JP2020074703A - Green algal mutants with reduced lipid decomposition ability and enhanced lipid productivity and uses thereof - Google Patents

Abstract

To provide eukaryotic microalgae with increased lipid productivity.SOLUTION: Disclosed is a eukaryotic microalgae mutant containing an amino acid sequence which is at least 50% identical to a specific amino acid sequence of the conserved region of a lipid droplet protein, in which a function of a protein localized on the membrane surface of a lipid droplet is reduced, and the amount of intracellular lipid accumulation and lipid productivity are increased and lipid decomposition ability is reduced as compared with the parent strain thereof.SELECTED DRAWING: Figure 1

Description

  TECHNICAL FIELD The present invention relates to a green alga mutant in which oil and fat degrading ability is decreased due to a mutation in an oil droplet protein gene, and thus an oil and fat accumulation amount and an oil and fat productivity are increased, and use thereof.

  Products such as biodiesel and biojet fuel are produced from raw materials such as triacylglycerol (hereinafter referred to as "TAG") produced by unicellular eukaryotic photosynthetic organisms (hereinafter referred to as "eukaryotic microalgae"). Although researches have been widely conducted worldwide, production costs are high at present and production on a commercial basis is difficult (Non-Patent Document 1). Therefore, further technological development is being continued, and one of them is the improvement of oil and fat productivity of algae.

  It is known that eukaryotic microalgae accumulate carbohydrates and lipids in cells under stress conditions such as nitrogen, phosphorus, or sulfur deficiency. Accumulated oils and fats (mainly TAG) are accumulated in intracellular organelles surrounded by phospholipid monolayers called lipid droplets. A large number of proteins called oil droplet proteins (hereinafter referred to as “LDP”) are present in the phospholipid monolayer of oil droplets in cells. Various types of LDP exist depending on species such as animals, fungi, and plants, and it is becoming clear that they have functions related to the formation of oil droplets and lipid metabolism, but the LDP of algae remains unknown. There are many cases (Non-Patent Document 2).

  Among algae, Chlamydomonas, Dunaliella, Haematococcus, and Major lipid droplet protein (MLDP) belong to the green subdivisions (Viridiplantae), Chlorophyta, and Chlorophyceae. Alternatively, LDP called Oil globule protein (OGP) and the like has been found (Non-patent Documents 3, 4, and 5). It has been shown that LDPs of these green algae have sequence homology and that the expression of genes increases at the same time as the accumulation of oils and fats at the time of nitrogen deficiency, and is localized in the oil droplet membrane (Non-patent Documents 3 and 4). 5). It was shown that suppressing the gene expression of MLDP (CHLREDRAFT_192823; NCBI accession number XP_001697668) identified in Chlamydomonas increased the oil droplet diameter, but the amount of accumulated fats and oils did not change (Non-Patent Document 3).

  As a strain belonging to the subdivision of green plants (Viridiplantae), Chlorophyta, Chlorophyta, Trebuxiophyceae, and Coccomyxa, Coccomyxa sp.Obi strain, and Coccomyxa sp.KJ strain (hereinafter Obi strain and KJ strain) (Known as stock). A protein (Oil globule protein) having sequence similarity to MLDP has been found as an oil droplet protein in the alga Lobosphaera, which belongs to the same Treborgiaphyceae (Trebouxiophyceae) (Non-Patent Document 6).

  The Obi strain is the same strain as the unicellular green alga Pseudochoricystis ellipsoidea MBIC11204 strain described in Patent Document 1, and has been deposited under accession number FERM BP-10484. KJ strain, which is closely related to Obi strain, has about twice as much oil productivity as Obi strain (oil fat production rate per culture solution volume), and is described in Patent Document 2 as a Pseudococcomyxa genus KJ strain. Have been described. This strain has been deposited under accession number FERM BP-22254. The Obi strain and the KJ strain grow well even in a medium having a pH of 3.5 or less, can be cultivated by the open culture system shown in Patent Document 3, and can continuously produce oil and fat outdoors by the method shown in Patent Document 4. It can be carried out.

  The present inventors have deciphered the genome sequences of the Obi strain and the KJ strain, and have been working on improving their breeding and culture techniques. For breeding a strain having improved fat and oil productivity, for example, a method for improving the photoutilization efficiency of photosynthesis (Patent Document 5), a method for promoting the activity of an enzyme involved in fat and oil production (Patent Document 6), or a fat and oil A method of suppressing decomposition (Patent Document 7) and the like can be considered. So far, by treatment with a mutagenic agent, a strain having a mutation in the TAG lipase gene having sequence similarity to Sugar-dependent 1 (SDP1) of higher plants has been isolated from Obi strain and KJ strain. Patent Document 7). The SDP1 mutant in the KJ strain suppressed the decomposition of fats and oils, but did not show the increase in fat and oil productivity at the time of nitrogen deficiency.

Japanese Patent No. 4748154 Japanese Patent No. 6088375 Japanese Patent No. 6235210 Japanese Patent No. 5810831 JP 2013-102715 JP JP 2017-046643 JP JP 2017-046645 JP

Chisti Y. (2013) Constraints to commercialization of algal fuels. J. Biotechnol. 167: 201-214. Huang AHC. (2018) Plant lipid droplets and their associated proteins: potential for rapid advances. Plant Physiol. 176: 1894-1918. Moellering ER, Benning C. (2010) RNA Interference silencing of a major lipid droplet protein affects lipid droplet size in Chlamydomonas reinhardtii. Eukaryot Cell. 9: 97-106. Davidi L, Katz A, Pick U. (2012) Characterization of major lipid droplet proteins from Dunaliella. Planta. 236: 19-33. Peled E, Leu S, Zarka A, Weiss M, Pick U, Khozin-Goldberg I, Boussiba S. (2011) Isolation of a novel oil globule protein from the green alga Haematococcus pluvialis (Chlorophyceae). Lipids. 46: 851-861 . Siegler H, Valerius O, Ischebeck T, Popko J, Tourasse NJ, Vallon O, Khozin-Goldberg I, Braus GH, Feussner I. (2017) Analysis of the lipid body proteome of the oleaginous alga Lobosphaera incisa.BMC Plant Biol. 17 : 98.

  Increasing the oil and fat productivity of eukaryotic microalgae can be considered as a powerful means of cost reduction necessary for the practical application of biofuel production. In the cells, decomposition of fats and oils occurs simultaneously with the synthesis of fats and oils, but in eukaryotic microalgae mutants in which the ability to decompose fats and oils (oil and fat decomposition ability) is reduced, the amount of fats and oils accumulated (weight of fats and oils per algal dry weight) increases, As a result, oil and fat productivity may increase. Further, by culturing such a eukaryotic microalgal mutant, it becomes possible to reduce the production cost of fats and oils to be supplied to biofuel and the like.

  Therefore, an object of the present invention is to provide eukaryotic microalgae in which the ability to decompose fats and oils is reduced, and the amount of fats and oils accumulated and the productivity of fats and oils are improved.

  As a result of intensive research to solve the above problems, in eukaryotic microalgae in which a gene encoding a specific LDP (LDP1) is mutated, fat and oil degradability is decreased, and fat and oil accumulation and fat and fat productivity are improved. Heading out, the present invention has been completed.

That is, the present invention includes the following.
(1) A eukaryote having an amino acid sequence having at least 50% sequence identity with the conserved region of LDP shown in SEQ ID NO: 7 or SEQ ID NO: 8 and having a reduced function of a protein localized on the oil droplet membrane surface. The eukaryotic microalgae mutant, which is a microalgae mutant, in which the amount of accumulated oils and fats in the cells and the productivity of oils and fats are reduced and the ability to decompose fats and oils is decreased as compared with the parent strain.
(2) The eukaryotic microalgae mutant according to (1), wherein the gene encoding the protein is disrupted.
(3) The eukaryotic microalgal mutant according to (1), which has reduced expression of a gene encoding the protein.
(4) The eukaryotic microalgae mutant according to (1), wherein the translation efficiency of the gene encoding the protein is reduced.
(5) The eukaryotic microalgae mutant according to any one of (1) to (4), which belongs to Chlorophyta.
(6) The eukaryotic microalgae mutant according to (5), which belongs to the Trebouxiophyceae.
(7) The eukaryotic microalgal mutant according to (6), which belongs to the genus Coccomyxa.
(8) A method for producing fats and oils, which comprises a step of culturing the eukaryotic microalgae mutant according to any one of (1) to (7).

  According to the present invention, it is possible to produce a eukaryotic microalgae mutant in which the amount of accumulated fats and oils in cells and the productivity of fats and oils are increased, and the degradability of fats and oils is decreased. Further, by culturing the eukaryotic microalgae mutant according to the present invention, it becomes possible to reduce the production cost of oils and fats used for biofuel and the like.

The antenna chlorophyll-decreasing mutant (5P strain) derived from the Obi strain and the LDP1 gene mutant (ldp1-1) derived from the 5P strain were cultured for 14 days in a test tube under nitrogen-deficient (1 / 2A7) medium, continuous light conditions. Then, the results are shown in which the amount of accumulated fats and oils was measured every 24 hours by transferring to a nitrogen-sufficient (MA5) medium and culturing for 3 days in the dark. In the graph, the vertical axis represents the amount of accumulated fats and oils, and the horizontal axis represents the time after transferring to a nitrogen-sufficient medium and transferring in a dark place. A list of sequences is shown. It is a continuation of FIG. It is a continuation of FIG. 2-2. It is a continuation of FIG. 2-3. It is a continuation of FIG. 2-4. It is a continuation of FIG. 2-5.

  Hereinafter, the present invention will be described in detail.

  The present invention, by lowering the function of the LDP protein named LDP1, compared with the parent strain, the intracellular fat accumulation amount and fat productivity increased, and fat degrading eukaryotic microalgae mutation Regarding the body

  The Obi strain-derived mutant obtained by treating the mutagenizing agent N-methyl-N'-nitro-N-nitrosoguanidine (NTG) was mutated to the LDP1 gene (genomic sequence) shown in SEQ ID NO: 2. Had.

  Major lipid droplet protein (MLDP), which has been shown to be involved in the size of oil droplets in LDP of Chlamydomonas (non-patent document 3), Donaliella (Non-patent document 4), which is also a green alga, and Hematococcus (non-patent document) Two proteins each having an amino acid sequence identity with LDP of 5) of about 20 to 30% were found in the Obi strain and the KJ strain, respectively, and they were named LDP1 protein and LDP2 protein. The amino acid sequences of the LDP1 protein and the LDP2 protein of the KJ strain are shown in SEQ ID NO: 5 and SEQ ID NO: 9, respectively. The amino acid sequences of the LDP1 protein and LDP2 protein of the Obi strain are shown in SEQ ID NO: 6 and SEQ ID NO: 10, respectively.

  On the other hand, the nucleotide sequences of the LDP1 gene (genomic sequence) of the KJ strain and its CDS are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. The nucleotide sequences of the LDP1 gene (genomic sequence) of the Obi strain and its CDS are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

  The amino acid sequences of LDP1 of Obi strain and KJ strain show about 98% sequence identity with each other. The N-terminal 9 amino acid residues and the C-terminal 24 amino acid residues of the LDP1 proteins of the KJ strain and the Obi strain show no similarity to the amino acid sequences of other LDPs. Therefore, the central portion excluding these N-terminal and C-terminal portions is defined as the conserved region of the LDP1 protein. The amino acid sequences of the conserved regions of the LDP1 protein of the KJ strain and the Obi strain are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 and the amino acid sequence of LDP2 of Obi strain or KJ strain show approximately 44% (119/268) identity. Compared to Oil globule protein (non-patent document 6), which is LDP of the alga Lobos faera belonging to the genus Trevoxia, the conserved region of LDP2 protein is about 52% (139/266), and the conserved region of LDP1 protein is about 45% ( 121/268).

  It has been shown that the LDP genes of Chlamydomonas, Donaliella, and Lobosphaera increase in expression level when oil and fat are accumulated (Non-patent Documents 3 to 6). Similar to these known LDP genes, LDP2 gene was upregulated when nitrogen was deficient, but LDP1 gene was not upregulated when nitrogen was deficient.

  The mutant strain derived from the Obi strain and the gene-disrupted strain derived from the KJ strain in which the LDP1 gene was disrupted had increased intracellular fat and oil accumulation amount and increased fat and oil productivity. Furthermore, they have found that these LDP1 gene-disrupted strains suppress oil and fat decomposition, and completed the present invention.

  In the present invention, examples of the eukaryotic microalgae include green algae, diatoms (diatom or Bacillariophyceae), and eukaryotic microalgae belonging to the class Eustigmatophyceae.

  Examples of green algae include green algae belonging to the Treboxya algal network. Examples of green algae belonging to the Trevoxya algal web include, for example, the genus Trebouxia, the genus Chlorella, the genus Chlorella, the genus Botryococcus, the genus Coricystis, the genus Coccomyxa, and the genus Pseudodococ. Green algae belonging to. Specific strains belonging to the Trevoxya alga net include the Obi strain (accession number FERM BP-10484) and its mutant P. ellipsoidea 5P strain (accession number FERM BP-22179; hereinafter sometimes referred to as "5P strain"). ) And KJ strain (accession number FERM BP-22254). Obi stock was entrusted to National Institute of Advanced Industrial Science and Technology, Patent Biological Depository Center (February 15, 2005, 1-chome, 1-chome, 1-chome, 1-chome, Tsukuba, Ibaraki, 305-8566, Japan) It has been deposited under the number FERM P-20401 and has been transferred to the international deposit under the Budapest Treaty under the deposit number FERM BP-10484. The Obi strain is available from the Japan Patent Evaluation Organism Depositary (NITE-IPOD) (Institute for Product Evaluation Technology, National Institute for Product Evaluation (NITE-IPOD) (Room 2-5-8, Kazusa, Kamasa, Kisarazu, Chiba Prefecture, 292-0818, Japan)). The 5P shares were entrusted to the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, 1st, 1st, 1st, East Tsukuba, 305-8566, Japan) as of October 21, 2011. No. FERM P-22179, and at the Japan Institute for Product Evaluation Technology, Japan Patent Organism Depositary (NITE-IPOD) (Room 2-5-8 Kazusa, Kamasa, Kisarazu, Chiba Prefecture, 292-0818, Japan) It has been transferred to an international deposit under the Budapest Treaty under accession number FERM BP-22179. KJ Co., Ltd. was established on June 4, 2013 as the Japan Institute for Product Evaluation Technology, Japan Patent Organism Depositary (NITE-IPOD) (2-5 Kazusa Kamafoot, Kisarazu City, Chiba Prefecture, 292-0818 Japan). -8 room 120) with the deposit number FERM P-22254, and with the deposit number FERM BP-22254 has been transferred to the international deposit under the Budapest Treaty. In addition, the Obi strain and the 5P strain have the same gene encoding the LDP1 protein (genomic DNA base sequence: SEQ ID NO: 2, CDS base sequence: SEQ ID NO: 4, full-length amino acid sequence: SEQ ID NO: 6, conserved region amino acid sequence: SEQ ID NO: 8).

  Examples of green algae other than the green algae belonging to the Trevoxya algae net include, for example, Tetraselmis genus, Ankistrodesmus genus, Daniella (Dunalliella) genus, Neochloris genus, Chlamydomonas genus, Ikedamo (= Senedesmus: Scenedesmus). Examples include green algae belonging to the genus and the like.

  Further, examples of diatoms include eukaryotic microalgae belonging to the genus Fistulifera, the genus Pheoductilum, the genus Thalassiosira, the genus Cyclotella, the genus Cylindrotheca, the genus Skeletonema, and the like. be able to. In addition, examples of the true eye spot algae include the genus Nannochloropsis.

  The eukaryotic microalgae mutant according to the present invention is a eukaryotic microalgae mutant obtained by subjecting the above-described eukaryotic microalgae as a parent strain to a method for reducing the function of LDP1 protein. Here, the function of the LDP1 protein is localized on the membrane surface of oil droplets, for example, preventing the binding of oil droplets to each other, and / or in the oil droplets by lipolytic enzymes such as SDP1 lipase (Patent Document 7) Means to promote TAG degradation.

  In the present invention, the LDP1 protein has at least 50%, preferably at least 65%, and particularly preferably at least 80% of the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8 (that is, the amino acid sequence of the conserved region of the LDP1 protein). %, Most preferably at least 85%, at least 90%, at least 95%, proteins having an amino acid sequence with 100% sequence identity and localized on the membrane surface of the oil droplet.

  The LDP1 protein has at least 50%, preferably at least 65%, particularly preferably at least 80%, most preferably at least 85%, at least 90% of the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6. A protein consisting of an amino acid sequence having at least 95% and 100% sequence identity and localized on the membrane surface of oil droplets can be mentioned.

  Examples of the LDP1 gene include genes encoding the above LDP1 protein. The LDP1 gene has at least 50%, preferably at least 58%, particularly preferably at least 65%, at least 80%, and most preferably at least 85% of the coding region of the mRNA shown in SEQ ID NO: 3 or SEQ ID NO: 4. %, At least 90%, at least 95%, 100% sequence identity, and a gene encoding a protein localized on the membrane surface of oil droplets.

  In many eukaryotic microalgae, there may be a plurality of LDP1 genes, such as alleles, synonymous genes, etc., but in the present invention, at least one or more of these LDP1 genes is meant.

  In the present invention, the eukaryotic microalgae mutant of the present invention can be obtained by subjecting the eukaryotic microalgae having the LDP1 gene described above to a method of reducing the function of the LDP1 protein.

  Specifically, examples of the method for decreasing the function of the LDP1 protein according to the present invention include a method in which a mutation is randomly introduced with a drug, radiation, ultraviolet ray, or the like, and a strain having a mutation in the LDP1 gene is phenotypically selected. Be done. Examples of the method for selectively destroying the LDP1 gene include a gene disruption method by genome editing.

Furthermore, as a method for reducing the function of the LDP1 protein, for example,
(1) A mutation is introduced by targeting the LDP1 gene to destroy the gene;
(2) suppresses transcription of the LDP1 gene and reduces expression of the gene;
(3) suppresses the translation of the LDP1 gene and reduces the translation efficiency of the gene;
There is a method.

(1) Method of introducing mutation with LDP1 gene as a target
As a method for introducing a mutation targeting the LDP1 gene, a gene knockout method called ZFN, TALEN or CRISPR / Cas (Gaj T, Gersbach CA, Barbas CF 3rd. (2013) ZFN, TALEN, and CRISPR / Cas-based methods. For genome engineering. Trends Biotechnol. 31: 397-405.), a mutant lacking the gene can be produced.

(2) Method of suppressing transcription of LDP1 gene and decreasing expression of the gene
Examples of the method for suppressing the transcription of the LDP1 gene include a method for introducing a mutation into the promoter region of the target eukaryotic microalgae.

  In addition, there is a method of introducing a mutation into a gene involved in positive expression control of the gene to reduce the function thereof.

  Alternatively, there may be mentioned a method in which a mutation is introduced into a gene involved in the negative expression control of the gene so that the negative expression control always works.

(3) Method of suppressing translation of LDP1 gene and decreasing translation efficiency of the gene
Examples of methods for suppressing translation of the LDP1 gene include so-called RNA interference method (Cerutti H et al., 2011, Eukaryot Cell, 10, 1164) and antisense method.

  Furthermore, the present invention includes a method for producing fats and oils containing TAG by mass-culturing the eukaryotic microalgae mutant according to the present invention described above. Examples of the large-scale culturing method include an open culturing system shown in Patent Document 3 and a continuous culturing method shown in Patent Document 4. After the culturing, the TAG-containing fats and oils can be obtained by, for example, extracting with hexane from the culture.

  A list of sequences is shown in FIG. 2 together with the sequence listing.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the technical scope of the present invention is not limited to these examples.

[Example 1] Isolation of LDP1 gene mutant and evaluation of fat and oil productivity
N-Methyl-N'-nitro-N-nitrosoguanidine (NTG) using the 5P strain (Patent Document 5, Accession No. FERM BP-22179), which is a mutant with a reduced amount of antenna chlorophyll derived from the Obi strain, as a parent strain Mutagenesis treatment was carried out, and multiple mutants with increased oil and fat accumulation were obtained by screening using the oil and fat accumulation at the time of nitrogen deficiency as an index. In this screening, oils and fats accumulated in cells were fluorescently stained with Nile Red or BODIPY, and cells with high fluorescence intensity were selected using a cell sorter. As a result of comparative analysis of the genomic sequence of the strain with high oil and fat productivity with the parent strain 5P, the LDP1 gene (genomic sequence: SEQ ID NO: 2, CDS sequence: SEQ ID NO: 4, full-length amino acid sequence: SEQ ID NO: 6, conserved region) Amino acid sequence of: SEQ ID NO: 8) There is a single base deletion in the first half, and a frameshift mutation occurs after the 15th phenylalanine residue. Therefore, this mutation was named ldp1-1. In addition, the strain having the mutation was named as ldp1-1 mutant.

  5P strain and ldp1-1 mutant in a test tube under continuous light conditions in acidic (pH 3.5) nitrogen-deficient medium (1 / 2A7: Takahashi et al., 2017, Algal Res, 32, 300) for 13 days. Upon culturing and comparing the oil and fat productivity, as shown in Table 1, the amount of oil and fat accumulation in the ldp1-1 mutant increased to about 1.3 times that of the parent strain, and the oil and fat productivity in 13 days of culture was about 1.6 times that of the parent strain. Doubled.

  In order to further investigate the reason why the oil productivity was increased in the ldp1-1 mutant, the oil decomposition ability of both strains was examined. First, fats and oils were accumulated by growing in a nitrogen-deficient medium. When this strain is transferred to a nitrogen-sufficient medium and placed in a dark place, energy cannot be obtained by photosynthesis, and thus the accumulated fats and oils are degraded in the wild strain. Therefore, the 5P strain and the ldp1-1 mutant were transferred to a nitrogen-sufficient medium (MA5: Imamura et al., 2012, J Gen Appl Microbiol, 58, 1) and continuously cultured in the dark for 3 days. In the ldp1-1 mutant, fat decomposition was suppressed (Fig. 1). From this result, it was inferred that LDP1 protein is required for the decomposition of fats and oils in the dark, and the lack of LDP1 protein suppresses the decomposition of fats and oils, thereby improving the productivity of fats and oils.

[Example 2] Isolation of LDP1 gene-disrupted strain and evaluation of oil / fat productivity The ldp1-1 mutant shown in Example 1 had mutations in about 35 genes in addition to the ldp1-1 mutation. It was not possible to confirm that the causative gene mutation of the phenotype indicated by is ldp1-1. Therefore, we attempted to disrupt the LDP1 gene using the CRISPR / Cas9 system, which is one of the genome editing technologies.

  First, the LDP1 gene (genome sequence: SEQ ID NO: 1, CDS sequence: SEQ ID NO: 3, full-length amino acid sequence: SEQ ID NO: 5, conserved region amino acid sequence: SEQ ID NO: 7) of the KJ strain (accession number FERM BP-22254) is specific. We designed a gRNA target sequence for efficient cleavage and synthesized gRNA. After forming a complex between this gRNA and purified Cas9 protein, this complex was introduced into cells of the parent strain by electroporation. Next, the cell group subjected to the electroporation treatment was grown in a nitrogen-deficient medium to accumulate oil and fat in the cells. After that, the cell group accumulating oil and fat was transferred to a nitrogen-sufficient medium and cultured in a dark place. It was expected from the results of Example 1 that fat and oil decomposition rapidly occur in the parent strain by culturing in the dark, but fat and oil decomposition does not occur in the LDP1 gene-disrupted strain. From this, cells having a high oil and fat accumulation amount even after dark culture were selected from the above cell group cultured in the dark by a cell sorter, and the selected cells were seeded on a plate to obtain a large number of colonies.

  DNA was extracted from each of the strains derived from these colonies, and the gene region containing the gRNA target sequence was amplified by PCR. As shown in Table 2, in the gRNA target sequence, a recognition sequence for the restriction enzyme HinfI is present at a position one base away from the PAM sequence. Therefore, the obtained PCR fragment was treated with restriction enzyme HinfI and then electrophoresed to select colonies having mutant DNA in which the PCR fragment was not cleaved. As a result, a plurality of strains (kjldp1-1 to 1-4) having mutations in the target sequence of the LDP1 gene were obtained, and there were 4 kinds of mutations (Table 2). All of these strains had a frameshift mutation in the LDP1 gene.

  These strains were cultured in a test tube in an acidic nitrogen-deficient medium under continuous light conditions for 14 days, and the fat and oil productivity was compared. As shown in Table 3, the accumulated amount of fat and oil and fat and oil in all LDP1 gene-disrupted strains were shown. Productivity increased from the parent strain. When transferred to a nitrogen-rich medium and continuously cultured in the dark for 4 days, the amount of accumulated fats and oils in the KJ strain (KJ-WT) decreased from 52.6% to 28.4%, but after 4 days in the LDP1 gene-disrupted strain. The amount of accumulated fats and oils was about 60%, and the decomposition of fats and oils was suppressed (Table 3).

FERM BP-10484
FERM BP-22179
FERM BP-22254

Claims (8)

  A eukaryotic microparticle having an amino acid sequence having at least 50% sequence identity with the conserved region of the oil droplet protein shown in SEQ ID NO: 7 or SEQ ID NO: 8 and having a reduced function of the protein localized on the oil droplet membrane surface. The eukaryotic microalgae mutant, which is an algae mutant, in which the amount of accumulated oil and fat in the cell and the productivity of oil and fat are increased and the ability to decompose oil and fat is decreased, as compared with the parent strain.   The eukaryotic microalgae mutant according to claim 1, wherein the gene encoding the protein is disrupted.   The eukaryotic microalgae mutant according to claim 1, which has reduced expression of a gene encoding the protein.   The eukaryotic microalgae mutant according to claim 1, which has reduced translation efficiency of a gene encoding the protein.   The eukaryotic microalgae mutant according to any one of claims 1 to 4, which belongs to Chlorophyta.   The eukaryotic microalgae mutant according to claim 5, which belongs to the Trebouxiophyceae.   The eukaryotic microalgae mutant according to claim 6, which belongs to the genus Coccomyxa. A method for producing fats and oils, comprising a step of culturing the eukaryotic microalgae mutant according to any one of claims 1 to 7.

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