Lipid manufacturing methods

Abstract

The present invention provides a method for producing lipids that improve the buoyancy of microalgae and thus the recovery efficiency, and a transformed microalgae with improved buoyancy. [Solution] A method for producing lipids, comprising culturing a transformed microalgae that has undergone at least one modification selected from the group consisting of (A) to (C) below, producing fatty acids or lipids having fatty acids as components, recovering the transformed bodies by flotation separation, and obtaining lipids from the recovered transformed bodies. (A) Modifications that promote the expression of at least one gene encoding a triacylglycerol synthesis pathway-related protein. (B) Modifications that promote the expression of at least one gene encoding a Calvin cycle-related protein. (C) Modifications that suppress the expression of at least one gene encoding a cell wall synthesis pathway-related protein.

Description

[Technical Field]

[0001] This invention relates to a method for producing lipids. Furthermore, this invention relates to a transformed organism used in said method. [Background technology]

[0002] Fatty acids are a major component of lipids and constitute lipids such as triacylglycerols (hereinafter simply referred to as "TAGs"), which are produced in living organisms through esterification with glycerol. Furthermore, fatty acids are stored and utilized as an energy source in many plants and animals. Fatty acids and lipids stored in plants and animals are widely used for food or industrial purposes. For example, derivatives of higher alcohols obtained by reducing higher fatty acids with approximately 12 to 18 carbon atoms are used as surfactants. Alkyl sulfate salts and alkylbenzene sulfonates are used as anionic surfactants. Polyoxyalkylene alkyl ethers and alkyl polyglycosides are used as nonionic surfactants. All of these surfactants are used as detergents or disinfectants. Similarly, cationic surfactants such as alkylamine salts and mono- or dialkyl quaternary ammonium salts, which are also derivatives of higher alcohols, are routinely used as textile treatment agents, hair rinses, or disinfectants. Benzalkonium-type quaternary ammonium salts are also routinely used as disinfectants and preservatives. Furthermore, plant-derived oils and fats are used as raw materials for biodiesel fuel. Furthermore, long-chain fatty acids with 18 or more carbon atoms have different chemical properties depending on the number of carbon atoms and the degree of unsaturation. For example, many long-chain polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid are essential fatty acids that cannot be synthesized in the body of animals and are used in functional foods. As such, fatty acids and lipids have a wide range of uses. Therefore, attempts are being made to improve the productivity of fatty acids and lipids in living organisms such as plants.

[0003] In recent years, research on renewable energy has been promoted in order to realize a sustainable society. Photosynthetic microorganisms, in particular, are expected to have a carbon dioxide reduction effect and to serve as biofuel organisms that do not compete with crops. In recent years, algae have attracted particular attention for their usefulness in biofuel production. Algae are being hailed as a next-generation biomass resource because they can produce lipids usable as biodiesel fuel through photosynthesis, and they do not compete with food sources. Furthermore, some reports suggest that algae possess higher lipid production and storage capabilities compared to plants.

[0004] Generally, when producing lipids using microalgae, for example, the cultured microalgae are separated and recovered using a centrifuge, and the lipids are extracted from the recovered microalgae. However, this recovery method has the disadvantage of high manufacturing costs from the perspective of equipment investment. In addition, when separating and recovering algal bodies using a centrifuge, algal bodies with low oil production and high specific gravity are preferentially settled and recovered, while algal bodies with high oil production and low specific gravity are less likely to settle and are difficult to recover. Therefore, methods have been proposed that are lower cost, simpler, or selectively recover algal bodies with high oil production. For example, Patent Document 1 contains Pseudochorycystis ( Pseudochoricystis ) Genus or Corycystis ( Choricystis A method for separating and recovering hydrocarbon-producing microalgae belonging to the genus ) from raw water containing said microalgae is disclosed, comprising: a hydroxide generation step of adding a soluble metal salt that generates sparingly soluble hydroxides to the raw water containing the microalgae, and then adjusting the pH of the raw water to a level at which sparingly soluble hydroxides are generated; a flocculation step of flocculating the microalgae with the precipitated sparingly soluble hydroxides; and a solid-liquid separation step of separating the flocculated flocs from the solid-liquid. Furthermore, Patent Document 2 describes Botryococcus in liquid ( BotryococcusA method for separating oil-producing Botryococcus algae is disclosed, which includes the steps of irradiating the algae with a heavy ion beam or X-rays and collecting the suspended oil-producing Botryococcus algae. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2014-100121 [Patent Document 2] Japanese Patent Publication No. 2017-136000 [Overview of the project] [Problems that the invention aims to solve]

[0006] As mentioned above, methods for improving recovery efficiency by agglomerating microalgae have been disclosed. However, especially when performing outdoor cultivation using an open-pond system or large-scale cultivation using a large photobioreactor, the growth rate of individual microalgae varies greatly. Therefore, the method described in Patent Document 1 recovers not only microalgae that have accumulated sufficient lipids, but also microalgae that have not yet accumulated sufficient lipids, making it impossible to selectively recover microalgae that have accumulated sufficient lipids. Furthermore, while the method described in Patent Document 2 allows for the selective recovery of algal bodies with high oil production, it requires irradiation of the algal bodies with heavy ion beams or X-rays, resulting in high equipment costs and making it unsuitable for outdoor cultivation or large-scale cultivation.

[0007] The object of this invention is to provide a method for producing lipids that improves the buoyancy of microalgae and thereby improves the recovery efficiency. Furthermore, the present invention aims to provide a transformed organism in which the buoyancy of microalgae is improved. [Means for solving the problem]

[0008] In view of the above problems, the inventors conducted thorough research. As a result of a more detailed analysis of the buoyancy of microalgae, the inventors found that the buoyancy improved in proportion to the culture time of the microalgae. It was thought that this improvement in buoyancy occurred because the amount of lipids per cell increased with culture, causing the specific gravity to become lower than that of the culture medium. Based on this consideration, further research was conducted, and the inventors discovered for the first time that by using production strains with improved lipid production capacity or production strains with a lower specific gravity of the cells themselves, the buoyancy of these production strains could be improved, and microalgae that had accumulated sufficient lipids could be selectively, more easily, and at a lower cost. This invention was completed based on these findings.

[0009] The present invention relates to a method for producing lipids, comprising culturing a transformed microalgae that has undergone at least one modification selected from the group consisting of (A) to (C) below, producing fatty acids or lipids composed thereof, recovering the transformed bodies by flotation separation, and obtaining lipids from the recovered transformed bodies. (A) Modifications that promote the expression of at least one gene encoding a protein related to the triacylglycerol synthesis pathway (hereinafter also referred to as the "TAG synthesis pathway"). (B) Modifications that promote the expression of at least one gene encoding a Calvin cycle (hereinafter also referred to as the "CBB cycle")-related protein. (C) Modifications that suppress the expression of at least one gene encoding a cell wall synthesis pathway-related protein.

[0010] The present invention also relates to a method for recovering transformed microalgae, which are transformed microalgae that have undergone at least one modification selected from the group consisting of (A) to (C) below, to produce fatty acids or lipids composed of fatty acids, and to recover the transformed microalgae by flotation separation. (A) Modifications that promote the expression of at least one gene encoding a triacylglycerol synthesis pathway-related protein. (B) Modification to promote the expression of at least one gene encoding a Calvin cycle-related protein (C) Modification to suppress the expression of at least one gene encoding a cell wall synthesis pathway-related protein

[0011] The present invention also relates to a transformant of microalgae modified as described in (A) to (C) above.

Advantages of the Invention

[0012] According to the method for producing lipids and the method for recovering transformants of the present invention, by using a transformant of microalgae with improved buoyancy, algal cells with high lipid production can be recovered more efficiently. In addition, in the transformant of the present invention, the expression of genes encoding TAG synthesis pathway-related proteins and genes encoding CBB cycle-related proteins is promoted, and the expression of genes encoding cell wall synthesis pathway-related proteins is suppressed, so it has excellent buoyancy.

Brief Description of the Drawings

[0013]

Figure 1

Modes for Carrying Out the Invention

[0014] As used herein, "lipids" include neutral lipids (such as triacylglycerol), simple lipids such as waxes and ceramides; complex lipids such as phospholipids, glycolipids, and sulfolipids; and derivative lipids such as fatty acids (free fatty acids), alcohols, and hydrocarbons derived from these lipids. Generally, fatty acids classified as derived lipids refer to the fatty acids themselves and mean "free fatty acids." In this invention, the fatty acid portion in simple lipids and complex lipid molecules is referred to as a "fatty acid residue." Unless otherwise specified, "fatty acid" is used as a general term for both "free fatty acids" and "fatty acid residues" contained in salts or ester compounds, etc. In this specification, "fatty acids or lipids comprising them as components" is used collectively to refer to "free fatty acids" and "lipids having such fatty acid residues." Furthermore, in this specification, "fatty acid composition" means the ratio of the weight of each fatty acid to the weight of all fatty acids (total fatty acids), which is the sum of the free fatty acids and fatty acid residues. The weight (production amount) and fatty acid composition of fatty acids can be measured by the method used in the examples. In this specification, the term "fatty acid" is not particularly limited as long as it is an aliphatic carboxylic acid. For example, it may be a fatty acid with 2 to 22 carbon atoms in the acyl group, a fatty acid with 4 to 22 carbon atoms, a fatty acid with 6 to 22 carbon atoms, a fatty acid with 8 to 22 carbon atoms, a fatty acid with 10 to 22 carbon atoms, or a fatty acid with 12 to 20 carbon atoms. Furthermore, in this specification, when "Cx:y" is used to represent fatty acids or the acyl groups that make up fatty acids, it indicates that the number of carbon atoms is x and the number of double bonds is y. "Cx" represents fatty acids or acyl groups with x carbon atoms.

[0015] In this specification, the identity of base sequences and amino acid sequences is calculated using the Lipman-Pearson method (Science, 1985, vol. 227, pp. 1435-1441). Specifically, it is calculated by performing the homology analysis (Search homology) using the Genetyx-Win genetic information processing software with a Unit size to compare (ktup) of 2. Furthermore, in this specification, "stringent conditions" include, for example, the method described in Molecular Cloning - A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press]. For example, conditions include hybridizing 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 with a probe by incubating at 65°C for 8 to 16 hours. Furthermore, in the specification, the "upstream" of a gene refers to the region following the 5' end of the gene or region being considered, rather than its position from the translation initiation point. On the other hand, the "downstream" of a gene refers to the region following the 3' end of the gene or region being considered.

[0016] In this invention and specification, "buoyancy" means the property or performance of not settling by centrifugation or standing, or floating to the surface of a liquid. Furthermore, in this invention and specification, "improved buoyancy" means that, compared to negative control algal strains (host and wild strains) cultured under the same culture conditions for the same number of days, the proportion and amount of cells that do not settle after a certain centrifugation or standing treatment (including cells that float to the surface of the liquid) are improved, or the proportion and amount of cells that float to the surface of the liquid after a certain centrifugation or standing treatment are improved. The improved buoyancy in the transformants of the present invention can be confirmed by visual inspection or by measuring the number and weight of algal cells in each fraction after subjecting the cultured algal cells to centrifugation or standing. There are no particular restrictions on the centrifugation conditions; for example, centrifugation at 21600 × g for 10 minutes is an example.

[0017] As described above, the transformant used in the lipid production method of the present invention is a transformant obtained by subjecting microalgae to at least one modification selected from the group consisting of (A) to (C) below. (A) Modifications that promote the expression of at least one gene encoding a TAG synthesis pathway-related protein (hereinafter also referred to as the "TAG synthesis pathway gene"). (B) Modifications that promote the expression of at least one gene encoding a CBB circuit-related protein (hereinafter also referred to as the "CBB circuit gene"). (C) Modifications that suppress the expression of at least one gene encoding a cell wall synthesis pathway-related protein (hereinafter also referred to as the "cell wall synthesis pathway gene"). By making any of the above modifications (A) to (C), the resulting transformants have improved buoyancy, thus improving recovery efficiency, and can be suitably used in the lipid production method of the present invention. The transformed product used in the lipid production method of the present invention is preferably modified in two or more of the above (A) to (C) from the viewpoint of improving buoyancy, more preferably modified in the above (A) and / or (B) and the following (C), and even more preferably modified in all of the above (A) to (C) ((A), (B), and (C)).

[0018] The aforementioned "TAG synthesis pathway-related protein" is not particularly limited as long as it is a protein involved in the TAG synthesis pathway, but it is preferably an enzyme that constitutes the TAG synthesis pathway. In the transformed microalgae used in the present invention, by promoting the expression of genes encoding proteins involved in the TAG synthesis pathway, lipid productivity can be improved and buoyancy can be enhanced.

[0019] Examples of TAG synthesis pathway-related proteins include acyl-CoA synthetase (hereinafter also referred to as "ACS"), glycerol-3-phosphate dehydrogenase (hereinafter also referred to as "G3PDH"), glycerol-3-phosphate acyltransferase (hereinafter also referred to as "GPAT"), lysophosphatidic acid acyltransferase (hereinafter also referred to as "LPAAT"), and diacylglycerol acyltransferase (hereinafter also referred to as "DGAT"), as well as phosphatidic acid phosphatase (hereinafter also referred to as "PAP"). In particular, from the viewpoint of improving buoyancy, it is preferable that the expression of the gene encoding ACS or the gene encoding AT (preferably DGAT) is promoted, and it is more preferable that the expression of both the gene encoding ACS and the gene encoding AT (preferably DGAT) is promoted.

[0020] The AT that can be used in the present invention is not particularly limited and can be any protein having acyltransferase activity (hereinafter also referred to as "AT activity"). Here, "AT activity" refers to the activity that catalyzes the acylation of glycerol compounds such as glycerol triphosphate, lysophosphatidic acid, and diacylglycerol. ATs are proteins that catalyze the acylation of glycerol compounds such as glycerol triphosphate, lysophosphatidic acid, and diacylglycerol. Fatty acid acyl-CoA, or acyl-ACP, which is formed by the binding of CoA to a free fatty acid, is incorporated into the glycerol backbone by various ATs and accumulated as TAGs, in which three fatty acid molecules are ester-bonded to one glycerol molecule. Therefore, by promoting the expression of the gene encoding AT, the lipid productivity (especially fatty acid productivity) of the transformant can be improved. As the culture time progresses, the amount of lipids accumulated in the transformant increases, the specific gravity of the transformant decreases, and as a result, the buoyancy of the transformant can be improved.

[0021] The AT activity of the protein used in the present invention can be confirmed, for example, by using a system with a triacylglycerol synthesis gene knockout cell. Alternatively, it can be confirmed by introducing DNA in which the gene encoding the protein is linked downstream of a promoter that functions in the host cell into the host cell, culturing the cells under conditions in which the introduced gene is expressed, and then adding acyl-CoA, various phospholipids, various glycolipids, etc. as donors to the cell lysate along with one of the receptors, glycerol triphosphate (hereinafter also called "G3P"), lysophosphatidic acid (hereinafter also called "LPA"), or diacylglycerol (hereinafter also called "DAG"), and examining whether LPA is synthesized from G3P, DAG from LPA, and TAG from DAG.

[0022] The ATs that can be used in the present invention can be appropriately selected from ordinary ATs or proteins that are functionally equivalent thereto, depending on the type of host, etc. Preferred ATs in the present invention include the following proteins (D) or (E). (D) A protein consisting of the amino acid sequence represented by Sequence ID No. 1. (E) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (D) and having AT activity. The amino acid sequence information of the protein used in this invention, and the sequence information of the gene encoding it, can be obtained, for example, from the National Center for Biotechnology Information (NCBI). The protein (D) consisting of the amino acid sequence represented by Sequence ID No. 1 is Nannochloropsis ( Nannochloropsis Nannochloropsis oceanica, an alga belonging to the genus ( Nannochloropsis oceanica This is AT (DGAT2-8) derived from the NIES-2145 strain. The protein consisting of the amino acid sequence represented by Sequence ID No. 1 (the protein (D)) has AT activity.

[0023] Generally, the amino acid sequence encoding an enzyme protein does not necessarily require the entire sequence to be conserved for enzyme activity to be observed; it is known that there are regions where changes in the amino acid sequence do not affect enzyme activity. In such regions that are not essential for enzyme activity, the enzyme's original activity can be maintained even if mutations such as amino acid deletion, substitution, insertion, or addition are introduced. In this invention, too, a protein in which the desired enzyme activity is maintained and the amino acid sequence of the enzyme protein has been partially mutated can be used.

[0024] Methods for introducing mutations into amino acid sequences include, for example, introducing mutations into the base sequence that codes for the amino acid sequence. Site-directed mutagenesis is one such method. Specific site-directed mutagenesis methods include Splicing overlap extension (SOE) PCR (Horton et al., Gene 77, 61-68, 1989), ODA method (Hashimoto-Gotoh et al., Gene, 152, 271-276, 1995), and Kunkel method (Kunkel, TA, Proc. Natl. Acad. Sci. USA, 1985, 82, 488). Commercially available kits such as the Site-Directed Mutagenesis System Mutan-SuperExpress Km kit (Takara Bio), Transformer™ Site-Directed Mutagenesis kit (Clontech), and KOD-Plus-Mutagenesis Kit (Toyobo) can also be used. Furthermore, after introducing random gene mutations, the target gene can be obtained by evaluating enzyme activity and performing genetic analysis using an appropriate method.

[0025] In the protein (E), from the viewpoint of AT activity, the identity of the amino acid sequence of the protein (D) with that of the protein (E) is 60% or 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 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Furthermore, as protein (E), examples include proteins in which one or more amino acids (for example, 1 to 145, preferably 1 to 127, more preferably 1 to 108, more preferably 1 to 90, more preferably 1 to 72, more preferably 1 to 54, more preferably 1 to 36, more preferably 1 to 25, more preferably 1 to 18, more preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 3) are deleted, substituted, inserted, or added to the amino acid sequence of protein (D), and which have AT activity. Examples of the protein (E) include a protein consisting of the amino acid sequence represented by SEQ ID NO: 85, or a protein consisting of an amino acid sequence that is 75% (preferably 80%, preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) identical to the amino acid sequence represented by SEQ ID NO: 85, and that has AT activity. The protein consisting of the amino acid sequence represented by SEQ ID NO: 85 is Nannochloropsis gaditana ( Nannochloropsis gaditana This is AT (DGAT) derived from ). The amino acid sequence represented by SEQ ID NO: 85 and the amino acid sequence represented by SEQ ID NO: 1 (amino acid sequence of protein (D)) have an identity of 81% and a similarity of 90%.

[0026] Furthermore, the AT that can be used in the present invention may be a protein consisting of an amino acid sequence to which a signal peptide involved in protein transport, or a known amino acid sequence that enhances protein stability, is added to the amino acid sequences of proteins (D) and (E).

[0027] The aforementioned proteins (D) and (E) can be obtained by conventional chemical methods, genetic engineering methods, etc. For example, natural product-derived proteins can be obtained by isolating and purifying them from Nannochloropsis oceanica. Alternatively, the aforementioned proteins (D) and (E) can be obtained by artificial chemical synthesis based on the amino acid sequence information shown in Sequence ID No. 1. Or, the aforementioned proteins (D) and (E) may be produced as recombinant proteins by genetic engineering technology. When producing recombinant proteins, the gene encoding AT, described later, can be used. Furthermore, the AT used in this invention may be one type, or two or more types of AT may be used in combination. Algae such as Nannochloropsis oceanica can be obtained from private or public research institutes and other conservation facilities. For example, strain NIES-2145 of Nannochloropsis oceanica can be obtained from the National Institute for Environmental Studies (NIES).

[0028] Examples of genes encoding the aforementioned AT (preferably the aforementioned protein (D) or (E)) (hereinafter also referred to as the "AT gene") include genes consisting of the following DNA(d) or (e). (d) DNA consisting of the base sequence represented by Sequence ID No. 2. (e) DNA comprising a base sequence that is 60% or more identical to the base sequence of DNA(d) and encoding a protein having AT activity. The base sequence represented by Sequence ID No. 2 is the base sequence of the gene (hereinafter also referred to as the "DGAT2-8 gene") that codes for the protein (DGAT2-8) consisting of the amino acid sequence represented by Sequence ID No. 1.

[0029] In the DNA(e) mentioned above, from the viewpoint of AT activity, the identity with the base sequence of DNA(d) is 60% or 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 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Furthermore, as DNA(e), DNA is also preferred in which one or more bases (for example, 1 to 526 bases, preferably 1 to 436 bases, preferably 1 to 382 bases, more preferably 1 to 327 bases, more preferably 1 to 273 bases, more preferably 1 to 218 bases, more preferably 1 to 163 bases, more preferably 1 to 109 bases, more preferably 1 to 76 bases, more preferably 1 to 54 bases, more preferably 1 to 32 bases, more preferably 1 to 21 bases, more preferably 1 to 10 bases) are deleted, substituted, inserted, or added in the base sequence of DNA(d), and which encodes a protein having AT activity. Furthermore, as DNA(e), DNA that hybridizes with DNA having a base sequence complementary to DNA(d) under stringent conditions and encodes a protein having AT activity is also preferred. Examples of DNA(e) include DNA consisting of the base sequence represented by Sequence ID No. 86, or DNA encoding a protein having AT activity, consisting of a base sequence that is 80% (preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identical to the base sequence represented by Sequence ID No. 86. The DNA consisting of the base sequence represented by Sequence ID No. 86 is a gene encoding AT (DGAT) derived from Nannochloropsis gaditana. The identity between the base sequence represented by Sequence ID No. 86 and the base sequence represented by Sequence ID No. 2 (the base sequence of DNA(d)) is 75%.

[0030] Furthermore, the AT gene that can be used in the present invention may be a gene consisting of a base sequence to which a signal peptide involved in protein transport, or a known amino acid sequence that enhances protein stability, is added to the base sequence of DNA(d) or (e).

[0031] Mutations include base deletions, substitutions, additions, or insertions. Methods for introducing mutations into a base sequence include, for example, site-directed mutagenesis. Specific site-directed mutagenesis methods include SOE-PCR, ODA, and Kunkel. Commercially available kits such as the Site-Directed Mutagenesis System Mutan-SuperExpress Km kit (Takara Bio), Transformer™ Site-Directed Mutagenesis kit (Clontech), and KOD-Plus-Mutagenesis Kit (Toyobo) can also be used. Alternatively, after introducing random gene mutations, the target gene can be obtained by evaluating enzyme activity and performing gene analysis using an appropriate method.

[0032] The aforementioned DNA(d) and (e) can be obtained by conventional genetic engineering techniques. For example, the AT gene can be artificially synthesized based on the amino acid sequence represented by SEQ ID NO: 1 or the base sequence represented by SEQ ID NO: 2. The synthesis of the AT gene can be performed using services such as those provided by Invitrogen. Alternatively, it can be obtained by cloning from the genomes of algae or plants that possess the AT gene, such as Nannochloropsis oceanica. For example, this can be done by the method described in Molecular Cloning - A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press (2001)]. Furthermore, depending on the type of host used, a portion of the base sequence represented by Sequence ID No. 2 may be optimized. For example, Thermo Fisher Scientific's GeneArt artificial gene synthesis service can be used. Information on codons used by various organisms can be obtained from sources such as the Codon Usage Database (www.kazusa.or.jp / codon / ). Furthermore, the AT gene used in this invention may be a single gene, or two or more AT genes may be used in combination.

[0033] The ACS that can be used in this invention is not particularly limited and can be any protein having acyl-CoA synthetase activity (hereinafter also referred to as "ACS activity"). Here, "ACS activity" means the activity of binding free fatty acids and CoA to produce acyl-CoA. ACS is a protein that adds CoA to biosynthesized fatty acids (free fatty acids) and is involved in the production of acyl-CoA. Therefore, by promoting the expression of ACS, the lipid productivity (especially fatty acid productivity) of the transformants used in lipid production can be improved. As the culture time progresses, the amount of lipids accumulated in the transformants increases, the specific gravity of the transformants decreases, and the buoyancy of the transformants can be improved.

[0034] The ACS activity of the protein used in this invention can be confirmed, for example, by using a system with an ACS synthesis gene knockout cell. Alternatively, it can be confirmed by introducing DNA in which the gene encoding the protein is linked downstream of a promoter that functions in host cells into an ACS synthesis gene knockout cell, culturing it in a minimal salt medium with free fatty acids as the single carbon source, and examining whether growth can be restored (whether or not it can grow by utilizing (assimilating) the free fatty acids in the medium). Alternatively, it can be confirmed by preparing an ACS protein or a cell lysate containing it, reacting it with a reaction solution containing free fatty acids, CoA, ATP, Mg ions, etc., and measuring the decrease in CoA content using Elman's reagent (DTNB).

[0035] The ACS that can be used in the present invention can be appropriately selected from ordinary ACS and functionally equivalent proteins, depending on the host type, etc. Preferred ACS in the present invention include the following proteins (F) or (G). (F) A protein consisting of the amino acid sequence represented by Sequence ID No. 3. (G) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (F) and having ACS activity. The protein (F), consisting of the amino acid sequence represented by Sequence ID No. 3, is a long-chain acyl-CoA synthetase (hereinafter also simply referred to as "LACS") derived from Nannochloropsis oceanica NIES-2145 strain. The protein (protein (F)), consisting of the amino acid sequence represented by Sequence ID No. 3, has ACS activity.

[0036] In the protein (G), from the viewpoint of ACS activity, the identity of the amino acid sequence of the protein (F) with that of the protein (G) is 60% or 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 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Furthermore, as the protein (G), examples include proteins in which one or more amino acids (for example, 1 to 259, preferably 1 to 226, more preferably 1 to 194, more preferably 1 to 162, more preferably 1 to 129, more preferably 1 to 97, more preferably 1 to 64, more preferably 1 to 45, more preferably 1 to 32, more preferably 1 to 19, more preferably 1 to 12, more preferably 1 to 6) are deleted, substituted, inserted, or added to the amino acid sequence of the protein (F), and which have ACS activity. Examples of the protein (G) include a protein consisting of the amino acid sequence represented by SEQ ID NO: 87, or a protein consisting of an amino acid sequence that is 70% (preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) identical to the amino acid sequence represented by SEQ ID NO: 87, and that has ACS activity. The protein consisting of the amino acid sequence represented by SEQ ID NO: 87 is ACS (LACS) derived from Nannochloropsis gaditana. The amino acid sequence represented by SEQ ID NO: 87 and the amino acid sequence represented by SEQ ID NO: 3 (the amino acid sequence of protein (F)) have an identity of 88% and a similarity of 93%. One method for introducing mutations into an amino acid sequence is the method described above for AT.

[0037] Furthermore, the ACS that can be used in the present invention may be a protein consisting of an amino acid sequence to which a signal peptide involved in protein transport, or a known amino acid sequence that enhances protein stability, is added to the amino acid sequences of the proteins (F) and (G).

[0038] The aforementioned proteins (F) and (G) can be obtained by conventional methods, similar to AT described above. Furthermore, the ACS used in this invention may be one type or a combination of two or more types.

[0039] Examples of genes encoding the aforementioned ACS (preferably the aforementioned protein (F) or (G)) (hereinafter also referred to as the "ACS gene") include genes consisting of the following DNA(f) or (g). (f) DNA consisting of the base sequence represented by Sequence ID No. 4. (g) DNA comprising a base sequence that is 60% or more identical to the base sequence of DNA(f) above, and which encodes a protein having ACS activity. The base sequence represented by Sequence ID No. 4 is the base sequence of the gene that codes for the protein (LACS) consisting of the amino acid sequence represented by Sequence ID No. 3 (hereinafter also referred to as the "LACS gene").

[0040] In the DNA(g) mentioned above, from the viewpoint of ACS activity, the identity with the base sequence of DNA(f) is 60% or 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 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Furthermore, as DNA(g), DNA is also preferred in which one or more bases (for example, 1 to 778 bases, preferably 1 to 681 bases, more preferably 1 to 584 bases, more preferably 1 to 486 bases, more preferably 1 to 389 bases, more preferably 1 to 292 bases, more preferably 1 to 194 bases, more preferably 1 to 136 bases, more preferably 1 to 97 bases, more preferably 1 to 58 bases, more preferably 1 to 38 bases, more preferably 1 to 19 bases) are deleted, substituted, inserted, or added in the base sequence of DNA(f), and which encodes a protein having ACS activity. Furthermore, as DNA(g), DNA that hybridizes with DNA having a base sequence complementary to DNA(f) under stringent conditions and encodes a protein having ACS activity is also preferred. Examples of DNA(g) include DNA consisting of the base sequence represented by Sequence ID No. 88, or DNA encoding a protein having ACS activity, which consists of a base sequence that is 80% (preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identical to the base sequence represented by Sequence ID No. 88. The DNA consisting of the base sequence represented by Sequence ID No. 88 is a gene encoding ACS (LACS) derived from Nannochloropsis gaditana. The identity between the base sequence represented by Sequence ID No. 88 and the base sequence represented by Sequence ID No. 4 (the base sequence of DNA(f)) is 76%. One method for introducing mutations into a base sequence is the method described above for the AT gene.

[0041] Furthermore, the ACS gene that can be used in the present invention may be a gene consisting of a base sequence to which a signal peptide involved in protein transport, or a DNA encoding a known amino acid sequence that enhances protein stability, is added to the base sequence of DNA(f) or (g).

[0042] The aforementioned DNA(f) or (g) can be obtained by conventional methods, similar to the AT gene described above. Furthermore, the ACS gene used in this invention may be a single gene, or two or more ACS genes may be used in combination.

[0043] In addition to the above modification (A), it is preferable that the expression of genes encoding proteins involved in the fatty acid synthesis pathway is also promoted. Proteins involved in such fatty acid synthesis pathways include, for example, acetyl-CoA carboxylase (hereinafter also referred to as "ACC"), acyl carrier protein (hereinafter also referred to as "ACP"), holo-ACP synthase (phosphopantetheinyltransferase), ACP-malonyltransferase (hereinafter also referred to as "MAT"), β-ketoacyl-ACP synthase (hereinafter also referred to as "KAS"), β-ketoacyl-ACP reductase (hereinafter also referred to as "KAR"), hydroxyacyl-ACP dehydratase (hereinafter also referred to as "HD"), enoyl-ACP reductase (hereinafter also referred to as "EAR"), and acyl-ACP thioesterase (hereinafter also referred to as "TE"). In particular, from the viewpoint of improving buoyancy, it is preferable that the expression of the gene encoding TE (hereinafter also referred to as the "TE gene") is promoted.

[0044] The TE that can be used in this invention is not particularly limited and can be any protein having acyl-ACP thioesterase activity (hereinafter also referred to as "TE activity"). Here, "TE activity" refers to the activity of hydrolyzing the thioester bond of acyl-ACP. TE is an enzyme that hydrolyzes the thioester bond of acyl-ACP synthesized by fatty acid synthases such as KAS, producing free fatty acids. TE completes fatty acid synthesis on ACP, and the cleaved fatty acids are used for the synthesis of polyunsaturated fatty acids and TAGs, among others. Therefore, by promoting the expression of the TE gene in addition to the TAG synthesis pathway gene, the lipid productivity (especially fatty acid productivity) of the transformant can be further improved. As the culture time progresses, the amount of lipids accumulated in the transformant increases, the specific gravity of the transformant decreases, and the buoyancy of the transformant can be improved.

[0045] The presence of TE activity in a protein can be confirmed, for example, by introducing DNA with a TE gene linked downstream of a promoter that functions in host cells such as E. coli into host cells lacking a fatty acid degradation system, culturing the cells under conditions in which the introduced TE gene is expressed, and analyzing the changes in the fatty acid composition of the host cells or culture medium using methods such as gas chromatography. Furthermore, TE activity can be measured by introducing DNA with the TE gene linked downstream of a promoter that functions within host cells such as E. coli into host cells, culturing the cells under conditions that allow the introduced TE gene to be expressed, and then performing a reaction on the cell lysate using various acyl-ACPs prepared by Yuan et al. (Yuan L. et al., Proc. Natl. Acad. Sci. USA, 1995, vol. 92(23), p. 10639-10643) as substrates.

[0046] The TEs that can be used in the present invention can be appropriately selected from ordinary TEs or proteins that are functionally equivalent thereto, depending on the type of host, etc. Preferred TEs in the present invention include the following proteins (H) or (I). (H) A protein consisting of the amino acid sequence represented by Sequence ID No. 5. (I) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (H) and having TE activity. The protein (H) consisting of the amino acid sequence represented by Sequence ID No. 5 is TE derived from Nannochloropsis oceanica NIES-2145 strain. The protein (protein (H)) consisting of the amino acid sequence represented by Sequence ID No. 5 has TE activity.

[0047] In the protein (I), from the viewpoint of TE activity, the identity of the amino acid sequence of the protein (H) with that of the protein (I) is 60% or 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 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Furthermore, as the protein (I), examples include proteins in which one or more amino acids (for example, 1 to 139, preferably 1 to 121, more preferably 1 to 104, more preferably 1 to 87, more preferably 1 to 69, more preferably 1 to 52, more preferably 1 to 34, more preferably 1 to 24, more preferably 1 to 17, more preferably 1 to 10, more preferably 1 to 6, more preferably 1 to 3) are deleted, substituted, inserted, or added to the amino acid sequence of the protein (H), and which have TE activity. One method for introducing mutations into an amino acid sequence is the method described above for AT.

[0048] Furthermore, the TE that can be used in the present invention may be a protein consisting of an amino acid sequence in which a signal peptide involved in protein transport, or a known amino acid sequence that enhances protein stability, is added to the amino acid sequence of protein (H) or (I). Moreover, the TE that can be used in the present invention may be a protein consisting of an amino acid sequence in which the chloroplast translocation signal sequence, which is presumed to be located in the N-terminal region of the amino acid sequence of protein (H) or (I), is changed to another chloroplast translocation signal sequence that functions within the host.

[0049] The aforementioned proteins (H) and (I) can be obtained by conventional methods, similar to AT described above. Furthermore, the TE used in this invention may be one type or a combination of two or more types.

[0050] Examples of genes encoding the aforementioned TE (preferably the protein (H) or (I)) include genes consisting of the following DNA(h) or (i). (h) DNA consisting of the base sequence represented by Sequence ID No. 6. (i) DNA comprising a base sequence that is 60% or more identical to the base sequence of DNA(h) and that encodes a protein having TE activity. The base sequence represented by Sequence ID No. 6 is the base sequence of a gene that codes for a protein consisting of the amino acid sequence represented by Sequence ID No. 5.

[0051] In the DNA(i) described above, from the viewpoint of TE activity, the identity with the base sequence of the DNA(h) is 60% or 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 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Furthermore, as DNA(i), DNA is also preferred in which one or more bases (for example, 1 to 418 bases, preferably 1 to 366 bases, more preferably 1 to 314 bases, more preferably 1 to 261 bases, more preferably 1 to 209 bases, more preferably 1 to 157 bases, more preferably 1 to 104 bases, more preferably 1 to 73 bases, more preferably 1 to 52 bases, more preferably 1 to 31 bases, more preferably 1 to 20 bases, more preferably 1 to 10 bases) are deleted, substituted, inserted, or added in the base sequence of DNA(h), and which encodes a protein having TE activity. Furthermore, as DNA(i), DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to DNA(h) and encodes a protein having TE activity is also preferred. One method for introducing mutations into a base sequence is the method described above for the AT gene.

[0052] Furthermore, the TE gene that can be used in the present invention may be a gene consisting of a base sequence in which DNA encoding a signal peptide involved in protein transport, or a known amino acid sequence that enhances protein stability, is added to the base sequence of DNA(h) or (i). Moreover, the TE gene that can be used in the present invention may be a DNA consisting of a base sequence in which the base sequence encoding a chloroplast transition signal sequence, which is presumed to be located in the 5' region of the base sequence of DNA(h) or (i), is changed to a base sequence encoding another chloroplast transition signal sequence that functions within the host.

[0053] The aforementioned DNA(h) or (i) can be obtained by conventional methods, similar to the AT gene described above. Furthermore, the TE gene used in this invention may be a single gene, or a combination of two or more TE genes may be used.

[0054] The aforementioned "CBB cycle-related protein" is not particularly limited as long as it is a protein involved in the CBB cycle, but it is preferably an enzyme that constitutes the CBB cycle. Algae, including plants and photosynthetic microorganisms, are known to perform carbon fixation through photosynthesis via the CBB cycle. The CBB cycle consists of 13 reaction steps, with one molecule of CO2 fixed in each reaction cycle. The resulting photosynthetic products are used not only as building blocks for organisms but also as an energy source. Therefore, by strengthening the CBB cycle and increasing the photosynthetic capacity of microalgae, lipid productivity can be increased, and the buoyancy of microalgae can be improved.

[0055] Examples of CBB cycle-related proteins include transketolase (hereinafter also referred to as "TK"), fructose-1,6-bisphosphate aldolase (hereinafter also referred to as "FBA"), ribose-5-phosphate isomerase (hereinafter also referred to as "RPI"), ribulose-1,5-bisphosphate carboxylase / oxygenase, sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and triosephosphate isomerase. Examples include isomerase, fructose-1,6-bisphosphatase, ribulose-5-phosphate epimerase, and Rubisco activase. In particular, from the viewpoint of improving buoyancy, it is preferable that the expression of at least one gene selected from the group consisting of TK, FBA, and RPI is promoted. Furthermore, it is preferable that the expression of the gene encoding TK is promoted, more preferably that the expression of the gene encoding TK and the gene encoding FBA is promoted, and even more preferably that the expression of the gene encoding TK, the gene encoding FBA, and the gene encoding RPI is promoted.

[0056] The TK that can be used in the present invention is not particularly limited and any protein having transketolase activity (hereinafter also referred to as "TK activity") is acceptable. Here, "TK activity" means the activity of rearranging the ketol group of a ketose to the aldehyde group of an aldose. TK is a protein (enzyme) that catalyzes the reactions in the CBB cycle that produce erythritol-4-phosphate and xylulose-5-phosphate from fructose-6-phosphate and glyceraldehyde-3-phosphate, and the reactions that produce xylulose-5-phosphate and ribose-5-phosphate from sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate. Therefore, by promoting the expression of the gene encoding TK, the CBB circuit is strengthened, which increases the amount of lipids accumulated in the transformants over time, reducing the specific gravity of the transformants and consequently improving their buoyancy.

[0057] The TK activity of the protein used in this invention can be confirmed, for example, by the method described in Plant Physiol. (1989) 90, 814-819. Specifically, it can be confirmed by preparing a solution containing the target protein by conventional methods, mixing it with fructose-6-phosphate and glyceraldehyde-3-phosphate to produce erythrose-4-phosphate and xylulose-5-phosphate, or by mixing it with sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate to produce xylulose-5-phosphate and ribose-5-phosphate.

[0058] The TK that can be used in the present invention can be appropriately selected from ordinary TKs or proteins functionally equivalent thereto, depending on the host type, etc. Preferred TKs in the present invention include the following proteins (J) or (K). (J) A protein consisting of the amino acid sequence represented by Sequence ID No. 7. (K) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (J) and possessing TK activity. The protein (J) consisting of the amino acid sequence represented by Sequence ID No. 7 is a TK derived from Nannochloropsis oceanica NIES-2145 strain. The protein (protein (J)) consisting of the amino acid sequence represented by Sequence ID No. 7 has TK activity.

[0059] In the protein (K), from the viewpoint of TK activity, the identity of the amino acid sequence of the protein (J) with that of the protein (K) is 60% or 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 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Furthermore, as the protein (K), examples include proteins in which one or more amino acids (for example, 1 to 289, preferably 1 to 253, more preferably 1 to 216, more preferably 1 to 180, more preferably 1 to 144, more preferably 1 to 108, more preferably 1 to 72, more preferably 1 to 50, more preferably 1 to 36, more preferably 1 to 21, more preferably 1 to 14, more preferably 1 to 7) are deleted, substituted, inserted, or added to the amino acid sequence of the protein (J), and which have TK activity. Examples of the protein (K) include a protein consisting of the amino acid sequence represented by SEQ ID NO: 89, or a protein consisting of an amino acid sequence that is 70% (preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) identical to the amino acid sequence represented by SEQ ID NO: 89, and that has TK activity. The protein consisting of the amino acid sequence represented by SEQ ID NO: 89 is a TK derived from Nannochloropsis gaditana. The amino acid sequence represented by SEQ ID NO: 89 and the amino acid sequence represented by SEQ ID NO: 7 (the amino acid sequence of protein (J)) have an identity of 91% and a similarity of 95%. One method for introducing mutations into an amino acid sequence is the method described above for AT.

[0060] Furthermore, the TK that can be used in the present invention may be a protein consisting of an amino acid sequence in which a signal peptide involved in protein transport, or a known amino acid sequence that enhances protein stability, is added to the amino acid sequences of proteins (J) and (K). Moreover, the TK that can be used in the present invention may be a protein consisting of an amino acid sequence in which the chloroplast localization signal sequence, which is presumed to be located in the N-terminal region of the amino acid sequences of proteins (J) and (K), is changed to another chloroplast localization signal sequence that functions within the host. Localization prediction using ChloroP (www.cbs.dtu.dk / services / ChloroP / ) predicts that the amino acid sequence from positions 1 to 63 of SEQ ID NO: 7 is the chloroplast localization signal sequence, and the inventors have confirmed that by adding the amino acid sequence from positions 1 to 100 of SEQ ID NO: 7 to the N-terminus of the reporter protein, the reporter protein can be localized to chloroplasts.

[0061] The aforementioned proteins (J) and (K) can be obtained by conventional methods, similar to AT described above. Furthermore, the TK used in this invention may be one type or a combination of two or more types of TK.

[0062] Specific examples of the gene encoding the TK (preferably the protein (J) or (K)) (hereinafter also referred to as the "TK gene") include the gene consisting of the following DNA (j) or (k). (j) DNA consisting of the base sequence represented by Sequence ID No. 8. (k) DNA comprising a base sequence that is 60% or more identical to the base sequence of DNA(j) and encoding a protein having TK activity. The base sequence represented by Sequence ID No. 8 is the base sequence of the gene that codes for the protein (TK) consisting of the amino acid sequence represented by Sequence ID No. 7 (hereinafter also referred to as the "TK gene").

[0063] In the DNA(k) mentioned above, from the viewpoint of TK activity, the identity with the base sequence of the DNA(j) is 60% or 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 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Furthermore, as the DNA(k), a gene encoding a protein having TK activity is also preferred, in which one or more bases (for example, 1 to 868, preferably 1 to 760, more preferably 1 to 651, more preferably 1 to 543, more preferably 1 to 434, more preferably 1 to 325, more preferably 1 to 217, more preferably 1 to 152, more preferably 1 to 108, more preferably 1 to 65, more preferably 1 to 43, more preferably 1 to 21) are deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 8. Furthermore, as DNA(k), a gene encoding a protein having TK activity is also preferred, which hybridizes with DNA having a base sequence complementary to DNA(j) under stringent conditions. Examples of the DNA(k) include DNA consisting of the base sequence represented by Sequence ID No. 90, or DNA encoding a protein having TK activity, consisting of a base sequence that is 75% (preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) identical to the base sequence represented by Sequence ID No. 90. The DNA consisting of the base sequence represented by Sequence ID No. 90 is a gene encoding TK derived from Nannochloropsis gaditana. The identity between the base sequence represented by Sequence ID No. 90 and the base sequence represented by Sequence ID No. 8 (the base sequence of DNA(j)) is 83%. One method for introducing mutations into a base sequence is the method described above for the AT gene.

[0064] Furthermore, the TK gene that can be used in the present invention may be a gene consisting of a base sequence in which a DNA encoding a signal peptide involved in protein transport, or a known amino acid sequence that enhances protein stability, is added to the base sequence of DNA(j) or (k). Moreover, the TK gene that can be used in the present invention may be a DNA consisting of a base sequence in which the base sequence encoding a chloroplast localization signal sequence, which is presumed to be located in the 5' region of the base sequence of DNA(j) or (k), is changed to a base sequence encoding another chloroplast localization signal sequence that functions within the host. Localization prediction using ChloroP (www.cbs.dtu.dk / services / ChloroP / ) predicts that the base sequence from positions 1 to 189 of SEQ ID NO: 8 encodes a chloroplast localization signal sequence, and in fact, the inventors have confirmed that by adding the base sequence from positions 1 to 300 of SEQ ID NO: 8 to the 5' end of the base sequence encoding the reporter protein, the reporter protein can be localized to chloroplasts.

[0065] The aforementioned DNA(j) or (k) can be obtained by conventional methods, similar to the AT gene described above. Furthermore, the TK gene used in this invention may be a single type, or a combination of two or more TK genes may be used.

[0066] The FBA that can be used in the present invention is not particularly limited and may be any protein having fructose-1,6-bisphosphate aldolase activity (hereinafter also referred to as "FBA activity"). Here, "FBA activity" means the activity of condensing glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, or the activity of condensing erythrose-4-phosphate and dihydroxyacetone phosphate. FBA is a protein (enzyme) that catalyzes the reactions in the CBB cycle that produce fructose-1,6-bisphosphate from glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, and the reactions that produce sedoheptulose-1,7-bisphosphate from erythritol-4-phosphate and dihydroxyacetone phosphate. Therefore, by promoting the expression of the gene encoding FBA, the CBB circuit can be strengthened, and as the culture time progresses, the amount of lipids accumulated in the transformants increases, reducing the specific gravity of the transformants and consequently improving the buoyancy of the transformants.

[0067] The presence of FBA activity in the protein used in this invention can be confirmed, for example, by the method described in Plant Physiol. (1989) 90, 814-819. Specifically, this can be confirmed by preparing a solution containing the target protein by conventional methods, mixing it with glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, and analyzing whether fructose-1,6-bisphosphate is produced, or whether sedoheptulose-1,7-bisphosphate is produced from erythritol-4-phosphate and dihydroxyacetone phosphate.

[0068] The FBAs that can be used in this invention can be appropriately selected from ordinary FBAs or proteins that are functionally equivalent thereto, depending on the type of host, etc. Preferred FBAs in this invention include the following proteins (L) or (M). (L) A protein consisting of the amino acid sequence represented by Sequence ID No. 9. (M) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (L) and having FBA activity. The protein (L) consisting of the amino acid sequence represented by Sequence ID No. 9 is FBA derived from Nannochloropsis oceanica NIES-2145 strain. The protein (protein (L)) consisting of the amino acid sequence represented by Sequence ID No. 9 has FBA activity.

[0069] In the protein (M), from the viewpoint of FBA activity, the identity of the amino acid sequence with that of the protein (L) is 60% or 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 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Furthermore, as the protein (M), examples include proteins in which one or more amino acids (for example, 1 to 152, preferably 1 to 133, more preferably 1 to 114, more preferably 1 to 95, more preferably 1 to 76, more preferably 1 to 57, more preferably 1 to 38, more preferably 1 to 26, more preferably 1 to 19, more preferably 1 to 11, more preferably 1 to 7, more preferably 1 to 3) are deleted, substituted, inserted, or added to the amino acid sequence of the protein (L), and which have FBA activity. Examples of the protein (M) include a protein consisting of the amino acid sequence represented by SEQ ID NO: 91, or a protein having FBA activity and consisting of an amino acid sequence that is 65% (preferably 70%, more preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identical to the amino acid sequence represented by SEQ ID NO: 91. The protein consisting of the amino acid sequence represented by SEQ ID NO: 91 is FBA derived from Nannochloropsis gaditana. The amino acid sequence represented by SEQ ID NO: 91 and the amino acid sequence represented by SEQ ID NO: 9 (the amino acid sequence of protein (L)) have an identity of 94% and a similarity of 97%. One method for introducing mutations into an amino acid sequence is the method described above for AT.

[0070] Furthermore, the FBA that can be used in the present invention may be a protein consisting of an amino acid sequence to which a signal peptide involved in protein transport, or a known amino acid sequence that enhances protein stability, has been added to the amino acid sequences of proteins (L) and (M). Moreover, the FBA that can be used in the present invention may be a protein consisting of an amino acid sequence in which the chloroplast localization signal sequence, which is presumed to be located in the N-terminal region of the amino acid sequences of proteins (L) and (M), has been changed to another chloroplast localization signal sequence that functions within the host. Localization prediction using ChloroP (www.cbs.dtu.dk / services / ChloroP / ) predicts that the amino acid sequence from positions 1 to 20, or from positions 1 to 26, of SEQ ID NO: 9 is the chloroplast localization signal sequence, and in fact, the inventors have confirmed that by adding the amino acid sequence from positions 1 to 100 of SEQ ID NO: 9 to the N-terminus of the reporter protein, the reporter protein can be localized to chloroplasts.

[0071] The aforementioned proteins (L) and (M) can be obtained by conventional methods, similar to AT described above. Furthermore, the FBA used in this invention may be one type or a combination of two or more types.

[0072] Specific examples of the gene encoding the FBA (preferably the protein (L) or (M)) (hereinafter also referred to as the "FBA gene") include the gene consisting of the following DNA(l) or (m). (l) DNA consisting of the base sequence represented by Sequence ID No. 10. (m) DNA comprising a base sequence that is 60% or more identical to the base sequence of DNA(l) and encoding a protein having FBA activity. The base sequence represented by Sequence ID No. 10 is the base sequence of the gene that codes for the protein (FBA) consisting of the amino acid sequence represented by Sequence ID No. 9 (hereinafter also referred to as the "FBA gene").

[0073] In the DNA(m) mentioned above, from the viewpoint of FBA activity, the identity with the base sequence of the DNA(l) is 60% or 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 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Furthermore, as the DNA(m), a gene encoding a protein having FBA activity is also preferred, in which one or more bases (for example, 1 to 459, preferably 1 to 402, more preferably 1 to 344, more preferably 1 to 287, more preferably 1 to 229, more preferably 1 to 172, more preferably 1 to 114, more preferably 1 to 80, more preferably 1 to 57, more preferably 1 to 34, more preferably 1 to 22, more preferably 1 to 11) are deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 10. Furthermore, as DNA(m), a gene encoding a protein having FBA activity is also preferred, which hybridizes with DNA having a base sequence complementary to DNA(l) under stringent conditions. Examples of the DNA(m) include DNA consisting of the base sequence represented by Sequence ID No. 92, or DNA encoding a protein having FBA activity, consisting of a base sequence that is 75% (preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identical to the base sequence represented by Sequence ID No. 92. The DNA consisting of the base sequence represented by Sequence ID No. 92 is a gene encoding FBA derived from Nannochloropsis gaditana. The identity between the base sequence represented by Sequence ID No. 92 and the base sequence represented by Sequence ID No. 10 (the base sequence of DNA(l)) is 85%. One method for introducing mutations into a base sequence is the method described above for the AT gene.

[0074] Furthermore, the FBA gene that can be used in the present invention may be a gene consisting of a base sequence in which DNA encoding a signal peptide involved in protein transport, or a known amino acid sequence that enhances protein stability, is added to the base sequence of DNA(l) or (m). Moreover, the FBA gene that can be used in the present invention may be a DNA consisting of a base sequence in which the base sequence encoding a chloroplast localization signal sequence, which is presumed to be located in the 5' region of the base sequence of DNA(l) or (m), is changed to a base sequence encoding another chloroplast localization signal sequence that functions within the host. Localization prediction using ChloroP (www.cbs.dtu.dk / services / ChloroP / ) predicts that the base sequences from positions 1 to 60 or from positions 1 to 78 of SEQ ID NO: 10 encode a chloroplast localization signal sequence, and the inventors have confirmed that by adding the base sequence from positions 1 to 300 of SEQ ID NO: 10 to the 5' end of the base sequence encoding the reporter protein, the reporter protein can be localized to chloroplasts.

[0075] The aforementioned DNA(l) and (m) can be obtained by conventional methods, similar to the AT gene described above. Furthermore, the FBA gene used in this invention may be a single type, or two or more FBA genes may be used in combination.

[0076] The RPI that can be used in the present invention is not particularly limited and any protein having ribose-5-phosphate isomerase activity (hereinafter also referred to as "RPI activity") is acceptable. Here, "RPI activity" means the activity of converting the aldehyde group of an aldose to a keto group. RPI is a protein (enzyme) that catalyzes the reaction that converts ribose-5-phosphate to ribulose-5-phosphate. Therefore, by promoting the expression of the gene encoding RPI, the CBB circuit can be strengthened, increasing the amount of lipids accumulated in the transformants over time, reducing the specific gravity of the transformants, and consequently improving the buoyancy of the transformants.

[0077] The presence of RPI activity in the protein used in this invention can be confirmed, for example, by the method described in The Plant Journal (2006) 48, 606-618. Specifically, this can be confirmed by preparing a solution containing the target protein by conventional methods and analyzing whether ribulose-5-phosphate is produced when it is mixed with ribose-5-phosphate.

[0078] The RPIs that can be used in the present invention can be appropriately selected from ordinary RPIs or proteins that are functionally equivalent thereto, depending on the type of host, etc. Preferred RPIs in the present invention include the following proteins (N) or (O). (N) A protein consisting of the amino acid sequence represented by Sequence ID No. 11. (O) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (N) and having RPI activity. The protein (N), consisting of the amino acid sequence represented by Sequence ID No. 11, is an RPI derived from Nannochloropsis oceanica NIES-2145 strain. The protein (protein (N)), consisting of the amino acid sequence represented by Sequence ID No. 11, has RPI activity.

[0079] In the protein (O), from the viewpoint of RPI activity, the identity of the amino acid sequence of the protein (N) with the protein (O) is 60% or 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 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Furthermore, the protein (O) is a protein in which one or more amino acids (for example, 1 to 112, preferably 1 to 98, more preferably 1 to 84, more preferably 1 to 70, more preferably 1 to 56, more preferably 1 to 42, more preferably 1 to 28, more preferably 1 to 19, more preferably 1 to 14, more preferably 1 to 8, more preferably 1 to 5, more preferably 1 or 2) are deleted, substituted, inserted, or added to the amino acid sequence of the protein (N), and which has RPI activity. Examples of the protein (O) include a protein consisting of the amino acid sequence represented by SEQ ID NO: 93, or a protein consisting of an amino acid sequence that is 70% (preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) identical to the amino acid sequence represented by SEQ ID NO: 93, and that has RPI activity. The protein consisting of the amino acid sequence represented by SEQ ID NO: 93 is RPI derived from Nannochloropsis gaditana. The amino acid sequence represented by SEQ ID NO: 93 and the amino acid sequence represented by SEQ ID NO: 11 (the amino acid sequence of protein (N)) have an identity of 92% and a similarity of 95%. One method for introducing mutations into an amino acid sequence is the method described above for AT.

[0080] Furthermore, the RPI that can be used in the present invention may be a protein consisting of an amino acid sequence to which a signal peptide involved in protein transport, or a known amino acid sequence that enhances protein stability, has been added to the amino acid sequences of the proteins (N) and (O). Moreover, the RPI that can be used in the present invention may be a protein consisting of an amino acid sequence in which the chloroplast localization signal sequence, which is presumed to be located in the N-terminal region of the amino acid sequences of the proteins (N) and (O), has been changed to another chloroplast localization signal sequence that functions within the host. Localization prediction using ChloroP (www.cbs.dtu.dk / services / ChloroP / ) predicts that the amino acid sequence from positions 1 to 49 of SEQ ID NO: 11 is the chloroplast localization signal sequence, and the inventors have confirmed that by adding the amino acid sequence from positions 1 to 100 of SEQ ID NO: 11 to the N-terminus of the reporter protein, the reporter protein can be localized to chloroplasts.

[0081] The aforementioned proteins (N) and (O) can be obtained by conventional methods, similar to AT described above. Furthermore, the RPI used in this invention may be one type, or two or more types of RPI may be used in combination.

[0082] Specific examples of the gene encoding the aforementioned RPI (preferably the protein (N) or (O)) (hereinafter also referred to as the "RPI gene") include the following gene consisting of DNA(n) or (o). (n) DNA consisting of the base sequence represented by Sequence ID No. 12. (o) DNA comprising a base sequence that is 60% or more identical to the base sequence of DNA(n) and that encodes a protein having RPI activity. The base sequence represented by Sequence ID No. 12 is the base sequence of the gene that codes for the protein (RPI) consisting of the amino acid sequence represented by Sequence ID No. 11 (hereinafter also referred to as the "RPI gene").

[0083] In the DNA(o), from the viewpoint of RPI activity, the identity with the base sequence of the DNA(n) is 60% or 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 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Furthermore, as the DNA(o), a gene encoding a protein having RPI activity is also preferred, in which one or more bases (for example, 1 to 339, preferably 1 to 297, more preferably 1 to 254, more preferably 1 to 212, more preferably 1 to 169, more preferably 1 to 127, more preferably 1 to 84, more preferably 1 to 59, more preferably 1 to 42, more preferably 1 to 25, more preferably 1 to 16, more preferably 1 to 8) are deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 12. Furthermore, as DNA(o), a gene encoding a protein having RPI activity is also preferred, which hybridizes under stringent conditions with DNA having a base sequence complementary to DNA(n). Examples of the DNA(o) include DNA consisting of the base sequence represented by Sequence ID No. 94, or DNA encoding a protein having RPI activity, consisting of a base sequence that is 75% (preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identical to the base sequence represented by Sequence ID No. 94. The DNA consisting of the base sequence represented by Sequence ID No. 94 is a gene encoding RPI derived from Nannochloropsis gaditana. The identity between the base sequence represented by Sequence ID No. 94 and the base sequence represented by Sequence ID No. 12 (the base sequence of DNA(n)) is 80%. One method for introducing mutations into a base sequence is the method described above for the AT gene.

[0084] Furthermore, the RPI gene that can be used in the present invention may be a gene consisting of a base sequence in which DNA encoding a signal peptide involved in protein transport, or a known amino acid sequence that enhances protein stability, is added to the base sequences of DNA(n) and (o). Moreover, the RPI gene that can be used in the present invention may be a DNA consisting of a base sequence in which the base sequence encoding a chloroplast localization signal sequence, which is presumed to be located in the 5' region of the base sequences of DNA(n) and (o), is changed to a base sequence encoding another chloroplast localization signal sequence that functions within the host. Localization prediction using ChloroP (www.cbs.dtu.dk / services / ChloroP / ) predicts that the base sequence from positions 1 to 147 of SEQ ID NO: 12 encodes a chloroplast localization signal sequence, and in fact, the inventors have confirmed that by adding the base sequence from positions 1 to 300 of SEQ ID NO: 12 to the 5' end of the base sequence encoding the reporter protein, the reporter protein can be localized to chloroplasts.

[0085] The aforementioned DNA(n) and (o) can be obtained by conventional methods, similar to the AT gene described above. Furthermore, the RPI gene used in this invention may be a single gene, or a combination of two or more RPI genes may be used.

[0086] Transformed cells that have undergone the modifications (A) and / or (B) described above can be obtained, for example, by introducing the TAG synthesis pathway gene and / or the CBB circuit gene into a host by a conventional method. Specifically, they can be produced by preparing a vector or gene expression cassette, such as an expression vector (gene expression plasmid), that can express the gene in host cells, and then introducing this into host cells to transform the host cells. Furthermore, transformants that have undergone the modifications (A) and / or (B) can also be obtained, for example, by modifying the gene expression regulatory region in a host having the TAG synthesis pathway gene and / or CBB circuit gene on its genome using a conventional method to promote the expression of the gene. Specifically, this can be done by replacing the promoter sequence located upstream of the TAG synthesis pathway gene and / or CBB circuit gene present on the host genome with one that exhibits higher promoter activity. In this specification, a modified organism that promotes or suppresses the expression of the gene encoding the target protein is also referred to as a "transformed organism," while an organism that has not been modified to promote or suppress the expression of the gene encoding the target protein is also referred to as a "host organism" or "wild-type strain."

[0087] The transformants that have undergone the modifications (A) and / or (B) described above exhibit improved lipid productivity (particularly fatty acid productivity) compared to the host organism itself. As the culture time progresses, the amount of lipids accumulated within the transformant cells increases, and the specific gravity of the transformants decreases. As a result, the buoyancy of the transformants is improved. Therefore, these transformants can be suitably used in lipid production methods. Furthermore, the fatty acid and lipid productivity of the host and transformed organisms can be measured using the methods described in the examples.

[0088] The vector (plasmid) that serves as the basis for a gene expression plasmid or gene expression cassette can be any vector capable of introducing the gene encoding the target protein into a host and expressing the target gene within the host cell. For example, a vector having an expression regulatory region such as a promoter or terminator depending on the type of host used, and having a replication origin or selection marker, can be used. Furthermore, it may be a vector that autonomously replicates and grows outside the chromosome, such as a plasmid, or a vector that is incorporated into the chromosome.

[0089] Examples of vectors that can be preferably used in the present invention include pUC18 (Takara Bio Inc.), pUC19 (Takara Bio Inc.), pUC118 (Takara Bio Inc.), P66 (Chlamydomonas Center), P-322 (Chlamydomonas Center), pPha-T1 (see Journal of Basic Microbiology, 2011, vol. 51, pp. 666-672), or pJET1 (Cosmo Bio Inc.). In particular, pUC18, pPha-T1, or pJET1 are preferred. Furthermore, the host can be transformed using a DNA fragment (gene expression cassette) consisting of the target gene, promoter, and terminator, referring to the method described in Proceedings of the National Academy of Sciences of the United States of America, 2011, vol. 108(52).

[0090] Furthermore, the type of promoter used to regulate the expression of the gene encoding the target protein incorporated into the vector can be appropriately selected depending on the type of host used. Promoter types that can be preferably used in this invention include housekeeping gene promoters (e.g., tubulin promoter, actin promoter, ubiquitin promoter, etc.), heat shock protein promoters, promoters for Nannochloropsis-derived violaxanthin / chlorophyll α-binding protein genes (VCP1 promoter, VCP2 promoter) (Proceedings of the National Academy of Sciences of the United States of America, 2011, vol. 108(52)), and promoters for Nannochloropsis-derived oleosin-like protein LDSP (lipid droplet surface protein) genes (LDSP promoter) (PLOS Genetics, 2012; 8(11): e1003064. doi: 10. 1371) The promoters of the ACP gene (ACP promoter), the desaturase gene, the AT gene (AT promoter), the glutamine synthase gene from the Nannochloropsis genus (GS promoter), and the ammonium transporter gene from the Nannochloropsis genus (AMT promoter) can be preferably used. Furthermore, since algae belonging to the Nannochloropsis genus, for example, are generally known to efficiently produce lipids under nutrient (especially nitrogen) deficient conditions and high light conditions, it is more preferable to use promoters that strongly express these genes under these conditions. From the viewpoint of strong expression under nutrient deficient conditions and high light conditions, promoters of genes involved in fatty acid synthesis pathways and TAG synthesis pathways and promoters of genes involved in nitrogen assimilation are preferred, the promoters of the LDSP gene, the ACP promoter, the desaturase gene promoter, the AT promoter, the GS promoter, and the AMT promoter are more preferred, and the promoters of the LDSP gene, the GS promoter, and the AMT promoter are even more preferred.

[0091] Furthermore, the type of selection marker used to confirm the incorporation of the gene encoding the target protein can be appropriately selected depending on the type of host used. Preferred selection markers in this invention include drug resistance genes such as ampicillin resistance gene, chloramphenicol resistance gene, erythromycin resistance gene, neomycin resistance gene, kanamycin resistance gene, spectinomycin resistance gene, tetracycline resistance gene, blastosidine S resistance gene, bialafos resistance gene, zeosin resistance gene, paromomycin resistance gene, gentamicin resistance gene, and hygromycin resistance gene. Additionally, deletions of genes related to nutritional requirements can also be used as selection marker genes. Examples of genes related to nutritional requirements include genes involved in nitrate assimilation (nitrate reductase, nitrate transporter). The gene encoding the target protein can be introduced into the vector by conventional methods such as restriction enzyme digestion or ligation.

[0092] The selection of transformants into which the target gene fragment has been introduced can be performed using selection markers. For example, if a drug resistance gene is introduced into the host cell along with the target DNA fragment during transformation, the drug resistance acquired by the transformant can be used as an indicator. Furthermore, the introduction of the target DNA fragment can be confirmed using methods such as PCR with the genome as a template.

[0093] In a host having the aforementioned TAG synthesis pathway gene and / or CBB circuit gene on its genome, a method for promoting the expression of the gene by modifying its expression regulatory region will be described. The "expression regulatory region" refers to the promoter, terminator, and untranslated region, and these sequences are generally involved in regulating the expression levels (transcription and translation levels) of adjacent genes. In hosts that have the TAG synthesis pathway gene and / or CBB circuit gene on their genome, modifying the expression regulatory region of the gene to promote its expression can improve lipid productivity and enhance the buoyancy of the transformant.

[0094] One method for modifying the gene expression regulatory region is, for example, replacing the promoter. In a host having the TAG synthesis pathway gene and / or CBB circuit gene on its genome, the expression of the gene can be promoted by replacing the promoter of the gene with a promoter that has higher transcriptional activity. As the host, species that have the TAG synthesis pathway gene and / or CBB circuit gene in their genome can be preferably used from among the aforementioned species.

[0095] The promoter used for promoter replacement is not particularly limited and can be appropriately selected from those that have higher transcriptional activity than the promoters of the TAG synthesis pathway gene and / or CBB circuit gene and are suitable for lipid production. For example, the tubulin promoter, heat shock protein promoter, violaxanthin / chlorophyll α-binding protein gene promoter (VCP1 promoter, VCP2 promoter), the oleosin-like protein LDSP gene derived from Nannochloropsis, the ACP promoter, the desaturase gene promoter, the AT promoter, the GS promoter, and the AMT promoter can be preferably used. From the viewpoint of improving the productivity of fatty acids or lipids composed of fatty acids, promoters of genes involved in fatty acid synthesis pathways or TAG synthesis pathways and promoters of genes involved in nitrogen assimilation are preferred, the LDSP gene promoter, the ACP promoter, the desaturase gene promoter, the AT promoter, the GS promoter, and the AMT promoter are more preferred, and the LDSP gene promoter, the GS promoter, and the AMT promoter are even more preferred.

[0096] The aforementioned promoter modification can be carried out using conventional methods such as homologous recombination. Specifically, a linear DNA fragment containing the upstream and downstream regions of the target promoter, and containing a different promoter in place of the target promoter, is constructed. This fragment is then taken up by a host cell, and homologous recombination with two crossovers occurs on the upstream and downstream sides of the target promoter in the host genome. As a result, the target promoter on the genome is replaced with the other promoter fragment, thereby modifying the promoter. Such methods for modifying target promoters by homologous recombination can be carried out by referring to literature such as Methods in molecular biology, 1995, vol. 47, pp. 291-302. In particular, when the host is an alga belonging to the genus Nannochloropsis, specific regions in the genome can be modified by homologous recombination by referring to literature such as Proceedings of the National Academy of Sciences of the United States of America, 2011, vol. 108(52).

[0097] The aforementioned "cell wall synthesis pathway-related protein" is not particularly limited as long as it is a protein involved in the cell wall synthesis pathway, but it is preferably an enzyme that constitutes the cell wall synthesis pathway. In this invention and specification, "cell wall" is a concept that includes the arginane layer. In the transformed microalgae used in this invention, suppressing the expression of cell wall synthesis pathway genes inhibits the expression of proteins involved in cell wall synthesis. As a result, cell wall construction is inhibited in the microalgae, and it is believed that the specific gravity decreases, thereby improving the buoyancy of the transformeds. Furthermore, in transformants in which the expression of the cell wall synthesis pathway gene is suppressed, the construction of cell walls such as cellulose-derived cell walls and arginane layers is inhibited, thus improving the efficiency of lipid extraction (recovery efficiency) during the lipid production process. Specifically, it is presumed that cell destruction by physical or chemical treatment becomes easier, making it possible to obtain algae from which lipids can be easily recovered.

[0098] Examples of the aforementioned cell wall synthesis pathway-related proteins include cellulose synthase (hereinafter also referred to as "CES"), polyketide synthase (hereinafter also referred to as "PKS involved in alginane synthesis") which is thought to be involved in the construction of the alginane layer, and UDP-glucose pyrophosphorylase. In particular, from the viewpoint of improving buoyancy, it is preferable that the expression of the gene encoding CES is suppressed.

[0099] In the present invention and this specification, "suppressed gene expression" means that, when cultured under the same conditions, the expression of the target gene in the modified (C) transformant is reduced or lost compared to the expression of the target gene in the host that has not undergone modification (C). The degree of reduction is sufficient if it is lower than the amount of gene expression in the host that has not undergone modification (C). When the expression level is set to 100%, the amount of gene expression in the modified (C) transformant is preferably 90% or less, more preferably 80% or less, more preferably 60% or less, more preferably 40% or less, and even more preferably 20% or less.

[0100] Algal cells are covered by a structure called a cell wall, the main component of which is cellulose. Cellulose is synthesized by a cellulose synthase complex consisting of multiple subunits. The active site of this complex is thought to be subunit A (hereinafter referred to as "CESA"). Therefore, by reducing or eliminating the expression of CES (preferably one of the subunits constituting CES, preferably CESA) in algae, cellulose synthesis is inhibited, which inhibits the construction of the cell wall derived from cellulose, resulting in a lower specific gravity and thus a transformant with improved buoyancy. In this invention, "cellulose synthase" refers to an enzyme (or complex) consisting of a protein expressed from the CES gene, which is involved in the biosynthesis of cellulose. Furthermore, in this specification, "cellulose synthesis activity" (hereinafter also referred to as "CES activity") means the activity that catalyzes the cellulose synthesis reaction.

[0101] The CES whose expression is reduced or lost in this invention is not particularly limited as long as it is a protein (enzyme) that exhibits CES activity. In this invention, the CES whose expression is reduced or lost can be selected as a candidate by, for example, analyzing genomic information with Blast and selecting those annotated as CES. Alternatively, amino acid sequences can be analyzed with Blastp and those annotated as CES can also be selected as candidates. Furthermore, the protein may be confirmed by culturing algae in which the gene encoding the selected protein has been disrupted or deleted and examining the effect on the cellulose layer of the cell wall.

[0102] The CES can be appropriately selected from ordinary CES or functionally equivalent proteins depending on the host type, etc. Preferred CES in the present invention include the following proteins (P) or (Q). (P) A protein consisting of the amino acid sequence represented by Sequence ID No. 13. (Q) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (P) and having CES activity. The protein (P) consisting of the amino acid sequence represented by Sequence ID No. 13 is a protein (hereinafter also referred to as "CES1") that constitutes subunit A of the cellulose synthase complex derived from Nannochloropsis oceanica NIES-2145 strain. The protein (P) consisting of the amino acid sequence represented by Sequence ID No. 13 has CES activity.

[0103] In the protein (Q), from the viewpoint of CES activity, the identity of the amino acid sequence of the protein (P) with the protein (Q) is 60% or 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 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Furthermore, the protein (Q) is a protein in which one or more amino acids (for example, 1 to 268, preferably 1 to 234, more preferably 1 to 201, more preferably 1 to 167, more preferably 1 to 134, more preferably 1 to 100, more preferably 1 to 67, more preferably 1 to 46, more preferably 1 to 33, more preferably 1 to 20, more preferably 1 to 13, more preferably 1 to 6) are deleted, substituted, inserted, or added to the amino acid sequence of the protein (P), and which has CES activity. Examples of the protein (Q) include a protein consisting of the amino acid sequence represented by SEQ ID NO: 95, or a protein having CES activity and consisting of an amino acid sequence that is 75% (preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identical to the amino acid sequence represented by SEQ ID NO: 95. The protein consisting of the amino acid sequence represented by SEQ ID NO: 95 is CES derived from Nannochloropsis gaditana. The amino acid sequence represented by SEQ ID NO: 95 and the amino acid sequence represented by SEQ ID NO: 13 (the amino acid sequence of protein (P)) have an identity of 83% and a similarity of 90%.

[0104] Examples of genes encoding CES (preferably the proteins (P) and (Q)) (hereinafter also referred to as "CES genes") include genes consisting of the following DNA(p) and (q). (p) DNA consisting of the base sequence represented by Sequence ID No. 14. (q) DNA comprising a base sequence that is 60% or more identical to the base sequence of DNA(p) and that encodes a protein having CES activity. The base sequence represented by Sequence ID No. 14 is the base sequence of the gene that codes for the protein (CES1) consisting of the amino acid sequence represented by Sequence ID No. 13 (hereinafter also referred to as the "CES1 gene").

[0105] In the DNA(q), from the viewpoint of CES activity, the identity with the base sequence of the DNA(p) is 60% or 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 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Furthermore, as DNA(q), a gene encoding a protein having CES activity is also preferred, in which one or more bases (for example, 1 to 806 bases, preferably 1 to 705 bases, more preferably 1 to 604 bases, more preferably 1 to 504 bases, more preferably 1 to 403 bases, more preferably 1 to 302 bases, more preferably 1 to 201 bases, more preferably 1 to 141 bases, more preferably 1 to 100 bases, more preferably 1 to 60 bases, more preferably 1 to 40 bases, more preferably 1 to 20 bases) are deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 14. Furthermore, as DNA(q), a gene encoding a protein having CES activity is also preferred, which hybridizes under stringent conditions with DNA having a base sequence complementary to DNA(p). Examples of the DNA(q) include DNA consisting of the base sequence represented by Sequence ID No. 96, or DNA encoding a protein having CES activity, consisting of a base sequence that is 85% (preferably 90%, more preferably 95%, and even more preferably 98%) or more identical to the base sequence represented by Sequence ID No. 96. The DNA consisting of the base sequence represented by Sequence ID No. 96 is a gene encoding CES derived from Nannochloropsis gaditana. The identity between the base sequence represented by Sequence ID No. 96 and the base sequence represented by Sequence ID No. 14 (the base sequence of DNA(p)) is 74%.

[0106] The transformed organism that has undergone the above modification (C) can be obtained, for example, by the following method. This invention describes a method for suppressing the expression of cell wall synthesis pathway genes. In this invention, the method for suppressing the expression of cell wall synthesis pathway genes can be appropriately selected from conventional methods. For example, methods include deleting (partially or in its entirety) the cell wall synthesis pathway gene, downregulating the cell wall synthesis pathway gene, modifying the promoter of the cell wall synthesis pathway gene, and using techniques such as antisense and promoter competition. Among these, it is preferable to suppress the expression of cell wall synthesis pathway genes by deleting or downregulating the cell wall synthesis pathway gene. Alternatively, instead of suppressing the expression of cell wall synthesis pathway genes, methods such as inactivating these genes, or introducing deletions or mutations in cell wall synthesis pathway-related proteins themselves can be appropriately selected. The effects obtained by these methods are equivalent to those obtained by suppressing the expression of cell wall synthesis pathway genes.

[0107] This paper describes a method for suppressing the expression of cell wall synthesis pathway genes by deleting or inactivating those genes. Methods for deleting or inactivating cell wall synthesis pathway genes can be appropriately selected from conventional methods. For example, cell wall synthesis pathway genes can be deleted or inactivated by general methods such as gene disruption methods utilizing the homologous recombination ability of the algae themselves, methods utilizing genome editing technologies such as transcription activator-like effector nucleases (TALENs) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat), and methods for introducing mutations using spontaneous mutations. Specifically, by cloning a DNA fragment containing a portion of the cell wall synthesis pathway gene into a suitable plasmid (vector) and then introducing the resulting circular recombinant plasmid into the cells of algae, it is possible to delete or disrupt the cell wall synthesis pathway gene in the genome through homologous recombination in a portion of the cell wall synthesis pathway gene, thereby inactivating the cell wall synthesis pathway gene. Furthermore, cell wall synthesis pathway genes can be randomly inactivated by methods such as using mutagenic agents like N-methyl-N'-nitro-N-nitrosoguanidine, inducing mutations in cell wall synthesis pathway genes by irradiation with ultraviolet light or gamma rays, inducing site-directed point mutations (e.g., frameshift mutations, in-frame mutations, stop codon insertions, etc.) in cell wall synthesis pathway genes, or replacing all or part of the cell wall synthesis pathway genes with any other DNA fragment (e.g., any select marker). In the present invention, it is preferable to delete or inactivate cell wall synthesis pathway genes on the genome by homologous recombination.

[0108] When deleting or inactivating cell wall synthesis pathway genes by homologous recombination, a homologous recombination plasmid (vector) or homologous recombination DNA cassette of the cell wall synthesis pathway genes is introduced into the algae. The plasmid or DNA cassette for homologous recombination of the cell wall synthesis pathway gene used here targets all or part of the cell wall synthesis pathway gene as the target region. It is preferable to construct a plasmid or DNA cassette having a base sequence homologous to a portion of the upstream part of the genome encoding the target region and a base sequence homologous to a portion of the downstream part of the genome, and then introduce this into the algae. Information on the upstream and downstream nucleotide sequences of the cell wall synthesis pathway genes necessary for homologous recombination can be obtained from sources such as the National Center for Biotechnology Information (NCBI).

[0109] The selection of algae in which cell wall synthesis pathway genes are deleted or inactivated can be performed using selection markers. For example, drug resistance acquired by algae as a result of the introduction of drug resistance genes into host cells can be used as an indicator. Furthermore, the introduction of a target DNA fragment can be confirmed using PCR methods with genomic DNA as a template.

[0110] Furthermore, in order to confirm that the cell wall synthesis pathway gene has been replaced with a homologous recombination plasmid or homologous recombination DNA cassette and that the cell wall synthesis pathway gene has been deleted or inactivated, a selection marker can be appropriately selected from commonly used selection markers to be incorporated into the homologous recombination plasmid or homologous recombination DNA cassette. Preferred selection markers for use in this invention include drug resistance genes such as ampicillin resistance gene, chloramphenicol resistance gene, erythromycin resistance gene, neomycin resistance gene, kanamycin resistance gene, spectinomycin resistance gene, tetracycline resistance gene, blastosidine S resistance gene, biafos resistance gene, zeosin resistance gene, paromomycin resistance gene, and hygromycin resistance gene. In addition, genes related to nutritional requirements can also be used as marker genes. Examples of genes related to nutritional requirements include genes involved in nitrate assimilation (nitrate reductase, nitrate transporter). The introduction of selection markers into vectors can be carried out by conventional methods such as restriction enzyme digestion or ligation.

[0111] The homologous recombination plasmid or homologous recombination DNA cassette used to delete or inactivate cell wall synthesis pathway genes can be prepared using commonly used plasmids (vectors). Examples of usable plasmids include pUC18 (Takara Bio Inc.), pUC19 (Takara Bio Inc.), pUC118 (Takara Bio Inc.), P66 (Chlamydomonas Center), P-322 (Chlamydomonas Center), pPha-T1 (see Journal of Basic Microbiology, 2011, vol. 51, pp. 666-672), or pJET1 (Cosmo Bio Inc.). In particular, pUC18, pPha-T1, or pJET1 are preferred. The size of homologous recombination plasmids or DNA cassettes used for deletion or inactivation of cell wall synthesis pathway genes can be appropriately set considering factors such as the efficiency of introduction into algae and the efficiency of homologous recombination. For example, the upstream or downstream base sequences of the target region used as homologous sequences are preferably 300 bp or more, and more preferably 500 bp or more. The upper limit is preferably 2.5 kbp, and more preferably 2 kbp. The transformation method for introducing the homologous recombination plasmid or homologous recombination DNA cassette into algae can be carried out in the same manner as described above.

[0112] This section describes a method for downregulating cell wall synthesis pathway genes to suppress their expression. For example, by identifying and deleting or inactivating the promoter located upstream of the CES gene, the expression level of the cell wall synthesis pathway gene decreases (downregulation of the cell wall synthesis pathway gene). When the expression level of the cell wall synthesis pathway gene decreases, the expression of proteins involved in cell wall synthesis is inhibited. As a result, it is presumed that cell wall construction is inhibited in the aforementioned algae. Therefore, by reducing the expression level of the cell wall synthesis pathway gene, cell wall synthesis is partially inhibited, and the specific gravity decreases, which is thought to improve the buoyancy of the transformants.

[0113] Methods for downregulating cell wall synthesis pathway genes can be appropriately selected from conventional methods. Examples include methods that induce mutations in the promoter or transcription / translation initiation region of cell wall synthesis pathway genes by mutagenic agents such as N-methyl-N'-nitro-N-nitrosoguanidine, irradiation with UV or gamma rays, methods that insert other arbitrary DNA fragments (e.g., arbitrary repressors, arbitrary selection markers, etc.) into the promoter sequence or transcription / translation initiation region of cell wall synthesis pathway genes, methods that replace all or part of the promoter sequence or transcription / translation initiation region of cell wall synthesis pathway genes with other arbitrary DNA fragments (e.g., arbitrary repressors, arbitrary selection markers, etc.), antisense methods, RNA interference methods, promoter competition, etc.

[0114] The host for the transformed organism can be appropriately selected from those commonly used. The host that can be used in this invention is preferably an alga belonging to the division Heterokonta, and more preferably an alga belonging to the class Euospotacea among the algae belonging to the division Heterokonta. Specific examples of algae belonging to the class Euospotacea include algae of the genus Nannochloropsis, Monodopsis ( Monodopsis Algae of the genus Bisqueria ( Vischeria Algae of the genus Chlorobotris ( Chlorobotrys Algae of the genus Goniochloris ( Goniochloris Examples include algae of the genus Nannochloropsis. Among these, algae of the genus Nannochloropsis are more preferred from the viewpoint of lipid productivity. Specific examples of algae belonging to the genus Nannochloropsis include Nannochloropsis oceanica and Nannochloropsis occulata. Nannochloropsis oculata ), Nannochloropsis gaditana, Nannochloropsis salina ( Nannochloropsis salina ), Nannochloropsis limnetica ( Nannochloropsis limnetica ), Nannochloropsis granulata ( Nannochloropsis granulata ), Nannochloropsis sp. ( NannochloropsisExamples include sp.). Among these, Nannochloropsis oceanica or Nannochloropsis gaditana are preferred from the viewpoint of lipid productivity, and Nannochloropsis oceanica is more preferred.

[0115] The transformation method can be appropriately selected from conventional methods depending on the type of host used. Examples include transformation methods using calcium ions, general competent cell transformation methods, protoplast transformation methods, electroporation methods, LP transformation methods, methods using Agrobacterium, and particle gun methods. When using algae of the genus Nannochloropsis as the host, transformation can also be performed using the electroporation method described in Randor Radakovits, et al., Nature Communications, DOI:10.1038 / ncomms1688, 2012, etc.

[0116] The selection of transformants into which the target gene fragment has been introduced can be performed using selection markers. For example, if a drug resistance gene is introduced into the host cell along with the target DNA fragment during transformation, the drug resistance acquired by the transformant can be used as an indicator. Furthermore, the introduction of the target DNA fragment can be confirmed using methods such as PCR with the genome as a template.

[0117] The transformant used in the lipid production method of the present invention exhibits improved buoyancy compared to the host that has not undergone the modifications (A) to (C) described above. Therefore, by culturing the transformant of the present invention under appropriate conditions and then recovering fatty acids or lipids composed of fatty acids from the resulting culture, fatty acids or lipids composed of fatty acids can be efficiently produced. Here, "culture" refers to the culture medium and transformed organisms after cultivation.

[0118] The culture conditions for the transformant of the present invention can be appropriately selected according to the host of the transformant, and culture conditions commonly used for that host can be used. Furthermore, from the viewpoint of fatty acid production efficiency, glycerol, acetic acid, glucose, etc., as precursors involved in the fatty acid biosynthesis pathway may be added to the culture medium.

[0119] The culture medium used for the above-mentioned cultivation may be based on natural or artificial seawater, or a commercially available culture medium may be used. Specific examples of culture media include f / 2 medium, ESM medium, Daigo IMK medium, L1 medium, MNK medium, etc. Among these, f / 2 medium, ESM medium, or Daigo IMK medium are preferred from the viewpoint of improving lipid productivity and nutrient concentration, f / 2 medium or Daigo IMK medium are more preferred, and f / 2 medium is even more preferred. To promote algal growth and improve fatty acid productivity, nitrogen sources, phosphorus sources, metal salts, vitamins, trace metals, etc., may be added to the culture medium as appropriate. Furthermore, if sodium nitrate is included as a nitrogen source, using a culture medium in which the sodium nitrate is replaced with ammonium bicarbonate (so that the molar amount of nitrogen atoms of the included sodium nitrate is equal to the molar amount of nitrogen atoms of the ammonium bicarbonate) can improve the growth of microalgae and lipid productivity, and as a result, improve buoyancy.

[0120] The amount of transformants inoculated into the culture medium can be selected as appropriate, with 1-50% (vol / vol) per medium being preferred and 1-10% (vol / vol) more preferred from the viewpoint of growth. The culture temperature is not particularly limited as long as it does not adversely affect the growth of algae, but is usually in the range of 5-40°C. From the viewpoint of promoting algal growth, improving fatty acid productivity, and reducing production costs, it is preferably 10-35°C, and more preferably 15-30°C. Furthermore, the cultivation is preferably carried out under light irradiation to enable photosynthesis. The light irradiation can be any condition that allows photosynthesis, and may be artificial light or sunlight. From the viewpoint of promoting algal growth and improving fatty acid productivity, the light intensity during light irradiation is preferably 1 to 4,000 μmol / m². 2In the range of / s, more preferably 10 to 2,500 μmol / m³ 2 In the range of / s, more preferably 100 to 2,500 μmol / m³ 2 range of / s, more preferably 200 to 2,500 μmol / m³ 2 In the range of / s, more preferably 250 to 2,500 μmol / m³ 2 In the range of / s, more preferably 300 to 2,500 μmol / m³ 2 The interval is in the range of / s. Furthermore, there are no particular restrictions on the interval of light irradiation, but from the same viewpoint as above, it is preferable to perform it in the light-dark cycle, with the light period being preferably 8 to 24 hours, more preferably 10 to 18 hours, and even more preferably 12 hours within a 24-hour period. Furthermore, the cultivation is preferably carried out in the presence of a gas containing carbon dioxide to enable photosynthesis, or in a culture medium containing a carbonate such as sodium bicarbonate. The concentration of carbon dioxide in the gas is not particularly limited, but from the viewpoint of promoting growth and improving fatty acid productivity, it is preferably 0.03 (similar to atmospheric conditions) to 10%, more preferably 0.05 to 5%, even more preferably 0.1 to 3%, and even more preferably 0.3 to 1%. The concentration of the carbonate is not particularly limited, but for example, when using sodium bicarbonate, it is preferably 0.01 to 5% by mass, more preferably 0.05 to 2% by mass, and even more preferably 0.1 to 1% by mass, from the viewpoint of promoting growth and improving fatty acid productivity. The culture time is not particularly limited and may be carried out for a long period (for example, about 150 days) to allow algae that accumulate lipids at high concentrations to grow at high concentrations. From the viewpoint of promoting algal growth, improving fatty acid productivity, and reducing production costs, the culture period is preferably 3 to 90 days, more preferably 7 to 30 days, and even more preferably 14 to 21 days. The culture may be carried out by aerated stirring culture, shaking culture, or static culture, but aerated stirring culture is preferred from the viewpoint of improving aeration.

[0121] Furthermore, the transformed algae used in the lipid production method of the present invention have improved buoyancy, making it easy to recover the algae and enabling efficient lipid production even in large-scale cultivation. Examples of such large-scale cultivation include the so-called open-pond method using natural ponds or artificial raceway ponds, and the closed-type method using a photobioreactor. In particular, when culturing microorganisms such as transformants on a small scale in a laboratory, for example, light is irradiated from multiple directions, allowing all cells to efficiently obtain light energy regardless of their degree of buoyancy. However, in an open-pond system, sunlight is usually irradiated from above the culture tank, so sufficient light for photosynthesis does not reach the lower layer of the culture tank. In contrast, the transformants used in the lipid production method of the present invention have improved buoyancy compared to the host, allowing them to float to near the surface of the culture medium at an earlier stage, and thus obtain light energy more efficiently even in an open-pond system, thereby improving lipid productivity. Furthermore, because the transformants used in the lipid production method of the present invention have improved buoyancy, the transformants that have accumulated lipids float in the upper layer (near the liquid surface) of the culture medium. Therefore, even without recovering the entire culture medium, transformants with a high lipid content can be recovered efficiently and simply by recovering only the upper layer of the culture medium. The open-pond system has advantages such as being simpler and having lower equipment costs than a closed-type photobioreactor. Furthermore, when the lipid production method of the present invention is carried out using a closed-type photobioreactor, it has the advantages of requiring less energy for recovery, easy control of culture conditions, and high production efficiency. In particular, when culturing algal bodies by continuous culture (chemostat) as described later, a closed-type photobioreactor allows for more efficient recovery of algal bodies, and also allows for appropriate control of the growth rate of algal bodies in the culture medium and the nutrients in the culture medium. The shape of the above-mentioned closed-type photobioreactor is not particularly limited, and various shapes can be applied, such as tubular, flat panel, Christmas tree, plate, horizontal, model, and porous substrate types.

[0122] The present invention provides a method for producing lipids, which includes a step of recovering algal bodies (transformed microalgae) by flotation separation. In this invention and specification, "flotation separation" means a method of separating the transformants whose specific gravity is lighter than that of the culture medium, as the transformants, whose specific gravity is lighter than that of the culture medium, move to the upper layer of the culture medium due to the difference in specific gravity between the transformants and the culture medium. The method for recovering algal bodies by flotation separation is not particularly limited, and recovery methods by flotation separation used in lipid production methods can be appropriately applied. For example, after centrifuging or allowing the culture solution to stand, the algal bodies that float to the upper layer can be recovered by decantation, or the algal bodies can be recovered by aspirating the upper layer of culture solution, or the algal bodies can be scooped up and recovered using a net or the like. In order to efficiently perform flotation separation, it is preferable to appropriately stir or aerate the culture solution during cultivation. Furthermore, when growing algal bodies in an open pond system, it is preferable to perform the above recovery process at night when sunlight does not reach the algal bodies, from the viewpoint of cultivating the algal bodies during the day. The conditions for the above centrifugation are not particularly limited, and any conditions that allow the algal bodies (especially those with high lipid accumulation) to concentrate in the upper layer are acceptable. For example, the centrifugal acceleration (g) is preferably 100 to 30,000 × g, and more preferably 1,000 to 20,000 × g. The centrifugation time is preferably 0.5 to 240 minutes, and more preferably 1 to 30 minutes. Furthermore, the above-mentioned conditions for standing are not particularly limited; any conditions that allow the algal bodies (especially those with high lipid accumulation) to concentrate in the upper layer are acceptable. For example, standing for 1 hour to 10 days is preferable, and standing for 6 hours to 2 days is more preferable. Also, if stirring or aeration is performed during cultivation, it is preferable not to stir or aerate under standing conditions.

[0123] Furthermore, because the transformed organisms of the present invention have improved buoyancy, it becomes unnecessary to recover the entire culture medium by, for example, recovering only the algal bodies located near the surface through flotation separation. This allows for the selective recovery of algal bodies in the upper layer that have accumulated sufficient oil, without recovering algal bodies with low oil accumulation and poor buoyancy. Algal bodies with poor buoyancy can be recovered after further cultivation to accumulate sufficient oil and float to the upper layer. By selectively recovering algal bodies that have floated to the upper layer and by not recovering the entire culture medium, the cost of replacing the culture medium can be significantly reduced. In other words, the lipid production method of the present invention can be performed in a batch culture manner, where all the necessary nutrients are added to the culture medium at the start of cultivation and all of the culture medium is recovered at the end of cultivation. Alternatively, it can be performed in a chemostat manner, where fresh culture medium is continuously supplied to the culture tank and the same amount of culture medium along with the algae is discharged from the system at the same time to recover the algae, thus recovering the algae while culturing in a steady state. When culturing algae in a continuous culture manner, it is preferable to preferentially recover algae with a high oil content by flotation separation and to re-cultivate algae with a low oil content. In the lipid production method of the present invention, it is preferable to cultivate algae by continuous culture from the viewpoint of reducing the cost of changing the culture medium. Furthermore, from the viewpoint of improving the efficiency of algae recovery, it is preferable to recover the algae by discharging the upper layer of culture medium in the case of continuous culture.

[0124] Methods for recovering lipids from cultures can be appropriately selected from conventional methods. For example, after disrupting the cells of the culture by methods such as heat treatment, high pressure treatment, high temperature and high pressure treatment, physical disruption using beads, compression such as hand pressing, acid or alkali treatment, or chemical treatment with digestive enzymes, lipids can be recovered by isolating the lipid components by filtration, centrifugation, gel filtration chromatography, ion exchange chromatography, or solvent extraction such as chloroform / methanol extraction, hexane extraction, and ethanol extraction. Furthermore, in the case of larger-scale cultivation, lipids can be obtained by recovering the oil from the culture by pressing and / or solvent extraction, followed by general purification such as degumming, deacidification, decolorization, dewaxing, and deodorization. After isolating the lipid components in this way, fatty acids can be obtained by hydrolyzing the isolated lipids. Methods for isolating fatty acids from lipid components include, for example, treatment at a high temperature of about 70°C in an alkaline solution, lipase treatment, or decomposition using high-pressure hot water.

[0125] The lipids produced in the manufacturing method of the present invention preferably contain fatty acids or fatty acid compounds, and more preferably contain fatty acids or fatty acid ester compounds, from the viewpoint of their usability. From the viewpoint of productivity, the fatty acid ester compound is preferably a simple lipid or a complex lipid, more preferably a simple lipid, and even more preferably a TAG.

[0126] The fatty acids obtained by the manufacturing method of the present invention can be used not only as food, but also as plasticizers, emulsifiers for cosmetics, detergents for soaps and detergents, textile treatment agents, hair rinses, or disinfectants and preservatives.

[0127] The present invention also provides modification plasmids or modification DNA cassettes for use in modifications (A) to (C) described above. The modification plasmid or modification DNA cassette used in modification (A) is a vector or DNA cassette containing a TAG synthesis pathway gene. The modification plasmid or modification DNA cassette used in modification (B) is a vector or DNA cassette containing a CBB circuit gene. The modification plasmid or modification DNA cassette used in modification (C) is a homologous recombination vector or homologous recombination DNA cassette having a base sequence homologous to a portion of the upstream side of a region consisting of the base sequence of a cell wall synthesis pathway gene and its upstream base sequence, and a base sequence homologous to a portion of the downstream side of a region consisting of the base sequence of a cell wall synthesis pathway gene on the genome and its downstream base sequence. These modification plasmids or modification DNA cassettes can be suitably used to produce microalgae modified in the manner described above (A) to (C). Furthermore, a kit for producing transformants, including the modification plasmid or modification DNA cassette, is also provided. The kit of the present invention may also include other elements necessary for detecting the production of transformants, such as a host, reagents commonly used to transform the host with the vector, a transformation buffer, and an indicator reagent for selecting transformants.

[0128] With regard to the embodiments described above, the present invention further discloses the following transformants and methods.

[0129] <1> A method for producing lipids, comprising culturing a transformed microalgae that has been modified in at least one way selected from the group consisting of (A) to (C) below, preferably at least two ways selected from the group consisting of (A) to (C) below, more preferably (A) and / or (B) and (C) below, even more preferably (A), (B) and (C) below, producing fatty acids or lipids composed of fatty acids, recovering the transformed bodies by flotation separation, and obtaining lipids from the recovered transformed bodies. (A) Modifications that promote the expression of at least one gene encoding a TAG synthesis pathway-related protein. (B) Modifications that promote the expression of at least one gene encoding a CBB circuit-related protein. (C) Modifications that suppress the expression of at least one gene encoding a cell wall synthesis pathway-related protein.

[0130] <2> A method for recovering transformed organisms, comprising culturing a transformed microalgae that has been modified according to at least one selected from the group consisting of (A) to (C) below, preferably at least two selected from the group consisting of (A) to (C) below, more preferably (A) and / or (B) and (C) below, even more preferably (A), (B) and (C) below, to produce fatty acids or lipids composed of them, and recovering the transformed organisms by flotation separation. (A) Modifications that promote the expression of at least one gene encoding a TAG synthesis pathway-related protein. (B) Modifications that promote the expression of at least one gene encoding a CBB circuit-related protein. (C) Modifications that suppress the expression of at least one gene encoding a cell wall synthesis pathway-related protein.

[0131] <3> The expression of the gene encoding the TAG synthesis pathway-related protein is promoted within the cells of the microalgae, thereby promoting the expression of the TAG synthesis pathway-related protein. <1> or <2> The method described in the section. <4> The gene encoding the TAG synthesis pathway-related protein is introduced into the microalgae, and the expression of the introduced gene encoding the TAG synthesis pathway-related protein is promoted. <1> ~ <3> The method described in any one of the items. <5> The gene encoding the TAG synthesis pathway-related protein is a gene encoding at least one selected from the group consisting of ACS, G3PDH, AT (GPAT, LPAAT, DGAT, etc.), more preferably a gene encoding at least one selected from the group consisting of ACS and AT (GPAT, LPAAT, DGAT, etc.), even more preferably a gene encoding ACS and a gene encoding AT (GPAT, LPAAT, and DGAT, etc.), and even more preferably a gene encoding ACS and a gene encoding DGAT. <1> ~ <4> The method described in any one of the items.

[0132] <6> The aforementioned AT is the following protein (D) or (E), <5> The method described in the section. (D) A protein consisting of the amino acid sequence represented by Sequence ID No. 1. (E) A protein having AT activity, having an amino acid sequence that is 60% or more identical to the amino acid sequence of protein (D), preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more. <7> The protein (E) is a protein in which one or more amino acids, preferably 1 to 145, preferably 1 to 127, more preferably 1 to 108, more preferably 1 to 90, more preferably 1 to 72, more preferably 1 to 54, more preferably 1 to 36, more preferably 1 to 25, more preferably 1 to 18, more preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 3 are deleted, substituted, inserted, or added to the amino acid sequence of the protein (D), and is a protein having AT activity. <6> The method described in the section. <8> The protein (E) is a protein consisting of the amino acid sequence represented by SEQ ID NO: 85, or a protein having AT activity and having an amino acid sequence that is 75% (preferably 80%, preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identical to the amino acid sequence represented by SEQ ID NO: 85. <6> or <7> The method described in the section. <9> The gene encoding AT is a gene consisting of the following DNA(d) or (e), <5> ~ <8> The method described in any one of the items. (d) DNA consisting of the base sequence represented by Sequence ID No. 2. (e) DNA encoding a protein having AT activity, having a base sequence that is 60% or more identical to the base sequence of DNA(d), preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more. <10> The DNA(e) is a DNA encoding a protein having AT activity, wherein the base sequence of the DNA(d) has one or more bases deleted, substituted, inserted, or added, preferably 1 to 526, preferably 1 to 436, preferably 1 to 382, ​​more preferably 1 to 327, more preferably 1 to 273, more preferably 1 to 218, more preferably 1 to 163, more preferably 1 to 109, more preferably 1 to 76, more preferably 1 to 54, more preferably 1 to 32, more preferably 1 to 21, more preferably 1 to 10. <9> The method described in the section. <11> The DNA(e) is a DNA consisting of the base sequence represented by Sequence ID No. 86, or a DNA that has 80% (preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identity with the base sequence represented by Sequence ID No. 86, and encodes a protein having AT activity. <9> or <10> The method described in the section.

[0133] <12> The ACS is the following protein (F) or (G), <5> ~ <11> The method described in any one of the items. (F) A protein consisting of the amino acid sequence represented by Sequence ID No. 3. (G) A protein having ACS activity, having an amino acid sequence that is 60% or more identical to the amino acid sequence of protein (F), preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more. <13> The protein (G) is a protein in which one or more amino acids, preferably 1 to 259, preferably 1 to 226, more preferably 1 to 194, more preferably 1 to 162, more preferably 1 to 129, more preferably 1 to 97, more preferably 1 to 64, more preferably 1 to 45, more preferably 1 to 32, more preferably 1 to 19, more preferably 1 to 12, more preferably 1 to 6, is deleted, substituted, inserted, or added to the amino acid sequence of the protein (F), and is a protein having ACS activity. <12> The method described in the section. <14> The protein (G) is a protein consisting of the amino acid sequence represented by SEQ ID NO: 87, or a protein consisting of an amino acid sequence that is 70% (preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) identical to the amino acid sequence represented by SEQ ID NO: 87, and which has ACS activity. <12> or <13> The method described in the section. <15> The gene encoding the aforementioned ACS is a gene consisting of the following DNA(f) or (g), <5> ~ <14> The method described in any one of the items. (f) DNA consisting of the base sequence represented by Sequence ID No. 4. (g) DNA encoding a protein having ACS activity, having a base sequence that is 60% or more identical to the base sequence of DNA(f), preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more. <16> The DNA(g) is a DNA encoding a protein having ACS activity, wherein the base sequence of the DNA(f) has one or more bases deleted, substituted, inserted, or added, preferably 1 to 778, preferably 1 to 681, more preferably 1 to 584, more preferably 1 to 486, more preferably 1 to 389, more preferably 1 to 292, more preferably 1 to 194, more preferably 1 to 136, more preferably 1 to 97, more preferably 1 to 58, more preferably 1 to 38, more preferably 1 to 19. <15> The method described in the section. <17> The DNA(g) is a DNA consisting of a base sequence represented by Sequence ID No. 88, or a DNA that has an identity of 80% (preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more with the base sequence represented by Sequence ID No. 88, and encodes a protein having ACS activity. <15> or <16> The method described in the section.

[0134] <18> In addition to the above modification (A), the expression of genes encoding proteins involved in the fatty acid synthesis pathway is promoted. <1> ~ <17> The method described in any one of the items. <19> The expression of genes encoding proteins involved in the fatty acid synthesis pathway is promoted within the cells of the microalgae, thereby promoting the expression of proteins involved in the fatty acid synthesis pathway. <18> The method described in the section. <20> The gene encoding a protein involved in the fatty acid synthesis pathway is introduced into the microalgae, and the expression of the gene encoding the protein involved in the fatty acid synthesis pathway that was introduced is promoted. <18> or <19> The method described in the section. <21> The gene encoding the protein involved in the fatty acid synthesis pathway is a gene encoding at least one selected from the group consisting of ACC, ACP, holo-ACP synthase, MAT, KAS, KAR, HD, EAR, and TE, preferably a gene encoding TE. <18> ~ <20> The method described in any one of the items.

[0135] <22> The TE is the following protein (H) or (I), <21> The method described in the section. (H) A protein consisting of the amino acid sequence represented by Sequence ID No. 5. (I) A protein having TE activity, having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (H), preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more. <23> The protein (I) is a protein in which one or more amino acids, preferably 1 to 139, preferably 1 to 121, more preferably 1 to 104, more preferably 1 to 87, more preferably 1 to 69, more preferably 1 to 52, more preferably 1 to 34, more preferably 1 to 24, more preferably 1 to 17, more preferably 1 to 10, more preferably 1 to 6, more preferably 1 to 3 are deleted, substituted, inserted, or added to the amino acid sequence of the protein (H), and is a protein having TE activity. <22> The method described in the section. <24> The gene encoding the aforementioned TE is a gene consisting of the following DNA(h) or (i), <21> ~ <23> The method described in any one of the items. (h) DNA consisting of the base sequence represented by Sequence ID No. 6. (i) DNA encoding a protein having TE activity, having a base sequence that is 60% or more identical to the base sequence of DNA(h), preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more. <25> The DNA(i) is a DNA encoding a protein having TE activity, wherein the base sequence of the DNA(h) has one or more bases deleted, substituted, inserted, or added, preferably 1 to 418, preferably 1 to 366, more preferably 1 to 314, more preferably 1 to 261, more preferably 1 to 209, more preferably 1 to 157, more preferably 1 to 104, more preferably 1 to 73, more preferably 1 to 52, more preferably 1 to 31, more preferably 1 to 20, more preferably 1 to 10. <24> Method of description.

[0136] <26> The expression of the gene encoding the CBB circuit-related protein is promoted within the cells of the microalgae, thereby promoting the expression of the CBB circuit-related protein. <1> ~ <25> The method described in any one of the items. <27> The gene encoding the CBB circuit-related protein is introduced into the microalgae, and the expression of the introduced gene encoding the CBB circuit-related protein is promoted. <1> ~ <26> The method described in any one of the items. <28> The gene encoding the CBB cycle-related protein is a gene encoding at least one selected from the group consisting of TK, FBA, RPI, ribulose-1,5-bisphosphate carboxylase / oxygenase, sedoheptulose-1,7-bisphosphatase, phospholibrokinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, fructose-1,6-bisphosphatase, ribulose-5-phosphate epimerase, and Rubisco activase; more preferably a gene encoding at least one selected from the group consisting of TK, FBA, and RPI; even more preferably a gene encoding TK; even more preferably a gene encoding TK and a gene encoding FBA; even more preferably a gene encoding TK, a gene encoding FBA, and a gene encoding RPI. <1> ~ <27> The method described in any one of the items.

[0137] <29> The TK is the following protein (J) or (K), <28> Method of description. (J) A protein consisting of the amino acid sequence represented by Sequence ID No. 7. (K) A protein having TK activity, having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (J), preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more. <30> The protein (K) is a protein in which one or more amino acids, preferably 1 to 289, preferably 1 to 253, more preferably 1 to 216, more preferably 1 to 180, more preferably 1 to 144, more preferably 1 to 108, more preferably 1 to 72, more preferably 1 to 50, more preferably 1 to 36, more preferably 1 to 21, more preferably 1 to 14, more preferably 1 to 7, is deleted, substituted, inserted, or added to the amino acid sequence of the protein (J), and is a protein having TK activity. <29> The method described in the section. <31> The protein (K) is a protein consisting of the amino acid sequence represented by SEQ ID NO: 89, or a protein having TK activity and having an amino acid sequence that is 70% (preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) identical to the amino acid sequence represented by SEQ ID NO: 89. <29> or <30> The method described in the section. <32> The gene encoding TK is a gene consisting of the following DNA(j) or (k), <28> ~ <31> One of the methods described above. (j) DNA consisting of the base sequence represented by Sequence ID No. 8. (k) DNA encoding a protein having TK activity, having a base sequence that is 60% or more identical to the base sequence of DNA(j), preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more. <33> The DNA(k) is a DNA encoding a protein having TK activity, wherein the base sequence of the DNA(j) has one or more bases deleted, substituted, inserted, or added, preferably 1 to 868, preferably 1 to 760, more preferably 1 to 651, more preferably 1 to 543, more preferably 1 to 434, more preferably 1 to 325, more preferably 1 to 217, more preferably 1 to 152, more preferably 1 to 108, more preferably 1 to 65, more preferably 1 to 43, more preferably 1 to 21. <32> Method of description. <34> The DNA(k) is a DNA consisting of the base sequence represented by Sequence ID No. 90, or a DNA that has a base sequence of 75% (preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identity with the base sequence represented by Sequence ID No. 90, and encodes a protein having TK activity. <32> or <33> Method of description.

[0138] <35> The FBA is the following protein (L) or (M), <28> ~ <34> The method described in any one of the items. (L) A protein consisting of the amino acid sequence represented by Sequence ID No. 9. (M) A protein having FBA activity, having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (L), preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more. <36> The protein (M) is a protein in which one or more amino acids, preferably 1 to 152, preferably 1 to 133, more preferably 1 to 114, more preferably 1 to 95, more preferably 1 to 76, more preferably 1 to 57, more preferably 1 to 38, more preferably 1 to 26, more preferably 1 to 19, more preferably 1 to 11, more preferably 1 to 7, more preferably 1 to 3 are deleted, substituted, inserted, or added to the amino acid sequence of the protein (L), and is a protein having FBA activity. <35> The method described in the section. <37> The protein (M) is a protein consisting of the amino acid sequence represented by SEQ ID NO: 91, or a protein having an amino acid sequence that is 65% (preferably 70%, more preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identical to the amino acid sequence represented by SEQ ID NO: 91, and that has FBA activity. <35> or <36> The method described in the section. <38> The gene encoding the FBA is a gene consisting of the following DNA(l) or (m), <28> ~ <37> The method described in any one of the items. (l) DNA consisting of the base sequence represented by Sequence ID No. 10. (m) DNA encoding a protein having FBA activity, having a base sequence that is 60% or more identical to the base sequence of DNA(l), preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more. <39> The DNA(m) is a DNA encoding a protein having FBA activity, wherein the base sequence of the DNA(l) has one or more bases deleted, substituted, inserted, or added, preferably 1 to 459, preferably 1 to 402, more preferably 1 to 344, more preferably 1 to 287, more preferably 1 to 229, more preferably 1 to 172, more preferably 1 to 114, more preferably 1 to 80, more preferably 1 to 57, more preferably 1 to 34, more preferably 1 to 22, more preferably 1 to 11. <38> The method described in the section. <40> The DNA(m) is a DNA consisting of the base sequence represented by Sequence ID No. 92, or a DNA that has a base sequence of 75% (preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identity with the base sequence represented by Sequence ID No. 92, and encodes a protein having FBA activity. <38> or <39> The method described in the section.

[0139] <41> The RPI is the following protein (N) or (O), <28> ~ <40> The method described in any one of the items. (N) A protein consisting of the amino acid sequence represented by Sequence ID No. 11. (O) A protein having RPI activity, having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (N), preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more. <42> The protein (O) is a protein in which one or more amino acids, preferably 1 to 112, preferably 1 to 98, more preferably 1 to 84, more preferably 1 to 70, more preferably 1 to 56, more preferably 1 to 42, more preferably 1 to 28, more preferably 1 to 19, more preferably 1 to 14, more preferably 1 to 8, more preferably 1 to 5, more preferably 1 or 2 amino acids are deleted, substituted, inserted, or added to the amino acid sequence of the protein (N), and is a protein having RPI activity. <41> The method described in the section. <43> The protein (O) is a protein consisting of the amino acid sequence represented by SEQ ID NO: 93, or a protein consisting of an amino acid sequence that is 70% (preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) identical to the amino acid sequence represented by SEQ ID NO: 93, and which has RPI activity. <41> or <42> Method of description. <44> The gene encoding the RPI is a gene consisting of the following DNA(n) and (o), <28> ~ <43> One of the methods described above. (n) DNA consisting of the base sequence represented by Sequence ID No. 12. (o) DNA encoding a protein having RPI activity, having a base sequence that is 60% or more identical to the base sequence of DNA(n), preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more. <45> The DNA(o) is a DNA encoding a protein having RPI activity, wherein the base sequence of the DNA(n) has one or more bases deleted, substituted, inserted, or added, preferably 1 to 339, preferably 1 to 297, more preferably 1 to 254, more preferably 1 to 212, more preferably 1 to 169, more preferably 1 to 127, more preferably 1 to 84, more preferably 1 to 59, more preferably 1 to 42, more preferably 1 to 25, more preferably 1 to 16, more preferably 1 to 8. <44> The method described in the section. <46> The DNA(o) is a DNA consisting of the base sequence represented by Sequence ID No. 94, or a DNA that encodes a protein having RPI activity, and which has a base sequence of 75% (preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identity with the base sequence represented by Sequence ID No. 94. <44> or <45> The method described in the section.

[0140] <47> The expression of the cell wall synthesis pathway-related protein is reduced or lost by deleting or inactivating the gene encoding the cell wall synthesis pathway-related protein, or by downregulating the gene encoding the cell wall synthesis pathway-related protein. <1> ~ <46> The method described in any one of the items. <48> The gene encoding the cell wall synthesis pathway-related protein is a gene encoding at least one selected from the group consisting of CES, PKS involved in arginane synthesis, and UDP-glucose pyrophosphorylase, preferably a gene encoding CES. <1> ~ <47> The method described in any one of the items.

[0141] <49> The CES is the following protein (P) or (Q), <48> The method described in the section. (P) A protein consisting of the amino acid sequence represented by Sequence ID No. 13. (Q) A protein having CES activity, having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (P), preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more. <50> The protein (Q) is a protein in which one or more amino acids, preferably 1 to 268, preferably 1 to 234, more preferably 1 to 201, more preferably 1 to 167, more preferably 1 to 134, more preferably 1 to 100, more preferably 1 to 67, more preferably 1 to 46, more preferably 1 to 33, more preferably 1 to 20, more preferably 1 to 13, more preferably 1 to 6, is deleted, substituted, inserted, or added to the amino acid sequence of the protein (P), and is a protein having CES activity. <49> The method described in the section. <51> The protein (Q) is a protein consisting of the amino acid sequence represented by SEQ ID NO: 95, or a protein having CES activity and consisting of an amino acid sequence that is 75% (preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identical to the amino acid sequence represented by SEQ ID NO: 95. <49> or <50> The method described in the section. <52> The gene encoding the aforementioned CES is a gene consisting of the following DNA(p) or (q), <48> ~ <51> The method described in any one of the items. (p) DNA consisting of the base sequence represented by Sequence ID No. 14. (q) DNA encoding a protein having CES activity, having a base sequence that is 60% or more identical to the base sequence of DNA(p), preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more. <53> The DNA(q) is a DNA encoding a CES-active protein, wherein the base sequence of the DNA(p) has one or more bases deleted, substituted, inserted, or added, preferably 1 to 806, preferably 1 to 705, more preferably 1 to 604, more preferably 1 to 504, more preferably 1 to 403, more preferably 1 to 302, more preferably 1 to 201, more preferably 1 to 141, more preferably 1 to 100, more preferably 1 to 60, more preferably 1 to 40, more preferably 1 to 20. <52> The method described in the section. <54> The DNA(q) is a DNA consisting of the base sequence represented by Sequence ID No. 96, or a DNA that has 85% (preferably 90%, more preferably 95%, and even more preferably 98%) or more identity with the base sequence represented by Sequence ID No. 96, and encodes a protein having CES activity. <52> or <53> The method described in the section.

[0142] <55> The microalgae is an alga belonging to the division Heterokonta, preferably an alga belonging to the class Euophthalmophyceae, more preferably at least one alga selected from the group consisting of algae of the genus Nannochloropsis, algae of the genus Monodopsis, algae of the genus Bisqueria, algae of the genus Chlorobotris, and algae of the genus Goniochloris, and even more preferably an alga of the genus Nannochloropsis. <1> ~ <54> The method described in any one of the items. <56> The algae belonging to the genus Nannochloropsis is at least one alga selected from the group consisting of Nannochloropsis oceanica, Nannochloropsis occulata, Nannochloropsis gaditana, Nannochloropsis salina, Nannochloropsis limnetica, Nannochloropsis granulata, and Nannochloropsis sp., preferably Nannochloropsis gaditana or Nannochloropsis oceanica. <55> The method described in the section.

[0143] <57> The process includes recovering lipids from the recovered transformants by pressing and / or solvent extraction, preferably by pressing and solvent extraction. <1> ~ <56> The method described in any one of the items. <58> The culture of the transformants is carried out in an open-pond system or a closed-type photobioreactor. <1> ~ <57> The method described in any one of the items. <59> The culture of the transformed organism is carried out by batch culture or continuous culture, preferably by continuous culture. <1> ~ <58> The method described in any one of the items. <60> In the aforementioned continuous cultivation, algae with increased oil accumulation are collected by flotation separation, and algae with low oil accumulation are continued to be cultivated in a continuous manner. <59> The method described in the section.

[0144] <61> The lipid includes triacylglycerol, <1> ~ <60> The method described in any one of the items.

[0145] <62> A transformed microalgae having undergone at least one modification selected from the group consisting of (A) to (C), preferably at least two modifications selected from the group consisting of (A) to (C), more preferably (A) and / or (B) and (C), and even more preferably (A), (B) and (C). <63> The transformed organism exhibits improved buoyancy compared to a host that has not undergone any of the modifications (A) to (C), <62> The described transformant. <64> When the transformed organism and the host organism that has not undergone any of the modifications (A) to (C) are cultured under the same culture conditions and then centrifuged at 21600 × g for 10 minutes, the proportion of cells that do not precipitate is improved compared to the host organism that has not undergone any of the modifications (A) to (C). <62> or <63> The transformed organism described in the section.

[0146] <65> The expression of the gene encoding the TAG synthesis pathway-related protein is promoted within the cells of the microalgae, thereby promoting the expression of the TAG synthesis pathway-related protein. <62> ~ <64> A transformant as described in any one of the items. <66> By introducing the gene encoding the TAG synthesis pathway-related protein into the microalgae, the expression of the gene encoding the introduced TAG synthesis pathway-related protein is promoted. <62> ~ <65> A transformant as described in any one of the items. <67> The gene encoding the TAG synthesis pathway-related protein, or a plasmid or DNA cassette containing the gene encoding the TAG synthesis pathway-related protein, <62> ~ <66> A transformant as described in any one of the items. <68> The expression of the gene encoding the CBB circuit-related protein is promoted within the cells of the microalgae, thereby promoting the expression of the CBB circuit-related protein. <62> ~ <67> A transformant as described in any one of the items. <69> By introducing the gene encoding the CBB circuit-related protein into the microalgae, the expression of the introduced gene encoding the CBB circuit-related protein is promoted. <62> ~ <68> A transformant as described in any one of the items. <70> The gene encoding the CBB circuit-related protein, or a plasmid or DNA cassette containing the gene encoding the CBB circuit-related protein, <62> ~ <69> A transformant as described in any one of the items. <71> The gene encoding the cell wall synthesis pathway-related protein is deleted or inactivated, or the gene encoding the cell wall synthesis pathway-related protein is downregulated. <62> ~ <70> A transformant as described in any one of the items. <72> The expression of the cell wall synthesis pathway-related proteins is reduced or lost due to the deletion or inactivation of the gene encoding the cell wall synthesis pathway-related proteins, or due to the downregulation of the gene encoding the cell wall synthesis pathway-related proteins. <62> ~ <71> A transformant as described in any one of the items. <73> The plasmid or DNA cassette for deleting or inactivating the gene encoding the cell wall synthesis pathway-related protein, or for downregulating the gene encoding the cell wall synthesis pathway-related protein, <62> ~ <72> A transformant as described in any one of the items.

[0147] <74> A method for producing a transformed microalga by performing the above modifications (A) to (C) on microalgae. <75> The gene encoding the TAG synthesis pathway-related protein, or a plasmid or DNA cassette containing the gene encoding the TAG synthesis pathway-related protein, is introduced into the microalgae. <74> Method for preparing the transformed organism described in the section. <76> The gene encoding the CBB circuit-related protein, or a plasmid or DNA cassette containing the gene encoding the CBB circuit-related protein, is introduced into the microalgae. <74> or <75> The method for producing the transformed organism described in the section. <77> The process involves introducing a plasmid or DNA cassette into the microalgae to delete or inactivate the gene encoding the cell wall synthesis pathway-related protein, or to downregulate the gene encoding the cell wall synthesis pathway-related protein. <74> ~ <76> A method for producing a transformed organism as described in any one of the items.

[0148] <78> A kit for producing transformed microalgae for carrying out the above modifications (A) to (C). <79> The aforementioned <75> ~ <77> The plasmid or DNA cassette described in any one of the above items <78> A kit for preparing transformed microalgae described in the section.

[0149] <80> The gene encoding the TAG synthesis pathway-related protein is a gene encoding at least one selected from the group consisting of ACS, G3PDH, AT (GPAT, LPAAT, DGAT, etc.), and PAP; more preferably a gene encoding at least one selected from the group consisting of ACS and AT (GPAT, LPAAT, and DGAT, etc.); even more preferably a gene encoding ACS and a gene encoding AT (GPAT, LPAAT, and DGAT, etc.); and even more preferably a gene encoding ACS and a gene encoding DGAT. <62> ~ <79> A transformant according to any one of the items, a method for producing the same, or a kit for producing a transformant. <81> The DGAT or the gene encoding the DGAT <6> ~ <11> The protein or gene specified in any one of the above paragraphs <80> The transformant described in the section, a method for producing the same, or a kit for producing the transformant. <82> The ACS or the gene encoding the ACS <12> ~ <17> The protein or gene specified in any one of the above paragraphs <80> or <81> The transformant described in the section, a method for producing the same, or a kit for producing the transformant. <83> The gene encoding the CBB cycle-related protein is a gene encoding at least one selected from the group consisting of TK, FBA, RPI, ribulose-1,5-bisphosphate carboxylase / oxygenase, sedoheptulose-1,7-bisphosphatase, phospholibrokinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, fructose-1,6-bisphosphatase, ribulose-5-phosphate epimerase, and Rubisco activase; more preferably, a gene encoding at least one selected from the group consisting of TK, FBA, and RPI; even more preferably, a gene encoding TK; even more preferably, a gene encoding TK and a gene encoding FBA; even more preferably, a gene encoding TK, a gene encoding FBA, and a gene encoding RPI. <62> ~ <82> A transformant according to any one of the items, a method for producing the same, or a kit for producing a transformant. <84> The TK or the gene encoding the TK <29> ~ <34> The protein or gene specified in any one of the above paragraphs <83> The transformant described in the section, a method for producing the same, or a kit for producing the transformant. <85> The FBA or the gene encoding the FBA <35> ~ <40> The protein or gene specified in any one of the above paragraphs <83> or <84> The transformant described in the section, a method for producing the same, or a kit for producing the transformant. <86> The RPI or the gene encoding the RPI <41> ~ <46> The protein or gene specified in any one of the above paragraphs <83> ~ <85> A transformant according to any one of the items, a method for producing the same, or a kit for producing a transformant. <87> The gene encoding the cell wall synthesis pathway-related protein is preferably a gene encoding at least one selected from the group consisting of CES, PKS involved in arginane synthesis, and UDP-glucose pyrophosphorylase, more preferably a gene encoding CES. <62> ~ <86> A transformant according to any one of the items, a method for producing the same, or a kit for producing a transformant. <88> The CES or the gene encoding the CES <49> ~ <54> The protein or gene specified in any one of the above paragraphs <87> The transformant described in the section, a method for producing the same, or a kit for producing the transformant.

[0150] <89> In the transformed organism, the expression of genes encoding proteins involved in the fatty acid synthesis pathway is promoted. <62> ~ <88> A transformant as described in any one of the items. <90> The expression of the gene encoding the protein involved in the fatty acid synthesis pathway is promoted within the cells of the microalgae, thereby promoting the expression of the protein involved in the fatty acid synthesis pathway. <89> The transformed organism described in the section. <91> By introducing a gene encoding a protein involved in the fatty acid synthesis pathway into the microalgae, the expression of the gene encoding the protein involved in the fatty acid synthesis pathway that was introduced is promoted. <89> or <90> The transformed organism described in the section. <92> The aforementioned comprising a gene encoding a protein involved in the fatty acid synthesis pathway, or a plasmid or DNA cassette containing a gene encoding a protein involved in the fatty acid synthesis pathway, <89> ~ <91> A transformant as described in any one of the items. <93> The process involves introducing a gene encoding a protein involved in the fatty acid synthesis pathway, or a plasmid or DNA cassette containing a gene encoding a protein involved in the fatty acid synthesis pathway, into the microalgae. <74> ~ <92> A method for producing a transformed organism as described in any one of the items. <94> The plasmid or DNA cassette containing a gene encoding a protein involved in the fatty acid synthesis pathway, <78> ~ <93> A kit for preparing transformants as described in any one of the items.

[0151] <95> The gene encoding the protein involved in the fatty acid synthesis pathway is a gene encoding a protein selected from the group consisting of ACC, ACP, holo-ACP synthase, MAT, KAS, KAR, HD, EAR, and TE, preferably a gene encoding TE. <89> ~ <94> A transformant according to any one of the items, a method for producing the same, or a kit for producing a transformant. <96> The TE or the gene encoding the TE, <22> ~ <25> The protein or gene specified in any one of the above paragraphs <95> The transformant described in the section, a method for producing the same, or a kit for producing the transformant.

[0152] <97> The microalgae is an alga belonging to the division Heterokonta, preferably an alga belonging to the class Euophthalmophyceae, more preferably at least one alga selected from the group consisting of algae of the genus Nannochloropsis, algae of the genus Monodopsis, algae of the genus Bisqueria, algae of the genus Chlorobotris, and algae of the genus Goniochloris, and even more preferably an alga of the genus Nannochloropsis. <62> ~ <96> A transformant according to any one of the items, a method for producing the same, or a kit for producing a transformant. <98> The algae belonging to the genus Nannochloropsis is at least one alga selected from the group consisting of Nannochloropsis oceanica, Nannochloropsis occulata, Nannochloropsis gaditana, Nannochloropsis salina, Nannochloropsis limnetica, Nannochloropsis granulata, and Nannochloropsis sp., preferably Nannochloropsis gaditana or Nannochloropsis oceanica. <97> The transformant described in the section, a method for producing the same, or a kit for producing the transformant.

[0153] <99> For producing lipids, <62> ~ <98> Use of a transformant described in any one of the items, a transformant prepared by the method for preparing such a transformant, or a kit for preparing a transformant. [Examples]

[0154] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto. Herein, the base sequences of the primers used in these examples are shown in Table 1.

[0155] [Table 1]

[0156] Preparation Example 1: Preparation of expression plasmids for CBB circuit genes and TAG synthesis pathway genes derived from Nannochloropsis oceanica, and gene disruption plasmids encoding cell wall synthesis pathway-related proteins. (1) Construction of plasmids for expressing zeosin resistance genes The zeosin resistance gene (SEQ ID NO: 15) and the tubulin promoter sequence (SEQ ID NO: 18) derived from Nannochloropsis gaditana CCMP526 strain, as described in the literature (Randor Radakovits, et al., Nature Communications, DOI:10.1038 / ncomms1688, 2012), were artificially synthesized. Using the synthesized DNA fragments as templates, PCR was performed using primer pairs 28 (SEQ ID NO: 28) and 29 (SEQ ID NO: 29), and primer pairs 34 (SEQ ID NO: 34) and 35 (SEQ ID NO: 35) as shown in Table 1, to amplify the zeosin resistance gene fragment and the tubulin promoter sequence fragment, respectively. In addition, using the genome of Nannochloropsis oceanica NIES2145 strain as a template, PCR was performed using primer pairs 36 (SEQ ID NO: 36) and 37 (SEQ ID NO: 37) as shown in Table 1, to amplify the heat shock protein terminator sequence fragment (SEQ ID NO: 19). Furthermore, using the plasmid vector pUC19 (Takara Bio Inc.) as a template, PCR was performed using primer pairs 38 (SEQ ID NO: 38) and 39 (SEQ ID NO: 39) shown in Table 1 to amplify the plasmid vector pUC19 fragment. These four amplified fragments were then subjected to restriction enzymes. DpnThe samples were processed with I (manufactured by Toyobo Co., Ltd.) and purified using the High Pure PCR Product Purification Kit (manufactured by Roche Applied Science). Subsequently, the four resulting fragments were fused using the In-Fusion HD Cloning Kit (manufactured by Clontech) to construct a plasmid for zeosin resistance gene expression. This expression plasmid consists of an insert sequence (zeosin resistance gene expression cassette) in which the tubulin promoter sequence, zeosin resistance gene, and heat shock protein terminator sequence are linked in that order, and a pUC19 vector sequence.

[0157] (2) Obtaining TAG synthesis pathway genes and CBB circuit genes from Nannochloropsis oceanica, and constructing plasmids for gene expression. Total RNA from Nannochloropsis oceanica strain NIES2145 was extracted, and cDNA was obtained by reverse transcription using SuperScript® III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). Using this cDNA as a template, PCR reactions were performed using the primer pairs shown in Table 1: primer 59 (SEQ ID NO: 59) and primer 60 (SEQ ID NO: 60), primer 61 (SEQ ID NO: 61) and primer 62 (SEQ ID NO: 62), primer 63 (SEQ ID NO: 63) and primer 64 (SEQ ID NO: 64), primer 65 (SEQ ID NO: 65) and primer 66 (SEQ ID NO: 66), primer 67 (SEQ ID NO: 67) and primer 68 (SEQ ID NO: 68), and primer 69 (SEQ ID NO: 69) and primer 70 (SEQ ID NO: 70), respectively, to synthesize DGAT2-8 (amino acid sequence: sequence number), a TAG synthesis pathway-related protein. DNA fragments were obtained for each of the following genes: the gene encoding gene 1) (DGAT2-8 gene, sequence: SEQ ID NO. 2), the gene encoding LACS2 (amino acid sequence: SEQ ID NO. 3) (LACS2 gene, sequence: SEQ ID NO. 4), the gene encoding TE2 (amino acid sequence: SEQ ID NO. 5) (TE2 gene, sequence: SEQ ID NO. 6), the gene encoding TK1 (amino acid sequence: SEQ ID NO. 7), a CBB cycle-related protein (TK1 gene, sequence: SEQ ID NO. 8), the gene encoding FBA2 (amino acid sequence: SEQ ID NO. 9) (FBA2 gene, sequence: SEQ ID NO. 10), and the gene encoding RPI (amino acid sequence: SEQ ID NO. 11) (RPI gene, sequence: SEQ ID NO. 12). Furthermore, using the genome of Nannochloropsis oceanica strain NIES2145 as a template, PCR was performed using primer pairs 40 (SEQ ID NO: 40) and 41 (SEQ ID NO: 41), as well as primer pairs 42 (SEQ ID NO: 42) and 43 (SEQ ID NO: 43), as shown in Table 1, to obtain the LDSP promoter fragment (SEQ ID NO: 20) and the VCP1 terminator fragment (SEQ ID NO: 21), respectively. Furthermore, using the zeosin resistance gene expression plasmid as a template, PCR was performed using primer pairs of primer 44 (SEQ ID NO: 44) and primer 39 (SEQ ID NO: 39) shown in Table 1 to amplify a fragment consisting of a zeosin resistance gene expression cassette (tubulin promoter sequence, zeosin resistance gene, heat shock protein terminator sequence) and a pUC19 sequence. DNA fragments of each TAG synthesis pathway gene or CBB circuit gene, along with an LDSP promoter fragment, a VCP1 terminator fragment, and a fragment consisting of a zeosin resistance gene expression cassette and a pUC19 sequence, were fused using the same method as described above to construct plasmids for DGAT2-8 gene expression (zeosin resistance), LACS2 gene expression (zeosin resistance), TE2 gene expression (zeosin resistance), TK1 gene expression (zeosin resistance), FBA2 gene expression (zeosin resistance), and RPI gene expression (zeosin resistance). Each expression plasmid consists of an insert sequence linked in the following order: LDSP promoter sequence, each TAG synthesis pathway gene or CBB circuit gene, VCP1 terminator sequence, tubulin promoter sequence, zeosin resistance gene, and heat shock protein terminator sequence, and a pUC19 vector sequence.

[0158] (3) Construction of plasmids for co-expression of the DGAT2-8-LACS2 gene and the RPI-TK1-FBA2 gene. Using the aforementioned LACS2 gene expression plasmid (zeosin-resistant) and DGAT2-8 gene expression plasmid (zeosin-resistant) as templates, PCR was performed using primer pairs of primer 49 (sequence number 49) and primer 39 (sequence number 39), and primer pairs of primer 71 (sequence number 71) and primer 72 (sequence number 72), respectively, to obtain each DNA fragment. In addition, using the genome of Nannochloropsis oceanica strain NIES2145 as a template, PCR was performed using primer pairs of primer 45 (sequence number 45) and primer 46 (sequence number 46), and primer pairs of primer 47 (sequence number 47) and primer 48 (sequence number 48), respectively, to obtain the glutamine synthase (GS) promoter fragment (sequence number 22) and the LDSP terminator fragment (sequence number 23). These four fragments were fused in the same manner as described above to construct the DGAT2-8-LACS2 gene co-expression plasmid (zeosin-resistant). This expression plasmid consists of an insert sequence ligated in the following order: GS promoter sequence, DGAT2-8 gene, LDSP terminator sequence, LDSP promoter sequence, LACS2 gene, VCP1 terminator sequence, tubulin promoter sequence, zeosin resistance gene, and heat shock protein terminator sequence, along with a pUC19 vector sequence.

[0159] Similarly, using the FBA2 gene expression plasmid (zeosin resistance) and the TK1 gene expression plasmid (zeosin resistance) as templates, PCR was performed using primer pairs of primer 49 (SEQ ID NO: 49) and primer 39 (SEQ ID NO: 39), and primer pairs of primer 73 (SEQ ID NO: 73) and primer 74 (SEQ ID NO: 74) shown in Table 1, respectively, to obtain each DNA fragment. These two DNA fragments were fused with the GS promoter fragment and the LDSP terminator fragment in the same manner as described above to construct the TK1-FBA2 gene co-expression plasmid (zeosin resistance). This expression plasmid consists of an insert sequence linked in the following order: GS promoter sequence, TK1 gene, LDSP terminator sequence, LDSP promoter sequence, FBA2 gene, VCP1 terminator sequence, tubulin promoter sequence, zeosin resistance gene, and heat shock protein terminator sequence, and a pUC19 vector sequence. Using the obtained TK1-FBA2 gene co-expression plasmid (zeosin-resistant) and the RPI gene expression plasmid (zeosin-resistant) as templates, PCR was performed using the primer pairs of primer 54 (sequence number 54) and primer 39 (sequence number 39) and primer 75 (sequence number 75) and primer 76 (sequence number 76) shown in Table 1 to obtain each DNA fragment. In addition, using the genome of Nannochloropsis oceanica strain NIES2145 as a template, PCR was performed using the primer pairs of primer 50 (sequence number 50) and primer 51 (sequence number 51) and primer 52 (sequence number 52) and primer 53 (sequence number 53) shown in Table 1 to obtain the ammonium transporter (AMT) promoter fragment (sequence number 24) and the Δ9 desaturase (Δ9DES) terminator fragment (sequence number 25). These four fragments were fused using the same method as described above to construct a plasmid for co-expression of the RPI-TK1-FBA2 gene (zeosin resistance). This expression plasmid consists of an insert sequence linked in the following order: AMT promoter sequence, RPI gene, Δ9DES terminator, GS promoter sequence, TK1 gene, LDSP terminator sequence, LDSP promoter sequence, FBA2 gene, VCP1 terminator sequence, tubulin promoter sequence, zeosin resistance gene, and heat shock protein terminator sequence, along with a pUC19 vector sequence.

[0160] (4) Construction of plasmids for TE2 gene expression (paromomycin resistance), DGAT2-8-LACS2 gene co-expression (hygromycin resistance), RPI-TK1-FBA2 gene co-expression (hygromycin resistance), and RPI-TK1-FBA2 gene co-expression (paromomycin resistance) Using the aforementioned TE2 gene expression plasmid (zeosin resistance), the DGAT2-8-LACS2 gene co-expression plasmid (zeosin resistance), and the RPI-TK1-FBA2 gene co-expression plasmid (zeosin resistance) as templates, PCR was performed using primer pairs of primer 35 (SEQ ID NO: 35) and primer 36 (SEQ ID NO: 36) shown in Table 1 to obtain each DNA fragment. In addition, using the artificially synthesized paromomycin resistance gene (SEQ ID NO: 16) and hygromycin resistance gene (SEQ ID NO: 17) as templates, PCR was performed using primer pairs of primer 30 (SEQ ID NO: 30) and primer 31 (SEQ ID NO: 31), and primer pairs of primer 32 (SEQ ID NO: 32) and primer 33 (SEQ ID NO: 33) shown in Table 1 to obtain each DNA fragment. The obtained fragments were appropriately fused using the same method as described above to construct plasmids for TE2 gene expression (paromomycin resistance), DGAT2-8-LACS2 gene co-expression (hygromycin resistance), RPI-TK1-FBA2 gene co-expression (hygromycin resistance), and RPI-TK1-FBA2 gene co-expression (paromomycin resistance).

[0161] (5) Construction of a plasmid for disrupting the cell wall synthesis pathway gene derived from Nannochloropsis oceanica In order to disrupt the gene (CES gene, nucleotide sequence: SEQ ID NO: 14) encoding CES (amino acid sequence: SEQ ID NO: 13), which is one of the cell wall synthesis pathway proteins, by homologous recombination, using the genome of Nannochloropsis oceanica NIES2145 strain as a template, a primer pair of primer 77 (SEQ ID NO: 77) and primer 78 (SEQ ID NO: 78) shown in Table 1, and a primer pair of primer 79 (SEQ ID NO: 79) and primer 80 (SEQ ID NO: 80) were used for PCR to amplify homologous sequence 1 (SEQ ID NO: 26) and homologous sequence 2 (SEQ ID NO: 27), respectively. Further, using the plasmid for zeocin resistance gene expression as a template, PCR was performed using the primer pair of primer 81 (SEQ ID NO: 81) and primer 82 (SEQ ID NO: 82) shown in Table 1 to obtain a DNA fragment. The three obtained fragments and the pUC19 fragment were fused by the same method as described above to construct a plasmid for CES gene disruption (zeocin resistance). The plasmid for gene disruption consists of an insert sequence linked in the order of homologous sequence 1, tubulin promoter sequence, zeocin resistance gene, heat shock protein terminator sequence, and homologous sequence 2, and a pUC19 vector sequence.

[0162] (6) Preparation of PCR fragment for electroporation Using the plasmid for TE2 gene expression (paromomycin resistance) as a template, PCR was performed using the primer pair of primer 55 (SEQ ID NO: 55) and primer 58 (SEQ ID NO: 58) shown in Table 1 to amplify a cassette for TE2 gene expression (paromomycin resistance). The cassette for TE2 gene expression (paromomycin resistance) consists of an LDSP promoter sequence, TE2 gene, VCP1 terminator sequence, tubulin promoter sequence, paromomycin resistance gene, and heat shock protein terminator sequence. Furthermore, using the DGAT2-8-LACS2 gene co-expression plasmid (zeosin resistance), the DGAT2-8-LACS2 gene co-expression plasmid (hygromycin resistance), and the RPI-TK1-FBA2 gene co-expression plasmid (paromomycin resistance) as templates, PCR was performed using primer pairs of primer 56 (SEQ ID NO: 56) and primer 58 (SEQ ID NO: 58) shown in Table 1 to amplify the DGAT2-8-LACS2 gene co-expression cassette (zeosin resistance), the DGAT2-8-LACS2 gene co-expression cassette (hygromycin resistance), and the TK1-FBA2 gene co-expression cassette (paromomycin resistance). This expression cassette consists of a GS promoter sequence, a DGAT2-8 gene or a TK1 gene, an LDSP terminator sequence, an LDSP promoter sequence, a LACS2 gene or a FBA2 gene, a VCP1 terminator sequence, a tubulin promoter sequence, a hygromycin resistance gene or a paromomycin resistance gene, and a heat shock protein terminator sequence.

[0163] Furthermore, using the aforementioned RPI-TK1-FBA2 gene co-expression plasmid (hygromycin resistance) as a template, PCR was performed using primer pairs 57 (SEQ ID NO: 57) and 58 (SEQ ID NO: 58) shown in Table 1 to amplify the RPI-TK1-FBA2 gene co-expression cassette (hygromycin resistance). This expression cassette consists of an AMT promoter sequence, the RPI gene, the Δ9DES terminator, the GS promoter sequence, the TK1 gene, the LDSP terminator sequence, the LDSP promoter sequence, the FBA2 gene, the VCP1 terminator sequence, the tubulin promoter sequence, the hygromycin resistance gene, and the heat shock protein terminator sequence. Furthermore, using the plasmid for CES gene disruption (zeocin resistance) as a template, PCR was performed using the primer pair of primer 83 (SEQ ID NO: 83) and primer 84 (SEQ ID NO: 84) shown in Table 1 to amplify the cassette for CES gene disruption. This disruption cassette consists of homologous sequence 1, tubulin promoter sequence, zeocin resistance gene, heat shock protein terminator sequence, and homologous sequence 2. The amplified fragment was purified using the High Pure PCR Product Purification Kit (manufactured by Roche Applied Science). For elution during purification, sterile water was used instead of the elution buffer included in the kit.

[0164] Production of transformant with TAG synthesis pathway gene and CBB circuit gene introduced into Nannochloropsis, and lipid production by the transformant (1) Production of transformant with TAG synthesis pathway gene and CBB circuit gene introduced into Nannochloropsis, and culture of the transformant About 1×10 9Nannochloropsis oceanica NIES2145 cells were washed with 384 mM sorbitol solution to remove salts and used as host cells for transformation. Approximately 500 ng of the DGAT2-8-LACS2 gene co-expression cassette (zeosin resistant) amplified in Preparation Example 1 was mixed with the host cells, and electroporation was performed under conditions of 50 μF, 500 Ω, and 2,200 V / 2 mm. After 24 hours of recovery culture in f / 2 liquid medium (NaNO3 75 mg, NaH2PO4·2H2O 6 mg, vitamin B12 0.5 μg, biotin 0.5 μg, thiamine 100 μg, Na2SiO3·9H2O 10 mg, Na2EDTA·2H2O 4.4 mg, FeCl3·6H2O 3.16 mg, CoSO4·7H2O 12 μg, ZnSO4·7H2O 21 μg, MnCl2·4H2O 180 μg, CuSO4·5H2O 7 μg, Na2MoO4·2H2O 7 μg / 1 L of artificial seawater), the samples were spread onto f / 2 agar medium containing 2 μg / mL of zeosin and cultured for 2-3 weeks at 25°C under a 0.3% CO2 atmosphere in 12 h / 12 ​​h light / dark conditions. The obtained colonies were seeded in 2 mL of N5P5 medium (a medium with 5 times the nitrogen concentration and 5 times the phosphorus concentration of f / 2 medium) containing 2 μg / mL of zeosin (in a 24-well plate) and cultured with shaking for 3 weeks at 25°C under 0.3% CO2 conditions with 12 h / 12 ​​h light / dark intervals. The resulting culture solution was seeded in 18 mL of N15P5 medium (a medium with 15 times the nitrogen concentration and 5 times the phosphorus concentration of f / 2 medium) and cultured at 20°C under 12 h / 12 ​​h light / dark intervals. Next, using a transformant into which the DGAT2-8-LACS2 gene co-expression cassette had been introduced (hereinafter also referred to as "DGAT2-8-LACS2 strain") as the parent strain, the TE2 gene expression cassette (paromomycin-resistant) amplified in Preparation Example 1 was introduced using the same method as described above. Transformant selection was performed using f / 2 agar medium containing 2 μg / mL zeosin and 100 μg / mL paromomycin. The resulting colonies were seeded in 2 mL of N5P5 medium containing 100 μg / mL paromomycin and cultured using the same method as described above. The resulting culture medium was then seeded in 18 mL of N15P5 medium and cultured using the same method as described above. Next, using the transformant into which the DGAT2-8-LACS2 gene co-expression cassette (zeocin resistance) and the TE2 gene expression cassette (paromomycin resistance) were introduced (hereinafter also referred to as the "TE2 on DGAT2-8-LACS2 strain") as the parental strain, the RPI-TK1-FBA2 gene co-expression cassette (hygromycin resistance) amplified in Preparation Example 1 was introduced in the same manner as described above. The selection of the transformant was performed on f / 2 agar medium containing 500 μg / mL hygromycin. The obtained colonies were seeded in 2 mL of N5P5 medium containing 500 μg / mL hygromycin and cultured in the same manner as described above. The obtained culture solution was seeded in 18 mL of N15P5 medium and cultured in the same manner as described above. For the Nannochloropsis oceanica NIES2145 strain (hereinafter also referred to as the "wild strain"), the TE2 on DGAT2-8-LACS2 strain, and the transformant into which the DGAT2-8-LACS2 gene co-expression cassette (zeocin resistance), the TE2 gene expression cassette (paromomycin resistance), and the RPI-TK1-FBA2 gene co-expression cassette (hygromycin resistance) were introduced (hereinafter also referred to as the "RPI-TK1-FBA2 on TE2 on DGAT2-8-LACS2 strain"), they were subcultured in 18 mL of N5P5 medium and cultured with shaking for 5 to 7 days at 25°C under a 0.3% CO2 atmosphere, light intensity of about 100 μmol / m 2 / s (normal light conditions), and 12 h / 12 h light / dark conditions to obtain the pre-preculture solution. Using a 96-well plate and Infinite M200 PRO (TECAN), the turbidity at 750 nm (hereinafter also referred to as "OD750") was measured. The pre-preculture solution was seeded into 18 mL of N5P5 medium so that the OD750 reached a final concentration of 0.1 and cultured under the same conditions for 5 days to obtain the pre-culture solution. Similarly, the pre-culture solution was seeded into 18 mL of N5P5 medium so that the OD750 reached a final concentration of 0.1 and the main culture was performed under the same conditions. Also, the culture was performed in the same manner under strong light conditions with the light intensity adjusted to about 300 μmol / m 2 / s and the CO2 concentration adjusted to 0.6% (the pre-preculture was under normal light conditions, and the culture from the pre-culture was under strong light conditions). The culture was performed with N = 2 to 4 for each strain.

[0165] (2) Extraction of lipids from Nannochloropsis culture solution and analysis of lipids After the start of the culture, sampling was performed over time, and lipid extraction was carried out using the following method. To 0.25 mL of culture medium, 50 μL of 1 mg / mL glyceryl triheptadecanoate (Sigma-Aldrich) chloroform solution was added as an internal standard. Then, 0.5 mL of chloroform and 1 mL of methanol were added to the culture medium, and the mixture was vigorously stirred and left to stand for 10 minutes. Subsequently, 0.5 mL of chloroform and 0.5 mL of 1.5% KCl were added and stirred, and the mixture was centrifuged at 3,000 rpm for 5 minutes. The chloroform layer (lower layer) was collected using a Pasteur pipette. The obtained chloroform layer was blown with nitrogen gas to dry it, and then redissolved in 50 μL of chloroform. 0.5 mL of 14% boron trifluoride solution (SIGMA) was added and stirred, and the mixture was incubated at 80°C for 30 minutes. Then, 0.5 mL of hexane and 0.5 mL of saturated saline were added and vigorously stirred. After standing at room temperature for 10 minutes, the upper layer, the hexane layer, was collected to obtain the fatty acid ester.

[0166] The obtained fatty acid esters were subjected to gas chromatography analysis. The measurement conditions are as follows. <Gas chromatography conditions> Analyzer: 7890A (Agilent technology) Capillary column: DB-1 MS 30m x 200μm x 0.25μm (J&W Scientific) Mobile phase: High-purity helium, Oven temperature: 150℃, hold for 0.5 minutes → 150~220℃ (increase temperature by 40℃ / min) → 220~320℃ (increase temperature by 20℃ / min) → 320℃, hold for 2 minutes (post-run for 2 minutes). Inlet temperature: 300℃, Injection method: Split injection (split ratio: 75:1) Injection volume: 1μL, Washing vial: methanol / chloroform, Detection method: FID Detector temperature: 300℃

[0167] Furthermore, fatty acid methyl esters were identified by comparing their retention times after subjecting various fatty acid methyl ester standards to gas chromatography under the same conditions. Gas chromatography-mass spectrometry analysis was also performed as needed. The amount of methyl esters in each fatty acid was quantified from the peak area of ​​the waveform data obtained by gas chromatography analysis. Inter-sample correction was performed by comparing each peak area with the peak area of ​​C17 fatty acid methyl ester derived from an internal standard, and the amount of each fatty acid per liter of culture medium was calculated. Furthermore, the sum of the amounts of each fatty acid was defined as the total fatty acid amount, and the weight percentage of each fatty acid amount in the total fatty acid amount was calculated. Table 2 shows the results under normal light conditions, and Table 3 shows the results under high light conditions. In the table below, the wild-type strain is indicated as "WT". Total fatty acid content ("TFA productivity" in the table) is shown in the format of mean ± standard deviation.

[0168] [Table 2]

[0169] [Table 3]

[0170] As is clear from Tables 2 and 3, both the TE2 on DGAT2-8-LACS2 strain and the RPI-TK1-FBA2 on TE2 on DGAT2-8-LACS2 strain showed higher lipid productivity compared to the wild-type strain under both normal and strong light conditions. In particular, the improvement in lipid productivity was even more pronounced in the RPI-TK1-FBA2 on TE2 on DGAT2-8-LACS2 strain.

[0171] (3) Re-culturing of wild-type strain and RPI-TK1-FBA2 on TE2 on DGAT2-8-LACS2 strain, and analysis of their ability to float. For the wild strain and the RPI-TK1-FBA2 on TE2 on DGAT2-8-LACS2 strain, cultivation was carried out again under normal light conditions and strong light conditions respectively in the same manner as the above-mentioned method (cultivation was performed with N = 3 for each strain), and sampling was carried out at 0.25 mL and 1 mL over time. Lipid extraction and analysis were performed on the 0.25 mL sampling solution in the same manner as the above-mentioned method to calculate the total fatty acid amount (TFA). Also, for the 1 mL sampling solution, centrifugation was carried out at 15000 rpm (21600×g) for 10 minutes using a CF15RXII centrifuge and a T15A43 rotor (both manufactured by Hitachi Koki Co., Ltd., now "Eppendorf High-Tech Technologies Co., Ltd."), and the entire amount of the centrifuged supernatant (including cells that did not precipitate by centrifugation and cells that floated on the water surface by centrifugation) was recovered. Lipid extraction and analysis were performed on 0.25 mL of the obtained centrifuged supernatant in the same manner as the above-mentioned method to calculate the fatty acid amount (SUP-FA) contained in the cells that did not precipitate by centrifugation (hereinafter also referred to as "floating cells" or "floatable cells"). The ratio of the fatty acid amount of the floating cells to the total fatty acid amount was calculated and used as the floating rate (SUP-FA / TFA). The results of cultivation under normal light conditions are shown in Table 4, and the results of cultivation under strong light conditions are shown in Table 5.

[0172]

Table 4

[0173]

Table 5

[0174] As is clear from Tables 4 and 5, the RPI-TK1-FBA2 on TE2 on DGAT2-8-LACS2 strain showed higher fatty acid content and buoyancy rate from floating cells compared to the wild-type strain under all culture conditions and durations, indicating improved buoyancy. The higher total fatty acid content compared to the wild-type strain under all conditions suggests that the enhanced TAG synthesis pathway and CBB cycle improved lipid content, resulting in a lower specific gravity and thus improved buoyancy. Furthermore, it was found that the buoyancy rate increased more rapidly under high-light conditions, which tend to increase lipid content.

[0175] Example 2: Preparation of transformants by introducing TAG synthesis pathway genes and CBB circuit genes into Nannochloropsis and disrupting cell wall synthesis pathway genes, and production of lipids by the transformants. (1) Create transformants by introducing the TAG synthesis pathway gene and the CBB circuit gene into Nannochloropsis and disrupting the cell wall synthesis pathway gene, culture the transformants, extract lipids, and analyze lipids. In the same manner as in Example 1, the Nannochloropsis oceanica NIES2145 strain was used as the parent strain, and the CES gene disruption cassette amplified in Preparation Example 1 was introduced. From the resulting colonies, transformants in which the CES gene was disrupted (hereinafter also referred to as "ΔCES strain") were selected by PCR. Next, using the ΔCES strain as the parent strain, the TK1-FBA2 gene co-expression cassette (paromomycin-resistant) amplified in Preparation Example 1 was introduced using the same method as described above. Transformants in which the CES gene was disrupted and the TK1-FBA2 gene co-expression cassette (paromomycin-resistant) was introduced (hereinafter also referred to as "TK1-FBA2 on ΔCES strain") were selected using the same method as in Example 1. Furthermore, using the TK1-FBA2 on ΔCES strain as the parent strain, the DGAT2-8-LACS2 gene co-expression cassette (hygromycin-resistant) amplified in Preparation Example 1 was introduced using the same method as described above. Transformants (hereinafter also referred to as "DGAT2-8-LACS2 on TK1-FBA2 on ΔCES strain") in which the CES gene was disrupted and the TK1-FBA2 gene co-expression cassette (paromomycin-resistant) and the DGAT2-8-LACS2 gene co-expression cassette (hygromycin-resistant) were introduced were selected using the same method as in Example 1. Wild-type strain, ΔCES strain, TK1-FBA2 on ΔCES strain, and DGAT2-8-LACS2 on TK1-FBA2 on ΔCES strain (N=1 each) were cultured in the same manner as in Example 1 (under normal light conditions), and lipid extraction and analysis were performed. The results are shown in Table 6.

[0176] [Table 6]

[0177] As is clear from Table 6, the ΔCES strain showed lower lipid productivity than the wild-type strain, but the TK1-FBA2 on ΔCES strain showed higher lipid productivity than the wild-type strain. Furthermore, it was revealed that the DGAT2-8-LACS2 on TK1-FBA2 on ΔCES strain showed even higher lipid productivity than the wild-type strain and the TK1-FBA2 on ΔCES strain.

[0178] (2) Re-culturing of wild-type strain, RPI-TK1-FBA2 on TE2 on DGAT2-8-LACS2 strain, and DGAT2-8-LACS2 on TK1-FBA2 on ΔCES strain, and analysis of their flotation properties. The wild-type strain, the RPI-TK1-FBA2 on TE2 on DGAT2-8-LACS2 strain, and the DGAT2-8-LACS2 on TK1-FBA2 on ΔCES strain were cultured under normal light and high light conditions using the same method as in Example 1 (N=3 cultures for each strain), and their flotation ability was analyzed. The results for culture under normal light conditions are shown in Table 7, and the results for culture under high light conditions are shown in Table 8. Furthermore, flotation cells were collected from the culture medium after 17 days of normal light culture and 14 days of high light culture by centrifugation, resuspended, and allowed to stand for 24 hours. Photographs before and after standing are shown in Figure 1.

[0179] [Table 7]

[0180] [Table 8]

[0181] As is clear from Tables 7 and 8, the DGAT2-8-LACS2 on TK1-FBA2 on ΔCES strain showed improved buoyancy compared to the wild-type strain, with higher levels of fatty acids derived from buoyant cells and higher buoyancy rates under all culture conditions and durations. Furthermore, compared to the RPI-TK1-FBA2 on TE2 on DGAT2-8-LACS2 strain, which does not suppress / disrupt the cell wall synthesis pathway gene, the DGAT2-8-LACS2 on TK1-FBA2 on ΔCES strain showed superior buoyancy, particularly at day 14 under normal light conditions and at days 7 and 10 under strong light conditions. Furthermore, as shown in Figure 1, the RPI-TK1-FBA2 on TE2 on DGAT2-8-LACS2 strain and the DGAT2-8-LACS2 on TK1-FBA2 on ΔCES strain were found to float to the surface of the liquid after standing, in the later stages of culture when the total fatty acid content is high (more algal bodies were visible on the surface). In particular, under these conditions, most of the cells of the DGAT2-8-LACS2 on TK1-FBA2 on ΔCES strain floated to the surface of the liquid after 24 hours of standing, indicating a very high buoyancy.

[0182] It was thought that the enhanced TAG synthesis pathway and CBB cycle improved the lipid content, and that the suppression of the cell wall synthesis pathway reduced the high-density cell wall components (such as cellulose), further increasing the lipid content. As a result, the specific gravity decreased significantly, leading to a substantial improvement in buoyancy.

[0183] As described above, by making at least one modification to microalgae, such as a modification that promotes the expression of at least one gene encoding a triacylglycerol synthesis pathway-related protein, a modification that promotes the expression of at least one gene encoding a Calvin cycle-related protein, and a modification that suppresses the expression of at least one gene encoding a cell wall synthesis pathway-related protein, it is possible to obtain a microalgae transformant with improved buoyancy, and by using this transformant, it is possible to provide a method for producing lipids that improves the recovery efficiency of the transformant.

Claims

1. A method for recovering a transformant, comprising culturing a microalgae transformant that has undergone at least one modification selected from the group consisting of (A) to (C) below, producing fatty acids or lipids composed of fatty acids, and recovering the transformant by flotation separation. (A) Modifications that promote the expression of at least one gene encoding a triacylglycerol synthesis pathway-related protein. (B) Modifications that promote the expression of at least one gene encoding a Calvin cycle-related protein. (C) Modifications that suppress the expression of at least one gene encoding a cell wall synthesis pathway-related protein.

2. A method for producing lipids, comprising culturing a transformed microalgae that has undergone at least one modification selected from the group consisting of (A) to (C) below, producing fatty acids or lipids composed of fatty acids, recovering the transformed bodies by flotation separation, and obtaining lipids from the recovered transformed bodies. (A) Modifications that promote the expression of at least one gene encoding a triacylglycerol synthesis pathway-related protein. (B) Modifications that promote the expression of at least one gene encoding a Calvin cycle-related protein. (C) Modifications that suppress the expression of at least one gene encoding a cell wall synthesis pathway-related protein.

3. The method according to claim 1 or 2, wherein the transformed microalga is a transformed microalga that has undergone at least two modifications selected from the group consisting of (A) to (C).

4. The method according to any one of claims 1 to 3, wherein the transformed microalga is a transformed microalga that has undergone the modification of (A) and / or (B) and the modification of (C).

5. The triacylglycerol synthesis pathway-related protein is at least one protein selected from the group consisting of diacylglycerol acyltransferase and long-chain acyl-CoA synthetase. The Calvin cycle-related protein is at least one protein selected from the group consisting of a gene encoding transketolase and fructose-1,6-bisphosphate aldolase. The aforementioned cell wall synthesis pathway-related protein is cellulose synthase. The method according to any one of claims 1 to 4.

6. The diacylglycerol acyltransferase is the following protein (D) or (E): The long-chain acyl-CoA synthetase is the following protein (F) or (G): The transketolase is the following protein (J) or (K): The fructose-1,6-bisphosphate aldolase is the following protein (L) or (M): The cellulose synthase is the following protein (P) or (Q): The method according to claim 5. (D) A protein consisting of the amino acid sequence represented by Sequence ID No.

1. (E) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (D) and having acyltransferase activity. (F) A protein consisting of the amino acid sequence represented by Sequence ID No.

3. (G) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (F) and having acyl-CoA synthetase activity. (J) A protein consisting of the amino acid sequence represented by Sequence ID No.

7. (K) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (J) and having transketolase activity. (L) A protein consisting of the amino acid sequence represented by Sequence ID No.

9. (M) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (L) and having fructose-1,6-bisphosphate aldolase activity. (P) A protein consisting of the amino acid sequence represented by Sequence ID No.

13. (Q) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (P) and having cellulose synthase activity.

7. The method according to any one of claims 1 to 6, wherein the transformed microalga is a transformed microalga that has been modified according to (A), (B), and (C) above.

8. The transformants exhibit enhanced expression of the gene encoding diacylglycerol acyltransferase, the gene encoding long-chain acyl-CoA synthetase, the gene encoding transketolase, and the gene encoding fructose-1,6-bisphosphate aldolase. The expression of the gene encoding cellulose synthase is suppressed. The method according to any one of claims 1 to 7.

9. The method according to any one of claims 1 to 8, wherein the microalgae are algae belonging to the division Heterokonta.

10. The method according to claim 9, wherein the alga belonging to the division Heterokonta is an alga belonging to the genus Nannochloropsis.

11. The method according to any one of claims 1 to 10, comprising the step of recovering lipids from the recovered transformants by pressing and / or solvent extraction.

12. The method according to any one of claims 1 to 11, wherein the culture of the transformants is carried out in an open-pond system or a closed-type photobioreactor.

13. The method according to any one of claims 1 to 12, wherein the culture of the transformed organism is carried out by continuous culture.

14. In the aforementioned continuous culture, Algal bodies with increased oil accumulation are recovered by flotation separation. Continue culturing algae with low levels of accumulated oil. The method according to claim 13.

15. Transformed microalgae that have undergone the following modifications (A), (B), and (C). (A) Modifications that promote the expression of at least one gene encoding a triacylglycerol synthesis pathway-related protein. (B) Modifications that promote the expression of at least one gene encoding a Calvin cycle-related protein. (C) Modifications that suppress the expression of at least one gene encoding a cell wall synthesis pathway-related protein.

16. The transformant according to claim 15, wherein the transformant has improved buoyancy compared to a host that has not undergone any of the modifications (A) to (C).

17. The transformant according to claim 15 or 16, wherein when the transformant and a host that has not undergone any of the modifications (A) to (C) are cultured under the same culture conditions and then centrifuged at 21600 × g for 10 minutes, the proportion of cells that do not precipitate is improved compared to the host that has not undergone any of the modifications (A) to (C).

18. The triacylglycerol synthesis pathway-related protein is at least one protein selected from the group consisting of diacylglycerol acyltransferase and long-chain acyl-CoA synthetase. The Calvin cycle-related protein is at least one protein selected from the group consisting of a gene encoding transketolase and fructose-1,6-bisphosphate aldolase. The aforementioned cell wall synthesis pathway-related protein is cellulose synthase. The transformed body according to any one of claims 15 to 17.

19. The diacylglycerol acyltransferase is the following protein (D) or (E): The long-chain acyl-CoA synthetase is the following protein (F) or (G): The transketolase is the following protein (J) or (K): The fructose-1,6-bisphosphate aldolase is the following protein (L) or (M): The cellulose synthase is the following protein (P) or (Q): The transformed body according to claim 18. (D) A protein consisting of the amino acid sequence represented by Sequence ID No.

1. (E) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (D) and having acyltransferase activity. (F) A protein consisting of the amino acid sequence represented by Sequence ID No.

3. (G) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (F) and having acyl-CoA synthetase activity. (J) A protein consisting of the amino acid sequence represented by Sequence ID No.

7. (K) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (J) and having transketolase activity. (L) A protein consisting of the amino acid sequence represented by Sequence ID No.

9. (M) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (L) and having fructose-1,6-bisphosphate aldolase activity. (P) A protein consisting of the amino acid sequence represented by Sequence ID No.

13. (Q) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of the protein (P) and having cellulose synthase activity.

20. The transformants exhibit enhanced expression of the gene encoding diacylglycerol acyltransferase, the gene encoding long-chain acyl-CoA synthetase, the gene encoding transketolase, and the gene encoding fructose-1,6-bisphosphate aldolase. The expression of the gene encoding cellulose synthase is suppressed. A transformed body according to any one of claims 15 to 19.

21. The transformed organism according to any one of claims 15 to 20, wherein the microalgae is an alga belonging to the division Heterokonta.

22. The transformed organism according to claim 21, wherein the alga belonging to the division Heterokonta is an alga belonging to the genus Nannochloropsis.

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