin, p909Pnapus-ZmKASII-Tnapin-Pnapus-VnaKAsII-Tnap Using Agrobacterium tumefaciens GV3101 strain into which Arabidopsis was introduced, Arabidopsis thaliana (WT :: CTE) into which plasmid p909CTE was introduced was transformed. The inflorescences of Arabidopsis grown about 1.5 months after sowing were excised, and further Agrobacterium introduced with each plasmid was infected with the inflorescences that were grown for 6 to 7 days and regenerated.
Seeds obtained after the Agrobacterium infection treatment were sown on MS agar medium (containing 100 μg / mL kraforan and 7 μg / mL bialaphos), and transformants were selected.
Wild-type Arabidopsis thaliana (WT), Arabidopsis thaliana introduced with plasmid p909CTE (WT :: CTE), and the selected transformants were grown at room temperature of 22 ° C. under fluorescent light illumination for 24 hours, respectively. After months of cultivation, the seeds were harvested.

(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. After centrifugation at room temperature and 1500 rpm for 5 minutes, the lower layer portion was collected and dried with nitrogen gas.
To the dried sample, 100 μL of 0.5N potassium hydroxide-methanol solution was added, and the mixture was incubated at 70 ° C. for 30 minutes to hydrolyze triacylglycerol. 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 perform fatty acid methyl esterification treatment. 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 → 150-320 ° C (20 ° C / min temperature rise) → 320 ° C holding 2 minutes Inlet detector temperature: 300 ° C
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

  The amount of methyl ester of each fatty acid was quantified from the peak area of the waveform data obtained by GC analysis. 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 Tables 5 and 6.

As shown in Table 5, in the seeds of the transformant prepared by introducing the KASII gene defined in the present invention into wild-type Arabidopsis thaliana, an increase in the amount of medium chain fatty acids cannot be confirmed.
On the other hand, as shown in Table 6, in the seeds of the transformant produced by introducing the CTE gene into Arabidopsis thaliana, the ratio of C12: 0 amount was about 3% and C14: 0 compared to wild type Arabidopsis thaliana. The proportion of the amount increases by about 13%, and the proportion of the C16: 0 amount increases by about 15%. Then, by introducing the KAS gene derived from various plants into the transformant into which the CTE gene has been introduced, the ratio of C12: 0 amount is compared with that of Arabidopsis thaliana (WT :: CTE) into which the CTE gene has been introduced, Further increased. Furthermore, C10: 0 fatty acid accumulation was also confirmed for some of the transformants of the present invention (CTE + RiKASII, CTE + BrKASII, CTE + VvKASII, and CTE + CsKASII).

  Generally, KAS II is known to catalyze a chain extension reaction from acyl-ACP having 16 carbon atoms to acyl-ACP having 18 carbon atoms. However, this finding is not consistent with the effect of introducing the KAS II gene on the TE gene-introduced strain confirmed in the above test example. Therefore, the plant-derived KAS II gene, which does not contain medium-chain fatty acids as the main accumulated lipid, is used in combination with TE that has substrate specificity for medium-chain acyl-ACP. It was suggested that there is an effect to increase.

  As described above, by introducing the KAS gene and TE gene defined in the present invention into a host, a transformant with improved productivity of medium chain fatty acids can be produced. By culturing this transformant, the productivity of medium chain fatty acids can be improved.

Claims (19)

  Transformation by introducing a plant-derived gene encoding β-ketoacyl-ACP synthase that does not contain medium-chain fatty acids as the main accumulated lipid, and a gene encoding acyl-ACP thioesterase having substrate specificity for medium-chain acyl-ACP A method for producing a lipid, comprising culturing a body to produce a fatty acid or a lipid comprising this as a constituent.   Transformation by introducing a plant-derived gene encoding β-ketoacyl-ACP synthase that does not contain medium-chain fatty acids as the main accumulated lipid, and a gene encoding acyl-ACP thioesterase having substrate specificity for medium-chain acyl-ACP A method for producing a lipid, which improves the productivity of a medium-chain fatty acid produced by culturing a body or a lipid comprising the medium-chain fatty acid.   Transformation by introducing a plant-derived gene encoding β-ketoacyl-ACP synthase that does not contain medium-chain fatty acids as the main accumulated lipid, and a gene encoding acyl-ACP thioesterase having substrate specificity for medium-chain acyl-ACP A method for modifying a fatty acid composition, wherein the body is cultured and the proportion of medium chain fatty acids in the total fatty acids produced is increased.   The method according to any one of claims 1 to 3, wherein the β-ketoacyl-ACP synthase is β-ketoacyl-ACP synthase II. The method according to any one of claims 1 to 4, wherein the β-ketoacyl-ACP synthase is any one of the following proteins (A) to (R).
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 60.
(B) A protein comprising 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.
(C) A protein consisting of the amino acid sequence represented by SEQ ID NO: 61.
(D) A protein 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 consisting of the amino acid sequence represented by SEQ ID NO: 62.
(F) A protein comprising an amino acid sequence having 85% or more identity with the protein (E) and having β-ketoacyl-ACP synthase activity.
(G) A protein consisting of the amino acid sequence represented by SEQ ID NO: 63.
(H) A protein comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (G) and having β-ketoacyl-ACP synthase activity.
(I) A protein comprising the amino acid sequence represented by SEQ ID NO: 64.
(J) A protein comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (I) and having β-ketoacyl-ACP synthase activity.
(K) A protein consisting of the amino acid sequence represented by SEQ ID NO: 65.
(L) A protein comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (K) and having β-ketoacyl-ACP synthase activity.
(M) A protein consisting of the amino acid sequence represented by SEQ ID NO: 66.
(N) A protein comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (M) and having β-ketoacyl-ACP synthase activity.
(O) A protein consisting of the amino acid sequence represented by SEQ ID NO: 67.
(P) A protein comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (O) and having β-ketoacyl-ACP synthase activity.
(Q) A protein consisting of the amino acid sequence represented by SEQ ID NO: 68.
(R) A protein comprising an amino acid sequence having 85% or more identity with the protein (Q) and having β-ketoacyl-ACP synthase activity. The method according to claim 5, wherein the gene encoding any one of the proteins (A) to (R) is a gene consisting of any one of the following DNA (a) to (r).
(A) DNA consisting of the base sequence represented by SEQ ID NO: 51.
(B) A DNA encoding a protein having a base sequence having an identity of 80% or more with the base sequence of the DNA (a) and having a β-ketoacyl-ACP synthase activity.
(C) DNA consisting of the base sequence represented by SEQ ID NO: 52.
(D) DNA encoding a protein comprising a base sequence having an identity of 80% or more with the base sequence of DNA (c) and having β-ketoacyl-ACP synthase activity.
(E) DNA consisting of the base sequence represented by SEQ ID NO: 53.
(F) DNA encoding a protein comprising a base sequence having 80% or more identity with the base sequence of DNA (e) and having β-ketoacyl-ACP synthase activity.
(G) DNA consisting of the base sequence represented by SEQ ID NO: 54.
(H) DNA encoding a protein comprising a base sequence having an identity of 80% or more with the base sequence of DNA (g) and having β-ketoacyl-ACP synthase activity.
(I) DNA consisting of the base sequence represented by SEQ ID NO: 55.
(J) DNA encoding a protein comprising a base sequence having 80% or more identity with the base sequence of DNA (i) and having β-ketoacyl-ACP synthase activity.
(K) DNA consisting of the base sequence represented by SEQ ID NO: 56.
(L) A DNA encoding a protein comprising a base sequence having 80% or more identity with the base sequence of the DNA (k) and having β-ketoacyl-ACP synthase activity.
(M) DNA consisting of the base sequence represented by SEQ ID NO: 57.
(N) A DNA encoding a protein comprising a base sequence having 80% or more identity with the base sequence of the DNA (m) and having β-ketoacyl-ACP synthase activity.
(O) DNA consisting of the base sequence represented by SEQ ID NO: 58.
(P) A DNA encoding a protein comprising a base sequence having 80% or more identity with the base sequence of the DNA (o) and having β-ketoacyl-ACP synthase activity.
(Q) DNA consisting of the base sequence represented by SEQ ID NO: 59.
(R) A DNA encoding a protein having a base sequence with 80% or more identity with the base sequence of the DNA (q) and having β-ketoacyl-ACP synthase activity.   The acyl-ACP thioesterase is a protein comprising the amino acid sequence represented by SEQ ID NO: 69 or 73, an amino acid sequence having 50% or more identity with the amino acid sequence of the protein, and medium-chain acyl-ACP A protein having an acyl-ACP thioesterase activity against or an amino acid sequence of the protein in which one or several amino acids are deleted, substituted, inserted or added, and an acyl-ACP thioesterase activity against medium chain acyl-ACP The method of any one of Claims 1-6 which is a protein which has.   The gene encoding the acyl-ACP thioesterase comprises a DNA comprising the nucleotide sequence represented by SEQ ID NO: 70 or SEQ ID NO: 74, or a nucleotide sequence having 50% or more identity with any one nucleotide sequence of the DNA And a DNA encoding a protein having acyl-ACP thioesterase activity for medium-chain acyl-ACP, one or several base sequences are deleted, substituted, inserted or added to any one base sequence of the DNA, And hybridizes under stringent conditions with a DNA encoding a protein having acyl-ACP thioesterase activity for medium-chain acyl-ACP, or a DNA comprising a base sequence complementary to any one of the base sequences. And encoding a protein having acyl-ACP thioesterase activity for medium-chain acyl-ACP The method according to any one of claims 1 to 7, which is a gene comprising DNA.   The method according to claim 1, wherein the transformant is a plant transformant.   The method according to claim 9, wherein the plant is Arabidopsis thaliana.   The method of any one of claims 1 to 10, wherein the lipid comprises a free medium chain fatty acid or a fatty acid ester compound thereof.   A gene encoding a plant-derived β-ketoacyl-ACP synthase that does not contain medium-chain fatty acids as the main accumulation lipid, and a gene encoding an acyl-ACP thioesterase having substrate specificity for medium-chain acyl-ACP, Transformant.   The transformant according to claim 12, wherein the β-ketoacyl-ACP synthase is β-ketoacyl-ACP synthase II. The transformant according to claim 12 or 13, wherein the β-ketoacyl-ACP synthase is any one of the following proteins (A) to (R).
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 60.
(B) A protein comprising 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.
(C) A protein consisting of the amino acid sequence represented by SEQ ID NO: 61.
(D) A protein 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 consisting of the amino acid sequence represented by SEQ ID NO: 62.
(F) A protein comprising an amino acid sequence having 85% or more identity with the protein (E) and having β-ketoacyl-ACP synthase activity.
(G) A protein consisting of the amino acid sequence represented by SEQ ID NO: 63.
(H) A protein comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (G) and having β-ketoacyl-ACP synthase activity.
(I) A protein comprising the amino acid sequence represented by SEQ ID NO: 64.
(J) A protein comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (I) and having β-ketoacyl-ACP synthase activity.
(K) A protein consisting of the amino acid sequence represented by SEQ ID NO: 65.
(L) A protein comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (K) and having β-ketoacyl-ACP synthase activity.
(M) A protein consisting of the amino acid sequence represented by SEQ ID NO: 66.
(N) A protein comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (M) and having β-ketoacyl-ACP synthase activity.
(O) A protein consisting of the amino acid sequence represented by SEQ ID NO: 67.
(P) A protein comprising an amino acid sequence having 85% or more identity with the amino acid sequence of the protein (O) and having β-ketoacyl-ACP synthase activity.
(Q) A protein consisting of the amino acid sequence represented by SEQ ID NO: 68.
(R) A protein comprising an amino acid sequence having 85% or more identity with the protein (Q) and having β-ketoacyl-ACP synthase activity. The transformant according to claim 14, wherein the gene encoding any one of the proteins (A) to (R) is a gene consisting of any one of the following DNA (a) to (r).
(A) DNA consisting of the base sequence represented by SEQ ID NO: 51.
(B) A DNA encoding a protein having a base sequence having an identity of 80% or more with the base sequence of the DNA (a) and having a β-ketoacyl-ACP synthase activity.
(C) DNA consisting of the base sequence represented by SEQ ID NO: 52.
(D) DNA encoding a protein comprising a base sequence having an identity of 80% or more with the base sequence of DNA (c) and having β-ketoacyl-ACP synthase activity.
(E) DNA consisting of the base sequence represented by SEQ ID NO: 53.
(F) DNA encoding a protein comprising a base sequence having 80% or more identity with the base sequence of DNA (e) and having β-ketoacyl-ACP synthase activity.
(G) DNA consisting of the base sequence represented by SEQ ID NO: 54.
(H) DNA encoding a protein comprising a base sequence having an identity of 80% or more with the base sequence of DNA (g) and having β-ketoacyl-ACP synthase activity.
(I) DNA consisting of the base sequence represented by SEQ ID NO: 55.
(J) DNA encoding a protein comprising a base sequence having 80% or more identity with the base sequence of DNA (i) and having β-ketoacyl-ACP synthase activity.
(K) DNA consisting of the base sequence represented by SEQ ID NO: 56.
(L) A DNA encoding a protein comprising a base sequence having 80% or more identity with the base sequence of the DNA (k) and having β-ketoacyl-ACP synthase activity.
(M) DNA consisting of the base sequence represented by SEQ ID NO: 57.
(N) A DNA encoding a protein comprising a base sequence having 80% or more identity with the base sequence of the DNA (m) and having β-ketoacyl-ACP synthase activity.
(O) DNA consisting of the base sequence represented by SEQ ID NO: 58.
(P) A DNA encoding a protein comprising a base sequence having 80% or more identity with the base sequence of the DNA (o) and having β-ketoacyl-ACP synthase activity.
(Q) DNA consisting of the base sequence represented by SEQ ID NO: 59.
(R) A DNA encoding a protein having a base sequence with 80% or more identity with the base sequence of the DNA (q) and having β-ketoacyl-ACP synthase activity.   The acyl-ACP thioesterase is a protein comprising the amino acid sequence represented by SEQ ID NO: 69 or 73, an amino acid sequence having 50% or more identity with the amino acid sequence of the protein, and medium-chain acyl-ACP A protein having an acyl-ACP thioesterase activity against or an amino acid sequence of the protein in which one or several amino acids are deleted, substituted, inserted or added, and an acyl-ACP thioesterase activity against medium chain acyl-ACP The transformant of any one of Claims 12-15 which is protein which has.   The gene encoding the acyl-ACP thioesterase comprises a DNA comprising the nucleotide sequence represented by SEQ ID NO: 70 or SEQ ID NO: 74, or a nucleotide sequence having 50% or more identity with any one nucleotide sequence of the DNA And a DNA encoding a protein having acyl-ACP thioesterase activity for medium-chain acyl-ACP, one or several base sequences are deleted, substituted, inserted or added to any one base sequence of the DNA, And hybridizes under stringent conditions with a DNA encoding a protein having acyl-ACP thioesterase activity for medium-chain acyl-ACP, or a DNA comprising a base sequence complementary to any one of the base sequences. And encoding a protein having acyl-ACP thioesterase activity for medium-chain acyl-ACP The transformant of any one of Claims 12-16 which is a gene which consists of DNA.   The transformant according to any one of claims 12 to 17, which is a transformant of a plant.   The transformant according to claim 18, wherein the plant is Arabidopsis thaliana.