Mutant green alga with increased lipid productivity and application thereof

Abstract

To provide an alga belonging to Chlorophyta with an increased lipid content and improved lipid productivity.SOLUTION: Provided is a mutant alga derived from a parental strain belonging to Chlorophyta, having a plastid ATP / ADP transporter that comprises an amino acid partial sequence having at least 80% sequence identity to a specific amino acid sequence. Compared with the parental strain, the mutant alga exhibits reduced function of the plastid ATP / ADP transporter and increased lipid content and lipid productivity.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a green algae mutant having increased fat / oil content and fat / oil productivity, and uses thereof. [Background technology]

[0002] One of the measures to reduce greenhouse gases is the development of biofuels. In response to the need to develop biofuels that do not compete with food, the production of bioethanol made from inedible cellulose and biodiesel fuel made from oils and fats from microalgae has been proposed, and these are called second-generation and third-generation biofuels, respectively. However, large-scale production of second-generation biofuels has yet to be realized, and technological development and demonstration projects are still ongoing.

[0003] Microalgae can be cultivated in land unsuitable for cultivation, so competition with food is not an issue. In addition, because they have a higher oil productivity than oil crops, they have attracted attention as a raw material for biodiesel and biojet fuel. Since 2007, several US companies have begun industrial production of microalgae fuel, but the high costs of cultivation and of recovering algae bodies from the culture solution have led to companies withdrawing one after another. In other words, there is currently no prospect of practical application of third-generation biomass fuels.

[0004] The high production cost of microalgae biofuel is the biggest barrier to its practical application, and therefore efforts to reduce costs are necessary in each step of cultivation, recovery, oil extraction, and fuel production. Of these, the cost of cultivation is said to account for about 70% of the total cost, so the most important issue for practical application is to improve the efficiency and reduce the cost of cultivation. The first method of reducing cultivation costs is to use microalgae with high oil productivity. If the amount of oil obtained from one cultivation increases, the cultivation cost will decrease in inverse proportion.

[0005] Microalgae is a general term for organisms consisting of a single cell or a few cells that perform oxygenic photosynthesis, and includes eukaryotes such as cyanobacteria (prokaryotes), diatoms, xanthophytes, red algae, and green algae. Although there is a wide variety of microalgae, only a limited number of species accumulate the oils and fats (mainly triglycerides) that are used as raw materials for biodiesel fuel. A survey of approximately 200 papers in international journals revealed that most of the microalgae that showed high oil and fat productivity (amount of oil produced per unit time) were green algae closely related to the chlorella species (genus Chlorella, Parachlorella) and the Desmodesmus species (genus Scenedesmus, genera).

[0006] The genus Parachlorella has a high oil-producing ability (Non-Patent Document 1). A study was conducted to isolate mutants with improved oil productivity by inducing mutations in Parachlorella NIES-2152 strain (Parachlorella kessleri) (hereinafter, referred to as the "wild strain" unless there is a risk of confusion) by irradiating it with a heavy ion beam, and the mutant strain PK4 with improved oil content (oil weight per dry weight of cells) was isolated (Non-Patent Documents 2 and 3). In order to identify the mutations in this high-oil-accumulating mutant PK4, in addition to analyzing the genome base sequences of the wild strain and the PK4 strain, transcriptome analysis of the wild strain was also performed (Non-Patent Document 4). As a result of comparing the genome base sequences of the wild strain and the PK4 strain, it was confirmed that amino acid substitutions, deletions, and nonsense mutations were present in the three genes represented by gene numbers 8741, 9067, and 9934, respectively (Non-Patent Document 4). This suggests that the increased fat and oil content of the PK4 strain may have been caused by one of these three gene mutations. However, since no genetic engineering methods, including genetic recombination, have been developed for Parachlorella, it was virtually impossible to identify which of these three genes was responsible for the increased fat and oil content. [Prior art documents] [Non-patent literature]

[0007]

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[0008] The main reason why the commercial use of oils and fats produced by eukaryotic microalgae has not progressed is the high production cost. One possible way to reduce the cost of oil and fat production is to genetically improve microalgae that originally have high oil and fat productivity to produce strains with higher oil and fat productivity. One specific way to achieve this is to identify the genetic mutation that caused the trait of improved oil and fat content in the above-mentioned PK4 strain, and then introduce an equivalent mutation into various green algae to improve the oil and fat content of each green algae. In addition, by culturing such green algae mutants, it is possible to reduce the cost of producing oils and fats for use in biofuels, etc.

[0009] Therefore, the present invention aims to (1) identify a genetic mutation that improves the oil content of the PK4 strain, and (2) provide a green alga with an improved oil content by introducing the genetic mutation into other green algae. [Means for solving the problem]

[0010] There are several methods to confirm which of the three mutations in the PK4 strain produced the trait of increased fat content. For example, by introducing a wild-type allele (denoted by A) into a mutant gene (denoted by a) in the PK4 strain, if the partial diploid (A / a) produced by this operation shows the trait of the wild strain, it can be concluded that the trait of the PK4 strain, increased fat content, was acquired by a recessive mutation in the A gene. To carry out such an experiment, a technique is required to integrate artificial DNA prepared in a test tube into the genome, that is, a technique to isolate a transformant. Another method is to destroy the wild-type allele A of the wild strain, and if the destroyed strain shows the trait of increased fat content of the PK4 strain, it can be concluded that the trait of increased fat content of the PK4 strain was acquired by a mutation in the A gene. To carry out such an experiment, a technique is required to introduce a mutation into a specific gene in the genome, for example, genome editing technology.

[0011] Considering future versatility, it seemed that there would be great merit in establishing a technology that can apply genome editing to Parachlorella. Therefore, we aimed to identify the genetic mutations responsible for the trait of increased oil content by disrupting the 8741, 9067, and 9934 genes of the wild strain using genome editing methods.

[0012] As a result of intensive research conducted to solve the above problems, it was found that a genome-edited strain in which the 9067 gene of a wild-type strain having the genomic DNA sequence of SEQ ID NO: 2 and encoding a plastidic ATP / ADP transporter having the amino acid sequence shown in SEQ ID NO: 8 was disrupted has improved oil content and oil productivity.

[0013] On the other hand, many green algae have multiple plastid ATP / ADP transporter genes on one genome. Analysis of these genes revealed that they are phylogenetically divided into three clades. Proteins belonging to different clades may have the same function, but in some cases, genes that emerged through gene duplication may form new clades in the process of acquiring new functions. The plastid ATP / ADP transporter gene encoded by the 9067 gene in the NIES-2152 strain belongs to clade-2, and may therefore have a different function from clade-1 and clade-3. On the other hand, it can be assumed that the plastid ATP / ADP transporters belonging to clade-2 in other green algae and the plastid ATP / ADP transporter encoded by the 9067 gene have the same function. The amino acid sequence of the plastid ATP / ADP transporter in green algae contains a characteristic sequence (conserved sequence) shown in SEQ ID NO: 12, but the conservation of this sequence was high (>90%) in clade-2 and weaker (<80%) in other clades. In other words, the present inventors discovered a gene encoding a plastid ATP / ADP transporter that is highly homologous to sequence number 12, and found that disrupting this gene can improve fat and oil productivity, thereby completing the present invention.

[0014] That is, the present invention includes the following. (1) A green algae mutant having as a parent strain an algae (living in a single cell or colony) belonging to the Chlorophyta division (Chlorophyta) having a plastid ATP / ADP transporter having an amino acid partial sequence that has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:12, wherein the green algae mutant has reduced function of the plastid ATP / ADP transporter and increased oil content and oil productivity compared to the parent strain. (2) The green alga mutant according to (1), in which a gene encoding the plastid ATP / ADP transporter is disrupted. (3) The green alga mutant according to (1), in which the expression of a gene encoding the plastid ATP / ADP transporter is reduced. (4) The green alga mutant according to (1), in which the translation efficiency of mRNA encoding the plastid ATP / ADP transporter is reduced. (5) The green alga mutant according to any one of (1) to (4), which belongs to the Trebouxiophyceae. (6) The green alga mutant according to (5), which belongs to the genus Parachlorella. (7) A method for producing fats and oils, comprising a step of culturing the green algae mutant according to any one of (1) to (6). Effect of the Invention

[0015] According to the present invention, it is possible to produce a green algae mutant having an increased intracellular oil content and oil productivity. Furthermore, by culturing the green algae mutant of the present invention, it is possible to reduce the production costs of oils to be used for biofuels, etc. [Brief description of the drawings]

[0016] [Figure 1] This shows the patterns of nuclear division and cell division of Treboxia algae. The black circle represents the nucleus, and the one or two lines surrounding the cell represent the cell wall. The inner lines represent newly synthesized cell walls. The bottom of the figure shows the light-dark cycle. During the light period (light colored band), cells swell and cell division occurs. New cell walls (thin lines) are formed around the newly formed cells, but the old cells (thick lines) remain. Multiple cells surrounded by old cell walls are called endospores. During the dark period (black band), the old cell walls dissolve and endospores are released. [Diagram 2]The figure shows the change in cell size during light and dark culture. The preculture of NIES-2152 strain was started at a very low OD750 (OD750 < 0.01), cultured in light and dark (L / D = 16 / 8) for several days, and transferred to the main culture before OD750 exceeded 0.5. Five to seven days after the start of the main culture, the shape of the cells was observed under an optical microscope and photographed. Dark or Light xh in the figure means that sampling was performed x hours after the shift to the dark or light phase. The horizontal axis is the area (μm2) measured on the micrograph, and the vertical axis is the frequency of appearance. The dashed line indicates 15 μm2. As the dark phase progresses, the proportion of cells smaller than 15 μm2 increases, and even after the shift to the light phase, the peak of cells of that size is observed up to 2 hours after the shift to the light phase. Four hours after the start of the light phase, the number of cells smaller than 15 μm2 was significantly reduced. [Diagram 3] The expression construct used for transformation of the NIES-2152 strain is shown. ble is the zeocin (and bleomycin) resistance gene. HSP83_P and HSP83_T represent the promoter and terminator of the HSP83 gene. [Figure 4] The pulse conditions for electroporation are shown below. In electroporation using a NEPA electroporator, a perforation pulse (Pp) that opens holes in the cell membrane is combined with a transport pulse (Tp) that introduces DNA or proteins into the opened holes to efficiently transport the target substance into the cell. Therefore, the magnitude of the electric field (V / cm) of the perforation pulse, the pulse width (ms), and the magnitude of the electric field (V / cm) of the transport pulse were changed to see the effect on the transformation efficiency (number of colonies that appeared). In this experiment, the number of perforation pulses was fixed at 1 (with some exceptions), and the number of transport pulses was fixed at 5. A cuvette with a distance between the electrodes of 2 mm was also used. [Diagram 5] The positions of the c2 motif present in the protein encoded by the 9934 gene and the target sequence used for genome editing are shown. [Figure 6]Growth and oil production of the NIES-2152 strain, PK4 strain, and the 9934 gene disruptant (CR24, CR26). A. Vertical axis: biomass dry weight per culture volume (g / L). B. Vertical axis: oil content (oil weight per dry weight; g-oil / g-DW). The main component of oil is TAG (triacylglycerols). C. Vertical axis: oil production per culture volume (g-oil / L). The UP / 5 medium was used, and the strains were cultured at 25°C under continuous light (75 μmol photons m-2 s-1) with air containing 1% (v / v) CO2 bubbled in. [Figure 7] The motifs present in the protein encoded by the 9067 gene and the location of the target sequence used for genome editing are shown. [Figure 8] Growth and oil production of the NIES-2152 strain, PK4 strain, and the 9067 gene disruptant (CR12, CR97). A. Vertical axis: biomass dry weight per culture volume (g / L). B. Vertical axis: oil content (oil weight per dry weight; g-oil / g-DW). The main component of oil is TAG (triacylglycerols). C. Vertical axis: oil production per culture volume (g-oil / L). The UP / 5 medium was used, and the strains were cultured at 25°C under continuous light (75 μmol photons m-2 s-1) with air containing 1% (v / v) CO2 bubbled in. [Figure 9] Growth and oil production of the NIES-2152 strain, PK4 strain, and the 9067 gene disruptant (CR12, CR97). A. Vertical axis: biomass dry weight per culture volume (g / L). B. Vertical axis: oil content (oil weight per dry weight; g-oil / g-DW). The main component of oil is TAG (triacylglycerols). C. Vertical axis: oil production per culture volume (g-oil / L). The culture medium used was UP / 5, and the strains were cultured at 25°C under short-day conditions (10 h / 14 h L / D, 75 μmol photons m-2 s-1) with bubbling air containing 1% (v / v) CO2. [Figure 10] The motifs present in the protein encoded by the 8741 gene and the location of the target sequence used for genome editing are shown. [Figure 11] Growth and oil production of the NIES-2152 strain, PK4 strain, and the 8741 gene disruptant (CR189, CR193). A. Vertical axis: biomass dry weight per culture volume (g / L). B. Vertical axis: oil content (oil weight per dry weight; g-oil / g-DW). The main component of oil is TAG (triacylglycerols). C. Vertical axis: oil production per culture volume (g-oil / L). The UP / 5 medium was used, and the strains were cultured at 25°C under continuous light (75 μmol photons m-2 s-1) with air containing 1% (v / v) CO2 bubbled in. [Figure 12] Phylogenetic tree of 35 plastid ATP / ADP transporters found in 10 types of green algae. Phylogenetic tree estimated by maximum likelihood method. Small letters indicate percentages of bootstrap values. 9067_t, 4343_t, and 7829_t are plastid ATP / ADP transporters present in the NIES-2152 strain and have the amino acid sequences shown in SEQ ID NOs: 8, 10, and 11. The accession numbers of the other plastid ATP / ADP transporters corresponding to the numbers shown in bold font are shown in Table 3. TLC1_ARATH is the UniProtKB / Swiss-Prot entry name for one of the plastid ATP / ADP transporters found in Arabidopsis thaliana. [Figure 13] The amino acid sequences conserved in Clade-2 plastid ATP / ADP transporters are shown. The "Clade-2 motif" line shows the highly conserved amino acid sequences (consensus sequence; SEQ ID NO: 12) in Clade-2 plastid ATP / ADP transporters. X indicates a less conserved amino acid sequence. The following lines show the amino acid sequences of plastid ATP / ADP transporters with the indicated accession numbers. Amino acid residues that match the consensus sequence are shown in grey. The last column after each amino acid sequence shows the number of amino acid residues that mismatch with the consensus sequence. Plastid ATP / ADP transporters belonging to Clade-2 are surrounded by a thick line. In these cases, the number of amino acid residues that mismatch with the consensus sequence was 3 or less. [Figure 14-1] The sequence listing is shown below. [Figure 14-2] This is a continuation of Figure 14-1. [Figure 14-3] This is a continuation of Figure 14-2. [Figure 14-4] This is a continuation of Figure 14-3. [Figure 14-5] This is a continuation of Figure 14-4. [Figure 14-6] Continuation of Figure 14-5. [Figure 14-7] Continuation of Figure 14-6. [Figure 14-8] Continuation of Figure 14-7. [Figure 14-9] Continuation of Figure 14-8. [Figure 14-10] Continuation of Figure 14-9. [Figure 14-11] Continuation of Figure 14-10. [Figure 14-12] Continuation of Figure 14-11. [Figure 14-13] Continuation of Figure 14-12. [Figure 14-14] Continuation of Figure 14-13. [Figure 14-15] Continuation of Figure 14-14. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention will be described in detail below. The present invention relates to a green algae mutant that has increased oil content and oil productivity compared to a parent strain by reducing the function of a plastid ATP / ADP transporter possessed by the green algae. Here, "oil content" refers to the weight of oil per unit dry weight of algae. "Biomass production" refers to the dry weight of algae per unit volume of culture medium or per unit culture area, and an increase in biomass production per unit time is called "biomass productivity." The product of biomass production and oil content is called "oil production," and an increase in oil production per unit time is called "oil productivity."

[0018] A mutant in which the 9067 gene of Parachlorella kessleri NIES-2152 strain was disrupted using Cas9-gRNA had improved oil content and oil productivity compared to the parent strain. As shown in Example 3, there are many reports that the plastid ATP / ADP transporter in plants supplies ATP to plastids differentiated into chloroplasts, leucoplasts, etc., and promotes starch synthesis. On the other hand, there have been no reports in the past that the deficiency of plastid ATP / ADP transporters in plants or green algae improves the oil content stored in the cells. In Nannochloropsis, a true eyespot alga, oil production is induced under nitrogen deficiency conditions, and there is a report that the plastid ATP / ADP transporter is also induced accordingly (Non-Patent Document 11). In the present invention, on the contrary, we have succeeded in improving oil productivity by reducing the function of the plastid ATP / ADP transporter in green algae.

[0019] Many green algae have three or more plastid ATP / ADP transporter genes. Molecular phylogenetic analysis based on the amino acid sequences of these plastid ATP / ADP transporters showed that the plastid ATP / ADP transporters were divided into three clades (Figure 12). Furthermore, the multiple plastid ATP / ADP transporters present in one strain often belong to different clades. In other words, many of the multiple plastid ATP / ADP transporter genes present in one strain are paralogs that arose by gene duplication before the speciation of green algae. Genes that are in a paralogous relationship with each other often encode proteins with different functions. On the other hand, proteins that belong to the same clade are usually thought to have the same function. Since the plastid ATP / ADP transporter gene encoded by the 9067 gene of the NIES-2152 strain belongs to clade-2, it can be inferred that the oil productivity of other green algae can be improved by disrupting the plastid ATP / ADP transporter genes that belong to clade-2 present in these green algae (Figure 12).

[0020] The amino acid sequences of plastid ATP / ADP transporter genes belonging to clade-2 conserve the characteristic sequence shown in SEQ ID NO: 12 (Figure 13). This makes it possible to determine whether a plastid ATP / ADP transporter gene belongs to clade-2 by comparing the sequences without constructing a phylogenetic tree.

[0021] From the above results, we have discovered a gene encoding a plastid ATP / ADP transporter having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NO: 12, and have found that disrupting this gene can improve fat and oil productivity, thereby completing the present invention.

[0022] In the present invention, examples of green algae (algae that belong to the Chlorophyta division and live in a single cell or colony) include green algae that belong to the Trebouxiophyceae class. Examples of green algae that belong to the Trebouxiophyceae class include green algae that belong to the genera Parachlorella, Trebouxia, Chlorella, Botryococcus, Choricystis, Coccomyxa, and Pseudococcomyxa. Examples of green algae other than those belonging to the class Treboxia include green algae belonging to the genera Tetraselmis, Ankistrodesmus, Dunalliella, Neochloris, Chlamydomonas, Scenedesmus, etc. Green algae including the NIES-2152 strain can be selected from the list of preserved strains at the Microbial Culture Collection of the National Institute for Environmental Studies and distributed.

[0023] The green algae mutant of the present invention can be produced by using the above-mentioned green algae as a parent strain and reducing the function of a plastid ATP / ADP transporter having an amino acid partial sequence that is highly homologous to the amino acid sequence shown in SEQ ID NO:12.

[0024] In the present invention, examples of plastid ATP / ADP transporters include proteins having an amino acid partial sequence that has at least 80%, preferably at least 85%, particularly preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 12 (i.e., the amino acid sequence of the conserved region of the plastid ATP / ADP transporter belonging to clade-2 shown in Figures 12 and 13), and that have the function of a plastid ATP / ADP transporter.

[0025] The amino acid sequence shown in SEQ ID NO:12: AKFSLFKPAEEMVYIX1LDX2X3X4RTX5GKAAX6DVX7GX8QX9GKX 10 X 11 X 12 SX 13 X 14 QQX 15 L In the formula, X may be any amino acid, and examples of each X include the following amino acids: X1: G / T / S, X2: D / E, X3: E / A / D / Q, X4: S / G, X5: K / R, X6: I, X7: V, X8: A / S, X9: S / T / A / V, X 10 : S / Q, X 11 : G / A / I / C, X 12 : G / I / S, X 13 : V / L / M / G / I, X 14 : L / S, X 15 : A / V / G / T, and each X is preferably the first amino acid among the amino acids listed here.

[0026] Further, examples of plastid ATP / ADP transporters include proteins consisting of an amino acid sequence having at least 50%, preferably at least 65%, particularly preferably at least 80%, and most preferably at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:8, and having the function of a plastid ATP / ADP transporter.

[0027] Furthermore, examples of genes encoding plastid ATP / ADP transporters include genes encoding the above-mentioned plastid ATP / ADP transporters, such as genes consisting of a base sequence having at least 50%, preferably at least 65%, particularly preferably at least 80%, and most preferably at least 85%, at least 90%, at least 95%, or 100% sequence identity to the coding region of the mRNA shown in SEQ ID NO:5 (or the base sequence in which u is t) and encoding a protein having the function of a plastid ATP / ADP transporter.

[0028] Here, the "function of the plastid ATP / ADP transporter" refers to the function of the plastid ATP / ADP transporter, which is present in the inner membrane of the plastid of green algae and transports ATP produced in the mitochondria and released into the cytoplasm into the plastid, and at the same time transports ADP from the plastid to the cytoplasm in a counter-transport manner.

[0029] In the present invention, a green algae mutant of the present invention can be obtained by subjecting a green alga having a gene encoding the plastid ATP / ADP transporter described above to a method for reducing the function of the plastid ATP / ADP transporter.

[0030] Specifically, examples of the method for reducing the function of the plastid ATP / ADP transporter according to the present invention include a method for randomly introducing mutations using drugs, radiation, ultraviolet light, etc., and selecting strains having mutations in the plastid ATP / ADP transporter gene by phenotype, etc. Also, examples of the method for selectively disrupting the plastid ATP / ADP transporter gene include gene disruption methods using genome editing.

[0031] Furthermore, methods for decreasing the function of plastid ATP / ADP transporters include, for example, (1) Introducing a mutation into the plastid ATP / ADP transporter gene to disrupt the gene; (2) suppressing the transcription of the plastid ATP / ADP transporter gene and decreasing the expression of the gene; (3) suppressing the translation of the plastid ATP / ADP transporter gene and reducing the translation efficiency of the gene; Methods include:

[0032] (1) Introducing a mutation into the plastid ATP / ADP transporter gene Methods for targeting plastid ATP / ADP transporter genes and introducing mutations include gene knockout methods 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.), which can be used to create mutants lacking the gene.

[0033] (2) A method for suppressing the transcription of a plastid ATP / ADP transporter gene and decreasing the expression of the gene. Methods for suppressing the transcription of a plastid ATP / ADP transporter gene include introducing a mutation into the promoter region of the gene in a target green alga.

[0034] Another method includes introducing a mutation into a gene involved in the positive expression control of the gene, thereby reducing the function of the gene.

[0035] Alternatively, a method can be used in which a mutation is introduced into a gene involved in negative expression regulation of the gene, thereby making the negative expression regulation constantly active.

[0036] (3) A method for suppressing the translation of a plastid ATP / ADP transporter gene and decreasing the translation efficiency of the gene. Methods for suppressing the translation of plastid ATP / ADP transporter genes include the so-called RNA interference method (Cerutti H et al., 2011, Eukaryot Cell, 10, 1164) and the antisense method.

[0037] Furthermore, the present invention includes a method for producing fats and oils containing TAG (triacylglycerol) by mass-cultivating the green algae mutant of the present invention described above. After cultivation, fats and oils containing TAG can be obtained from the culture by, for example, hexane extraction. EXAMPLES

[0038] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[0039] [Example 1] Development of a transformation method applicable to Parachlorella genus green algae By using genome editing technology to introduce a mutation into a specific gene, it is possible to change the traits that the gene carries. Currently, the mainstream genome editing technology is to introduce a complex of Cas9 protein and guide RNA (gRNA) (Cas9-gRNA complex) into the nucleus. In this method, a complex of Cas9 protein, which acts as a pair of scissors to cut the genome, and gRNA, which guides Cas9 protein to the target gene (Cas9-gRNA complex; hereafter, this is referred to as RNP, an abbreviation for ribonucleoprotein), is directly introduced into the cell. Cas9 protein has a nuclear localization sequence (NLS), and the RNP that has been transferred to the nucleus selectively cuts the target sequence in the target gene. In this method, it is necessary to efficiently introduce RNP into the cell, and methods such as electroporation and particle guns are used for small cells such as green algae.

[0040] It was technically difficult to measure the efficiency of RNP introduction into wild-type cells by electroporation. Therefore, we optimized the electroporation conditions by electroporating a DNA fragment that encodes a drug resistance gene and has a molecular weight several times that of RNP, and comparing the frequency of the phenomenon (transformation) in which the introduced DNA is integrated into chromosomal DNA with a certain probability and expresses the trait of drug resistance.

[0041] In many green algae, including Parachlorella, a new cell wall is created inside the original cell wall (mother cell wall) during cell division. After 2 to 3 cell divisions, the mother cell wall dissolves and 4 to 8 cells (endospores) are released. As a result, the cell is surrounded by a double cell wall during most of the cell cycle (Figure 1). In this way, it was predicted that it would be difficult to transfer polymers into the cells of green algae surrounded by a double cell wall using techniques such as electroporation. However, cells immediately after the release of endospores have a relatively thin single cell wall, so it is highly likely that DNA and other substances can be introduced more easily. On the other hand, it has been reported that in light and dark culture with day and night alternations, the dissolution of the mother cell wall occurs during the dark period (Non-Patent Document 5). Therefore, the wild strain was cultured under light and dark conditions of 16 h light and 8 h dark, and the size distribution of cells (μm 2 ) was measured from micrographs. As shown in Figure 2, as the dark period progressed, the size of the nuclei increased by 40 μm. 2 The frequency of cells with sizes of 20 μm or more decreased, and around 8 h of the dark period (0 h of the light period), 2 The frequency of appearance of the following cells was the highest. From this result, it was found that the dissolution of the mother cell wall occurs within 8 hours of the dark period, and small cells appear. Therefore, we decided to perform electroporation using cells 0 to 3 h after the shift to the light period.

[0042] Since the frequency of transformation is very low, it is common to introduce a drug resistance gene into cells and select transformants that have become antibiotic resistant. In this example, we decided to use a gene (ble) that is resistant to an antibiotic called zeocin. In order for the zeocin resistance gene to be expressed in the host and for the host to become drug resistant, it is necessary to place a promoter sequence before the zeocin resistance gene and a terminator sequence after it. In plants, including green algae, there is a mechanism (silencing) that suppresses the expression of the introduced gene (Non-Patent Document 6). Therefore, it is appropriate to use the promoter and terminator of a gene that is highly expressed in the host for this purpose. Therefore, we searched for genes that are highly expressed in wild strains from RNA-sec data (Non-Patent Document 4). Next, we selected genes that could be estimated with high accuracy from the base sequences around the highly expressed genes. In this way, we finally selected the promoter and terminator of the HSP83 gene and used them in transformation experiments. The structure of the artificial gene (expression construct) for expressing the ble gene, constructed using the HSP83 gene promoter and terminator, is shown in Figure 3. The DNA fragment corresponding to the expression construct having this structure (hereafter referred to as bleHH DNA) was amplified by PCR using appropriate primers and used in transformation experiments.

[0043] In electroporation, the magnitude and duration of the electric field optimal for DNA introduction must be optimized according to the type of green algae. The number of pulses and the pulse shape also affect the efficiency of DNA introduction. The NEPA electroporator used in this example combines a perforation pulse (Pp) that opens holes in the cell membrane with a transport pulse (Tp) that introduces DNA or proteins into the opened holes to achieve efficient intracellular transport of the target substance (Figure 4).

[0044] In this example, the wild-type strain was cultured in BG11 medium (https: / / mcc.nies.go.jp / medium / en / bg11.pdf) with a light / dark cycle of (16 h light) / (8 h dark), and the OD 750The cells were cultured until the β-amino acid concentration reached 0.175–2. Within 3 hours of the start of the light phase, the cells were harvested by centrifugation (2,900 × g, 5 min), washed twice with 5 mM MES buffer (pH 5.3), then washed with Max Efficiency Transformation Reagent for Alga (TRA, Thermo Fisher), and cultured at a cell density of 1 × 10 in TRA buffer containing 10 mM glucose. 9 cells mL -1 The cells (1.5–3 × 10 7 After mixing 1 μg of bleHH DNA with 1 μg of bleHH cells, electroporation was performed. The electroporation conditions were fixed at one Pp pulse, and the magnitude and pulse width of the electric field were varied. For Tp, the pulse width was fixed at 50 ms, the number of pulses was fixed at five, and the magnitude of the electric field was varied. Next, according to Non-Patent Document 7, the cells were left to stand on ice for 10 minutes, suspended in BG11 medium containing 55 mM glucose, and cultured in the dark at 25°C for 24 hours, after which 35 μg mL -1 Selection was performed on BG11 agar medium containing Zeocin, and the transformation efficiency was calculated from the number of Zeocin-resistant colonies that appeared 10 days later, and the conditions under which this was maximized were identified (Table 1).

[0045] [Table 1]

[0046] Table 1. Transformation efficiency of the NIES-2152 strain The NIES-2152 strain was cultured under a 16-h light / 8-h dark condition, and electroporation was performed using cells 0-3 h after the shift to the light period on the 3rd or 4th day after the start of culture. The amount of DNA placed in the cuvette per electroporation was 1 μg. Cell / cuvette is the number of cells placed in the cuvette per electroporation. For Pp and Tp, see Figure 4. The number of Tp pulses was 5 in all electroporation experiments.

[0047] As shown in Table 1, when bleHH DNA was not introduced, no zeocin-resistant colonies appeared (Table 1: rows 1, 7, 14, 21, 26, 32, 36, 41). The longer the Pp pulse length, the higher the transformation efficiency (Table 1: rows 8, 9 vs. 10; rows 17 vs. 19; rows 22 vs. 23; rows 24 vs. 25; rows 27 vs. 28; rows 29 vs. 30; rows 37 vs. 39). In other words, the transformation efficiency was highest at 15 ms, the longest Pp pulse length among the conditions used. In experiments in which the Pp electric field was varied, the higher the Pp, the higher the transformation efficiency, with the highest transformation efficiency observed at the highest electric field used, Pp = 2,500 V / cm (Table 1: rows 2 vs. 3; rows 5 vs. 6; rows 9, 11 vs. 12; rows 15 vs. 16; rows 30 vs. 31; rows 42, 43, 44 vs. 45). In most experiments, only one Pp pulse was used. Two Pp pulses did not increase the transformation efficiency (Table 1: rows 2 vs. 6). On the other hand, when the electric field strength of Tp was changed, the transformation efficiency was highest at 250 V / cm, which was 1.4 times that at 100 V / cm (Table 1: row 9 vs. 13; row 22 vs. 24; row 23 vs. 25; row 27 vs. 29; row 28 vs. 30; row 34 vs. 35, row 37 vs. 38, row 39 vs. 40). However, when Tp was increased to 500 V / cm, the efficiency decreased to about half that at 100 V / cm (Table 1: row 18).

[0048] OD of the culture 750 When wild-type cells with a Pp of more than 1 were used (experiments No. 1 and 3), the transformation efficiency was low. From these results, we concluded that the highest transformation efficiency was obtained under the following conditions: Pp = 2,500 V / cm, Pp pulse width 15 ms, Pp number of times 1, Tp = 250 V / cm, Tp pulse width 50 ms, Tp number of times 5. DNA was extracted from the obtained Zeocin-resistant colonies, and PCR was used to check whether the ble gene was inserted into the chromosome. It was confirmed that almost all Zeocin-resistant clones possessed the ble gene.

[0049] [Example 2] Knockout of wild-type Parachlorella genes using genome editing As mentioned above, the PK4 strain is a mutant strain with improved fat and oil content derived from a wild-type Parachlorella strain. As a result of genome analysis of this strain, it was revealed that the PK4 strain has mutations in at least three genes (8741, 9934, 9067) (Non-Patent Document 4), and it is highly likely that one of these mutations is the cause of the improved fat and oil content. The genomic DNA sequences from the initiation codon to the termination codon of the 8741, 9067, and 9934 gene sequences of the wild-type NIES-2152 strain are shown in SEQ ID NOs: 1 to 3, respectively. The mRNA sequences corresponding to the CDSs transcribed from these sequences are shown in SEQ ID NOs: 4 to 6, respectively, and the amino acid sequences of the proteins translated from the CDSs are shown in SEQ ID NOs: 7 to 9, respectively. These genes were disrupted using genome editing technology as described below, and the growth and fat and oil content of each gene-disrupted strain were examined.

[0050] First, the CRISPR / Cas9 target sequences (20 bases) in each of the gene sequences of SEQ ID NOs: 1 to 3 were searched for using CRISPRdirect (http: / / crispr.dbcls.jp / ) software. Among the hit target sequences, three sequences with a Tm value of 75-80°C and a GC content of 60-65% were selected as CRISPR / Cas9 target sequences. Next, CRISPR RNA (crRNA) having a sequence complementary to the selected target sequence was chemically synthesized, and 200 pmol of crRNA was mixed with 200 pmol of trans-activating RNA (tracrRNA), heated at 95°C for 5 minutes, and then slowly cooled to room temperature to form a hybrid. The Cas9 protein solution (Nippon Gene) was concentrated 10-fold using an Amicon Ultra centrifugal ultrafiltration filter (100 kD, Millipore), and the salt concentration in the Cas9 protein stock solution was reduced by adding 9-fold of RNase-free water. The above crRNA-tracrRNA hybrid was then mixed with 10 μg of Cas9 protein and allowed to stand at room temperature for 30 minutes or more to form RNPs.

[0051] The NIES-2152 strain carrying RNP was grown in BG11 medium under a 16-h light / 8-h dark cycle at OD 750 The preculture was performed until the OD was approximately 1.0. 750 The algae were inoculated into 50 mL of fresh BG11 medium so that the chromatin density was 0.005, and the medium was further cultured under a 16-h light / 8-h dark condition for one week. After one week, within 3 hours of switching to the light period, the cells were harvested by centrifugation (2,900 × g, 5 min), washed twice with 5 mM MES buffer (pH 5.3), then washed with Max Efficiency Transformation Reagent for Alga (TRA, Thermo Fisher), and cultured at a cell density of 1 × 10 in TRA buffer containing 10 mM glucose. 9 cells mL -1 The mixture was suspended in a 50 ml solution.

[0052] 30 μL of cell suspension (3 × 10 7 cells), the RNP, and 1 μg of Zeocin resistance gene expression cassette (bleHH) DNA synthesized by PCR amplification were mixed, and RNP was introduced by electroporation under the same conditions as DNA introduction for transformation (Pp = 2,500 V / cm, Pp pulse width 15 ms, Pp number of times 1, Tp = 250 V / cm, Tp pulse width 50 ms, Tp number of times 5). After electroporation, the cells were left on ice for 10 minutes, suspended in BG11 medium containing 55 mM glucose, and cultured with shaking at 37°C for 6 hours in the dark. They were then transferred to 25°C. After 24 hours of culture, 35 μg mL -1 Selection was carried out on BG11 agar medium containing Zeocin.

[0053] Colonies that were resistant to Zeocin were isolated, and each clone was analyzed by PCR using primers specific to each gene region to determine whether or not bleHH DNA had been inserted into the CRISPR / Cas9 target gene. Several clones with PCR fragment sizes in the target region that were different from those in the original strain were selected, and the base sequence of the target region was analyzed to confirm whether or not the gene had been disrupted.

[0054] Below, we explain the genome editing results for three genes, 9934, 9067, and 8741, and the properties of the genome-edited strains for each gene.

[0055] Disruption of the 9934 gene The protein encoded by the 9934 gene has a C2 domain at the N-terminus that is conserved in protein kinase C and other proteins. This domain is thought to bind to the cell membrane and be involved in signal transduction. The loss of this gene function may have altered the expression of enzymes related to oil production, resulting in improved oil productivity in PK4. Therefore, three sequences in the C2 domain region were selected as target sequences for disruption of the 9934 gene, and a crRNA sequence that acts on these sequences was designed (Table 2). Next, a complex of gRNA and Cas9 protein (RNP) containing this crRNA sequence was introduced into NIES-2152 cells under the optimized electroporation conditions described above.

[0056] [Table 2]

[0057] Table 2. crRNA sequences used for gene disruption and their characteristics 〇 indicates that the gene disruption strain was obtained using the crRNA. - indicates that the gene disruption strain was not obtained.

[0058] Only when gene editing was attempted using the third sequence (GC 65%, Tm 79.5) of the three crRNA sequences for 9934 gene disruption shown in Table 2, two gene disruption strains (CR24, CR26) were obtained. In these disruption strains, bleHH DNA was inserted at the mutation site (Figure 5).

[0059] Therefore, we investigated the increase in dry weight per unit volume of culture medium (growth) and the oil content, i.e., the oil content per dry weight, of the NIES-2152 strain, the PK4 strain, and the 9934 gene-disrupted strains (CR24, CR26) (Fig. 6). Growth was slightly lower in the PK4 strain. On the other hand, the oil content of the PK4 strain was higher than that of the wild-type strain. The characteristics of the 9934 gene-disrupted strains (CR24, CR26) were similar to those of the wild-type strain, but different from those of the PK4 strain (Fig. 6). From these results, we concluded that the 9934 gene mutation was not the cause of the improved oil content.

[0060] Disruption of the 9067 gene Next, the 9067 gene was disrupted. From the amino acid sequence of the protein encoded by the 9067 gene, it was predicted to be a plastidic ATP / ADP transporter gene. The conserved region of this protein is divided into the N-terminal side and the C-terminal side, except for the central part (Figure 7). As the target sequence, one site in the conserved region on the N-terminal side and two sites in the central part with low conservation were selected, and gene disruption was attempted. Regardless of the target used, gene disruption strains were obtained by inserting the bleHH fragment. Therefore, the growth under continuous light, the oil content, and the oil production amount obtained by the product of growth and the oil content were examined for the CR12 strain, in which an insertion was confirmed in sequence 1 shown in Table 2 and Figure 7, and the CR97 strain, 2152 strain, and PK4 strain, in which an insertion was confirmed in sequence 3 shown in Table 2 and Figure 7 (Figure 8). The growth of the 9067 gene disruption strains (CR12, CR97) was higher than that of the PK4 strain and was equivalent to that of the wild type strain. In addition, the lipid content of the 9067 gene disruptant (CR12, CR97) was significantly higher than that of the wild type and was comparable to that of the PK4 strain. This result suggested that the improved lipid content of the PK4 strain was due to the 9067 gene mutation. In addition, the lipid production on the 11th day of culture was approximately 10% higher than that of the wild type and approximately 20% higher than that of the PK4 strain.

[0061] Next, culture was performed under day and night conditions. Growth and fat content of 2152, PK4, and 9067 gene disruptant strains (CR12, CR97) under short-day conditions (10 h / 14 h light / dark (L / D)) were examined (Fig. 9). Growth of the 9067 gene disruptant strain was almost the same as that of the wild-type strain, as was the case when cultured under continuous light. The fat content was also significantly higher than that of the wild-type strain, and fat productivity on the 11th day of culture was about 40% higher than that of the wild-type strain and the PK strain. Although not shown, similar results were obtained when cultured under 12 h / 12 ​​h L / D or 14 h / 10 h L / D conditions (data not shown). In other words, it was revealed that the difference in fat content between the 9067 gene-deficient strain and the wild-type strain was higher when cultured under day and night conditions than when cultured under continuous light.

[0062] Disruption of the 8741 gene The 8741 gene is predicted to be Endo-1,4-β-mannanase based on the amino acid sequence it encodes, and a domain conserved in cellulase is present from the center of the gene toward the C-terminus (Figure 10). Three locations in this region were selected as targets for gene disruption, and genome editing was attempted. Only when gene editing was attempted for the second sequence (GC 60%, Tm 75.5) shown in Table 2 and Figure 10, strains (CR189, CR193) in which the bleHH fragment was inserted into the target region were obtained.

[0063] Therefore, we investigated the growth and TAG amount per dry weight of the 2152 strain, PK4 strain, and 8741 gene-disrupted strains (CR189, CR193) (Figure 11). The growth of the 8741 gene-disrupted strain was equivalent to that of the PK4 strain and slower than the wild-type strain. Meanwhile, the oil content of the 8741 gene-disrupted strain was almost the same as that of the wild-type strain. From these results, we concluded that the 8741 gene mutation in the PK4 strain is not the cause of the improved oil content.

[0064] From these results, we concluded that of the three gene mutations in the PK4 strain, the 9067 gene mutation is responsible for the improved oil content. On the other hand, when cultured in UP / 5 medium, the PK4 strain grew less well than the wild-type strain, and we speculated that this was due to the 8741 gene mutation. In other words, we concluded that the two traits of the PK4 strain, the improved oil content and the reduced growth, are brought about by mutations in the 9067 gene and the 8741 gene, respectively.

[0065] Example 3 The plastid ATP / ADP transporter is present in the inner membrane of plastids of plants and algae, and plays a role in transporting ATP produced in mitochondria and exported to the cytoplasm into the plastid, while ADP is counter-transported from the plastid to the cytoplasm. Proteins with a structure similar to the plastid ATP / ADP transporter are also present in parasitic bacteria such as Chlamydia and Rickettsia, which can only grow inside host cells, and fungi such as Microsporidia.

[0066] The model plant Arabidopsis thaliana has two plastid ATP / ADP transporter genes. Mutants lacking these two genes are highly resistant to ATP / ADP transport under low light (125 μmol photons m -2 s -1 ), and grew normally under long-day conditions (16 h / 8 h L / D), but grew slowly under short-day conditions (10 h / 14 h L / D) (Non-Patent Document 8). Under the latter conditions, ATP production by photosynthesis was reduced, and as a result, starch required for nighttime activity could not be accumulated in sufficient amounts, which is thought to be one of the reasons for the growth inhibition. In non-photosynthetic organs, the role of the plastid ATP / ADP transporter is probably more important, and knocking down this gene in potato reduced the starch content in tubers by half (Non-Patent Document 9). This potato knockdown strain induced a defense response against fungal infection more quickly than the wild type by using cell wall components (elicitors) possessed by pathogenic fungi and pathogenic bacteria. The mechanism is unknown, but it is possible that a signal transduction mechanism triggered by a decrease in ATP concentration in the plastid is involved (Non-Patent Document 10).

[0067] The genome of the NIES-2152 strain contains two plastid ATP / ADP transporter genes in addition to the gene encoding the plastid ATP / ADP transporter shown in SEQ ID NO: 2, and the amino acid sequences of the proteins encoded by these genes are shown in SEQ ID NOs: 10 and 11. To investigate whether multiple plastid ATP / ADP transporter genes exist in other green algae, the amino acid sequence (TLC1_ARATH) of the plastid ATP / ADP transporter encoded by the AATP1 gene of Arabidopsis thaliana was used as a query to search nine types of green algae proteomes (Table 3) registered in the Uniprot Proteomes database (https: / / www.uniprot.org / proteomes / ) using the blastp search engine.

[0068] [Table 3]

[0069] Table 3. Accession numbers of plastid ATP / ADP transporter proteins present in various green algae, scientific name of the green algae, gene accession number, number of amino acids in the protein, and number indicating position in the phylogenetic tree in Figure 12 Note 1 in the table: This protein has an exceptional sequence; the N-terminus has a sequence similar to the plastid ATP / ADP transporter, but the C-terminus (from amino acid residue 563 onwards) has a sequence with the motif of glutathione S-transferase. Note 2: The full-length sequence has not been obtained.

[0070] As a result, as shown in Table 3 and Figure 12, the proteome of each green alga contained three or more types of amino acid sequences of plastid ATP / ADP transporters, which is more than that of Arabidopsis thaliana. These multiple plastid ATP / ADP transporters may have different functional roles. Therefore, their molecular phylogenetic relationships were examined using the maximum likelihood method (Figure 12). In this case, three proteins (A0A0D2NFA4, A0A0D2MTH2, A0A7J7Q1S0) that did not have the full-length plastid ATP / ADP transporter sequence and the sequence of E1Z4I7, a fusion protein with glutathione S-transferase, were excluded from the analysis. As a result, the plastid ATP / ADP transporters were divided into three clades, and the plastid ATP / ADP transporter encoded by the 9067 gene belonged to clade 2. Eight of the 10 types of green algae, including the NIES-2152 strain, had plastid ATP / ADP transporters belonging to clade-2. Furthermore, only the base sequence of the N-terminal coding portion of the gene encoding A0A7J7Q1S0 in Scenedesmus sp. strain NREL 46B-D3 has been determined, but when a phylogenetic tree was created using only the sequence of this N-terminal portion, it was found that A0A7J7Q1S0 belongs to clade-2. In other words, all green algae except Chlorella sorokiniana strain UTEX 1602 have plastid ATP / ADP transporters belonging to clade-2, and they are considered to have a common function. From this, it is expected that the oil productivity of other green algae will be improved by disrupting the gene of the clade-2 plastid ATP / ADP transporter of those green algae.

[0071] Therefore, we extracted a total of 39 sequences (including 8 sequences belonging to clade-2 shown in Figure 12) similar to the amino acid sequence of the plastid ATP / ADP transporter encoded by the 9067 gene from among the green algae proteins registered in Uniprot, and compared them by creating sequence alignments, and found that the amino acid sequence shown in SEQ ID NO: 12 is well conserved in the plastid ATP / ADP transporter belonging to clade-2. This sequence is also partially conserved in clade-1 and clade-3, but the number of mismatches is 10 or more, which is clearly different from the clade-2 sequence, which has 3 or less mismatches (Figure 13).

Claims

1. An algal mutant having as a parent strain an alga belonging to the Chlorophyta division having a plastid ATP / ADP transporter having an amino acid partial sequence that has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:12, wherein the algal mutant has reduced function of the plastid ATP / ADP transporter and increased oil content and oil productivity compared to the parent strain.

2. The algal mutant of claim 1 , wherein the gene encoding the plastid ATP / ADP transporter is disrupted.

3. The algal mutant of claim 1 , wherein the expression of a gene encoding the plastid ATP / ADP transporter is reduced.

4. The algal mutant of claim 1 , wherein the translation efficiency of the mRNA encoding the plastid ATP / ADP transporter is reduced.

5. The algae mutant according to any one of claims 1 to 4, which belongs to the Trebouxiophyceae.

6. The algae mutant of claim 5, which belongs to the genus Parachlorella.

7. A method for producing fats and oils, comprising the step of culturing the algal mutant of claim 1.

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