Algae with modified lipase and SPX2 genes, and a method for producing triacylglycerol using the same.

Abstract

This invention provides a means to increase the amount of triacylglycerol accumulated in high-lipid algae. [Solution] Algae characterized by the following (1) and (2), (1) Decreased expression of class 3 lipase genes, (2) Reduced expression of the SPX2 gene.

Description

[Technical Field]

[0001] The present invention relates to algae that accumulate triacylglycerols in large quantities, and to a method for producing triacylglycerols using the same. [Background technology]

[0002] Algal biomass is larger than that of plants, offering advantages in biofuel production and the production of useful lipids (Non-Patent Literature 1). Research is underway worldwide to produce industrial and food products, including biofuels, using biomass such as triacylglycerol (TAG) derived from algae as raw materials. However, high production costs are currently a problem for commercialization. Therefore, further technological development is essential to reduce production costs.

[0003] Nutrient reductions in many plants and algae lead to decreased growth and accumulation of lipids such as TAG. SPX proteins, which possess an SPX domain, are known to be involved in the absorption, transport, and storage of inorganic phosphate and the signaling that controls these processes (Non-Patent Literature 2). The true eyespot alga Nannochloropsis (hereinafter referred to as "Nannochloropsis") is an ultramicroalga that accumulates a certain amount of TAG during normal cultivation, but it has been reported that it accumulates even more TAG under nutrient-deficient conditions, and that its lipid composition is simple and suitable for fuel (Non-Patent Literature 1, Non-Patent Literature 3). It also has the characteristics of being able to be cultured in seawater, enabling low-cost production due to high-density cultivation, and for which genetic modification technology has already been established (Non-Patent Literature 4, Non-Patent Literature 5). The present inventor previously discovered that in Nannochloropsis, the accumulation of triacylglycerol increases when the function of SPX2, which responds to phosphorus deficiency, is lost (Patent Literature 1).

[0004] Lipid accumulation is regulated by lipid degradation and lipid synthesis. Lipases are known proteins involved in lipid degradation. In budding yeast and higher plants, the major known TAG lipases, TGL and SDP1, both possess a patatin-like domain (Non-Patent Literature 6, 7, and 8). There is a report that when the expression level of the AtSDP1 homolog gene in the diatom (Phaeodactylum tricornutum) is suppressed to 20%-40% by RNAi, lipid accumulation doubles (Non-Patent Literature 9). Nannochloropsis has lipases TGL1 and TGL2, which have the same domain structure. When a TGL1 / TGL2 double mutant was created, an increase in lipid accumulation was confirmed on the second day of early culture (Non-Patent Literature 10). However, since an increase in TAG accumulation was not confirmed in the later stages of culture or under nutrient-deficient conditions, it is thought that the main TAG degradation in Nannochloropsis is carried out by an unknown oil droplet-localized lipase other than TGL1 and TGL2, but this has not been identified to date. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2020 / 050412 [Non-patent literature]

[0006] [Non-Patent Document 1] Plant J. 54, 621-639, (2008) [Non-Patent Document 2] Liu et al., Open Biol. 2018-Jan 3; 8(1): 170231 [Non-Patent Document 3] Biotechnol. Bioeng. 102, 100-112, (2009) [Non-Patent Document 4] Proc. Natl. Acad. Sci. USA 108, 21265-21269, (2011) [Non-Patent Document 5] Genes Cells 25, 695-702, (2020) [Non-Patent Document 6] Plant Cell 18, 665-675, (2006) [Non-Patent Document 7] J. Biol. Chem. 278, 23317-23323, (2003) [Non-Patent Document 8] J. Biol. Chem. 280, 37301-37309, (2005) [Non-Patent Document 9] Biochim. Biophys. Acta 1861, 239-248, (2016) [Non-Patent Document 10] Biochim. Biophys. Acta 1864, 1185-1193, (2019) [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Nannochloropsis is a highly lipid-accumulating algae compared to other algae, but further technological development is desired to reduce production costs. This invention was made against this backdrop and aims to provide a means to increase the amount of TAG accumulated in lipid-accumulating algae such as Nannochloropsis. [Means for solving the problem]

[0008] Previous reports have shown that spx2 gene knockout strains accumulate more oil under phosphorus-deficient conditions than wild-type strains (International Publication No. 2020 / 050412). Furthermore, it has been shown that TGL1 / TGL2 double knockout strains, which are major TAG lipase homologs in plants, do not lead to increased oil production at a practical level (Biochim. Biophys. Acta 1864, 1185-1193, (2019)). Therefore, in order to achieve the above objectives, the inventors have diligently conducted research and focused on class 3 lipase (Pfam#PF01764) as a new TAG lipase that functions on the surface of oil droplets. This structure represents a domain containing an α / β hydrolase fold, such as ferloylesterase A from Aspergillus niger (J. Mol. Biol. 338, 495-506, (2004)), triacylglycerol lipase OBL1 from Arabidopsis thaliana (New Phytol 217, 1062-1076, (2018)), and diacylglycerol lipase α from human (Proc Natl Acad Sci USA 113, 26-33, (2016)). From the results of the Pfam domain search, 23 class 3 lipases were found in Nannochloropsis. Therefore, for No3LIP7 and No3LIP14, we created gene disruption strains by genome editing using a parent strain in which the SPX2 gene was disrupted by genome editing. As a result, the double knockout strains of the spx2 No3lip7 and spx2 No3lip14 genes successfully increased lipid accumulation under phosphorus-deficient conditions.

[0009] The present invention provides the following [1] to [6]. [1] Algae characterized by the following (1) and (2), (1) Decreased expression of class 3 lipase genes, (2) Reduced expression of the SPX2 gene.

[0010] [2] The algae described in [1], characterized in that the algae belong to the genus Nannochloropsis.

[0011] 〔3〕The algae according to 〔1〕, wherein the class 3 lipase gene is a gene encoding a protein of any one of the following (a), (b), or (c). (a) A protein consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8 (b) A protein consisting of an amino acid sequence in which 1 to 50 amino acid residues are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8, and having lipase activity (c) A protein consisting of an amino acid sequence having 40% or more homology with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8, and having lipase activity

[0012] 〔4〕The algae according to 〔1〕, wherein the SPX2 gene is a gene encoding a protein of any one of the following (a), (b), or (c), and is a gene whose expression level increases under phosphorus deficiency conditions (a) A protein consisting of the amino acid sequence represented by SEQ ID NO: 10 (b) A protein consisting of an amino acid sequence in which 1 to 50 amino acid residues are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 10 (c) A protein consisting of an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 10

[0013] 〔5〕A method for producing triacylglycerol, comprising culturing the algae according to any one of 〔1〕 to 〔4〕, producing triacylglycerol in the algae, and collecting the produced triacylglycerol

[0014] 〔6〕The method for producing triacylglycerol according to 〔5〕, comprising culturing the algae under phosphorus deficiency conditions

Advantages of the Invention

[0015] This invention provides a novel alga. This alga accumulates TAG in high quantities, making it useful for TAG production. Furthermore, since this alga does not contain foreign genes, it does not fall under the category of genetically modified organisms and can be cultivated outdoors. [Brief explanation of the drawing]

[0016] [Figure 1] Diagram showing the light and temperature conditions for culture. A. Light and temperature control program used for culture. Solid lines indicate light intensity, and dashed lines indicate temperature. B. Culture conditions for pre-culture and main culture. [Figure 2] This figure compares wild-type strains, spx2 No3lip strain 7, and spx2 No3lip strain 14 under phosphorus-deficient conditions. A. Changes in cell density in culture medium. B. Changes in biomass per cell. C. Changes in TAG accumulation per culture medium. D. Changes in TAG accumulation per biomass. E. Changes in TAG accumulation per cell. F. Changes in TAG accumulation efficiency. [Figure 3] This figure compares the spx2 strain and the spx2 No3lip14 strain under phosphorus-deficient conditions. A. Change in cell density in culture medium. B. Change in biomass per unit of culture medium. C. Change in biomass per unit of cell. D. Change in TAG accumulation per unit of culture medium. E. Change in TAG accumulation per unit of cell. F. Change in TAG accumulation per unit of biomass. G. Change in TAG accumulation efficiency. * indicates a statistically significant difference between the spx2 strain and the spx2 No3lip14 strain. n=4, error bars represent se. p < 0.05 (Tukey test). [Figure 4] This figure compares the growth of wild-type, No3lip14, spx2, and spx2 No3lip14 strains under phosphorus-deficient conditions. A. Changes in cell density in culture medium. B. Changes in biomass per unit of culture medium. C. Changes in biomass per unit of cell. D. Changes in forward scatter light (FSC). E. Fluorescence micrographs (Nile Red and chlorophyll) and DIC micrographs of cells after culturing. [Figure 5]This figure compares the TAG accumulation levels of wild-type, No3lip14, spx2, and spx2 No3lip14 strains under phosphorus-deficient conditions. A. Change in TAG accumulation per culture medium. B. Change in TAG accumulation per biomass. C. Change in TAG accumulation per cell. D. Change in TAG accumulation efficiency. [Modes for carrying out the invention]

[0017] The present invention will be described in detail below. The algae of the present invention are preferably algae belonging to the genus Nannochloropsis, but other algae may also be used. Algae belonging to the genus Nannochloropsis include Nannochloropsis oceanica, Nannochloropsis gaditana, Nannochloropsis salina, Nannochloropsis oculata, Nannochloropsis atomus, Nannochloropsis maculata, Nannochloropsis granulata, Nannochloropsis limnetica, Nannochloropsis maritima, and Nannochloropsis australis.

[0018] The algae of the present invention have the following two characteristics. The first characteristic is the reduced expression of the class 3 lipase gene. Class 3 lipase has a domain containing an α / β hydrolase fold. Therefore, it is possible to identify the class 3 lipase gene in algae based on this domain (for example, accession number: PF01764 in the Pfam database). The class 3 lipase gene is preferably class 3 triacylglycerol lipase.

[0019] Specific examples of class 3 lipase genes include No3LIP7, No3LIP14, No3LIP6, No3LIP10, or the genes corresponding to these genes in each alga. The nucleotide sequences of No3LIP7, No3LIP14, No3LIP6, and No3LIP10 are shown in SEQ ID NOs. 1, 3, 5, and 7, respectively, and the amino acid sequences of the proteins encoded by these genes are shown in SEQ ID NOs. 2, 4, 6, and 8, respectively. Examples of genes corresponding to No3LIP7, No3LIP14, No3LIP6, or No3LIP10 include (b) genes encoding a protein having lipase activity, consisting of an amino acid sequence in which 1 to 50 amino acid residues are substituted, added, or deleted from the amino acid sequence represented by SEQ ID NOs. 2, 4, 6, or 8, and (c) genes encoding a protein having lipase activity, consisting of an amino acid sequence having 40% or more homology to the amino acid sequence represented by SEQ ID NOs. 2, 4, 6, or 8.

[0020] In the protein of (b), the number of amino acid residues to be substituted, added, or deleted is not particularly limited as long as it is between 1 and 50, but is preferably around 1 to 30, more preferably around 1 to 10, even more preferably around 1 to 5, and particularly preferably around 1, 2, 3, or 4.

[0021] In the protein of (c), the homology with the amino acid sequence represented by SEQ ID NOs: 2, 4, 6, or 8 is not particularly limited as long as it is 40% or more, but is preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, and particularly preferably 80% or more. The homology value may be even higher, for example, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more. The homology of the amino acid sequence can be calculated using the BLASTP program provided by NCBI (National Center of Biotechnology Information).

[0022] A Blast search was performed on the amino acid sequences represented by SEQ ID NOs: 2, 4, 6, and 8. The results showed that for SEQ ID NO: 2 (No3LIP7), there is a protein with approximately 70% homology to Nanochloropsis gaditana; for SEQ ID NO: 4 (No3LIP14), there is a protein with approximately 40% homology to both Nanochloropsis gaditana and Nanochloropsis salina; for SEQ ID NO: 6 (No3LIP6), there is a protein with approximately 80% homology to both Nanochloropsis gaditana and Nanochloropsis salina; and for SEQ ID NO: 8 (No3LIP10), there is a protein with approximately 60% homology to Nanochloropsis gaditana and approximately 70% homology to Nanochloropsis salina. From this, it is thought that the genes encoding the protein in (c) (genes with more than 40% homology at the amino acid level) include genes corresponding to No3LIP7, No3LIP14, No3LIP6, and No3LIP10 in algae belonging to the genus Nannochloropsis.

[0023] In the algae of the present invention, the expression of the class 3 lipase gene is reduced. Here, "reduced expression of the class 3 lipase gene" means that the expression level of the class 3 lipase gene is lower compared to the wild type, and includes cases where the class 3 lipase gene is not expressed at all. Furthermore, the reduction in expression may be due to an effect on genes in the genome (e.g., gene modification by genome editing, mutation introduction by radiation), or it may be due to a method that does not affect genes in the genome (e.g., suppression of gene expression by RNAi or antisense method).

[0024] Modifications of class 3 lipase genes include, for example, gene deletion (gene disruption), introduction of mutations in the protein-coding region, and introduction of mutations in the expression regulatory region.

[0025] The second characteristic is the reduced expression of the SPX2 gene. The SPX2 gene is described in many publications (for example, Liu et al., Open Biol. 2018-Jan 3; 8(1): 170231, International Publication No. 2020 / 050412, etc.), so it is possible to identify the SPX2 gene in algae based on these publications.

[0026] Specific examples of the SPX2 gene include the SPX2 gene of the Nannochloropsis NIES-2145 strain, or the corresponding gene in various algae. The nucleotide sequence of the SPX2 gene of the Nannochloropsis NIES-2145 strain is shown in SEQ ID NO: 9, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO: 10. Examples of genes corresponding to the SPX2 gene of the Nannochloropsis NIES-2145 strain include genes whose expression level increases under phosphorus-deficient conditions, and which encode proteins consisting of (b) amino acid sequences in which 1 to 50 amino acid residues are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 10, or (c) amino acid sequences having 60% or more homology to the amino acid sequence represented by SEQ ID NO: 10.

[0027] In the protein of (b), the number of amino acid residues to be substituted, added, or deleted is not particularly limited as long as it is between 1 and 50, but is preferably around 1 to 30, more preferably around 1 to 10, even more preferably around 1 to 5, and particularly preferably around 1, 2, 3, or 4.

[0028] In the protein of (c), the homology with the amino acid sequence represented by SEQ ID NO: 10 is not particularly limited as long as it is 60% or more, but is preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more. The homology value may be even higher, for example, 97% or more, 98% or more, or 99% or more.

[0029] Whether a gene's expression level increases under phosphorus-deficient conditions can be determined by culturing the algae containing that gene under normal conditions (conditions without phosphorus deficiency) and under phosphorus-deficient conditions, and comparing the gene's expression levels under both conditions.

[0030] "Decreased SPX2 gene expression" means, as with class 3 lipase genes, that the expression level of the SPX2 gene is lower compared to the wild type, and includes cases where the SPX2 gene is not expressed at all. Furthermore, the decrease in expression may be due to an effect on genes in the genome (e.g., gene modification by genome editing) or not (e.g., suppression of gene expression by RNAi or antisense methods). Examples of SPX2 gene modification include gene deletion (gene disruption), introduction of mutations in the protein-coding region, and introduction of mutations in the expression regulatory region.

[0031] The present invention's method for producing TAG is characterized by culturing the algae described above, allowing the algae to produce TAG, and collecting the produced TAG.

[0032] The algae of the present invention may be cultured under normal conditions (conditions that do not deplete phosphorus), but it is preferable to culture them under phosphorus-deficient conditions in order to increase the amount of TAG accumulated. Culture conditions other than phosphorus can be appropriately selected depending on the type of algae. For example, when culturing algae of the genus Nannochloropsis, F2N medium, HD medium, or mediums from which phosphorus has been removed can be used as the culture medium, the culture temperature can be set to about 15 to 25°C, and the light intensity during cultivation can be 10 to 1000 μmol photons / m². 2 It can be set to / sec.

[0033] Methods for collecting TAG produced in algae include methods commonly used to recover TAG accumulated within cells, such as drying, freezing, crushing, filtering, centrifugation, and solvent extraction of algal cells. [Examples]

[0034] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0035] Experimental materials The true eyespot alga Nannochloropsis NIES-2145 (hereinafter referred to as "N. 2145") was used. This algal strain is available from the National Institute for Environmental Studies (http: / / www.nies.go.jp / ).

[0036] The genes used As novel lipase genes, we searched the transcript data of N. oceanica CCMP1779 v2.0 (https: / / phycocosm.jgi.doe.gov / Nanoce1779_2 / Nanoce1779_2.home.html) for those possessing a lipase class 3 domain (PF01764), and selected 23 candidates. Of these, we focused on No3LIP7 and No3LIP14 based on their expression profiles. We also used the SPX2 gene (International Public Release No. 2020 / 050412), a gene whose expression level increases under phosphorus-deficient conditions.

[0037] The sequences of each gene are listed in the sequence listing. The sequences of No3LIP7, No3LIP14, and SPX2 are listed as sequence numbers 1, 3, and 9, respectively.

[0038] Experimental Procedure 1 Culture conditions HD medium was used as the standard liquid culture medium for culturing N. 2145. 222 mg of ZnSO4·7H2O, 79 mg of CuSO4·5H2O, 15 mg of MoO3, 2.86 g of H3BO3, and 1.81 g of MnCl2·4H2O were dissolved in 1 L of deionized water and stored at 4°C as A-5 stock. 2.5 g of KNO3, 0.25 g of Na2HPO4, 0.075 g of Fe-EDTA, and 5 ml of A-5 stock were dissolved in 500 ml of deionized water. This mixture, along with Daigo Artificial Seawater SP (FUJIFILM) dissolved in 500 ml of deionized water, was sterilized by autoclaving and then mixed. 2 ml of vitamin mixture was added before using it as HD medium. The vitamin mixture contains Vitamin B 120.6 μg of phosphate, 0.3 μg of biotin, and 60 μg of thiamine HCl were dissolved in 10 ml of deionized water, filtered, and then used. A phosphorus-deficient medium was prepared by removing Na2HPO4 from HD medium, and a phosphorus-half medium was prepared by adding 0.125 g of Na2HPO4 to HD medium. For normal culture, 5 ml of culture medium cultured for 7 days was added to 50 ml of HD medium, resulting in a concentration of 700 μmol photons / m². 2 Cells were cultured at 25°C with aeration of 15 ml / min of 2% CO2 at a rate of / sec. Cell counts were measured using a flow cytometer RF-500 (Sysmex).

[0039] 2. Method for producing spx2-disrupting strains and lipase-disrupting strains To disrupt the class 3 lipase genes No3LIP14 and NoLIP7, and the phosphate metabolism gene SPX2, we used a genome editing tool called transcriptional activator-like effector (TALE) nucleases (TALENs). TALENs are artificial nucleases that fuse the TALE domain, a DNA-binding protein derived from plant pathogenic bacteria, with the nuclease domain of FokI, a nuclease derived from marine bacteria, allowing for free target design. The TALE domain has 16 to 18 sequences of 34 amino acids called TALE repeats, and each TALE repeat identifies a single base of DNA. A single DNA double-strand break can be introduced into a target sequence sandwiched between two TALENs, L-TALEN and R-TALEN. In this experiment, we used the Platinum TALEN (PtTALEN) system, which further enhanced activity by modifying the 4th and 32nd amino acids of the TALE repeat. Target sequences for PtTALENs were selected using a web tool called TAL Effector Nucleotide Targeter 2.0 (https: / / tale-nt.cac.cornell.edu). Activity was examined using cultured cells, and all-in-one PtTALEN vectors for Nannochloropsis expression were constructed for TALENs showing high activity and used to introduce mutations into each target gene. For SPX2 disruption PtTALENs, two PtTALEN pairs showed mutagenesis activity, so all-in-one PtTALEN vectors for Nannochloropsis expression were constructed, introduced into Nannochloropsis, and the sequences near the target were amplified by PCR. Mutagenesis was then examined by direct sequencing.The target sequences are SPX_A (L-TALEN: TGCTAGAAGCCGCACCGC, spacer: TAGCATCATGAAGTT, R-TALEN: TGGTCTGTATCTGCGCGA) and SPX_D (L-TALEN: TGCAATACGACAAACTAA, spacer: AGCGAATGATTCGCA, R-TALEN: TTCTGGCCGAAGTGGAGA). Using an all-in-one PtTALEN vector targeting SPX_A, strains 4-11-3 (7 base deletion) were obtained, and using an all-in-one PtTALEN vector targeting SPX_D, strains 5-9-2 (5 base deletion) and 5-19-2 (4 base insertion) were obtained. Furthermore, all-in-one PtTALEN vectors targeting SPX_A and SPX_D were also developed. By simultaneously introducing the PtTALEN vector, we obtained a total of four SPX2 frameshift mutant strains, 8-4-1 (82 base pair deletion).

[0040] Furthermore, we also constructed double knockout strains for each gene. We introduced an all-in-one PtTALEN vector targeting LIP7_D (L-TALEN: TTCCAGCAGCAGCCAGTA, Spacer: TCATCGTCGCTCACC, R-TALEN: ACCAAGCCCGCAGCACCA), which had previously been detected to have gene disruption activity, into the SPX2 knockout strain 8-4-1. This resulted in obtaining two double knockout strains for SPX2 and No3LIP7: strain 2-4-4-1 (SPX2: 82 base deletion, No3LIP7: 2 base deletion) and strain 2-8-5-1 (SPX2: 82 base deletion, No3LIP7: 1 base insertion). In addition, an all-in-one PtTALEN vector targeting LIP14_A (L-TALEN: TCAGTCTGCGGCATGCCC, spacer: TTGTGTCGGGCGCGC, LR-TALEN: CAGCCGCCGTGGCTGCGA), which had previously been detected to exhibit mutagenesis activity, was introduced into strain 8-4-1. This resulted in obtaining two strains with double knockout of SPX2 and No3LIP14: strain 13-12 (SPX2: 82 base deletion, No3LIP14: 7 base deletion) and strain 13-15 (SPX2: 82 base deletion, No3LIP14: 10 base deletion).

[0041] In addition to the above, we also constructed strains 6B-6 (No3LIP14: 13 base deletion) and 6B-10 (No3LIP14: 17 base deletion) in which only the class 3 lipase gene was disrupted. For the construction of the single class 3 lipase gene disruption strains, strain 8-4-1 was replaced with the wild-type strain, and the other strains were constructed using the same method as for the double disruption strains. The sequences used for mutation introduction are listed in sequence numbers 11-22 of the sequence listing.

[0042] 3. Culturing under phosphorus-deficient conditions In Experiment 1, cells from the wild-type strain, strain 2-4-4-1 (SPX2: 82 base deletion, No3LIP7: 2 base deletion), and strain 13-12 (SPX2: 82 base deletion, No3LIP14: 7 base deletion), cultured under normal conditions for 7 days, were each measured in 1 × 10⁶ cells. 8Cells were subcultured into 500 ml of HD medium to a density of 2 1×10 cells / ml, and incubated under a 16-hour light period (700 μmol photons / m 8 2 / sec, unilateral irradiation), an 8-hour dark period, and aerated with 2% CO2 at 300 ml / min at 25°C for 4 days. Subsequently, the cells were subcultured into 500 ml of phosphate-deficient medium to a density of 1×10 2 cells / ml, aerated with 2% CO2 at 450 ml / min, and incubated under a 16-hour light period (500 μmol photons / m 2 2 / sec, bilateral irradiation, 22°C), an 8-hour dark period (25°C) for 2 days, and then under a 16-hour light period (1000 μmol photons / m

[0043] 2 / sec, bilateral irradiation, 15°C), an 8-hour dark period (25°C) for 10 days. 8 In Experiment 2, cells of the 8-4-1 strain (SPX2: 82-base deletion) and the 13-12 strain (SPX2: 82-base deletion, No3LIP14: 7-base deletion) that had been normally cultured for 7 days were subcultured into 500 ml of 1 / 2 phosphate medium to a density of 1×10 8 cells / ml, and incubated under the light irradiation conditions of Figure 1A, the set temperature of the incubator, and aerated with 2% CO2 at 450 ml / min for 4 days. Subsequently, the cells were subcultured into 500 ml of phosphate-deficient medium to a density of 2×10

[0044] cells / ml, with 4 samples each, and incubated under the light irradiation conditions of Figure 1A, the set temperature of the incubator, and aerated with 2% CO2 at 450 ml / min for 10 days. 8 In Experiment 3, cells of the wild strain, the 6B-6 strain (No3LIP14: 13-base deletion), the 8-4-1 strain (SPX2: 82-base deletion), and the 13-12 strain (SPX2: 82-base deletion, No3LIP14: 7-base deletion) that had been normally cultured for 7 days were subcultured into 500 ml of 1 / 2 phosphate medium to a density of 1×10 8Four samples were subcultured into 500 ml of phosphorus-deficient medium to achieve a cells / ml ratio. The samples were then cultured for 10 days under the light irradiation conditions and incubator temperature settings shown in Figure 1A, with 2% CO2 aeration at 450 ml / min.

[0045] 4 Lipid extraction Ten mL of culture medium was collected at each day and centrifuged at 4670G for 10 minutes at 25°C to precipitate the cultured cells. After centrifugation, the supernatant was removed, and the precipitated cultured cells were rapidly frozen in liquid nitrogen and stored at -80°C.

[0046] Frozen cells were suspended in 0.8 ml of deionized water, 1 ml of chloroform and 2 ml of methanol were added, and the mixture was stirred and left at room temperature for 1 hour. 1 ml of chloroform and 1 ml of deionized water were added to the suspension and centrifuged at 1000 × g for 5 minutes using a swing rotor. The water-methanol layer (upper layer) was removed, and the chloroform layer (lower layer) was transferred to a new glass test tube. Meanwhile, 1.5 ml of chloroform was added to the original glass test tube and suspended. Both the original test tube with this suspension and the new test tube containing the chloroform layer were centrifuged at 1000 × g for 5 minutes using a swing rotor. After centrifugation, the chloroform layer from the new test tube was transferred to another test tube whose weight was measured. The chloroform layer from the original test tube was recovered and centrifuged at 1000 × g for 5 minutes using a swing rotor. The chloroform layer was recovered and combined with the previously prepared chloroform extract to obtain the lipid extract. This lipid extract was dried using a vacuum concentrator, dissolved in chloroform to a concentration of 10 mg / ml, and then stored at -20°C.

[0047] 5 Lipid analysis Lipid extracts were spotted onto thin-layer silica plates and developed for 35 minutes with a developing solution of 160 mL of hexane, 40 mL of diethyl ether, and 4 mL of acetic acid. Tags were identified under UV irradiation using 0.001% (w / v) primuline. The silica containing the tags was scraped off, and 10 μL of 5 mM henoeicosanoic acid and 2.5 ml of 1.5 M hydrochloric acid / methanol were added to suspend the mixture. The mixture was then allowed to stand at 85°C for 2.5 hours to methylate the fatty acids. 2.5 ml of hexane was added, the mixture was suspended, and the upper layer of methylated fatty acids was collected. The collected methylated fatty acids were dried and dissolved in 100 μL of hexane to prepare the gas chromatography sample. Gas chromatography was performed using a SHIMADZU GC-2014 with an HR-SS-10 (0.25 φ x 25 m) (SHINWA CHEMICAL INDUSTRIES, LTD.) microscope.

[0048] 6. Measurement of biomass (dry weight of cells) Ten milliliters of culture medium cultured in phosphorus-deficient medium were collected at each day, transferred to a 50 ml tube, and centrifuged at 4670 G for 10 minutes at 25°C. After centrifugation, the supernatant was carefully removed, taking care not to remove any cells. H2O was added to the remaining precipitate to suspend it, and it was transferred to a weighed 1.5 ml tube. Centrifuged at 7000 G for 10 minutes at 25°C. The supernatant was carefully removed, taking care not to remove any cells, and the tube was placed in a high-temperature drying oven. With the cap removed, it was dried at 105°C for 5 hours. The 1.5 ml tube was removed from the high-temperature drying oven, and the biomass was weighed using an electronic balance.

[0049] Experimental results 1. Experiment 1: Comparison of wild-type, spx2 No3lip7 strain, and spx2 No3lip14 strain under phosphorus-deficient conditions. As shown in Figure 2A, the spx2 No3lip7 strain had a slightly higher cell density compared to the wild-type strain at days 3, 6, 7, and 10 of culture under phosphorus-deficient conditions. The spx2 No3lip14 strain had a lower cell density than the wild-type strain throughout the culture period. As shown in Figure 2B, the spx2 No3lip14 strain had a higher biomass per cell compared to the wild-type strain at days 6, 7, and 10 of culture, while the spx2 No3lip7 strain had a slightly lower biomass per cell compared to the wild-type strain. As shown in Figures 2C, 2D, and 2F, the spx2 No3lip7 and spx2 No3lip14 strains had higher TAG accumulation per culture medium and per biomass unit, as well as higher TAG accumulation efficiency, compared to the wild-type strain. As shown in Figure 2E, the TAG accumulation per cell in the spx2 No3lip7 strain was similar to that of the wild-type strain, but in the spx2 No3lip14 strain it was higher than that of the wild-type strain.

[0050] 2. Experiment 2: Comparison of spx2 strain and spx2 No3lip14 strain under phosphorus-deficient conditions. As shown in Figure 3, the cell density of the spx2 No3lip14 strain was significantly lower than that of the spx2 strain under phosphorus-deficient conditions. Biomass per culture medium and per cell was significantly higher in the spx2 No3lip14 strain than in the spx2 strain from day 1 of culture onward. TAG accumulation per culture medium was significantly higher in the spx2 No3lip14 strain than in the spx2 strain from day 6 of culture onward, and TAG accumulation per cell was significantly higher in the spx2 No3lip14 strain than in the spx2 strain from day 1 of culture onward. TAG accumulation per biomass was significantly lower in the spx2 No3lip14 strain than in the spx2 strain on days 1 and 3 of culture, but there was no difference from day 6 onward. TAG accumulation efficiency was significantly higher in the spx2 No3lip14 strain than in the spx2 strain from day 6 of culture onward.

[0051] 3. Experiment 3: Comparison of wild-type strain, No3lip14 strain, spx2 strain, and spx2 No3lip14 strain under phosphorus-deficient conditions. As shown in Figure 4A, the spx2 No3lip14 strain had a lower cell density under phosphorus-deficient conditions compared to the wild-type strain, No3lip14 strain, and spx2 strain, but had higher biomass per culture medium and per cell, as shown in Figures 4B and 4C. Furthermore, as shown in Figure 4D, the forward scattered light values ​​of cells measured by flow cytometry were also higher for the spx2 No3lip14 strain compared to the wild-type strain, No3lip14 strain, and spx2 strain, suggesting that the cells were larger than those of the other strains. Microscopic observation revealed a high frequency of cells with larger diameters. As shown in Figures 5A, 5C, and 5D, the spx2 No3lip14 strain had higher TAG accumulation per culture medium and per cell, as well as higher TAG accumulation efficiency, compared to the wild-type strain, No3lip14 strain, and spx2 strain. 4. Summary The results above clearly show that strains in which the SPX2 gene and a novel lipase gene (No3LIP7 or No3LIP14) were disrupted by genome editing accumulated TAGs more than wild-type strains under phosphorus-deficient conditions. In particular, the spx2 No3lip14 strain showed increased biomass per cell and increased TAG accumulation, making it suitable for TAG production under phosphorus-deficient conditions. Compared with spx2-only genome-edited strains, the spx2 No3lip14 strain also showed increased biomass per culture medium and per cell, as well as higher TAG accumulation. This indicates that disrupting both the SPX2 gene and the lipase gene No3LIP14 by genome editing results in higher TAG accumulation capacity than disrupting the SPX2 gene alone. [Industrial applicability]

[0052] This invention can be used in industrial fields related to fuels and the like.

Claims

1. Algae characterized by the following (1) and (2), (1) Decreased expression of class 3 lipase genes, (2) Reduced expression of the SPX2 gene.

2. The algae according to claim 1, characterized in that the algae belong to the genus Nannochloropsis.

3. The algae according to claim 1, characterized in that the class 3 lipase gene is a gene that encodes one of the following proteins (a), (b), or (c). (a) A protein consisting of an amino acid sequence represented by SEQ ID NOs: 2, 4, 6, or 8, (b) A protein having lipase activity, consisting of an amino acid sequence in which 1 to 50 amino acid residues are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NOs: 2, 4, 6, or 8, (c) A protein having lipase activity and consisting of an amino acid sequence having 40% or more homology to the amino acid sequence represented by Sequence ID No. 2, 4, 6, or 8.

4. The algae according to claim 1, characterized in that the SPX2 gene is a gene that encodes one of the following proteins (a), (b), or (c), and whose expression level increases under phosphorus-deficient conditions. (a) A protein consisting of the amino acid sequence represented by Sequence ID No. 10, (b) A protein consisting of an amino acid sequence in which 1 to 50 amino acid residues are substituted, added, or deleted in the amino acid sequence represented by Sequence ID No.

10. (c) A protein consisting of an amino acid sequence having 60% or more homology to the amino acid sequence represented by Sequence ID No.

10.

5. A method for producing triacylglycerol, characterized by culturing algae according to any one of claims 1 to 4, causing the algae to produce triacylglycerol, and collecting the produced triacylglycerol.

6. A method for producing triacylglycerol according to claim 5, characterized by culturing algae under phosphorus-deficient conditions.

Images