Lipid production method

Abstract

To provide a method for producing lipids that improves the buoyancy of microalgae and improves recovery efficiency, and to provide a transformant with the improved buoyancy of microalgae.SOLUTION: There is provided a method for producing lipids comprising: culturing transformants of microalgae with at least one modification selected from the group consisting of the following (A) to (C) to produce fatty acids or lipids composed of the same; and recovering the transformants by flotation to obtain lipids from the recovered transformants, (A) a modification that promotes the expression of at least one gene encoding a triacylglycerol synthesis pathway-related protein; (B) a modification that promotes the expression of at least one gene encoding a Calvin cycle-related protein; and (C) a modification that suppresses the expression of at least one gene encoding a cell wall synthesis pathway-related protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing lipids. The present invention also relates to a transformant used in the method. [Background technology]

[0002] Fatty acids are one of the main components of lipids, and in vivo they form lipids such as triacylglycerols (hereinafter simply referred to as "TAGs"), which are formed by esterification with glycerin. Furthermore, in many plants and animals, fatty acids are also stored and used as an energy source. Fatty acids and lipids stored in plants and animals are widely used for food and industrial purposes. For example, derivatives of higher alcohols obtained by reducing higher fatty acids having 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 in detergents or disinfectants. Similarly, cationic surfactants such as alkylamine salts and mono- or di-alkyl quaternary ammonium salts, which are derivatives of higher alcohols, are routinely used in fabric treatment agents, hair rinses, and disinfectants. Benzalkonium-type quaternary ammonium salts are routinely used in disinfectants and antiseptics. Furthermore, plant-derived oils and fats are also used as raw materials for biodiesel fuel. In addition, 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 living animals, and are used in functional foods, etc. As described above, fatty acids and lipids are used in a wide variety of ways, and therefore attempts have been made to improve the in vivo productivity of fatty acids and lipids in plants and other organisms.

[0003] In recent years, research into renewable energy has been progressing in an effort to realize a sustainable society. In particular, photosynthetic microorganisms are expected to not only reduce carbon dioxide emissions but also serve as biofuel organisms that do not compete with crops. In particular, in recent years, algae have been attracting attention as a potential biofuel source due to their potential for biodiesel production. Because algae can produce lipids that can be used as biodiesel fuel through photosynthesis without competing with food, they are attracting attention as a potential next-generation biomass resource. Furthermore, it has been reported that algae have a higher lipid production and accumulation capacity than plants.

[0004] Generally, when lipids are produced using microalgae as the algae, for example, a method is adopted in which cultured microalgae are separated and collected using a centrifuge or the like, and lipids are extracted from the collected microalgae. However, such a collection method has the disadvantage of high production costs in terms of capital investment, etc. In addition, when separating and collecting algal cells using a centrifuge or the like, there is also the disadvantage that algal cells with low oil production and high specific gravity are preferentially precipitated and easily recovered by centrifugation, while algal cells with high oil production and low specific gravity are less likely to precipitate and be recovered. For this reason, methods have been proposed that are easier and cheaper to use, or that selectively collect algal cells with high oil production. For example, Patent Document 1 discloses Pseudochoricystis ( Pseudochoricystis ) genus or Corycystis ( Choricystis The present invention discloses a method for separating and recovering hydrocarbon-producing microalgae belonging to the genus Micrococcus from raw water containing the microalgae, the method comprising the steps of: adding a soluble metal salt that produces a poorly soluble hydroxide to the raw water containing the microalgae; adjusting the pH of the raw water to a level at which the poorly soluble hydroxide is produced; flocculating the microalgae by the precipitated poorly soluble hydroxide; and solid-liquid separation of the flocs produced in the flocculating step. In addition, Patent Document 2 discloses a method for treating Botryococcus ( Botryococcus) A method for isolating highly oil-producing Botryococcus algae cells is disclosed, which includes the steps of irradiating the algae cells with a heavy ion beam or X-rays and collecting the floating highly oil-producing Botryococcus algae cells. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-100121 [Patent Document 2] JP 2017-136000 A Summary of the Invention [Problem to be solved by the invention]

[0006] As mentioned above, a method has been disclosed for improving recovery efficiency by aggregating microalgae. However, when outdoor cultivation using an open pond method or mass cultivation using a large photobioreactor is performed, there is a large variation in the growth rate of individual microalgae. 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, and it is not possible to selectively recover microalgae that have accumulated sufficient lipids. Furthermore, although the method described in Patent Document 2 allows selective recovery of algal cells with high oil production, it requires irradiating the algal cells with heavy ion beams or X-rays, which increases equipment costs and is not suitable for outdoor or large-scale cultivation.

[0007] An object of the present invention is to provide a method for producing lipids that improves the floating property of microalgae and thereby improves recovery efficiency. Another object of the present invention is to provide a transformant of microalgae with improved floating ability. [Means for solving the problem]

[0008] In view of the above-mentioned problems, the present inventors conducted extensive research. As a result of a more detailed analysis of the floating property of microalgae, the inventors found that the floating property improves in proportion to the culture time of the microalgae. It was believed that this improvement in floating property occurs because the amount of oil per cell increases with culture, resulting in a lower specific gravity than the medium. Based on this observation, further research was conducted and the inventors discovered for the first time that by using a production strain with improved lipid production ability or a production strain with a lower specific gravity of the cells themselves, the floating property of the production strain can be improved, and microalgae with sufficient lipid accumulation can be selectively recovered more simply and at low cost. The present invention has been completed based on these findings.

[0009] The present invention relates to a method for producing lipids, comprising culturing a transformant of microalgae that has been subjected to at least one modification selected from the group consisting of the following (A) to (C), producing fatty acids or lipids containing fatty acids as constituent components, recovering the transformant by floatation separation, and obtaining lipids from the recovered transformant: (A) A modification that enhances 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) A modification that enhances the expression of at least one gene encoding a protein related to the Calvin cycle (hereinafter also referred to as the "CBB cycle"). (C) a modification that suppresses 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 a transformant, comprising culturing a transformant of microalgae that has been subjected to at least one modification selected from the group consisting of the following (A) to (C), causing the transformant to produce fatty acids or lipids containing fatty acids as constituent components, and recovering the transformant by floatation separation: (A) A modification that enhances the expression of at least one gene encoding a protein involved in the triacylglycerol synthesis pathway. (B) a modification that enhances the expression of at least one gene encoding a Calvin cycle-related protein. (C) a modification that suppresses 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 that has been modified as described above in (A) to (C). [Effects of the Invention]

[0012] According to the lipid production method and transformant recovery method of the present invention, by using a microalgae transformant with improved floating ability, algae that are highly producing lipids can be recovered more efficiently. Furthermore, the transformant of the present invention has excellent buoyancy because the expression of genes encoding proteins related to the TAG synthesis pathway and genes encoding proteins related to the CBB cycle is promoted, and the expression of genes encoding proteins related to the cell wall synthesis pathway is suppressed. [Brief explanation of the drawings]

[0013]

Figure 1

[0014] As used herein, "lipid" includes simple lipids such as neutral lipids (such as triacylglycerols), waxes, and ceramides; complex lipids such as phospholipids, glycolipids, and sulfolipids; and derived 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, meaning "free fatty acids." In the present invention, the fatty acid moiety in simple lipid and complex lipid molecules is referred to as "fatty acid residue." Unless otherwise specified, "fatty acid" is used as a general term for "free fatty acids" and "fatty acid residues" contained in salts or ester compounds, etc. In this specification, "fatty acids or lipids containing fatty acids as constituent components" is used to collectively refer to "free fatty acids" and "lipids having the 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) of fatty acids and fatty acid composition can be measured by the methods 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, the acyl group may be a fatty acid having from 2 to 22 carbon atoms, from 4 to 22 carbon atoms, from 6 to 22 carbon atoms, from 8 to 22 carbon atoms, from 10 to 22 carbon atoms, or from 12 to 20 carbon atoms. Furthermore, in this specification, when a fatty acid or an acyl group constituting a fatty acid is represented by "Cx:y," it means that the number of carbon atoms is x and the number of double bonds is y. "Cx" represents a fatty acid or acyl group having x carbon atoms.

[0015] In this specification, the identity of nucleotide sequences and amino acid sequences is calculated by the Lipman-Pearson method (Science, 1985, vol. 227, pp. 1435-1441). Specifically, the identity is calculated by performing analysis using the homology analysis (Search homology) program of the genetic information processing software Genetyx-Win, with the unit size to compare (ktup) set to 2. As used herein, "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, hybridization conditions include incubating a solution containing 6×SSC (1×SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5×Denhardt's, and 100 mg / mL herring sperm DNA together with a probe at 65°C for 8 to 16 hours. Furthermore, in the specification, "upstream" of a gene does not refer to the position from the translation start point, but refers to the region following the 5' side of the gene or region of interest, while "downstream" of a gene refers to the region following the 3' side of the gene or region of interest.

[0016] In the present invention and this specification, the term "floatability" refers to the property or ability to not settle upon centrifugation or standing still, or to float to the surface of the liquid. Furthermore, in the present invention and this specification, "improved floating ability" means that, compared to a negative control algal strain (host, wild-type strain) cultured under the same culture conditions for the same number of days, the proportion or amount of cells that do not settle after a certain amount of centrifugation or standing (including cells that float to the liquid surface) is improved, or the proportion or amount of cells that float to the liquid surface after a certain amount of centrifugation or standing is improved. The improved floating ability of the transformant of the present invention can be confirmed by centrifuging the cultured algal cells or standing them still, and then visually observing or measuring the cell count and weight of the algal cells in each fraction. The centrifugation conditions are not particularly limited, and examples include centrifugation at 21,600 × g for 10 minutes.

[0017] As described above, the transformant used in the lipid production method of the present invention is a microalgae transformant having at least one modification selected from the group consisting of the following (A) to (C): (A) A modification that promotes the expression of at least one gene encoding a TAG synthesis pathway-related protein (hereinafter also referred to as a "TAG synthesis pathway gene"). (B) Modification that promotes the expression of at least one gene encoding a CBB cycle-related protein (hereinafter also referred to as a "CBB cycle gene"). (C) Modification that suppresses the expression of at least one gene encoding a cell wall synthesis pathway-related protein (hereinafter also referred to as a "cell wall synthesis pathway gene"). By carrying out any of the modifications (A) to (C) above, the resulting transformant has improved floatation properties and therefore improved recovery efficiency, and can be suitably used in the lipid production method of the present invention. From the viewpoint of improving buoyancy, the transformant used in the method for producing lipids of the present invention preferably has undergone two or more of the modifications (A) to (C) above, more preferably has undergone the modifications (A) and / or (B) above and the following (C), and even more preferably has undergone all of the modifications (A) to (C) above ((A), (B), and (C)).

[0018] The "TAG synthesis pathway-related protein" is not particularly limited as long as it is a protein involved in the TAG synthesis pathway, and is preferably an enzyme that constitutes the TAG synthesis pathway. In the microalgae transformant used in the present invention, promoting the expression of a gene encoding a protein involved in the TAG synthesis pathway can improve lipid productivity and flotation.

[0019] Examples of the TAG synthesis pathway-related proteins include acyltransferases (hereinafter also referred to as "AT") such as 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"), and phosphatidic acid phosphatase (hereinafter also referred to as "PAP"). In particular, from the viewpoint of improving buoyancy, it is preferable that the expression of a gene encoding an ACS or a gene encoding an AT (preferably a DGAT) is promoted, and it is more preferable that the expression of a gene encoding an ACS and a gene encoding an AT (preferably a DGAT) is promoted.

[0020] The AT that can be used in the present invention is not particularly limited, and may be any protein having acyltransferase activity (hereinafter also referred to as "AT activity"). Here, "AT activity" refers to the activity of catalyzing 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 acyl-CoA, or acyl-ACP, in which CoA is bound to a free fatty acid, is incorporated into the glycerol backbone by various ATs and accumulated as TAG, which consists of one glycerol molecule esterified with three fatty acid molecules. Therefore, by promoting the expression of the gene encoding AT, the lipid productivity (particularly fatty acid productivity) of the transformant can be improved, and the amount of lipid accumulated in the transformant increases over the course of culture time, reducing the specific gravity of the transformant and, as a result, improving the buoyancy of the transformant.

[0021] The AT activity of the protein used in the present invention can be confirmed, for example, in a system using a strain lacking a triacylglycerol synthesis gene. Alternatively, DNA in which a gene encoding the protein is linked downstream of a promoter that functions in the host cell is introduced into the host cell, the cells are cultured under conditions in which the introduced gene is expressed, and then the cell lysate is added with any of the acceptors glycerol triphosphate (hereinafter also referred to as "G3P"), lysophosphatidic acid (hereinafter also referred to as "LPA"), and diacylglycerol (hereinafter also referred to as "DAG"), along with acyl-CoA, various phospholipids, various glycolipids, etc. as donors, and the synthesis of LPA from G3P, DAG from LPA, and TAG from DAG can be confirmed in this system.

[0022] The AT that can be used in the present invention can be appropriately selected from conventional ATs and proteins functionally equivalent thereto depending on the type of host, etc. Preferred ATs in the present invention include the following proteins (D) and (E). (D) A protein consisting of the amino acid sequence represented by SEQ 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 that has AT activity. Information on the amino acid sequences of the proteins used in the present invention and information on the sequences of the genes encoding them can be obtained from, for example, the National Center for Biotechnology Information (NCBI). The protein (D) consisting of the amino acid sequence represented by SEQ ID NO: 1 is isolated from Nannochloropsis ( Nannochloropsis Nannochloropsis oceanica (), an algae belonging to the genus Nannochloropsis oceanica ) AT (DGAT2-8) derived from the NIES-2145 strain. The protein consisting of the amino acid sequence represented by SEQ ID NO: 1 (the protein (D)) has AT activity.

[0023] In general, it is known that an amino acid sequence encoding an enzyme protein does not necessarily have to conserve the entire sequence in order to exhibit enzymatic activity, and that there are regions in which changes in the amino acid sequence do not affect the enzymatic activity. In such regions that are not essential for the enzymatic activity, the original activity of the enzyme can be maintained even if mutations such as deletion, substitution, insertion, or addition of amino acids are introduced. In the present invention, a protein that retains the desired enzymatic activity and has a partial mutation in the amino acid sequence of the enzyme protein can also be used.

[0024] Methods for introducing mutations into amino acid sequences include, for example, methods for introducing mutations into nucleotide sequences encoding the amino acid sequence. Examples of methods for introducing mutations include site-specific mutagenesis. Specific methods for introducing site-specific mutations include methods using splicing overlap extension (SOE) PCR (Horton et al., Gene 77, 61-68, 1989), the ODA method (Hashimoto-Gotoh et al., Gene 152, 271-276, 1995), and the Kunkel method (Kunkel, TA, Proc. Natl. Acad. Sci. USA, 1985, 82, 488). Alternatively, commercially available kits such as the Site-Directed Mutagenesis System Mutan-SuperExpress Km Kit (Takara Bio), the Transformer™ Site-Directed Mutagenesis Kit (Clontech), and the KOD-Plus-Mutagenesis Kit (Toyobo) can also be used. Alternatively, the target gene can be obtained by randomly mutating the gene and then evaluating the enzyme activity and analyzing the gene by an appropriate method.

[0025] In terms of AT activity, the identity of the protein (E) with the amino acid sequence of the protein (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. Further, examples of the protein (E) 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 the 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 having 75% (preferably 80%, preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identity to the amino acid sequence represented by SEQ ID NO: 85 and having AT activity. The protein consisting of the amino acid sequence represented by SEQ ID NO: 85 is derived from Nannochloropsis gaditana ( Nannochloropsis gaditana The amino acid sequence represented by SEQ ID NO: 85 is an AT (DGAT) derived from the ribosomal protein (D). The identity and similarity between the amino acid sequence represented by SEQ ID NO: 1 (amino acid sequence of protein (D)) are 81% and 90%, respectively.

[0026] Furthermore, the AT 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 has been added to the amino acid sequence of the above proteins (D) and (E).

[0027] The proteins (D) and (E) can be obtained by conventional chemical techniques, genetic engineering techniques, and the like. For example, proteins derived from natural products can be obtained by isolating and purifying them from Nannochloropsis oceanica. Alternatively, the proteins (D) and (E) can be obtained by artificial chemical synthesis based on the amino acid sequence information shown in SEQ ID NO: 1. Alternatively, the proteins (D) and (E) can be produced as recombinant proteins by genetic engineering techniques. When producing recombinant proteins, a gene encoding AT, described below, can be used. The AT used in the present 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 collections of private or public research institutes. For example, Nannochloropsis oceanica strain NIES-2145 can be obtained from the National Institute for Environmental Studies (NIES).

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

[0029] In terms of AT activity, the identity of the DNA (e) with the base sequence of the 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, the DNA (e) is also preferably a DNA in which one or more bases (for example, 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) have been deleted, substituted, inserted, or added in the base sequence of the DNA (d), and which encodes a protein having AT activity. Furthermore, the DNA (e) is also preferably a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA (d) and encodes a protein having AT activity. Examples of the DNA (e) include DNA consisting of the nucleotide sequence shown in SEQ ID NO: 86, or DNA consisting of a nucleotide sequence having 80% (preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identity to the nucleotide sequence shown in SEQ ID NO: 86 and encoding a protein having AT activity. The DNA consisting of the nucleotide sequence shown in SEQ ID NO: 86 is a gene encoding an AT (DGAT) derived from Nannochloropsis gaditana. The nucleotide sequence shown in SEQ ID NO: 86 has 75% identity with the nucleotide sequence shown in SEQ ID NO: 2 (the nucleotide sequence of DNA (d)).

[0030] Furthermore, the AT 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 the DNA (d) or (e).

[0031] Examples of mutations include deletion, substitution, addition, or insertion of bases. Methods for introducing mutations into a base sequence include, for example, site-specific mutagenesis. Specific methods for introducing site-specific mutations include methods using SOE-PCR, the ODA method, and the Kunkel method. Commercially available kits such as the Site-Directed Mutagenesis System Mutan-SuperExpress Km Kit (Takara Bio Inc.), the Transformer™ Site-Directed Mutagenesis Kit (Clontech), and the KOD-Plus-Mutagenesis Kit (Toyobo Co., Ltd.) can also be used. Alternatively, a target gene can be isolated by randomly introducing a gene mutation and then evaluating the enzyme activity and performing genetic analysis using an appropriate method.

[0032] The DNAs (d) and (e) can be obtained by conventional genetic engineering techniques. For example, an AT gene can be artificially synthesized based on the amino acid sequence represented by SEQ ID NO: 1 or the nucleotide sequence represented by SEQ ID NO: 2. The AT gene can be synthesized using services provided by, for example, Invitrogen. Alternatively, the AT gene can be obtained by cloning from the genome of algae or plants that have an AT gene on their genome, such as Nannochloropsis oceanica. For example, this can be achieved by the method described in Molecular Cloning—A Laboratory Manual Third Edition [Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press (2001)]. Furthermore, a portion of the nucleotide sequence represented by SEQ ID NO: 2 may be optimized depending on the type of host used. For example, Thermo Fisher Scientific's GeneArt artificial gene synthesis service can be used. Information on codons used by various organisms is available from the Codon Usage Database (www.kazusa.or.jp / codon / ) and other sources. The AT gene used in the present invention may be one type, or two or more types of AT genes may be used in combination.

[0033] The ACS that can be used in the present invention is not particularly limited, and may be any protein having acyl-CoA synthetase activity (hereinafter also referred to as "ACS activity"). Here, "ACS activity" refers to the activity of binding free fatty acids and CoA to produce acyl-CoA. ACS is a protein involved in the production of acyl-CoA by adding CoA to biosynthesized fatty acids (free fatty acids). Therefore, by promoting the expression of ACS, it is possible to improve the lipid productivity (particularly fatty acid productivity) of the transformant used for lipid production, and the amount of lipid accumulated in the transformant increases over the course of culture time, reducing the specific gravity of the transformant and improving the buoyancy of the transformant.

[0034] The ACS activity of the protein used in the present invention can be confirmed, for example, using a system using an ACS synthetic gene-deficient strain. Alternatively, DNA in which a gene encoding the protein is ligated downstream of a promoter functional in the host cell is introduced into the ACS synthetic gene-deficient strain, followed by culturing in a minimal salt medium containing free fatty acids as the sole carbon source and examining whether growth is restored (whether the strain can utilize (assimilate) the free fatty acids in the medium to grow). Alternatively, ACS protein or a cell lysate containing the protein can be prepared, reacted with a reaction solution containing free fatty acids, CoA, ATP, Mg ions, etc., and the decrease in CoA level can be measured using Ellman's reagent (DTNB).

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

[0036] In terms of ACS activity, the identity of the protein (G) with the amino acid sequence of the protein (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. Further, examples of the protein (G) 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) have been 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 set forth in SEQ ID NO: 87, or a protein consisting of an amino acid sequence that has 70% (preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identity to the amino acid sequence set forth in SEQ ID NO: 87 and that has ACS activity. The protein consisting of the amino acid sequence set forth in SEQ ID NO: 87 is an ACS (LACS) derived from Nannochloropsis gaditana. The amino acid sequence set forth in SEQ ID NO: 87 and the amino acid sequence set forth in SEQ ID NO: 3 (the amino acid sequence of protein (F)) share 88% identity and 93% similarity. Methods for introducing mutations into the amino acid sequence include, for example, the methods 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 in 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 sequence of the above proteins (F) and (G).

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

[0039] Examples of the gene (hereinafter also referred to as "ACS gene") encoding the ACS (preferably the protein (F) or (G)) include genes consisting of the following DNA (f) or (g): (f) DNA consisting of the base sequence represented by SEQ ID NO: 4. (g) A DNA having a base sequence that is 60% or more identical to the base sequence of the DNA (f) and that encodes a protein having ACS activity. The base sequence shown in SEQ ID NO: 4 is the base sequence of the gene (hereinafter also referred to as "LACS gene") that encodes the protein (LACS) consisting of the amino acid sequence shown in SEQ ID NO: 3.

[0040] In terms of ACS activity, the identity of the DNA (g) with the base sequence of the 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, the DNA (g) is also preferably a DNA in which one or more bases (for example, 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) have been deleted, substituted, inserted, or added in the base sequence of the DNA (f), and which encodes a protein having ACS activity. Furthermore, the DNA (g) is also preferably a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA (f) and encodes a protein having ACS activity. Examples of the DNA (g) include DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 88, or DNA consisting of a nucleotide sequence having 80% (preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identity to the nucleotide sequence set forth in SEQ ID NO: 88 and encoding a protein having ACS activity. The DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 88 is a gene encoding ACS (LACS) derived from Nannochloropsis gaditana. The nucleotide sequence set forth in SEQ ID NO: 88 has 76% identity with the nucleotide sequence set forth in SEQ ID NO: 4 (the nucleotide sequence of DNA (f)). Methods for introducing a mutation into a nucleotide sequence include, for example, the methods 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 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 the DNA (f) or (g).

[0042] The DNA (f) or (g) can be obtained by a conventional method, similar to the above-mentioned AT gene. The ACS gene used in the present invention may be one type, or two or more types of ACS genes may be used in combination.

[0043] In addition to the modification (A), it is preferable that the expression of a gene encoding a protein 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 (phosphopantetheinyl transferase), 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"), acyl-ACP thioesterase (hereinafter also referred to as "TE"), and the like. In particular, from the viewpoint of improving buoyancy, it is preferable that the expression of a gene encoding a TE (hereinafter also referred to as a "TE gene") is promoted.

[0044] The TE that can be used in the present invention is not particularly limited, and may be any protein that has 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 to generate free fatty acids. The action of TE terminates fatty acid synthesis on ACP, and the released fatty acids are used for the synthesis of polyunsaturated fatty acids or TAGs. Therefore, by promoting the expression of the TE gene in addition to the TAG synthesis pathway gene, the lipid productivity (particularly fatty acid productivity) of the transformant can be further improved, and the amount of lipid accumulated in the transformant increases over the course of culture time, reducing the specific gravity of the transformant and improving the buoyancy of the transformant.

[0045] Whether a protein has TE activity can be confirmed, for example, by introducing DNA in which a TE gene is linked downstream of a promoter that functions in host cells such as Escherichia 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 changes in the fatty acid composition in the host cells or culture medium using methods such as gas chromatography analysis. Alternatively, DNA in which a TE gene is linked downstream of a promoter that functions in host cells such as Escherichia coli can be introduced into the host cells, and the cells can be cultured under conditions in which the introduced TE gene is expressed. TE activity can then be measured by reacting the cell lysate with various acyl-ACPs as substrates prepared by the method of Yuan et al. (Yuan L. et al., Proc. Natl. Acad. Sci. USA, 1995, vol. 92(23), pp. 10639-10643).

[0046] TEs that can be used in the present invention can be appropriately selected from conventional TEs and proteins functionally equivalent thereto depending on the type of host, etc. Preferred TEs in the present invention include the following proteins (H) and (I). (H) A protein consisting of the amino acid sequence represented by SEQ 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 that has TE activity. The protein (H) consisting of the amino acid sequence represented by SEQ ID NO: 5 is a TE derived from Nannochloropsis oceanica strain NIES-2145. The protein consisting of the amino acid sequence represented by SEQ ID NO: 5 (the protein (H)) has TE activity.

[0047] In terms of TE activity, the identity of the protein (I) with the amino acid sequence of the protein (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. Further, examples of the protein (I) 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) have been deleted, substituted, inserted, or added to the amino acid sequence of the protein (H), and which have TE activity. Methods for introducing mutations into the amino acid sequence include, for example, the methods described above for AT.

[0048] The TE that can be used in the present invention may also be a protein consisting of an amino acid sequence in which a signal peptide involved in protein transport, a known amino acid sequence that increases protein stability, etc. is added to the amino acid sequence of the protein (H) or (I). Furthermore, the TE that can be used in the present invention may also be a protein consisting of an amino acid sequence in which the chloroplast targeting signal sequence predicted to be present in the N-terminal region of the amino acid sequence of the protein (H) or (I) is replaced with another chloroplast targeting signal sequence that functions in a host.

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

[0050] Examples of the gene encoding the 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 SEQ ID NO: 6. (i) A DNA having a base sequence that is 60% or more identical to the base sequence of the DNA (h) and encoding a protein having TE activity. The base sequence shown in SEQ ID NO:6 is the base sequence of a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:5.

[0051] In terms of TE activity, the identity of the DNA (i) 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, the DNA (i) is also preferably a DNA in which one or more bases (for example, 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) have been deleted, substituted, inserted, or added in the base sequence of the DNA (h), and which encodes a protein having TE activity. Furthermore, the DNA (i) is also preferably a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA (h) and encodes a protein having TE activity. Methods for introducing a mutation into a nucleotide sequence include, for example, the methods described above for the AT gene.

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

[0053] The DNA (h) or (i) can be obtained by a conventional method, similar to the above-mentioned AT gene. Furthermore, the TE gene used in the present invention may be one type, or two or more types of TE genes may be used in combination.

[0054] The "CBB cycle-related protein" is not particularly limited as long as it is a protein involved in the CBB cycle, and is preferably an enzyme that constitutes the CBB cycle. Plants and algae, including photosynthetic microorganisms, are known to fix carbon dioxide through photosynthesis via the CBB cycle. The CBB cycle consists of 13 reaction steps, with one molecule of CO2 fixed per reaction cycle. The resulting photosynthetic products are used not only as components of living organisms but also as an energy source. Therefore, by strengthening the CBB cycle and increasing the photosynthetic ability of microalgae, lipid productivity can be increased, and the floating ability of microalgae can be improved.

[0055] Examples of the 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-bisphosphate, phosphoribulokinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and triosephosphate isomerase. isomerase, fructose-1,6-biosphosphatase, ribulose-5-phosphate epimerase, Rubisco activase, etc. In particular, from the viewpoint of improving buoyancy, it is preferable that the expression of a gene encoding at least one selected from the group consisting of TK, FBA, and RPI is promoted. Also, it is preferable that the expression of a gene encoding TK is promoted, more preferable that the expression of a gene encoding TK and a gene encoding FBA is promoted, and even more preferable that the expression of a gene encoding TK, a gene encoding FBA, and a gene encoding RPI is promoted.

[0056] The TK that can be used in the present invention is not particularly limited, and may be any protein having transketolase activity (hereinafter also referred to as "TK activity"). Here, "TK activity" refers to the activity of transferring 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 erythrose-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 cycle is strengthened, which increases the amount of lipids accumulated in the transformant over the course of culture time, reducing the specific gravity of the transformant and, as a result, improving the buoyancy of the transformant.

[0057] The TK activity of the protein used in the present invention can be confirmed, for example, by the method described in Plant Physiol. (1989) 90, 814-819. Specifically, a solution containing the target protein is prepared by a standard method, and the solution is mixed with fructose-6-phosphate and glyceraldehyde-3-phosphate to confirm the production of erythrose-4-phosphate and xylulose-5-phosphate, or with sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate to confirm the production of xylulose-5-phosphate and ribose-5-phosphate.

[0058] The TK that can be used in the present invention can be appropriately selected from conventional TKs and proteins functionally equivalent thereto depending on the type of host, etc. Preferred TKs in the present invention include the following proteins (J) and (K). (J) A protein consisting of the amino acid sequence represented by SEQ 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 that has TK activity. The protein (J) consisting of the amino acid sequence represented by SEQ ID NO: 7 is a TK derived from Nannochloropsis oceanica strain NIES-2145. The protein consisting of the amino acid sequence represented by SEQ ID NO: 7 (the protein (J)) has TK activity.

[0059] In terms of TK activity, the identity of the protein (K) with the amino acid sequence of the protein (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. Further, examples of the protein (K) 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 set forth in SEQ ID NO: 89, or a protein consisting of an amino acid sequence that has 70% (preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identity to the amino acid sequence set forth in SEQ ID NO: 89 and that has TK activity. The protein consisting of the amino acid sequence set forth in SEQ ID NO: 89 is TK derived from Nannochloropsis gaditana. The amino acid sequence set forth in SEQ ID NO: 89 and the amino acid sequence set forth in SEQ ID NO: 7 (the amino acid sequence of protein (J)) have 91% identity and 95% similarity, respectively. Methods for introducing mutations into the amino acid sequence include, for example, the methods described above for AT.

[0060] Furthermore, the TK that can be used in the present invention may be a protein consisting of the amino acid sequence of the proteins (J) and (K) to which a signal peptide involved in protein transport or a known amino acid sequence that enhances protein stability has been added. Furthermore, the TK that can be used in the present invention may be a protein consisting of an amino acid sequence in which the chloroplast targeting signal sequence presumed to be present in the N-terminal region of the amino acid sequence of the proteins (J) and (K) has been replaced with another chloroplast targeting signal sequence that functions in a 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 a chloroplast targeting signal sequence. The present inventors have confirmed that adding the amino acid sequence from positions 1 to 100 of SEQ ID NO: 7 to the N-terminus of a reporter protein can localize the reporter protein to chloroplasts.

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

[0062] Specific examples of the gene (hereinafter also referred to as "TK gene") encoding the TK (preferably the protein (J) or (K)) include genes consisting of the following DNA (j) or (k): (j) DNA consisting of the base sequence represented by SEQ ID NO: 8. (k) A DNA having a base sequence that is 60% or more identical to the base sequence of the DNA (j) and encoding a protein having TK activity. The base sequence shown in SEQ ID NO: 8 is the base sequence of a gene (hereinafter also referred to as "TK gene") that encodes a protein (TK) consisting of the amino acid sequence shown in SEQ ID NO: 7.

[0063] In terms of TK activity, the identity of the DNA (k) 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, the DNA (k) is preferably a gene 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) have been deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 8, and which encodes a protein having TK activity. Furthermore, the DNA (k) is preferably a gene that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA (j) and encodes a protein having TK activity. Examples of the DNA (k) include DNA consisting of the nucleotide sequence shown in SEQ ID NO: 90, or DNA consisting of a nucleotide sequence having 75% (preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identity to the nucleotide sequence shown in SEQ ID NO: 90 and encoding a protein having TK activity. The DNA consisting of the nucleotide sequence shown in SEQ ID NO: 90 is a gene encoding TK derived from Nannochloropsis gaditana. The nucleotide sequence shown in SEQ ID NO: 90 has 83% identity with the nucleotide sequence shown in SEQ ID NO: 8 (the nucleotide sequence of DNA (j)). Methods for introducing a mutation into a nucleotide sequence include, for example, the methods described above for the AT gene.

[0064] The TK gene that can be used in the present invention may also be a gene consisting of a nucleotide sequence in which DNA encoding a signal peptide involved in protein transport or a known amino acid sequence that enhances protein stability has been added to the nucleotide sequence of DNA (j) or (k). Furthermore, the TK gene that can be used in the present invention may also be a DNA consisting of a nucleotide sequence in which the nucleotide sequence encoding a chloroplast targeting signal sequence predicted to be present in the 5'-terminal region of the nucleotide sequence of DNA (j) or (k) has been replaced with a nucleotide sequence encoding another chloroplast targeting signal sequence that functions in a host. Localization prediction using ChloroP (www.cbs.dtu.dk / services / ChloroP / ) predicts that the nucleotide sequence from positions 1 to 189 of SEQ ID NO: 8 encodes a chloroplast targeting signal sequence. The present inventors have confirmed that adding the nucleotide sequence from positions 1 to 300 of SEQ ID NO: 8 to the 5'-terminus of a nucleotide sequence encoding a reporter protein can localize the reporter protein to chloroplasts.

[0065] The DNA (j) or (k) can be obtained by a conventional method, similar to the above-mentioned AT gene. The TK gene used in the present invention may be one type, or two or more types of TK genes may be used in combination.

[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" refers to the activity of condensing glyceraldehyde-3-phosphate with dihydroxyacetone phosphate or the activity of condensing erythrose-4-phosphate with dihydroxyacetone phosphate. FBA is a protein (enzyme) that catalyzes the reaction in the CBB cycle to produce fructose-1,6-bisphosphate from glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, as well as the reaction to produce sedoheptulose-1,7-bisphosphate from erythrose-4-phosphate and dihydroxyacetone phosphate. Therefore, by promoting the expression of the gene encoding FBA, the CBB cycle can be strengthened, and the amount of lipids accumulated in the transformant increases over the course of culture time, reducing the specific gravity of the transformant and, as a result, improving the buoyancy of the transformant.

[0067] Whether the protein used in the present invention has FBA activity can be confirmed, for example, by the method described in Plant Physiol. (1989) 90, 814-819. Specifically, a solution containing the target protein is prepared by a standard method, and mixed with glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, and analyzed to confirm the production of fructose-1,6-bisphosphate or the production of sedoheptulose-1,7-bisphosphate from erythrose-4-phosphate and dihydroxyacetone phosphate.

[0068] The FBA that can be used in the present invention can be appropriately selected from conventional FBAs and proteins functionally equivalent thereto depending on the type of host, etc. Preferred FBAs in the present invention include the following proteins (L) and (M). (L) A protein consisting of the amino acid sequence represented by SEQ 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 that has FBA activity. The protein (L) consisting of the amino acid sequence represented by SEQ ID NO: 9 is an FBA derived from Nannochloropsis oceanica strain NIES-2145. The protein consisting of the amino acid sequence represented by SEQ ID NO: 9 (the protein (L)) has FBA activity.

[0069] In terms of FBA activity, the identity of the protein (M) with the amino acid sequence 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. Further, examples of the protein (M) 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 set forth in SEQ ID NO: 91, or a protein consisting of an amino acid sequence that has 65% (preferably 70%, more preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identity to the amino acid sequence set forth in SEQ ID NO: 91 and that has FBA activity. The protein consisting of the amino acid sequence set forth in SEQ ID NO: 91 is an FBA derived from Nannochloropsis gaditana. The amino acid sequence set forth in SEQ ID NO: 91 and the amino acid sequence set forth in SEQ ID NO: 9 (the amino acid sequence of protein (L)) have 94% identity and 97% similarity. Methods for introducing mutations into the amino acid sequence include, for example, the methods 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 in 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 sequence of the proteins (L) and (M). Furthermore, the FBA that can be used in the present invention may be a protein consisting of an amino acid sequence in which a chloroplast targeting signal sequence predicted to be present in the N-terminal region of the amino acid sequence of the proteins (L) and (M) has been replaced with another chloroplast targeting signal sequence that functions in a host. Localization prediction using ChloroP (www.cbs.dtu.dk / services / ChloroP / ) predicts that the amino acid sequence from positions 1 to 20 or 1 to 26 of SEQ ID NO: 9 is a chloroplast targeting signal sequence. The present inventors have confirmed that adding the amino acid sequence from positions 1 to 100 of SEQ ID NO: 9 to the N-terminus of a reporter protein can localize the reporter protein to chloroplasts.

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

[0072] Specific examples of the gene (hereinafter also referred to as "FBA gene") encoding the FBA (preferably the protein (L) or (M)) include genes consisting of the following DNA (l) or (m): (l) DNA consisting of the base sequence represented by SEQ ID NO: 10. (m) A DNA having a base sequence that is 60% or more identical to the base sequence of the DNA (l) and encoding a protein having FBA activity. The base sequence shown in SEQ ID NO: 10 is the base sequence of a gene (hereinafter also referred to as "FBA gene") that encodes a protein (FBA) consisting of the amino acid sequence shown in SEQ ID NO: 9.

[0073] In terms of FBA activity, the identity of the DNA (m) 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, the DNA (m) is preferably a gene 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) have been deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 10, and which encodes a protein having FBA activity. Furthermore, the DNA (m) is preferably a gene that hybridizes under stringent conditions with a DNA having a base sequence complementary to the DNA (l) and encodes a protein having FBA activity. Examples of the DNA (m) include DNA consisting of the nucleotide sequence shown in SEQ ID NO: 92, or DNA consisting of a nucleotide sequence having 75% (preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identity to the nucleotide sequence shown in SEQ ID NO: 92 and encoding a protein having FBA activity. The DNA consisting of the nucleotide sequence shown in SEQ ID NO: 92 is a gene encoding FBA derived from Nannochloropsis gaditana. The nucleotide sequence shown in SEQ ID NO: 92 has 85% identity with the nucleotide sequence shown in SEQ ID NO: 10 (the nucleotide sequence of DNA (l)). Methods for introducing a mutation into a nucleotide sequence include, for example, the methods described above for the AT gene.

[0074] The FBA gene that can be used in the present invention may also be a gene consisting of a nucleotide sequence in which DNA encoding a signal peptide involved in protein transport or a known amino acid sequence that enhances protein stability has been added to the nucleotide sequence of DNA (l) or (m). Furthermore, the FBA gene that can be used in the present invention may also be a DNA consisting of a nucleotide sequence in which the nucleotide sequence encoding a chloroplast targeting signal sequence predicted to be present in the 5'-terminal region of the nucleotide sequence of DNA (l) or (m) has been replaced with a nucleotide sequence encoding another chloroplast targeting signal sequence that functions in the host. Localization prediction using ChloroP (www.cbs.dtu.dk / services / ChloroP / ) predicts that the nucleotide sequence from positions 1 to 60 or 1 to 78 of SEQ ID NO: 10 encodes a chloroplast targeting signal sequence. The present inventors have confirmed that adding the nucleotide sequence from positions 1 to 300 of SEQ ID NO: 10 to the 5'-terminus of a nucleotide sequence encoding a reporter protein can localize the reporter protein to chloroplasts.

[0075] The DNAs (l) and (m) can be obtained by conventional methods, similar to the above-mentioned AT gene. Furthermore, the FBA gene used in the present invention may be one type, or two or more types of FBA genes may be used in combination.

[0076] The RPI that can be used in the present invention is not particularly limited, and may be any protein that has ribose-5-phosphate isomerase activity (hereinafter also referred to as "RPI activity"). Here, "RPI activity" refers to the activity of converting the aldehyde group of an aldose to a keto group. RPI is a protein (enzyme) that catalyzes the reaction of converting ribose-5-phosphate into ribulose-5-phosphate. Therefore, by promoting the expression of the gene encoding RPI, the CBB cycle can be strengthened, and the amount of lipids accumulated in the transformant increases over the course of culture time, reducing the specific gravity of the transformant and, as a result, improving the buoyancy of the transformant.

[0077] The RPI activity of the protein used in the present invention can be confirmed, for example, by the method described in The Plant Journal (2006) 48, 606-618. Specifically, a solution containing the target protein is prepared by a standard method, and the solution is mixed with ribose-5-phosphate to analyze the production of ribulose-5-phosphate.

[0078] The RPI that can be used in the present invention can be appropriately selected from conventional RPIs and proteins functionally equivalent thereto depending on the type of host, etc. Preferred RPIs in the present invention include the following proteins (N) and (O). (N) A protein consisting of the amino acid sequence represented by SEQ 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 has RPI activity. The protein (N) consisting of the amino acid sequence represented by SEQ ID NO: 11 is an RPI derived from Nannochloropsis oceanica strain NIES-2145. The protein consisting of the amino acid sequence represented by SEQ ID NO: 11 (the protein (N)) has RPI activity.

[0079] In terms of RPI activity, the identity of the protein (O) with the amino acid sequence of the protein (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. Further, examples of the protein (O) include proteins 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) have been deleted, substituted, inserted or added to the amino acid sequence of the protein (N), and which have RPI activity. Examples of the protein (O) include a protein consisting of the amino acid sequence set forth in SEQ ID NO: 93, or a protein consisting of an amino acid sequence that has 70% (preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identity to the amino acid sequence set forth in SEQ ID NO: 93 and that has RPI activity. The protein consisting of the amino acid sequence set forth in SEQ ID NO: 93 is an RPI derived from Nannochloropsis gaditana. The amino acid sequence set forth in SEQ ID NO: 93 and the amino acid sequence set forth in SEQ ID NO: 11 (the amino acid sequence of protein (N)) have 92% identity and 95% similarity. Methods for introducing mutations into the amino acid sequence include, for example, the methods 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 in 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 sequence of the proteins (N) and (O). Furthermore, the RPI that can be used in the present invention may be a protein consisting of an amino acid sequence in which a chloroplast targeting signal sequence predicted to be present in the N-terminal region of the amino acid sequence of the proteins (N) and (O) has been replaced with another chloroplast targeting signal sequence that functions in a 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 a chloroplast targeting signal sequence. The present inventors have confirmed that adding the amino acid sequence from positions 1 to 100 of SEQ ID NO: 11 to the N-terminus of a reporter protein can localize the reporter protein to chloroplasts.

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

[0082] Specific examples of the gene (hereinafter also referred to as "RPI gene") encoding the RPI (preferably the protein (N) or (O)) include genes consisting of the following DNA (n) or (o): (n) DNA consisting of the base sequence represented by SEQ ID NO: 12. (o) A DNA having a base sequence that is 60% or more identical to the base sequence of the DNA (n) and encoding a protein having RPI activity. The base sequence shown in SEQ ID NO: 12 is the base sequence of a gene (hereinafter also referred to as "RPI gene") that encodes a protein (RPI) consisting of the amino acid sequence shown in SEQ ID NO: 11.

[0083] In terms of RPI activity, the identity of the DNA (o) 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. Also preferred as the DNA (o) is a gene 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) have been deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 12, and which encodes a protein having RPI activity. Furthermore, the DNA (o) is preferably a gene that hybridizes under stringent conditions with a DNA having a base sequence complementary to the DNA (n) and encodes a protein having RPI activity. Examples of the DNA (o) include DNA consisting of the nucleotide sequence shown in SEQ ID NO: 94, or DNA consisting of a nucleotide sequence having 75% (preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identity to the nucleotide sequence shown in SEQ ID NO: 94 and encoding a protein having RPI activity. The DNA consisting of the nucleotide sequence shown in SEQ ID NO: 94 is a gene encoding RPI derived from Nannochloropsis gaditana. The nucleotide sequence shown in SEQ ID NO: 94 has 80% identity with the nucleotide sequence shown in SEQ ID NO: 12 (the nucleotide sequence of DNA (n)). Methods for introducing a mutation into a nucleotide sequence include, for example, the methods described above for the AT gene.

[0084] The RPI gene that can be used in the present invention may also be a gene consisting of the nucleotide sequence of the DNAs (n) and (o) to which DNA encoding a signal peptide involved in protein transport or a known amino acid sequence that enhances protein stability has been added. Furthermore, the RPI gene that can be used in the present invention may also be a DNA consisting of a nucleotide sequence in which the nucleotide sequence encoding a chloroplast targeting signal sequence predicted to be present in the 5'-terminal region of the nucleotide sequence of the DNAs (n) and (o) has been changed to a nucleotide sequence encoding another chloroplast targeting signal sequence that functions in a host. Localization prediction using ChloroP (www.cbs.dtu.dk / services / ChloroP / ) predicts that the nucleotide sequence from positions 1 to 147 of SEQ ID NO: 12 encodes a chloroplast targeting signal sequence. The present inventors have confirmed that adding the nucleotide sequence from positions 1 to 300 of SEQ ID NO: 12 to the 5'-terminus of a nucleotide sequence encoding a reporter protein can localize the reporter protein to chloroplasts.

[0085] The DNAs (n) and (o) can be obtained by conventional methods, similar to the above-mentioned AT gene. Furthermore, the RPI gene used in the present invention may be one type, or two or more types of RPI genes may be used in combination.

[0086] A transformant having undergone the above-described modification (A) and / or (B) can be obtained, for example, by introducing the TAG synthesis pathway gene and / or the CBB cycle gene into a host by a conventional method. Specifically, the transformant can be produced by preparing a vector such as an expression vector (gene expression plasmid) capable of expressing the gene in a host cell or a gene expression cassette, and then introducing the vector into a host cell to transform the host cell. Alternatively, a transformant having undergone the above-described modifications (A) and / or (B) can be obtained by, for example, modifying the expression regulatory region of the TAG synthetic pathway gene and / or CBB cycle gene in a host having the gene on its genome by a standard method to promote the expression of the gene. Specifically, the transformant can be produced by, for example, replacing the promoter sequence located upstream of the TAG synthetic pathway gene and / or CBB cycle gene present on the genome of the host with one exhibiting higher promoter activity. In this specification, a transformant that has been modified to promote or suppress the expression of a gene encoding a protein of interest is also referred to as a "transformant," and a wild-type strain that has not been modified to promote or suppress the expression of a gene encoding a protein of interest is also referred to as a "host" or "wild-type strain."

[0087] The transformant having undergone the above-described modifications (A) and / or (B) has improved lipid productivity (particularly fatty acid productivity) compared to the host itself, and the amount of lipid accumulated in the transformant cells increases over the course of culture, reducing the specific gravity of the transformant. As a result, the transformant exhibits improved flotation. Therefore, the transformant can be suitably used in a method for producing lipids. The productivity of fatty acids and lipids of the host or transformant can be measured by the methods used in the Examples.

[0088] The vector (plasmid) that serves as the base for a gene expression plasmid or gene expression cassette may be any vector that can introduce a gene encoding a target protein into a host and express the target gene in the host cell. For example, a vector having an expression control region such as a promoter or terminator appropriate for the type of host used, and a vector having a replication origin, selection marker, etc., may be used. Furthermore, the vector may be a vector that autonomously replicates and replicates outside the chromosome, such as a plasmid, or a vector that is integrated into the chromosome.

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

[0090] The type of promoter that regulates the expression of the gene encoding the protein of interest incorporated into the vector can also be selected appropriately depending on the type of host used. Promoters that can be preferably used in the present invention include housekeeping gene promoters (e.g., tubulin promoter, actin promoter, ubiquitin promoter, etc.), heat shock protein promoters, promoters of the violaxanthin / chlorophyll a-binding protein gene derived from Nannochloropsis (VCP1 promoter, VCP2 promoter) (Proceedings of the National Academy of Sciences of the United States of America, 2011, vol. 108(52)), and promoters of the oleosin-like protein LDSP (lipid droplet surface protein) gene derived from Nannochloropsis (LDSP promoter) (PLOS Genetics, 2012; 8(11): e1003064. doi: 10.1016 / j.1999.001003064). 1371), the ACP gene promoter (ACP promoter), the desaturase gene promoter, the AT gene promoter (AT promoter), the glutamine synthetase gene promoter derived from Nannochloropsis (GS promoter), and the ammonium transporter gene promoter derived from Nannochloropsis (AMT promoter) can be preferably used. Furthermore, since algae belonging to the genus Nannochloropsis are generally known to efficiently produce lipids under conditions of nutrient deficiency (especially nitrogen) or high light intensity, it is more preferable to use a promoter that is highly expressed under these conditions. From the viewpoint of high expression under conditions of nutrient deficiency or high light intensity, promoters of genes involved in the fatty acid synthesis pathway or TAG synthesis pathway and promoters of genes involved in nitrogen assimilation are preferred, with the LDSP gene promoter, ACP promoter, desaturase gene promoter, AT promoter, GS promoter, and AMT promoter being more preferred, and the LDSP gene promoter, GS promoter, and AMT promoter being even more preferred.

[0091] Furthermore, the type of selection marker used to confirm that a gene encoding a protein of interest has been incorporated can be appropriately selected depending on the type of host used. Selection markers that can be preferably used in the present 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, blasticidin S resistance gene, bialaphos resistance gene, zeocin resistance gene, paromomycin resistance gene, gentamicin resistance gene, and hygromycin resistance gene. Furthermore, a deletion of a gene associated with auxotrophy can also be used as a selection marker gene. Examples of genes associated with auxotrophy include genes involved in nitrate assimilation (nitrate reductase, nitrate transporter). The gene encoding the protein of interest can be introduced into the vector by standard methods such as restriction enzyme treatment and ligation.

[0092] Transformants into which a target gene fragment has been introduced can be selected using a selection marker or the like. For example, a drug resistance gene can be introduced into host cells together with the target DNA fragment during transformation, and the drug resistance acquired by the transformant can be used as an indicator. Introduction of the target DNA fragment can also be confirmed by PCR or other methods using the genome as a template.

[0093] A method for promoting the expression of the TAG synthesis pathway genes and / or CBB cycle genes by modifying the expression regulatory regions of the genes in a host having the genes on its genome will be described below. The term "expression regulatory region" refers to a promoter, terminator, and non-translated region, and these sequences are generally involved in regulating the expression level (transcription level, translation level) of adjacent genes. In a host having the TAG synthesis pathway genes and / or CBB cycle genes on its genome, modifying the expression regulatory region of the genes to promote expression of the genes can improve lipid productivity and the buoyancy of transformants.

[0094] Methods for modifying the expression regulatory region include, for example, promoter replacement. In a host having the TAG synthesis pathway genes and / or CBB cycle genes on its genome, the promoter of the gene can be replaced with a promoter with higher transcriptional activity to promote the expression of the gene. As the host, among the above-mentioned species, those having the TAG synthesis pathway genes and / or CBB cycle genes on their genome can be suitably used.

[0095] The promoter used for promoter replacement is not particularly limited, and can be appropriately selected from those that have higher transcription activity than the promoters of the TAG synthesis pathway genes and / or CBB cycle genes and are suitable for lipid production. For example, tubulin promoters, heat shock protein promoters, violaxanthin / chlorophyll a-binding protein gene promoters (VCP1 promoter, VCP2 promoter), the promoter of the oleosin-like protein LDSP gene derived from the genus 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 containing fatty acids as constituents, promoters of genes involved in the fatty acid synthesis pathway or the TAG synthesis pathway and promoters of genes involved in nitrogen assimilation are preferred, with the LDSP gene promoter, ACP promoter, desaturase gene promoter, AT promoter, GS promoter, and AMT promoter being more preferred, and the LDSP gene promoter, GS promoter, and AMT promoter being even more preferred.

[0096] The promoter can be modified using conventional methods such as homologous recombination. Specifically, a linear DNA fragment containing the upstream and downstream regions of the target promoter and containing another promoter in place of the target promoter is constructed, and this is then introduced into a host cell, where double-crossover homologous recombination occurs between the upstream and downstream regions of the target promoter in the host genome. As a result, the target promoter on the genome is replaced with the other promoter fragment, resulting in a modified promoter. Such a method for modifying a target promoter by homologous recombination can be performed with reference 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, a specific region in the genome can be modified by homologous recombination with reference to literature such as Proceedings of the National Academy of Sciences of the United States of America, 2011, vol. 108(52).

[0097] The "cell wall synthesis pathway-related protein" is not particularly limited as long as it is a protein involved in the cell wall synthesis pathway, and is preferably an enzyme that constitutes the cell wall synthesis pathway. In the present invention and this specification, the term "cell wall" is a concept that includes the alginane layer. In the transformant of the microalgae used in the present invention, suppressing the expression of the cell wall synthesis pathway genes inhibits the expression of proteins involved in cell wall synthesis, which is thought to result in inhibiting the construction of the cell wall in the microalgae, reducing the specific gravity and improving the floating ability of the transformant. Furthermore, in transformants in which the expression of the cell wall synthesis pathway genes is suppressed, the construction of cell walls, such as cellulose-derived cell walls and alginate layers, is inhibited, which improves the lipid extraction efficiency (recovery efficiency) during the lipid production process. Specifically, it is presumed that cell disruption by physical or chemical treatment becomes easier, and therefore it is thought that algae from which lipids can be easily recovered can be obtained.

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

[0099] In the present invention and this specification, "gene expression is suppressed" means that, when cultured under the same conditions, the expression of the gene of interest in a transformant that has undergone modification (C) is reduced or lost compared to the expression of the gene of interest in a host that has not undergone modification (C). The degree of reduction may be any as long as it is lower than the expression level of the gene in a host that has not undergone modification (C), and when the expression level is taken as 100%, the expression level of the gene in a transformant that has undergone modification (C) 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 any one of the subunits constituting CES, preferably CESA) in algae, cellulose synthesis is inhibited, which in turn inhibits the construction of a cellulose-derived cell wall, reducing the specific gravity and allowing the production of transformants with improved floating properties. In the present invention, the term "cellulose synthase" refers to an enzyme (or complex) consisting of a protein expressed from a CES gene, which is involved in the biosynthesis of cellulose. In addition, in this specification, the term "cellulose synthesis activity" (hereinafter also referred to as "CES activity") means the activity of catalyzing the cellulose synthesis reaction.

[0101] In the present invention, the CES whose expression is reduced or eliminated is not particularly limited as long as it is a protein (enzyme) that exhibits CES activity. In the present invention, the CES whose expression is reduced or eliminated can be selected as a candidate by analyzing genome information using Blast and selecting those annotated as CES. Alternatively, the amino acid sequence can be analyzed using Blastp and those annotated as CES can also be selected as a candidate. Furthermore, the protein can 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 conventional CESs and proteins functionally equivalent thereto depending on the type of host, etc. Preferred CESs in the present invention include the following proteins (P) and (Q). (P) A protein consisting of the amino acid sequence represented by SEQ 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 that has CES activity. The protein (P) consisting of the amino acid sequence represented by SEQ 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 (the protein (P)) consisting of the amino acid sequence represented by SEQ ID NO: 13 has CES activity.

[0103] In terms of CES activity, the identity of the protein (Q) with the amino acid sequence of the protein (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. Further, examples of the protein (Q) include proteins 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) have been deleted, substituted, inserted or added to the amino acid sequence of the protein (P), and which have 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 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 and has CES activity. The protein consisting of the amino acid sequence represented by SEQ ID NO: 95 is a 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)) share 83% identity and 90% similarity.

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

[0105] In terms of CES activity, the identity of the DNA (q) 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. Also preferred as the DNA (q) is a gene in which one or more bases (for example, 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) have been deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 14, and which encodes a protein having CES activity. Furthermore, the DNA (q) is preferably a gene that hybridizes under stringent conditions with a DNA having a base sequence complementary to the DNA (p) and encodes a protein having CES activity. Examples of the DNA (q) include DNA consisting of the nucleotide sequence shown in SEQ ID NO: 96, or DNA consisting of a nucleotide sequence that has 85% (preferably 90%, more preferably 95%, and even more preferably 98%) or more identity to the nucleotide sequence shown in SEQ ID NO: 96 and encodes a protein having CES activity. The DNA consisting of the nucleotide sequence shown in SEQ ID NO: 96 is a gene encoding CES derived from Nannochloropsis gaditana. The nucleotide sequence shown in SEQ ID NO: 96 has 74% identity with the nucleotide sequence shown in SEQ ID NO: 14 (the nucleotide sequence of DNA (p)).

[0106] The transformant having undergone the above modification (C) can be obtained, for example, by the following method. Methods for suppressing the expression of cell wall synthesis pathway genes will now be described. In the present invention, the method for suppressing the expression of cell wall synthesis pathway genes can be appropriately selected from conventional methods. Examples include methods for deleting (partially or entirely) cell wall synthesis pathway genes, methods for downregulating cell wall synthesis pathway genes, methods for modifying the promoters of cell wall synthesis pathway genes, and methods 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 genes. Instead of suppressing the expression of a cell wall synthesis pathway gene, it is also possible to appropriately select a method of inactivating the cell wall synthesis pathway gene, or introducing a deletion or mutation into a cell wall synthesis pathway-related protein itself, etc. The effects obtained by these methods are equivalent to those obtained by suppressing the expression of a cell wall synthesis pathway gene.

[0107] A method for suppressing the expression of a cell wall synthesis pathway gene by deleting or inactivating the cell wall synthesis pathway gene will be described. The method for deleting or inactivating a cell wall synthesis pathway gene can be appropriately selected from conventional methods. For example, the cell wall synthesis pathway gene can be deleted or inactivated by a general method such as a gene disruption method utilizing the homologous recombination ability of the algae itself, a method utilizing genome editing technologies such as transcription activator-like effector nuclease (TALEN) or CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat), or a mutagenesis method utilizing mutation or the like. Specifically, a DNA fragment containing a portion of a cell wall synthesis pathway gene or a circular recombinant plasmid obtained by cloning into an appropriate plasmid (vector) is introduced into the cells of algae, and homologous recombination in a partial region of the cell wall synthesis pathway gene can be used to delete the cell wall synthesis pathway gene on the genome, or to disrupt the cell wall synthesis pathway gene and inactivate it. Alternatively, cell wall synthesis pathway genes can be randomly inactivated by using mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine, inducing mutations in cell wall synthesis pathway genes by irradiation with ultraviolet light or gamma rays, inducing site-specific point mutations in cell wall synthesis pathway genes (e.g., frameshift mutations, in-frame mutations, insertion of stop codons, etc.), or substituting all or part of a cell wall synthesis pathway gene with any other DNA fragment (e.g., any selection marker). In the present invention, it is preferable to delete or inactivate a cell wall synthesis pathway gene on the genome by homologous recombination.

[0108] When a cell wall synthesis pathway gene is deleted or inactivated by homologous recombination, a plasmid (vector) or a DNA cassette for homologous recombination of the cell wall synthesis pathway gene is introduced into the algae. The plasmid or DNA cassette for homologous recombination of the cell wall synthesis pathway gene used herein targets all or part of the cell wall synthesis pathway gene. Preferably, a plasmid or DNA cassette is constructed having a base sequence homologous to a portion of the upstream genome encoding the target region and a base sequence homologous to a portion of the downstream genome, and then introduced into the algae. Information on the upstream and downstream base sequences of cell wall synthesis pathway genes necessary for homologous recombination can be obtained from, for example, the National Center for Biotechnology Information (NCBI).

[0109] Algae in which cell wall synthesis pathway genes have been deleted or inactivated can be selected using a selection marker or the like. For example, drug resistance acquired by algae as a result of introducing a drug resistance gene into host cells can be used as an indicator. Introduction of the target DNA fragment can also be confirmed by PCR or other methods using genomic DNA as a template.

[0110] Furthermore, to confirm that the cell wall synthesis pathway gene has been replaced with the homologous recombination plasmid or the homologous recombination DNA cassette and the cell wall synthesis pathway gene has been deleted or inactivated, a selection marker to be incorporated into the homologous recombination plasmid or the homologous recombination DNA cassette can be appropriately selected from commonly used selection markers. Selection markers that can be preferably used in the present 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, blasticidin S resistance gene, bialaphos resistance gene, zeocin resistance gene, paromomycin resistance gene, and hygromycin resistance gene. Furthermore, genes associated with auxotrophy can also be used as marker genes. Examples of genes associated with auxotrophy include genes involved in nitrate assimilation (nitrate reductase, nitrate transporter). The selection marker can be introduced into a vector by standard methods such as restriction enzyme treatment or ligation.

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

[0112] A method for downregulating cell wall synthesis pathway genes to suppress the expression of cell wall synthesis pathway genes is described. 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 is reduced (downregulation of the cell wall synthesis pathway gene). When the expression level of the cell wall synthesis pathway gene is reduced, 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 algae. Therefore, it is thought that reducing the expression level of the cell wall synthesis pathway gene partially inhibits cell wall synthesis, reducing the specific gravity and improving the floating ability of the transformant.

[0113] The method for downregulating cell wall synthesis pathway genes can be appropriately selected from conventional methods, such as inducing mutations in the promoter or transcription / translation initiation region of a cell wall synthesis pathway gene using mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine or irradiation with UV or gamma rays, inserting any other DNA fragment (e.g., any repressor, any selection marker, etc.) into the promoter sequence or transcription / translation initiation region of a cell wall synthesis pathway gene, substituting all or part of the promoter sequence or transcription / translation initiation region of a cell wall synthesis pathway gene with any other DNA fragment (e.g., any repressor, any selection marker, etc.), antisense methods, RNA interference, promoter competition, etc.

[0114] The host for the transformant can be appropriately selected from those commonly used. The host that can be used in the present invention is preferably an algae belonging to the Heterokontophyta phylum, and among the algae belonging to the Heterokontophyta phylum, an algae belonging to the Euonymus phyceae is more preferable. Specific examples of algae belonging to the Euonymus phyceae include algae of the genus Nannochloropsis, Monodopsis ( Monodopsis ) genus of algae, Bisceria ( Vischeria ) algae of the genus Chlorobotrys ( Chlorobotrys ) genus of algae, Goniochloris ( Goniochloris Among them, algae of the genus Nannochloropsis are more preferred from the viewpoint of lipid productivity. Specific examples of the algae of the genus Nannochloropsis include Nannochloropsis oceanica, Nannochloropsis oculata ( Nannochloropsis oculata ), Nannochloropsis gaditana, Nannochloropsis salina ( Nannochloropsis salina ), Nannochloropsis limnetica ( Nannochloropsis limnetica ), Nannochloropsis granulata ( Nannochloropsis granulata ), Nannochloropsis sp. ( NannochloropsisAmong them, from the viewpoint of lipid productivity, Nannochloropsis oceanica or Nannochloropsis gaditana is preferred, 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, electroporation, LP transformation, methods using Agrobacterium, and particle gun methods. When algae of the genus Nannochloropsis are used 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, for example.

[0116] Transformants into which a target gene fragment has been introduced can be selected using a selection marker or the like. For example, a drug resistance gene can be introduced into host cells together with the target DNA fragment during transformation, and the drug resistance acquired by the transformant can be used as an indicator. Introduction of the target DNA fragment can also be confirmed by PCR or other methods using the genome as a template.

[0117] The transformant used in the method for producing lipids of the present invention has improved flotation ability compared to a host that has not undergone the modifications (A) to (C). Therefore, by culturing the transformant of the present invention under appropriate conditions and then recovering fatty acids or lipids containing fatty acids as constituents from the resulting culture, fatty acids or lipids containing fatty acids as constituents can be efficiently produced. Here, the term "culture" refers to the culture medium and transformants after culturing.

[0118] The culture conditions for the transformant of the present invention can be appropriately selected depending on the host of the transformant, and culture conditions generally used for that host can be used. In addition, from the viewpoint of fatty acid production efficiency, for example, glycerol, acetic acid, glucose, or the like may be added to the medium as a precursor involved in the fatty acid biosynthesis system.

[0119] The medium used for the culture may be based on natural seawater or artificial seawater, or a commercially available culture medium may be used. Specific examples of the medium include f / 2 medium, ESM medium, Daigo IMK medium, L1 medium, and MNK medium. Among them, from the viewpoint of improving lipid productivity and nutrient concentration, f / 2 medium, ESM medium, or Daigo IMK medium is preferred, f / 2 medium or Daigo IMK medium is more preferred, and f / 2 medium is even more preferred. In order to promote algae growth and improve fatty acid productivity, a nitrogen source, a phosphorus source, metal salts, vitamins, trace metals, and the like may be appropriately added to the medium. Furthermore, when sodium nitrate is added as a nitrogen source, the growth of microalgae and lipid productivity can be improved by using a medium in which the sodium nitrate is replaced with ammonium bicarbonate (the molar amount of nitrogen atoms in the added sodium nitrate is replaced with the molar amount of nitrogen atoms in the ammonium bicarbonate so that they are equal), which results in improved flotation.

[0120] The amount of transformant to be inoculated into the medium can be selected appropriately, and from the viewpoint of growth, it is preferably 1 to 50% (vol / vol) per medium, more preferably 1 to 10% (vol / vol). The culture temperature is not particularly limited as long as it does not adversely affect the growth of the algae, but is usually in the range of 5 to 40°C. From the viewpoints of promoting algal growth, improving fatty acid productivity, and reducing production costs, it is preferably 10 to 35°C, more preferably 15 to 30°C. The culture is preferably carried out under light irradiation so that photosynthesis can occur. The light irradiation may be under any conditions that allow photosynthesis, and may be artificial light or sunlight. The light intensity during light irradiation is preferably 1 to 4,000 μmol / m 2 from the viewpoint of promoting algae growth and improving fatty acid productivity. 2 / s, more preferably 10 to 2,500 μmol / m 2 / s, more preferably 100 to 2,500 μmol / m 2 / s, more preferably 200 to 2,500 μmol / m 2 / s, more preferably 250 to 2,500 μmol / m 2 / s, more preferably 300 to 2,500 μmol / m 2 The interval between light irradiations is not particularly limited, but from the same viewpoint as above, it is preferable to perform the irradiation in a 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 out of 24 hours. The culture is preferably carried out in the presence of a gas containing carbon dioxide to enable photosynthesis, or in a medium containing a carbonate such as sodium bicarbonate. The concentration of carbon dioxide in the gas is not particularly limited, but from the viewpoints 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 carbonate is not particularly limited, but when sodium bicarbonate is used, for example, 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 viewpoints of promoting growth and improving fatty acid productivity. The culture time is not particularly limited, and may be long (for example, about 150 days) so that algae that accumulate high concentrations of lipids can grow at high concentrations. From the viewpoints 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 aeration-agitation culture, shaking culture, or static culture, and aeration-agitation culture is preferred from the viewpoint of improving breathability.

[0121] Furthermore, the transformant used in the lipid production method of the present invention has improved floating properties, which facilitates the recovery of algal cells, and lipids can be efficiently produced even in large-scale culture. Examples of such large-scale culture include the so-called open pond method using natural ponds or artificial raceway ponds, and methods using closed photobioreactors. In particular, when culturing microorganisms such as transformants on a small scale in a laboratory, light is irradiated from multiple directions, allowing all cells to efficiently obtain light energy regardless of their degree of buoyancy. However, in open pond systems, 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 solution earlier, thereby allowing for more efficient acquisition of light energy even in open pond systems, thereby improving lipid productivity. Furthermore, since the transformants used in the lipid production method of the present invention have improved buoyancy, transformants that have accumulated lipids float in the upper layer of the culture solution (near the liquid surface). Therefore, transformants with high lipid content can be efficiently and easily recovered by recovering the upper layer of the culture solution without recovering the entire culture solution. The open pond system has advantages such as being simpler and having lower equipment costs than closed photobioreactors. Furthermore, when the lipid production method of the present invention is carried out using a closed photobioreactor, there are advantages in that less energy is required for recovery, culture conditions are easily controlled, and production efficiency is high. In particular, when culturing algal cells by continuous culture (chemostat) as described below, a closed photobioreactor allows for more efficient recovery of algal cells and also allows for appropriate control of the growth rate of the algal cells in the culture solution and the nutrients in the culture solution. The shape of the closed photobioreactor is not particularly limited, and various shapes can be used, such as tubular, flat panel, Christmas tree, plate, horizontal, model, and porous substrate shapes.

[0122] The method for producing lipids of the present invention includes a step of recovering algae (transformants of microalgae) by flotation separation. In the present invention and this specification, "flotation separation" refers to a method in which the transformants, whose specific gravity is lighter than that of the culture solution due to the difference in specific gravity between the transformants and the culture solution, move to the upper layer of the culture solution, thereby separating the transformants whose specific gravity is lighter than that of the culture solution. The method for recovering the algal cells by flotation separation is not particularly limited, and any recovery method by flotation separation used in lipid production methods can be applied as appropriate. For example, after centrifuging or leaving the culture solution to stand, the algal cells that float to the top layer can be recovered by decantation, or the algal cells can be recovered by suctioning the upper layer culture solution, or the algal cells can be recovered by scooping them up using a net or the like. Note that, in order to perform flotation separation efficiently, it is preferable to appropriately stir or aerate the culture solution during culture. Furthermore, when growing the algal cells in an open pond system, it is also preferable to perform the recovery process at night when the algal cells are not exposed to sunlight, in view of culturing the algal cells during the day. The conditions for the centrifugation are not particularly limited, as long as they allow the algae (especially those with high lipid accumulation) to concentrate in the upper layer. For example, the centrifugal acceleration (g) is preferably 100 to 30,000 × g, more preferably 1,000 to 20,000 × g. The centrifugation time is preferably 0.5 to 240 minutes, more preferably 1 to 30 minutes. The conditions for the standing are not particularly limited, and may be any conditions that allow the algae (especially those with high lipid accumulation) to concentrate in the upper layer. For example, standing is preferably for 1 hour to 10 days, and more preferably for 6 hours to 2 days. Furthermore, if agitation or aeration is performed during culture, it is also preferable not to perform agitation or aeration during the standing conditions.

[0123] Furthermore, because the transformant of the present invention has improved floating properties, it is possible to recover only the algal cells present near the surface by flotation separation, eliminating the need to recover the entire culture solution. This makes it possible to selectively recover the algal cells in the upper layer where sufficient oil has accumulated, without recovering the algal cells with low floating properties, for example. The algal cells with low floating properties can be continued to be cultured, allowing them to accumulate sufficient oil and float to the upper layer, and then recovered. By selectively recovering the algal cells that have floated to the upper layer in this way and not recovering all of the culture solution, the cost of replacing the culture solution can be significantly reduced. That is, the lipid production method of the present invention can be performed in a batch culture mode, in which all necessary nutrients are added to the culture medium at the start of culture and all of the culture medium is recovered at the end of culture. Alternatively, the method can be performed in a continuous culture mode (chemostat), in which fresh culture medium is continuously supplied to the culture tank and the same amount of culture medium is simultaneously discharged from the system along with the algae, thereby recovering the algae while culturing in a steady state. When culturing algae using continuous culture, it is preferable to preferentially recover algae with a high oil content by floatation separation, and to re-culture algae with a low oil content. In the lipid production method of the present invention, it is preferable to culture the algae using 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] The method for recovering lipids from the culture can be appropriately selected from conventional methods. For example, the cells of the culture can be disrupted by heat treatment, high-pressure treatment, high-temperature and high-pressure treatment, physical disruption using beads or the like, squeezing such as by hand pressing, acid or alkali treatment, chemical treatment using digestive enzymes or the like, and then the lipid components can be isolated by filtration, centrifugation, gel filtration chromatography, ion exchange chromatography, or solvent extraction such as chloroform / methanol extraction, hexane extraction, and ethanol extraction, thereby recovering lipids. When larger-scale cultivation is performed, lipids can be obtained by recovering oil from the culture by squeezing and / or solvent extraction, followed by general purification such as degumming, deacidification, bleaching, decolorization, dewaxing, and deodorization. After isolating the lipid components in this manner, fatty acids can be obtained by hydrolyzing the isolated lipids. Methods for isolating fatty acids from lipid components include, for example, treatment in an alkaline solution at a high temperature of about 70°C, lipase treatment, and decomposition using high-pressure hot water.

[0125] From the viewpoint of utility, the lipid produced by the production method of the present invention preferably contains a fatty acid or a fatty acid compound, and more preferably contains a fatty acid or a fatty acid ester compound. From the viewpoint of productivity, the fatty acid ester compound is preferably a simple lipid or a complex lipid, more preferably a simple lipid, and even more preferably TAG.

[0126] The fatty acids obtained by the production method of the present invention can be used as food, as well as for plasticizers, emulsifiers for cosmetics, cleaning agents such as soaps and detergents, fiber treatment agents, hair rinses, or disinfectants and preservatives.

[0127] The present invention also provides a modification plasmid or a modification DNA cassette used in the modifications (A) to (C). 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 a homologous recombination DNA cassette that has a base sequence homologous to a portion upstream 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 downstream of a region on the genome consisting of the base sequence of a cell wall synthesis pathway gene and its downstream base sequence. These modification plasmids or modification DNA cassettes can be suitably used to produce microalgae that have undergone the modifications (A) to (C). The present invention also provides a kit for producing a transformant, which includes the modification plasmid or modification DNA cassette. The kit of the present invention may also include, in addition to the vector, other elements necessary for detecting the production of a transformant, such as a host, reagents typically used for transforming a host with the vector, a transformation buffer, and reagents that serve as indicators for selecting transformants.

[0128] In relation to the above-mentioned embodiments, the present invention further discloses the following transformants and methods.

[0129] <1> A method for producing lipids, comprising culturing a transformant of microalgae that has been modified with at least one modification selected from the group consisting of the following (A) to (C), preferably at least two modifications selected from the group consisting of the following (A) to (C), more preferably the following (A) and / or (B) and the following (C), and even more preferably the following (A), (B) and (C), to produce fatty acids or lipids containing fatty acids as constituent components, recovering the transformant by floatation separation, and obtaining lipids from the recovered transformant. (A) A modification that enhances the expression of at least one gene encoding a protein involved in the TAG synthesis pathway. (B) a modification that enhances the expression of at least one gene encoding a CBB cycle-related protein (C) a modification that suppresses the expression of at least one gene encoding a cell wall synthesis pathway-related protein

[0130] <2> A method for recovering a transformant, comprising culturing a transformant of microalgae that has been modified in at least one manner selected from the group consisting of the following (A) to (C), preferably in at least two manners selected from the group consisting of the following (A) to (C), more preferably in the manner of the following (A) and / or (B) and the following (C), and even more preferably in the manner of the following (A), (B) and (C), to produce fatty acids or lipids containing fatty acids as constituent components, and recovering the transformant by floatation separation. (A) A modification that enhances the expression of at least one gene encoding a protein involved in the TAG synthesis pathway. (B) a modification that enhances the expression of at least one gene encoding a CBB cycle-related protein (C) a modification that suppresses 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 in the cells of the microalgae, thereby promoting the expression of the TAG synthesis pathway-related protein. <1> or <2> The method described in section. <4> A gene encoding the TAG synthesis pathway-related protein is introduced into the microalgae, and expression of the introduced gene encoding the TAG synthesis pathway-related protein is promoted. <1> ~ <3> 10. The method according to any one of claims 1 to 9. <5> The gene encoding a TAG synthetic 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, DGAT, etc.), even more preferably a gene encoding an ACS and a gene encoding an AT (GPAT, LPAAT, DGAT, etc.), and even more preferably a gene encoding an ACS and a gene encoding a DGAT. <1> ~ <4> 10. The method according to any one of claims 1 to 9.

[0132] <6> The AT is the following protein (D) or (E): <5> The method described in section. (D) A protein consisting of the amino acid sequence represented by SEQ ID NO: 1. (E) A protein having an amino acid sequence that is 60% or more, 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, even more preferably 99% or more identical to the amino acid sequence of the protein (D), and having AT activity. <7> the protein (E) is a protein in which one or more, 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, amino acids are deleted, substituted, inserted or added to the amino acid sequence of the protein (D), and the protein (E) has AT activity; <6> The method described in section. <8> The protein (E) is a protein consisting of the amino acid sequence represented by SEQ ID NO: 85, or a protein consisting of an amino acid sequence having an identity of 75% (preferably 80%, preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more to the amino acid sequence represented by SEQ ID NO: 85, and having AT activity. <6> or <7> The method described in section. <9> the gene encoding the AT is a gene consisting of the following DNA (d) or (e): <5> ~ <8> 10. The method according to any one of claims 1 to 9. (d) DNA consisting of the base sequence represented by SEQ ID NO: 2. (e) DNA having a base sequence that is 60% or more, 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, even more preferably 99% or more identical to the base sequence of DNA (d), and encoding a protein having AT activity. <10> the DNA (e) is a DNA in which one or more, 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, and more preferably 1 to 10 bases are deleted, substituted, inserted, or added to the base sequence of the DNA (d), and which encodes a protein having AT activity; <9> The method described in section. <11> the DNA (e) is a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 86, or a DNA consisting of a nucleotide sequence having an identity of 80% (preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more to the nucleotide sequence represented by SEQ ID NO: 86, and encoding a protein having AT activity; <9> or <10> The method described in section.

[0133] <12> The ACS is the following protein (F) or (G): <5> ~ <11> 10. The method according to any one of claims 1 to 9. (F) A protein consisting of the amino acid sequence represented by SEQ ID NO:3. (G) A protein having an amino acid sequence that is 60% or more, 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, even more preferably 99% or more identical to the amino acid sequence of the protein (F), and having ACS activity. <13> the protein (G) is a protein in which one or more, 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 amino acids are deleted, substituted, inserted or added to the amino acid sequence of the protein (F), and the protein (G) has ACS activity; <12> The method described in 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 having an identity of 70% (preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more to the amino acid sequence represented by SEQ ID NO: 87, and having ACS activity; <12> or <13> The method described in section. <15> the gene encoding the ACS is a gene consisting of the following DNA (f) or (g): <5> ~ <14> 10. The method according to any one of claims 1 to 9. (f) DNA consisting of the base sequence represented by SEQ ID NO: 4. (g) DNA having a base sequence that is 60% or more, 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, even more preferably 99% or more identical to the base sequence of DNA (f), and encoding a protein having ACS activity. <16> the DNA (g) is a DNA in which one or more, 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, and more preferably 1 to 19 bases are deleted, substituted, inserted, or added to the base sequence of the DNA (f), and which encodes a protein having ACS activity; <15> The method described in section. <17> the DNA (g) is a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 88, or a DNA consisting of a nucleotide sequence having an identity of 80% (preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more to the nucleotide sequence represented by SEQ ID NO: 88, and encoding a protein having ACS activity; <15> or <16> The method described in section.

[0134] <18> In addition to the modification (A), the expression of a gene encoding a protein involved in a fatty acid synthesis pathway is enhanced. <1> ~ <17> 10. The method according to any one of claims 1 to 9. <19> The expression of a gene encoding a protein involved in the fatty acid synthesis pathway is promoted in the cells of the microalgae, thereby promoting the expression of the protein involved in the fatty acid synthesis pathway. <18> The method described in section. <20> a gene encoding a protein involved in the fatty acid synthesis pathway is introduced into the microalgae, and expression of the introduced gene encoding the protein involved in the fatty acid synthesis pathway is promoted; <18> or <19> The method described in section. <21> The gene encoding a 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> 10. The method according to any one of claims 1 to 9.

[0135] <22> The TE is the following protein (H) or (I): <21> The method described in section. (H) A protein consisting of the amino acid sequence represented by SEQ ID NO: 5. (I) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of 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, even more preferably 99% or more, and having TE activity. <23> the protein (I) is a protein in which one or more, 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 amino acids are deleted, substituted, inserted or added to the amino acid sequence of the protein (H), and the protein (I) has TE activity; <22> The method described in section. <24> the gene encoding the TE is a gene consisting of the following DNA (h) or (i): <21> ~ <23> 10. The method according to any one of claims 1 to 9. (h) DNA consisting of the base sequence represented by SEQ ID NO: 6. (i) DNA having a base sequence that is 60% or more, 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, even more preferably 99% or more identical to the base sequence of DNA (h), and encoding a protein having TE activity. <25> the DNA (i) is a DNA in which one or more, 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 bases are deleted, substituted, inserted or added to the base sequence of the DNA (h), and which encodes a protein having TE activity; <24> The method described.

[0136] <26> The expression of a gene encoding the CBB cycle-related protein is promoted in the cells of the microalgae, thereby promoting the expression of the CBB cycle-related protein. <1> ~ <25> 10. The method according to any one of claims 1 to 9. <27> a gene encoding the CBB cycle-related protein is introduced into the microalgae, and expression of the introduced gene encoding the CBB cycle-related protein is promoted; <1> ~ <26> 10. The method according to any one of claims 1 to 9. <28> the gene encoding a 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, phosphoribulokinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate 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, and even more preferably a gene encoding TK, a gene encoding FBA, and a gene encoding RPI; <1> ~ <27> 10. The method according to any one of claims 1 to 9.

[0137] <29> The TK is the following protein (J) or (K): <28> The method described. (J) A protein consisting of the amino acid sequence represented by SEQ ID NO: 7. (K) A protein having an amino acid sequence that is 60% or more, 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, even more preferably 99% or more identical to the amino acid sequence of the protein (J), and having TK activity. <30> the protein (K) is a protein in which one or more, 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, amino acids are deleted, substituted, inserted or added to the amino acid sequence of the protein (J), and the protein has TK activity; <29> The method described in section. <31> The protein (K) is a protein consisting of the amino acid sequence represented by SEQ ID NO: 89, or a protein consisting of an amino acid sequence having 70% (preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more identity to the amino acid sequence represented by SEQ ID NO: 89, and having TK activity. <29> or <30> The method described in section. <32> the gene encoding TK is a gene consisting of the following DNA (j) or (k): <28> ~ <31> A method according to any one of the preceding claims. (j) DNA consisting of the base sequence represented by SEQ ID NO: 8. (k) A DNA having a base sequence that is 60% or more, 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, even more preferably 99% or more identical to the base sequence of the DNA (j), and encoding a protein having TK activity. <33> the DNA (k) is a DNA in which one or more, 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 bases are deleted, substituted, inserted or added to the base sequence of the DNA (j), and which encodes a protein having TK activity; <32> The method described. <34> the DNA (k) is a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 90, or a DNA consisting of a nucleotide sequence having an identity of 75% (preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more to the nucleotide sequence represented by SEQ ID NO: 90, and encoding a protein having TK activity; <32> or <33> The method described.

[0138] <35> The FBA is the following protein (L) or (M): <28> ~ <34> 10. The method according to any one of claims 1 to 9. (L) A protein consisting of the amino acid sequence represented by SEQ ID NO:9. (M) A protein having an amino acid sequence that is 60% or more, 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, even more preferably 99% or more identical to the amino acid sequence of the protein (L), and having FBA activity. <36> the protein (M) is a protein in which one or more, 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, amino acids are deleted, substituted, inserted or added to the amino acid sequence of the protein (L), and the protein (M) has FBA activity; <35> The method described in section. <37> The protein (M) is a protein consisting of the amino acid sequence represented by SEQ ID NO: 91, or a protein consisting of an amino acid sequence having an identity of 65% (preferably 70%, more preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more to the amino acid sequence represented by SEQ ID NO: 91, and having FBA activity. <35> or <36> The method described in section. <38> the gene encoding the FBA is a gene consisting of the following DNA (l) or (m): <28> ~ <37> 10. The method according to any one of claims 1 to 9. (l) DNA consisting of the base sequence represented by SEQ ID NO: 10. (m) DNA having a base sequence that is 60% or more, 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, even more preferably 99% or more identical to the base sequence of DNA (l), and encoding a protein having FBA activity. <39> The DNA (m) is a DNA in which one or more, 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 bases are deleted, substituted, inserted or added to the base sequence of the DNA (l), and which encodes a protein having FBA activity. <38> The method described in section. <40> the DNA (m) is a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 92, or a DNA consisting of a nucleotide sequence having an identity of 75% (preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more to the nucleotide sequence represented by SEQ ID NO: 92, and encoding a protein having FBA activity; <38> or <39> The method described in section.

[0139] <41> The RPI is the following protein (N) or (O): <28> ~ <40> 10. The method according to any one of claims 1 to 9. (N) A protein consisting of the amino acid sequence represented by SEQ ID NO: 11. (O) A protein having an amino acid sequence that is 60% or more identical to the amino acid sequence of 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, even more preferably 99% or more, and having RPI activity. <42> The protein (O) is a protein in which one or more, 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, and more preferably 1 or 2 amino acids are deleted, substituted, inserted or added to the amino acid sequence of the protein (N), and which has RPI activity. <41> The method described in 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 having an identity of 70% (preferably 75%, more preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more to the amino acid sequence represented by SEQ ID NO: 93, and having RPI activity. <41> or <42> The method described. <44> the gene encoding RPI is a gene consisting of the following DNAs (n) and (o): <28> ~ <43> A method according to any one of the preceding claims. (n) DNA consisting of the base sequence represented by SEQ ID NO: 12. (o) DNA having a base sequence that is 60% or more, 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, even more preferably 99% or more identical to the base sequence of DNA (n), and encoding a protein having RPI activity. <45> The DNA (o) is a DNA in which one or more, 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, and more preferably 1 to 8 bases are deleted, substituted, inserted, or added to the base sequence of the DNA (n), and which encodes a protein having RPI activity. <44> The method described in section. <46> the DNA (o) is a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 94, or a DNA consisting of a nucleotide sequence having an identity of 75% (preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more to the nucleotide sequence represented by SEQ ID NO: 94, and encoding a protein having RPI activity; <44> or <45> The method described in section.

[0140] <47> The expression of the cell wall synthesis pathway-related protein is reduced or eliminated 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> 10. The method according to any one of claims 1 to 9. <48> The gene encoding a cell wall synthesis pathway-related protein is a gene encoding at least one selected from the group consisting of CES, PKS involved in aldinane synthesis, and UDP-glucose pyrophosphorylase, preferably a gene encoding CES. <1> ~ <47> 10. The method according to any one of claims 1 to 9.

[0141] <49> The CES is the following protein (P) or (Q): <48> The method described in section. (P) A protein consisting of the amino acid sequence represented by SEQ 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), 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, even more preferably 99% or more, and having CES activity. <50> the protein (Q) is a protein in which one or more, 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 amino acids are deleted, substituted, inserted or added to the amino acid sequence of the protein (P), and the protein (Q) has CES activity; <49> The method described in section. <51> The protein (Q) is a protein consisting of the amino acid sequence represented by SEQ ID NO: 95, or a protein consisting of an amino acid sequence having an identity of 75% (preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 98%) or more to the amino acid sequence represented by SEQ ID NO: 95, and having CES activity. <49> or <50> The method described in section. <52> The gene encoding the CES is a gene consisting of the following DNA (p) or (q): <48> ~ <51> 10. The method according to any one of claims 1 to 9. (p) DNA consisting of the base sequence represented by SEQ ID NO: 14. (q) DNA having a base sequence that is 60% or more, 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, even more preferably 99% or more identical to the base sequence of DNA (p), and encoding a protein having CES activity. <53> the DNA (q) is a DNA in which one or more, 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 bases are deleted, substituted, inserted or added to the base sequence of the DNA (p), and which encodes a protein having CES activity; <52> The method described in section. <54> the DNA (q) is a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 96, or a DNA consisting of a nucleotide sequence having an identity of 85% (preferably 90%, more preferably 95%, and even more preferably 98%) or more to the nucleotide sequence represented by SEQ ID NO: 96, and encoding a protein having CES activity; <52> or <53> The method described in section.

[0142] <55> The microalgae are algae belonging to the phylum Heterokontophyta, preferably algae belonging to the class Euonymus, 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 Vischelia, algae of the genus Chlorobotrys, and algae of the genus Goniochloris, and even more preferably algae of the genus Nannochloropsis. <1> ~ <54> 10. The method according to any one of claims 1 to 9. <56> The algae belonging to the genus Nannochloropsis is at least one alga selected from the group consisting of Nannochloropsis oceanica, Nannochloropsis oculata, Nannochloropsis gaditana, Nannochloropsis salina, Nannochloropsis limnetica, Nannochloropsis granulata, and Nannochloropsis sp., preferably Nannochloropsis gaditana or Nannochloropsis oceanica. <55> The method described in section.

[0143] <57> the method of claim 1, further comprising the step of recovering lipids from the recovered transformant by compression and / or solvent extraction, preferably by compression and solvent extraction. <1> ~ <56> 10. The method according to any one of claims 1 to 9. <58> The transformant is cultured in an open pond system or a closed photobioreactor. <1> ~ <57> 10. The method according to any one of claims 1 to 9. <59> The transformant is cultured by batch culture or continuous culture, preferably continuous culture. <1> ~ <58> 10. The method according to any one of claims 1 to 9. <60> In the continuous culture, the algae cells with increased amounts of accumulated fats and oils are recovered by floatation separation, and the algae cells with less accumulated fats and oils are continuously cultured. <59> The method described in section.

[0144] <61> The lipid comprises triacylglycerol. <1> ~ <60> The method according to any one of the preceding claims.

[0145] <62> A microalgae transformant having 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 transformant has improved buoyancy compared to a host that has not undergone any of the modifications (A) to (C). <62> The transformant described above. <64> When the transformant and a host not subjected to any of the modifications (A) to (C) are cultured under the same culture conditions and then centrifuged at 21,600 × g for 10 minutes, the percentage of cells that do not precipitate is improved compared to a host not subjected to any of the modifications (A) to (C). <62> or <63> The transformant according to paragraph 1.

[0146] <65> The expression of the gene encoding the TAG synthesis pathway-related protein is promoted in the cells of the microalgae, thereby promoting the expression of the TAG synthesis pathway-related protein. <62> ~ <64> The transformant according to any one of the above. <66> By introducing the gene encoding the TAG synthesis pathway-related protein into the microalgae, the expression of the introduced gene encoding the TAG synthesis pathway-related protein is promoted. <62> ~ <65> The transformant according to any one of the above. <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> The transformant according to any one of the above. <68> the expression of the gene encoding the CBB cycle-related protein is promoted in the cells of the microalgae, thereby promoting the expression of the CBB cycle-related protein; <62> ~ <67> The transformant according to any one of the above. <69> By introducing the gene encoding the CBB cycle-related protein into the microalgae, the expression of the introduced gene encoding the CBB cycle-related protein is promoted. <62> ~ <68> The transformant according to any one of the above. <70> The gene encoding the CBB cycle-associated protein, or a plasmid or DNA cassette containing the gene encoding the CBB cycle-associated protein. <62> ~ <69> The transformant according to any one of the above. <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 down-regulated. <62> ~ <70> The transformant according to any one of the above. <72> The expression of the cell wall synthesis pathway-related protein is reduced or lost due to deletion or inactivation of the gene encoding the cell wall synthesis pathway-related protein, or downregulation of the gene encoding the cell wall synthesis pathway-related protein. <62> ~ <71> The transformant according to any one of the above. <73> The cell wall synthesis pathway-related protein-encoding gene is deleted or inactivated, or the cell wall synthesis pathway-related protein-encoding gene is down-regulated, comprising a plasmid or a DNA cassette. <62> ~ <72> The transformant according to any one of the above.

[0147] <74> A method for producing a transformant of microalgae, which comprises carrying out the above modifications (A) to (C) on microalgae. <75> A gene encoding the TAG synthesis pathway-related protein, or a plasmid or a DNA cassette containing the gene encoding the TAG synthesis pathway-related protein, is introduced into the microalgae. <74> A method for producing a transformant according to any one of the preceding paragraphs. <76> A gene encoding the CBB cycle-related protein, or a plasmid or a DNA cassette containing the gene encoding the CBB cycle-related protein, is introduced into the microalgae. <74> or <75> 2. A method for producing a transformant according to claim 1. <77> A plasmid or a DNA cassette for deleting or inactivating a gene encoding a cell wall synthesis pathway-related protein, or for down-regulating a gene encoding a cell wall synthesis pathway-related protein is introduced into the microalga. <74> ~ <76> The method for producing a transformant according to any one of the above.

[0148] <78> A kit for producing a microalgal transformant for carrying out the modifications (A) to (C). <79> The aforementioned <75> ~ <77> The plasmid or DNA cassette according to any one of claims 1 to 4, <78> A kit for producing a transformant of the microalgae described in paragraph 1.

[0149] <80> The gene encoding a TAG synthetic 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, DGAT, etc.), even more preferably a gene encoding an ACS and a gene encoding an AT (GPAT, LPAAT, DGAT, etc.), and even more preferably a gene encoding an ACS and a gene encoding a DGAT. <62> ~ <79> The transformant according to any one of the above, a method for producing the same, or a kit for producing the transformant. <81> The DGAT or the gene encoding the DGAT is <6> ~ <11> The protein or gene is defined in any one of the above. <80> 1. A transformant according to claim 1, a method for producing the same, or a kit for producing the transformant. <82> The ACS or the gene encoding the ACS is <12> ~ <17> The protein or gene is defined in any one of the above. <80> or <81> 1. A transformant according to claim 1, a method for producing the same, or a kit for producing the transformant. <83> the gene encoding a 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, phosphoribulokinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate 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, and even more preferably a gene encoding TK, a gene encoding FBA, and a gene encoding RPI; <62> ~ <82> The transformant according to any one of the above, a method for producing the same, or a kit for producing the transformant. <84> The TK or the gene encoding the TK is <29> ~ <34> The protein or gene is defined in any one of the above. <83> 1. A transformant according to claim 1, a method for producing the same, or a kit for producing the transformant. <85> The FBA or the gene encoding the FBA is <35> ~ <40> The protein or gene is defined in any one of the above. <83> or <84> 1. A transformant according to claim 1, a method for producing the same, or a kit for producing the transformant. <86> The RPI or the gene encoding the RPI is <41> ~ <46> The protein or gene is defined in any one of the above. <83> ~ <85> The transformant according to any one of the above, a method for producing the same, or a kit for producing the transformant. <87> The gene encoding a cell wall synthesis pathway-related protein is preferably a gene encoding at least one selected from the group consisting of CES, PKS involved in aldinane synthesis, and UDP-glucose pyrophosphorylase, more preferably a gene encoding CES. <62> ~ <86> The transformant according to any one of the above, a method for producing the same, or a kit for producing the transformant. <88> The CES or the gene encoding the CES is <49> ~ <54> The protein or gene is defined in any one of the above. <87> 1. A transformant according to claim 1, a method for producing the same, or a kit for producing the transformant.

[0150] <89> In the transformant, expression of a gene encoding a protein involved in a fatty acid synthesis pathway is promoted. <62> ~ <88> The transformant according to any one of the preceding claims. <90> The expression of a gene encoding a protein involved in the fatty acid synthesis pathway is promoted in the cells of the microalgae, thereby promoting the expression of the protein involved in the fatty acid synthesis pathway. <89> The transformant according to paragraph 1. <91> By introducing a gene encoding a protein involved in the fatty acid synthesis pathway into the microalgae, expression of the introduced gene encoding a protein involved in the fatty acid synthesis pathway is promoted. <89> or <90> The transformant according to paragraph 1. <92> The method comprises the step of: providing a gene encoding a protein involved in the fatty acid synthesis pathway; or providing a plasmid or DNA cassette containing a gene encoding a protein involved in the fatty acid synthesis pathway. <89> ~ <91> The transformant according to any one of the above. <93> A gene encoding a protein involved in a fatty acid synthesis pathway, or a plasmid or a DNA cassette containing a gene encoding a protein involved in a fatty acid synthesis pathway, is introduced into the microalga. <74> ~ <92> The method for producing a transformant according to any one of the above. <94> The method further comprises the step of preparing a method for preparing a fatty acid synthesis pathway comprising administering to a subject a method for preparing a fatty acid synthesis pathway comprising administering to the ... <78> ~ <93> A kit for producing a transformant according to any one of the above.

[0151] <95> The gene encoding a 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 a TE. <89> ~ <94> The transformant according to any one of the above, a method for producing the same, or a kit for producing the transformant. <96> The TE or the gene encoding the TE is <22> ~ <25> The protein or gene is defined in any one of the above. <95> 1. A transformant according to claim 1, a method for producing the same, or a kit for producing the transformant.

[0152] <97> The microalgae are algae belonging to the phylum Heterokontophyta, preferably algae belonging to the class Euonymus, 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 Vischelia, algae of the genus Chlorobotrys, and algae of the genus Goniochloris, and even more preferably algae of the genus Nannochloropsis. <62> ~ <96> The transformant according to any one of the above, a method for producing the same, or a kit for producing the transformant. <98> The algae belonging to the genus Nannochloropsis is at least one alga selected from the group consisting of Nannochloropsis oceanica, Nannochloropsis oculata, Nannochloropsis gaditana, Nannochloropsis salina, Nannochloropsis limnetica, Nannochloropsis granulata, and Nannochloropsis sp., preferably Nannochloropsis gaditana or Nannochloropsis oceanica. <97> 3. A transformant according to claim 1, a method for producing the same, or a kit for producing the transformant.

[0153] <99> For producing lipids, <62> ~ <98> Use of the transformant described in any one of the above, a transformant produced by the method for producing the transformant, or a kit for producing a transformant. [Example]

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

[0155] [Table 1]

[0156] Preparation Example 1: Preparation of plasmids for expressing CBB cycle genes and TAG synthesis pathway genes from Nannochloropsis oceanica, and plasmids for disrupting genes encoding cell wall synthesis pathway-related proteins (1) Construction of a plasmid for expressing the Zeocin resistance gene The zeocin 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. PCR was performed using the synthesized DNA fragment as a template and the primer pair of primer 28 (SEQ ID NO: 28) and primer 29 (SEQ ID NO: 29) shown in Table 1, as well as the primer pair of primer 34 (SEQ ID NO: 34) and primer 35 (SEQ ID NO: 35), to amplify the zeocin resistance gene fragment and the tubulin promoter sequence fragment, respectively. Furthermore, PCR was performed using the genome of Nannochloropsis oceanica NIES2145 strain as a template and the primer pair of primer 36 (SEQ ID NO: 36) and primer 37 (SEQ ID NO: 37) shown in Table 1 to amplify the heat shock protein terminator sequence fragment (SEQ ID NO: 19). Furthermore, using the plasmid vector pUC19 (manufactured by Takara Bio Inc.) as a template, PCR was performed using the primer pair of primer 38 (SEQ ID NO: 38) and primer 39 (SEQ ID NO: 39) shown in Table 1 to amplify the plasmid vector pUC19 fragments. These four amplified fragments were amplified using restriction enzymes DpnThe fragments were treated with PCR primer I (Toyobo Co., Ltd.) and purified using a High Pure PCR Product Purification Kit (Roche Applied Science). The four resulting fragments were then fused using an In-Fusion HD Cloning Kit (Clontech) to construct a plasmid for expressing the Zeocin resistance gene. This expression plasmid consists of an insert sequence (Zeocin resistance gene expression cassette) in which the tubulin promoter sequence, Zeocin resistance gene, and heat shock protein terminator sequence are linked in this order, and a pUC19 vector sequence.

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

[0158] (3) Construction of DGAT2-8-LACS2 gene co-expression plasmid and RPI-TK1-FBA2 gene co-expression plasmid PCR was performed using the LACS2 gene expression plasmid (zeocin-resistant) and the DGAT2-8 gene expression plasmid (zeocin-resistant) as templates, respectively, with the primer pair of primer 49 (SEQ ID NO: 49) and primer 39 (SEQ ID NO: 39) and the primer pair of primer 71 (SEQ ID NO: 71) and primer 72 (SEQ ID NO: 72) shown in Table 1 to obtain the respective DNA fragments. PCR was also performed using the genome of Nannochloropsis oceanica NIES2145 strain as a template with the primer pair of primer 45 (SEQ ID NO: 45) and primer 46 (SEQ ID NO: 46) and the primer pair of primer 47 (SEQ ID NO: 47) and primer 48 (SEQ ID NO: 48) shown in Table 1 to obtain the glutamine synthetase (GS) promoter fragment (SEQ ID NO: 22) and the LDSP terminator fragment (SEQ ID NO: 23). These four fragments were fused in the same manner as described above to construct a DGAT2-8-LACS2 gene co-expression plasmid (zeocin-resistant). This expression plasmid consists of an insert sequence in which the following sequences are linked in order: GS promoter sequence, DGAT2-8 gene, LDSP terminator sequence, LDSP promoter sequence, LACS2 gene, VCP1 terminator sequence, tubulin promoter sequence, Zeocin resistance gene, and heat shock protein terminator sequence, and a pUC19 vector sequence.

[0159] Similarly, PCR was performed using the FBA2 gene expression plasmid (zeocin-resistant) and the TK1 gene expression plasmid (zeocin-resistant) as templates, respectively, with the primer pair of primer 49 (SEQ ID NO: 49) and primer 39 (SEQ ID NO: 39), and the primer pair of primer 73 (SEQ ID NO: 73) and primer 74 (SEQ ID NO: 74) shown in Table 1 to obtain DNA fragments. These two DNA fragments, the GS promoter fragment, and the LDSP terminator fragment were fused in the same manner as described above to construct a TK1-FBA2 gene co-expression plasmid (zeocin-resistant). This expression plasmid consisted of an insert sequence ligated in this order: GS promoter sequence, TK1 gene, LDSP terminator sequence, LDSP promoter sequence, FBA2 gene, VCP1 terminator sequence, tubulin promoter sequence, zeocin resistance gene, and heat shock protein terminator sequence, as well as pUC19 vector sequence. PCR was performed using the resulting TK1-FBA2 gene coexpression plasmid (zeocin-resistant) and the RPI gene expression plasmid (zeocin-resistant) as templates, respectively, with the primer pair of primer 54 (SEQ ID NO: 54) and primer 39 (SEQ ID NO: 39), and the primer pair of primer 75 (SEQ ID NO: 75) and primer 76 (SEQ ID NO: 76) shown in Table 1 to obtain DNA fragments. PCR was also performed using the genome of Nannochloropsis oceanica NIES2145 strain as a template with the primer pair of primer 50 (SEQ ID NO: 50) and primer 51 (SEQ ID NO: 51), and the primer pair of primer 52 (SEQ ID NO: 52) and primer 53 (SEQ ID NO: 53) shown in Table 1 to obtain an ammonium transporter (AMT) promoter fragment (SEQ ID NO: 24) and a Δ9 desaturase (Δ9DES) terminator fragment (SEQ ID NO: 25). These four fragments were fused in the same manner as described above to construct a plasmid (Zeocin-resistant) for co-expression of the RPI-TK1-FBA2 genes. This expression plasmid consisted of an insert sequence consisting of the 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, Zeocin resistance gene, and heat shock protein terminator sequence, in this order, and the pUC19 vector sequence.

[0160] (4) Construction of a TE2 gene expression plasmid (paromomycin resistance), a DGAT2-8-LACS2 gene co-expression plasmid (hygromycin resistance), a RPI-TK1-FBA2 gene co-expression plasmid (hygromycin resistance), and a RPI-TK1-FBA2 gene co-expression plasmid (paromomycin resistance) PCR was performed using the TE2 gene expression plasmid (zeocin-resistant), the DGAT2-8-LACS2 gene co-expression plasmid (zeocin-resistant), and the RPI-TK1-FBA2 gene co-expression plasmid (zeocin-resistant) as templates, respectively, to obtain DNA fragments. PCR was also performed using the artificially synthesized paromomycin resistance gene (SEQ ID NO: 16) and hygromycin resistance gene (SEQ ID NO: 17) as templates, respectively, to obtain DNA fragments. The obtained fragments were fused appropriately using the same method as described above to construct a TE2 gene expression plasmid (paromomycin-resistant), a DGAT2-8-LACS2 gene co-expression plasmid (hygromycin-resistant), an RPI-TK1-FBA2 gene co-expression plasmid (hygromycin-resistant), and an RPI-TK1-FBA2 gene co-expression plasmid (paromomycin-resistant).

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

[0162] (6) Preparation of PCR fragments for electroporation Using the TE2 gene expression plasmid (paromomycin resistance) as a template, PCR was performed with a primer pair of primer 55 (SEQ ID NO: 55) and primer 58 (SEQ ID NO: 58) shown in Table 1 to amplify the TE2 gene expression cassette (paromomycin resistance). The TE2 gene expression cassette (paromomycin resistance) consists of an LDSP promoter sequence, a TE2 gene, a VCP1 terminator sequence, a tubulin promoter sequence, a paromomycin resistance gene, and a heat shock protein terminator sequence. Furthermore, PCR was performed using the DGAT2-8-LACS2 gene co-expression plasmid (zeocin-resistant), the DGAT2-8-LACS2 gene co-expression plasmid (hygromycin-resistant), and the RPI-TK1-FBA2 gene co-expression plasmid (paromomycin-resistant) as templates, respectively, with the primer pair primer 56 (sequence number 56) and primer 58 (sequence number 58) shown in Table 1, to amplify the DGAT2-8-LACS2 gene co-expression cassette (zeocin-resistant), the DGAT2-8-LACS2 gene co-expression cassette (hygromycin-resistant), and the TK1-FBA2 gene co-expression cassette (paromomycin-resistant). 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 an 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, PCR was performed using the RPI-TK1-FBA2 gene co-expression plasmid (hygromycin-resistant) as a template and the primer pair Primer 57 (SEQ ID NO: 57) and Primer 58 (SEQ ID NO: 58) shown in Table 1 to amplify the RPI-TK1-FBA2 gene co-expression cassette (hygromycin-resistant). This expression cassette consists of an 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, hygromycin-resistant gene, and heat shock protein terminator sequence. Furthermore, using the CES gene disruption plasmid (zeocin-resistant) as a template, PCR was performed with the primer pair of primer 83 (SEQ ID NO: 83) and primer 84 (SEQ ID NO: 84) shown in Table 1 to amplify a CES gene disruption cassette. This disruption cassette consists of homologous sequence 1, a tubulin promoter sequence, a zeocin-resistant gene, a heat shock protein terminator sequence, and homologous sequence 2. The amplified fragment was purified using a High Pure PCR Product Purification Kit (Roche Applied Science). Note that sterilized water was used for elution during purification, instead of the elution buffer included in the kit.

[0164] Example 1: Preparation of a transformant by introducing TAG synthesis pathway genes and CBB cycle genes into Nannochloropsis, and lipid production by the transformant (1) Preparation of transformants by introducing TAG synthesis pathway genes and CBB cycle genes into Nannochloropsis, and culturing the transformants. Approximately 1×10 9Nannochloropsis oceanica NIES2145 cells were washed with 384 mM sorbitol to remove salts and used as host cells for transformation. Approximately 500 ng of the DGAT2-8-LACS2 gene co-expression cassette (Zeocin resistance) amplified in Preparation Example 1 was mixed with the host cells and electroporated under conditions of 50 μF, 500 Ω, and 2,200 V / 2 mm. After 24 hours of recovery 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 / L of artificial seawater), the cells were plated onto f / 2 agar medium containing 2 μg / mL Zeocin and cultured at 25°C under a 0.3% CO2 atmosphere with a 12h / 12h light / dark cycle for 2–3 weeks. The resulting colonies were seeded into 2 mL of N5P5 medium (f / 2 medium with 5x the nitrogen and 5x the phosphorus concentration) containing 2 μg / mL Zeocin (24-well plate) and cultured at 25°C under 0.3% CO2 with a 12h / 12h light / dark cycle for 3 weeks. The resulting culture was seeded into 18 mL of N15P5 medium (f / 2 medium with 15x the nitrogen and 5x the phosphorus concentration) and cultured at 20°C under a 12h / 12h light / dark cycle. Next, the TE2 gene expression cassette (paromomycin resistance) amplified in Preparation Example 1 was introduced into the transformant (hereinafter also referred to as the "DGAT2-8-LACS2 strain") as a parent strain in the same manner as above. Transformants were selected on f / 2 agar medium containing 2 μg / mL Zeocin and 100 μg / mL paromomycin. The resulting colonies were inoculated into 2 mL of N5P5 medium containing 100 μg / mL paromomycin and cultured in the same manner as above. The resulting culture solution was inoculated into 18 mL of N15P5 medium and cultured in the same manner as above. Next, the RPI-TK1-FBA2 gene coexpression cassette (hygromycin resistance) amplified in Preparation Example 1 was introduced into a transformant (hereinafter also referred to as the "TE2 on DGAT2-8-LACS2 strain") containing a DGAT2-8-LACS2 gene coexpression cassette (zeocin resistance) and a TE2 gene expression cassette (paromomycin resistance) using the same method as above. Transformants were selected on f / 2 agar medium containing 500 μg / mL of hygromycin. The resulting colonies were plated on 2 mL of N5P5 medium containing 500 μg / mL of hygromycin and cultured in the same manner as above. The resulting culture was plated on 18 mL of N15P5 medium and cultured in the same manner as above. Nannochloropsis oceanica NIES2145 strain (hereinafter also referred to as the "wild strain"), the TE2 on DGAT2-8-LACS2 strain, and a transformant (hereinafter also referred to as the "RPI-TK1-FBA2 on TE2 on DGAT2-8-LACS2 strain") carrying a DGAT2-8-LACS2 gene co-expression cassette (zeocin resistance), a TE2 gene expression cassette (paromomycin resistance), and an RPI-TK1-FBA2 gene co-expression cassette (hygromycin resistance) were introduced into 18 mL of N5P5 medium and grown at 25°C in a 0.3% CO2 atmosphere under a light intensity of approximately 100 μmol / m 2 The pre-preculture was cultured with shaking under 1 / s (normal light conditions) and 12h / 12h light / dark conditions for 5 to 7 days to obtain the pre-preculture. The turbidity at 750 nm (hereinafter also referred to as "OD750") was measured using a 96-well plate and an Infinite M200 PRO (TECAN). The pre-preculture was inoculated into 18 mL of N5P5 medium so that the final OD750 concentration was 0.1, and the medium was cultured under the same conditions for 5 days to obtain the preculture. Similarly, the preculture was inoculated into 18 mL of N5P5 medium so that the final OD750 concentration was 0.1, and the main culture was performed under the same conditions. The light intensity was set at approximately 300 μmol / m 2 At 1 / s, the culture was also performed under high light conditions with a CO2 concentration of 0.6% (the pre-culture was performed under normal light conditions, and the pre-culture was performed under high light conditions from the pre-culture onwards). Each strain was cultured in N = 2-4.

[0165] (2) Extraction and analysis of lipids from Nannochloropsis culture medium After the start of the main culture, sampling was carried out over time, and lipid extraction was carried out by 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, vigorously stirred, and allowed to stand for 10 minutes. Then, 0.5 mL of chloroform and 0.5 mL of 1.5% KCl were added and stirred. The mixture was then centrifuged at 3,000 rpm for 5 minutes, and the chloroform layer (lower layer) was collected using a Pasteur pipette. The resulting chloroform layer was dried by blowing nitrogen gas over it and redissolved in 50 μL of chloroform. 0.5 mL of 14% boron trifluoride solution (Sigma) was added, stirred, and then incubated at 80°C for 30 minutes. Next, 0.5 mL of hexane and 0.5 mL of saturated saline were added, vigorously stirred, and allowed to stand at room temperature for 10 minutes. The upper hexane layer was then collected to obtain fatty acid esters.

[0166] The obtained fatty acid ester was subjected to gas chromatography analysis under the following measurement conditions. <Gas chromatography conditions> Analyzer: 7890A (Agilent technology) Capillary column: DB-1 MS 30 m × 200 μm × 0.25 μm (J&W Scientific), Mobile phase: High purity helium, Oven temperature: 150°C, hold for 0.5 minutes → 150-220°C (heating rate: 40°C / min) → 220-320°C (heating rate: 20°C / min) → hold for 2 minutes at 320°C (post-run: 2 minutes) Inlet temperature: 300℃, Injection method: Split injection (split ratio: 75:1), Injection volume: 1μL, Washing vials: methanol and chloroform, Detection method: FID Detector temperature: 300℃

[0167] The fatty acid methyl esters were identified by subjecting various fatty acid methyl ester samples to gas chromatography under the same conditions and comparing their retention times. If necessary, they were also identified by gas chromatography-mass spectrometry. The amount of methyl ester of each fatty acid was quantified from the peak area of ​​the waveform data obtained by gas chromatography analysis. Corrections were made between samples by comparing each peak area with the peak area of ​​the C17 fatty acid methyl ester derived from the 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 taken as the total amount of fatty acids, and the weight percentage of each fatty acid relative to the total amount of fatty acids was calculated. The results for culture under normal light conditions are shown in Table 2, and the results for culture under high light conditions are shown in Table 3. In the following tables, the wild-type strain is abbreviated as "WT." The total fatty acid content ("TFA productivity" in the tables) is shown in the form of mean ± standard deviation.

[0168] [Table 2]

[0169] [Table 3]

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

[0171] (3) Re-cultivation of wild-type and RPI-TK1-FBA2 on TE2 on DGAT2-8-LACS2 strains and analysis of their budding ability The wild-type strain and the RPI-TK1-FBA2 on TE2 on DGAT2-8-LACS2 strain were cultured under normal and high light conditions (N=3 for each strain) using the same method as described above, and 0.25 mL and 1 mL aliquots were taken over time. Lipids were extracted and analyzed from the 0.25 mL samples using the same method as described above, and total fatty acids (TFA) were calculated. The 1 mL samples were centrifuged at 15,000 rpm (21,600 × g) for 10 minutes using a CF15RXII centrifuge and a T15A43 rotor (both manufactured by Hitachi Koki Co., Ltd., now Eppendorf-Himac Technologies Co., Ltd.). The supernatant (containing both cells that remained and those that floated to the surface) was collected. Using 0.25 mL of the resulting supernatant, lipid extraction and analysis were performed using the same method as described above, and the amount of fatty acids (SUP-FA) contained in the cells that did not settle after centrifugation (hereafter referred to as "planktonic cells" or "planktonic cells") was calculated. The ratio of the amount of fatty acids in planktonic cells to the total amount of fatty acids was calculated and used as the floating rate (SUP-FA / TFA). The results for culture under normal light conditions are shown in Table 4, and the results for culture under high 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 had higher amounts of fatty acids derived from buoyant cells and higher floating rates than the wild-type strain under all culture conditions and days, indicating improved floating ability. Since the total fatty acid content was higher than that of the wild-type strain under all conditions, it is thought that the enhancement of the TAG synthesis pathway and CBB cycle improved the lipid content, resulting in a decrease in specific gravity and therefore improved floating ability. Furthermore, under high light conditions, which tend to increase lipid content, the floating rate increased earlier.

[0175] Example 2: Preparation of a transformant by introducing TAG synthesis pathway genes and CBB cycle genes into Nannochloropsis and disrupting cell wall synthesis pathway genes, and lipid production by the transformant (1) Creation of transformants by introducing TAG synthesis pathway genes and CBB cycle genes into Nannochloropsis and disrupting cell wall synthesis pathway genes, culturing the transformants, extracting lipids, and analyzing the lipids. The CES gene disruption cassette amplified in Preparation Example 1 was introduced into the Nannochloropsis oceanica NIES2145 strain as a parent strain in the same manner as in Example 1. From the resulting colonies, transformants in which the CES gene had been disrupted (hereinafter also referred to as "ΔCES strains") were selected by PCR. Next, using the ΔCES strain as a parent strain, the TK1-FBA2 gene co-expression cassette (paromomycin resistance) amplified in Preparation Example 1 was introduced using the same method as above. Using the same method as in Example 1, a transformant in which the CES gene had been disrupted and the TK1-FBA2 gene co-expression cassette (paromomycin resistance) had been introduced (hereinafter also referred to as the "TK1-FBA2 on ΔCES strain") was selected. Furthermore, the TK1-FBA2 on ΔCES strain was used as a parent strain and the DGAT2-8-LACS2 gene coexpression cassette (hygromycin resistance) amplified in Preparation Example 1 was introduced using the same method as above. Using the same method as in Example 1, a transformant in which the CES gene had been disrupted and the TK1-FBA2 gene coexpression cassette (paromomycin resistance) and the DGAT2-8-LACS2 gene coexpression cassette (hygromycin resistance) had been introduced (hereinafter also referred to as the "DGAT2-8-LACS2 on TK1-FBA2 on ΔCES strain") was selected. The wild-type strain, the ΔCES strain, the TK1-FBA2 on ΔCES strain, and the DGAT2-8-LACS2 on TK1-FBA2 on ΔCES strain (N=1 for each strain) were cultured (under normal light conditions) in the same manner as in Example 1, and lipid extraction and analysis were performed. The results are shown in Table 6.

[0176] [Table 6]

[0177] As shown in Table 6, although the ΔCES strain had lower lipid productivity than the wild-type strain, the TK1-FBA2 on ΔCES strain exhibited higher lipid productivity than the wild-type strain. Furthermore, the DGAT2-8-LACS2 on TK1-FBA2 on ΔCES strain was found to exhibit even higher lipid productivity than the wild-type strain and the TK1-FBA2 on ΔCES strain.

[0178] (2) Re-cultivation 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 budding ability 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 conditions and high light conditions (N=3 for each strain) in the same manner as in Example 1, and their buoyancy was analyzed. The results for the culture under normal light conditions are shown in Table 7, and the results for the culture under high light conditions are shown in Table 8. In addition, the culture medium on day 17 of the normal light culture and day 14 of the high light culture was centrifuged to recover buoyant cells, which were then resuspended and allowed to stand for 24 hours. Photographs of the cells before and after the incubation 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 had higher fatty acid content and a higher floating rate than the wild-type strain under all culture conditions and days. Furthermore, the DGAT2-8-LACS2 on TK1-FBA2 on ΔCES strain had a higher floating rate than the RPI-TK1-FBA2 on TE2 on DGAT2-8-LACS2 strain, which does not have cell wall synthesis pathway genes suppressed or disrupted, particularly on day 14 of culture under normal light conditions and on days 7 and 10 of culture under high light conditions, demonstrating its superior floating ability. 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 when left to stand during the later stages of cultivation when the total fatty acid content was high (the algae were more turbid at the surface). In particular, the DGAT2-8-LACS2 on TK1-FBA2 on ΔCES strain, under these conditions, showed that the majority of cells floated to the surface after 24 hours of standing, demonstrating its extremely high floating ability.

[0182] In addition to improving the oil content by strengthening the TAG synthesis pathway and CBB cycle, suppressing the cell wall synthesis pathway reduced cell wall components with high specific gravity (such as cellulose), further improving the oil content. As a result, the specific gravity was significantly reduced, which is thought to have greatly improved the flotation ability.

[0183] As described above, by performing at least one of the following modifications on microalgae: a modification that promotes the expression of at least one gene encoding a protein related to the triacylglycerol synthesis pathway; a modification that promotes the expression of at least one gene encoding a protein related to the Calvin cycle; and a modification that suppresses the expression of at least one gene encoding a protein related to the cell wall synthesis pathway, it is possible to obtain a microalgae transformant with improved floating properties, 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 transformant of microalgae that has been subjected to at least one modification selected from the group consisting of the following (A) to (C), causing the transformant to produce a fatty acid or a lipid containing the fatty acid as a constituent component, and recovering the transformant by floatation separation: A method for recovering a transformant, wherein the transformant of the microalgae has improved productivity of fatty acids or lipids containing fatty acids as components compared to the microalgae before transformation, and / or cell wall construction is inhibited. (A) A modification that enhances the expression of a gene encoding at least one enzyme selected from the group consisting of diacylglycerol acyltransferase and long-chain acyl-CoA synthetase. (B) A modification that enhances the expression of a gene encoding at least one enzyme selected from the group consisting of transketolase and fructose-1,6-bisphosphate aldolase. (C) Modification that suppresses the expression of a gene encoding a cellulose synthase

2. A method for producing lipids, comprising culturing a transformant of microalgae that has been subjected to at least one modification selected from the group consisting of the following (A) to (C), producing a fatty acid or a lipid containing the fatty acid as a constituent component, recovering the transformant by floatation separation, and obtaining lipids from the recovered transformant: A method for producing lipids, wherein the transformant of the microalgae has improved productivity of fatty acids or lipids containing fatty acids as constituents compared to the microalgae before transformation, and / or cell wall construction is inhibited. (A) A modification that enhances the expression of a gene encoding at least one enzyme selected from the group consisting of diacylglycerol acyltransferase and long-chain acyl-CoA synthetase. (B) A modification that enhances the expression of a gene encoding at least one enzyme selected from the group consisting of transketolase and fructose-1,6-bisphosphate aldolase. (C) Modification that suppresses the expression of a gene encoding a cellulose synthase

3. The method according to claim 1 or 2, wherein the microalgae transformant is a microalgae transformant having 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 microalgae transformant is a microalgae transformant that has undergone the modifications (A) and / or (B) and the modification (C).

5. 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 any one of claims 1 to 4. (D) A protein consisting of the amino acid sequence represented by SEQ ID NO:

1. (E) A protein having an amino acid sequence that is 90% 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 SEQ ID NO:

3. (G) A protein having an amino acid sequence that is 90% 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 SEQ ID NO:

7. (K) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of the protein (J) and that has transketolase activity. (L) A protein consisting of the amino acid sequence represented by SEQ ID NO:

9. (M) A protein having an amino acid sequence that is 90% 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 SEQ ID NO:

13. (Q) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of the protein (P) and having cellulose synthase activity.

6. The method according to any one of claims 1 to 5, wherein the microalgae transformant is a microalgae transformant modified by (A), (B), and (C).

7. the transformant has enhanced expression of a gene encoding diacylglycerol acyltransferase, a gene encoding long-chain acyl-CoA synthetase, a gene encoding transketolase, and a gene encoding fructose-1,6-bisphosphate aldolase; The expression of genes encoding cellulose synthases is suppressed. The method according to any one of claims 1 to 6.

8. The method according to any one of claims 1 to 7, wherein the microalgae are algae belonging to the Heterokontophyta phylum.

9. The method according to claim 8, wherein the algae belonging to the Heterokontophyta division are algae belonging to the genus Nannochloropsis.

10. The method according to any one of claims 1 to 9, comprising a step of recovering lipids from the recovered transformant by squeezing and / or solvent extraction.

11. The method according to any one of claims 1 to 10, wherein the transformant is cultured in an open pond system or a closed photobioreactor.

12. The method according to any one of claims 1 to 11, wherein the transformant is cultured by continuous culture.

13. In the continuous culture, The algae with increased oil accumulation are collected by flotation separation. Continue culturing algae with low levels of accumulated oil. The method of claim 12.

14. A microalgae transformant having the following modifications (A), (B), and (C): (A) A modification that enhances the expression of a gene encoding at least one enzyme selected from the group consisting of diacylglycerol acyltransferase and long-chain acyl-CoA synthetase. (B) A modification that enhances the expression of a gene encoding at least one enzyme selected from the group consisting of transketolase and fructose-1,6-bisphosphate aldolase. (C) Modification that suppresses the expression of a gene encoding a cellulose synthase

15. The transformant according to claim 14, wherein the transformant has improved buoyancy compared to a host that has not undergone any of the modifications (A) to (C).

16. The transformant according to claim 14 or 15, wherein when the transformant and a host not subjected to any of the modifications (A) to (C) are cultured under the same culture conditions and then centrifuged at 21,600 × g for 10 minutes, the percentage of cells that do not precipitate is improved compared to a host not subjected to any of the modifications (A) to (C).

17. 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 transformant according to any one of claims 14 to 16. (D) A protein consisting of the amino acid sequence represented by SEQ ID NO:

1. (E) A protein having an amino acid sequence that is 90% 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 SEQ ID NO:

3. (G) A protein having an amino acid sequence that is 90% 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 SEQ ID NO:

7. (K) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of the protein (J) and that has transketolase activity. (L) A protein consisting of the amino acid sequence represented by SEQ ID NO:

9. (M) A protein having an amino acid sequence that is 90% 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 SEQ ID NO:

13. (Q) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of the protein (P) and having cellulose synthase activity.

18. the transformant has enhanced expression of a gene encoding diacylglycerol acyltransferase, a gene encoding long-chain acyl-CoA synthetase, a gene encoding transketolase, and a gene encoding fructose-1,6-bisphosphate aldolase; The expression of genes encoding cellulose synthases is suppressed. The transformant according to any one of claims 14 to 17.

19. The transformant according to any one of claims 14 to 18, wherein the microalgae are algae belonging to the Heterokontophyta division.

20. The transformant according to claim 19, wherein the algae belonging to the Heterokontophyta division are algae belonging to the genus Nannochloropsis.

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