JP6627336B2 - Eukaryotic microalgal lipase mutants - Google Patents

Description

  The present invention provides a mutant eukaryotic microalgae capable of increasing triacylglycerol accumulation as compared with a wild-type or parent strain as a result of a decrease in triacylglycerol lipase activity and growing under conditions with a short day length, and uses thereof. About.

  Biodiesel fuel produced from fatty acids produced by unicellular photosynthetic organisms (hereinafter referred to as "microalgae") or compounds such as triacylglycerol (hereinafter referred to as "TAG") that releases fatty acids by hydrolysis Research on the production of industrial and food products, including, is being conducted worldwide. However, at present, TAG production costs are high, and it is difficult to produce biodiesel fuel and the like on a commercial basis (Non-Patent Document 1). For this reason, further technological development for reducing the production cost of biodiesel fuel and the like has been continued, and one of them is research on improving TAG productivity of microalgae. Much of the work to improve TAG productivity has focused on enhancing TAG biosynthesis. On the other hand, it is considered that the amount of accumulated TAG can be increased by suppressing the decomposition of the synthesized TAG.

  In fact, in plants, the amount of TAG accumulated has been successfully increased as shown below by reducing lipase activity. Rapeseed is an important source of vegetable oil along with palm and soy. Although oil and fat mainly composed of TAG accumulate in rapeseed seeds, the TAG content decreases by about 10% when the seeds mature. This reduction was thought to be due to lipase specific to TAG, and attempts were made to increase the oil content of rapeseed by suppressing this lipase activity. It is known that Arabidopsis thaliana has a TAG-specific lipase called SUGAR-DEPENDENT1 (SDP1), but rape also has a plurality of genes highly homologous to SDP1. In order to suppress the expression from these genes, an RNAi sequence targeting a nucleotide sequence well conserved in rape SDP1 was designed. Then, when this RNAi sequence was cloned downstream of a promoter specifically expressed in seeds and then introduced into rapeseed, the TAG content of the seeds was significantly increased in the introduced strain (Non-Patent Document 2). By a similar method, the TAG content of Jatropha seeds has also been successfully increased (Non-Patent Document 3).

  On the other hand, in eukaryotic microalgae, there is only one report that TAG accumulation was increased by decreasing lipase activity. Thalassiosira pseudonana, a type of diatom, accumulates TAG under nitrogen-deficient conditions, and among genes whose transcription is reduced under these culture conditions, one that has the possibility of encoding lipase was searched. Found a gene named. Thus, when an antisense RNA that suppresses the expression of Thaps3_264297 was expressed, the amount of accumulated TAG increased about three-fold (Non-Patent Document 4). Thaps3_264297 protein was expressed in Escherichia coli and biochemical analysis revealed that Thaps3_264297 protein had TAG lipase activity, phospholipase activity, and lysophosphatidic acid acyltransferase activity. The similarity between the Thaps3_264297 protein and the aforementioned SDP1 is limited.

  The unicellular green alga “Pseudochoricystis ellipsoidea Obi strain” (hereinafter sometimes referred to as “Obi strain”) is separated and analyzed by Sato et al. (Non-Patent Document 5; Patent Document 1). Was. The genus and species name of this strain are not named according to the International Algae, Fungi, and Plant Naming Convention, but are tentative names. Subsequent phylogenetic analysis using the gene indicates that they are closely related to the genera Coccomyxa and Pseudococcomyxa. This strain, like other microalgae, accumulates TAG in cells when the nitrogen source in the medium is depleted (Non-Patent Document 6). Since the Obi strain can grow even at a medium pH of 3.5 or less (Patent Document 2), large-scale cultivation outdoors can be performed while minimizing the growth of protozoa that prey on microalgae. The present inventors have been working on breeding of Obi strains and improvement of large-scale culture technology.

Patent No. 4748154 JP 2014-117202 A

Chisti Y. (2013) Constraints to commercialization of algal fuels. J. Biotechnol. 167: 201-214. Kelly AA, Shaw E, Powers SJ, Kurup S, Eastmond PJ. (2013) Suppression of the SUGAR-DEPENDENT1 triacylglycerol lipase family during seed development enhances oil yield in oilseed rape (Brassica napus L.). Plant Biotechnol J. 11: 355 -361. Kim MJ, Yang SW, Mao HZ, Veena SP, Yin JL, Chua NH. (2014) Gene silencing of Sugar-dependent 1 (JcSDP1), encoding a patatin-domain triacylglycerol lipase, enhances seed oil accumulation in Jatropha curcas.Biotechnol Biofuels .7: 36.doi: 10.1186 / 1754-6834-7-36. Trentacoste EM, Shrestha RP, Smith SR, Gle C, Hartmann AC, Hildebrand M, Gerwick WH. (2013) Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth.Proc Natl Acad Sci U S A. 110: 19748-19753. Satoh A, Kato M, Yamato T, Ikegami Y, Sekiguchi H, Kurano, N, Miyachi S. (2010) Characterization of the lipid accumulation in a new microalgal species, Pseudochoricystis ellipsoidea (Trebouxiophyceae). 2010, J. Jap. Inst. Energy 89, 909-913. Ito T, Tanaka M, Shinkawa H, Nakada T, Ano Y, Kurano N, Soga T, Tomita M. (2013) Metabolic and morphological changes of an oil accumulating trebouxiophycean alga in nitrogen-deficient conditions.Metabolomics. 9: 178-187 .

  It is desirable to improve eukaryotic microbial growth and TAG productivity and reduce TAG production costs. An object of the present invention is to separate eukaryotic microalgae capable of increasing the amount of TAG accumulated in cells and proliferating even under a short day length, and to provide a TAG production method using the microalgae.

  As a result of intensive studies to solve the above problems, the Obi strain in which the gene encoding a protein homologous to Arabidopsis TAG lipase Sugar dependent 1 (AtSDP1) (hereinafter referred to as `` SDP1 protein '') has been mutated In the mutant of the present invention, it was found that the amount of TAG accumulated in the cell was increased, and it was possible to proliferate under conditions with a short day length, thereby completing the present invention.

That is, the present invention includes the following.
(1) A mutant eukaryotic microalgae in which the TAG lipase activity of the SDP1 protein is reduced, and the amount of intracellular TAG accumulation is increased as compared to the parent strain, and / or growth under short-day culture conditions The eukaryotic microalgal variant, wherein the SDP1 protein is a protein having an amino acid sequence having at least 50% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 and having TAG lipase activity .
(2) The eukaryotic microalgal variant according to (1), wherein the gene encoding the SDP1 protein is disrupted.
(3) The eukaryotic microalgal mutant according to (1), wherein the expression of the gene encoding the SDP1 protein is reduced.
(4) The eukaryotic microalgal variant according to (1), wherein the translation efficiency of the gene encoding the SDP1 protein is reduced.
(5) The eukaryotic microalgal variant according to any one of (1) to (4), which belongs to the phylum Algae.
(6) The eukaryotic microalgal variant according to (5), which belongs to the treboxia algae web.
(7) The eukaryotic microalgal variant according to (6), which belongs to the genus Coccomyxa or the genus Pseudococcomyxa.
(8) A TAG production method, comprising 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 TAG in a cell is improved or the growth in a culture condition with a short day length is improved. Further, by culturing the eukaryotic microalgae mutant according to the present invention, it is possible to reduce the production cost of TAG used for biofuels and the like.

It is a graph which shows a change in dry weight (A) and TAG content (B) when Obi strain and a mutant derived from Obi strain are cultured in the dark. The mutants derived from Obi strains and Obi strain and photosynthetic conditions cultured OD ₇₅₀ when (A) is a graph showing changes in the TAG content (B). It is a graph which shows the growth of the Obi strain and the mutant derived from the Obi strain. Cultivating a mutant derived Obi strains and Obi strain under continuous light (A) or light conditions light period 8 hours and 16 hours darkness (B), to measure its growth at OD _750. Sampling was performed at the time indicated by the arrow in the figure.

  The present invention, by reducing the TAG lipase activity carried by SDP1 protein having homology to Sugar dependent 1 (AtSDP1) is a TAG lipase that acts at the time of germination of Arabidopsis seeds, compared to the parent strain (or wild strain), Eukaryotic microspheres with increased intracellular TAG accumulation and / or improved growth under short-day culture conditions (i.e., culture conditions with short photoperiods (e.g., 8 hours light and 16 hours dark)) Algal variants.

  One of the most important issues for reducing the production cost of biofuels and the like using microalgae-derived TAG as a raw material is to significantly improve the microalgae TAG productivity. The present inventors have proposed Pseudochoricystis ellipsoidea (P. ellipsoidea; P. ellipsoidea) (tentative name) belonging to the Chlorophyceae / Chlorophyta (hereinafter referred to as "green algae") (provisional name) Obi strain [Accession number FERM BP -10484; Patent Document 1 (referred to as "Pseudochoricystis ellipsoidea Sekiguchi et Kurano gen. Et sp. Nov.) MBIC11204 strain" in the patent). Deletion of the SDP1 protein consisting of the amino acid sequence represented by SEQ ID NO: 3 (DNA nucleotide sequence: SEQ ID NO: 1, mRNA nucleotide sequence: SEQ ID NO: 2) increases the amount of TAG accumulated in the cells of this green algae, The present inventors have found that growth under short conditions is improved, and have completed the present invention.

  The SDP1 protein is a TAG lipase widely present in plants, and is known to be involved in the degradation and translocation of stored TAG, especially during oil seed germination. Specifically, the TAG lipase activity refers to an enzyme activity that hydrolyzes one ester bond in triacylglycerol in which three fatty acids are ester-bonded to glycerol to produce diacylglycerol and a fatty acid. I do. Improving the intracellular TAG content of eukaryotic microalgae by reducing the TAG lipase activity carried by SDP1 using artificial genetic manipulation, and culturing a TAG lipase activity-reduced strain, It is possible to significantly reduce the production cost of TAGs to be provided.

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

Examples of the green algae include green algae belonging to the treboxia algae web. Examples of the green algae belonging to the treboxia algae network include, for example, the genus Treboxia (Trebouxia), the genus Chlorella (Chlorella), the genus Botryococcus (Botryococcus), the genus Corycystis (Choricystis), the genus Coccomyxa, the genus Pseudocoma (Pseudococ) Green algae belonging to Specific strains belonging to the treboxia algae network include P. ellipsoidea Obi strain (Accession No. FERM BP-10484; Patent Document 1) and its mutant P. ellipsoidea 5P strain ( Accession No. FERM BP-22179 ; No. 102715; hereinafter, sometimes referred to as “5P strain”). In addition, the P. ellipsoidea Obi strain and the P. ellipsoidea 5P strain share the same gene encoding the SDP1 protein (genomic DNA base sequence: SEQ ID NO: 1, mRNA base sequence: SEQ ID NO: 2, amino acid sequence: SEQ ID NO: 3). Have. Examples of green algae other than the green algae belonging to the treboxia algae network include, for example, the genus Tetraselmis, the genus Ankistrodesmus, the genus Dunalliella, the genus Neochloris, the genus Chlamydomonas, and the Ikedamo (= Scenedesmus). Green algae belonging to the genus and the like are included.

  Further, examples of diatoms include eukaryotic microalgae belonging to the genus Fistulifera, the genus Pheodactylum, the genus Thalassiosira, the genus Cyclotella, the genus Cylindrothica, the genus Skelletonema, and the like. be able to. Furthermore, as the euphoria alga, there may be mentioned the genus Nannochloropsis.

  The eukaryotic microalgae mutant according to the present invention is a eukaryotic microalgae mutant obtained by subjecting the above eukaryotic microalgae to a parent strain and subjecting it to a method for reducing TAG lipase activity.

  In the present invention, the SDP1 protein has at least 50%, preferably at least 50%, the amino acid sequence shown in SEQ ID NO: 4 (that is, a highly conserved amino acid sequence corresponding to the amino acid sequence at positions 172 to 625 in SEQ ID NO: 3). Proteins having an amino acid sequence with 65%, particularly preferably at least 80%, most preferably at least 85%, at least 90%, at least 95%, 100% sequence identity and having TAG lipase activity Can be

  In addition, the SDP1 protein has at least 50%, preferably at least 65%, particularly preferably at least 80%, most preferably at least 85%, at least 90%, at least 95%, the amino acid sequence shown in SEQ ID NO: 3. A protein consisting of an amino acid sequence having 100% sequence identity and having TAG lipase activity is exemplified.

  The SDP1 gene has at least 50%, preferably at least 58%, particularly preferably at least 65%, at least 80%, and most preferably the coding region of the mRNA represented by the nucleotide sequence from nucleotides 131 to 2419 in SEQ ID NO: 2. Preferably, a gene encoding an mRNA having at least 85%, at least 90%, at least 95%, 100% sequence identity and encoding a protein having TAG lipase activity, or 1 to 1 with respect to the mRNA base sequence A base sequence in which a partial base sequence of several places (for example, 10 places or less, preferably 5 places or less, more preferably 3 places or less, and still more preferably 2 places or less) is deleted, substituted, added or inserted. And a gene encoding a protein having a TAG lipase activity.

  The SDP1 gene may be, for example, at least 50%, preferably at least 65%, particularly preferably at least 80%, most preferably at least 85%, at least 90%, at least 95% the amino acid sequence shown in SEQ ID NO: 3. A gene consisting of an amino acid sequence having 100% sequence identity and encoding a protein having TAG lipase activity, or one to several (for example, 10 or less, preferably 5 Or less, more preferably 3 or less, and even more preferably 2 or less) amino acid subsequences comprising a deleted, substituted, added or inserted amino acid sequence and encoding a protein having TAG lipase activity Genes.

  In many eukaryotic microalgae, a plurality of SDP1 genes, such as alleles and synonyms, may be present. In the present invention, at least one or a plurality of SDP1 genes are meant.

  In the present invention, the eukaryotic microalgae according to the present invention is subjected to a method for reducing the TAG lipase activity carried by the SDP1 protein encoded by the SDP1 gene for eukaryotic microalgae having the SDP1 gene described above. Algal variants can be obtained.

  Specifically, a mutant eukaryotic microalgae having a reduced TAG lipase activity carried by the SDP1 protein according to the present invention can be produced according to the procedure described in Examples. That is, after applying a mutagen to the eukaryotic microalgae of the parent strain, screening mutants having an increased TAG content and confirming that a mutation has occurred in the SDP1 gene in the obtained mutant. Can produce. As a result, mutation involving base substitution, deletion, insertion and / or addition can be introduced into the DNA of the SDP1 gene region.

Other methods for reducing the TAG lipase activity carried by the SDP1 protein include, for example,
(1) introducing a mutation targeting the SDP1 gene and disrupting the gene;
(2) suppresses transcription of the SDP1 gene and reduces the expression of the gene;
(3) suppress the translation of the SDP1 gene, reduce the translation efficiency of the gene;
Method.

(1) Method for introducing a mutation targeting the SDP1 gene
As a method of introducing a mutation targeting the SDP1 gene, when the nucleotide sequence of the gene is known, ZFN, TALEN or a gene knockout method called CRISPR / Cas (Gaj T, Gersbach CA, Barbas CF 3rd. (2013 Using ZFN, TALEN, and CRISPR / Cas-based methods for genome engineering. Trends Biotechnol. 31: 397-405.), A mutant deficient in that gene can be created.

  When the nucleotide sequence of the gene is not known, the nucleotide sequence of the gene can be determined by the following method. Since eukaryotic microalgae and SDP1 protein present in plants have a very well-conserved amino acid sequence, the genome of the target eukaryotic microalgae can be analyzed using PCR primers designed based on that amino acid sequence. A PCR amplification reaction is performed using DNA as a template, and the amplified DNA fragment is cloned into Escherichia coli, and then the nucleotide sequence of the DNA fragment is determined. According to this method, the partial nucleotide sequence of the gene can be known, and the gene knockout method can be applied.

  One organism may contain more than one SDP1 gene, and the PCR-amplified DNA fragment should not be directly subjected to a sequencing reaction. If it is desired to further extend the SDP1 gene partial sequence obtained in this way, the inverse PCR method (Huang SH. (1994) Inverse polymerase chain reaction.An efficient approach to cloning cDNA ends.Mol Biotechnol. 12: 15-22. ) May be used.

  In recent years, the so-called next-generation sequencing technology has been developed, and it has become very easy to determine the entire nucleotide sequence of the genome. Therefore, the entire genome sequence of the target eukaryotic microalgae is determined first, and the gene sequence encoding the SDP1 protein may be extracted from the sequence.

(2) A method for suppressing transcription of the SDP1 gene and reducing the expression of the gene
Examples of a method for suppressing the transcription of the SDP1 gene include a method of introducing a mutation into the promoter region of the target eukaryotic microalgae.

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

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

(3) A method for suppressing the translation of the SDP1 gene and reducing the translation efficiency of the gene
As a method for suppressing the translation of the SDP1 gene, a so-called RNA interference method (Cerutti H et al., 2011, Eukaryot Cell, 10, 1164) can be mentioned.

  The amount of TAG accumulation of the eukaryotic microalgae mutant obtained as described above and the growth under culture conditions with a short day length were measured by the method described in Example 1 and the like, and the amount of TAG accumulation in cells was measured. If the increase or the improvement in the growth under culture conditions with a short day length can be confirmed, the production of the eukaryotic microalgal mutant having reduced TAG lipase activity carried by the SDP1 protein according to the present invention is completed.

As an example of the eukaryotic microalgae mutant according to the present invention, a TAG lipase mutant Obi-LOD68 strain derived from the Obi strain shown in the Examples is an independent administration on August 27, 2015 (2015). Deposited under the accession number FERM P-22293 at the Patent Organism Depositary Center (NITE-IPOD) (Room No. 2-5-8 Kazusa -Kamashita , Kisarazu-shi, Chiba) and further deposited under accession number FERM BP-22293 It has been transferred to an international deposit under the Budapest Treaty.

Further, the present invention includes a method for producing TAG by culturing the eukaryotic microalgae mutant according to the present invention described above in large quantities. As the mass culture method, a culture method or the like described in Non-Patent Document 6 which has already been established can be used. Specifically, there is a method of culturing microalgae using a medium having urea as a nitrogen source and a pH of 4 or less (Patent Document 2). According to this culturing method, by using urea as the nitrogen source, fluctuation of pH due to consumption of nitrogen is minimized. In addition, even when CO ₂ is introduced into a culture solution having a pH of 4 or less, bicarbonate ions are hardly generated, so that the pH of the culture solution does not easily fluctuate. As described above, the pH of the culture solution can be stably maintained at 4 or less, so that the growth of other microalgae and protists can be suppressed.
After the culture, lipids containing TAG can be obtained from the culture by, for example, extracting with hexane.

  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 TAG lipase mutant
The Obi strain or the 5P strain, which is a low chlorophyll mutant derived from the Obi strain (JP-A-2013-102715), was treated with a mutagenic agent, NTG (1-methyl-3-nitro-1-nitrosoguanidine). Then, 1/3 DENSO medium (0.29 mM (NH ₄ ) ₂ SO ₄ , 0.79 mM Urea, 130 μM MgSO ₄ , 90 μM KH ₂ PO ₄ , 90 μM K ₂ HPO ₄ , 20 μM CaCl ₂ , 1.2 μM CuSO ₄ , 0.38 μM H ₃ BO ₃ , 0.35 μM ZnSO ₄ , 0.2 μM MnSO ₄ , 0.1 μM CoCl ₂ , 4 nM Na ₂ MoO ₄ , 0.1% (v / v) Fe solution (3 g / L citric acid, 4.9 g / L ammonium ferric citrate, 0.5 g / L EDTA-2Na)] for 1 week. At this time, the culture solution was depleted of nitrogen, and TAG was accumulated in the cells. Thereafter, the cells were transferred to MA5 medium (Imamura et al., 2012, J Gen Appl Microbiol, 58, 1) and then placed in the dark for 4 days. When cells were placed in this state, i.e., in a state with nitrogen but not photosynthesis, the accumulated TAG was progressively degraded (in wild-type strains) (see FIG. 1B). Four days later, intracellular TAG was fluorescently stained with BODIPY 501/515, and cells with strong fluorescence (a large amount of accumulated TAG) were concentrated using FACS (fluorescence activated cell sorting). The cells subjected to FACS concentration were cultured again in 1/3 DENSO medium to accumulate TAG, then transferred to MA5 medium, and cultured in the dark for 4 days. Thereafter, FACS concentration was performed again. When this FACS concentration was performed a total of three times, a cell population that hardly decomposed TAG was obtained even after being placed in the dark for 4 days. This population was appropriately diluted and plated on MA5 agar to form colonies.

  As described above, when the Obi strain was cultured under nitrogen-deficient conditions, TAG was accumulated in the cells, but at the same time, the chlorophyll content of the cells was reduced. When cells with high TAG and low chlorophyll were transferred to fresh MA5 medium and placed in the dark for several days, it was found that the TAG content of the cells decreased and the chlorophyll content increased. This was thought to be because chlorophyll was newly synthesized using the energy and carbon content obtained by the decomposition of TAG.

  Therefore, the colonies of the Obi strain obtained after the FACS concentration were spread on a nitrocellulose membrane placed on an agar medium made of a nitrogen-free MA5 medium (MA5-N), and spread under a fluorescent lamp. For one week. Then, the yellow cells grew on the nitrocellulose membrane. In these cells, the TAG content is thought to be high and the chlorophyll content low. Next, the yellow cells together with the nitrocellulose membrane were transferred onto MA5 agar medium and cultured in the dark. The cell color of the wild Obi strain turned green within 4 days after being transferred to the dark. On the other hand, in many clones derived from colonies separated after FACS concentration, the color of the cells remained yellow even after being transferred to MA5 agar medium. As described below, these clones lacked the ability to degrade TAG in the dark.

[Example 2] TAG degradation of TAG lipase mutant Using the method described in Example 1, two mutants (Obi-LOD68 and Obi- LOD69) and 2 strains (5P-LOD11, 5P-LOD18) were isolated from the 5P strain.

  These mutants and their parent strains, Obi strain and 5P strain, were cultured for 1 week in 1/3 DENSO medium. Then, the cells having accumulated TAG were collected, resuspended in MA5 medium, and cultured in the dark for 4 days.

  FIG. 1 shows the results of changes in dry weight and TAG content when the Obi strain, 5P strain, Obi-LOD68 strain, 5P-LOD11 strain, and 5P-LOD18 strain were cultured in the dark. The dry weight of each strain placed in the dark was slightly reduced on the first day, but hardly changed after day 2 (FIG. 1A). On the other hand, the TAG content significantly decreased in the Obi strain and the 5P strain in the first two days. In contrast, none of the mutants reduced TAG content slightly (5P-LOD11 strain) or did not (FIG. 1B). Although not shown in the figure, the TAG content did not decrease in the Obi-LOD69 strain under the same conditions.

  After culturing Obi strain, 5P strain and two mutants (Obi-LOD68, 5P-LOD18) in 1/3 DENSO medium for one week and accumulating TAG in the cells, these cells were transferred to MA5 medium. After transfer, the cells were cultured under fluorescent light for 7 days. All of these strains grown under fluorescent light grew similarly (FIG. 2A). TAG of wild strains (Obi strain and 5P strain) rapidly decreased with the start of growth. On the other hand, in the mutant, TAG decreased slightly more slowly than in the wild type (FIG. 2B). From this result, it was expected that at least two TAG lipases were present in the Obi strain. One is a TAG lipase whose activity is induced only under photosynthetic conditions, and the other is a TAG lipase that is active even in the dark. The mutants isolated this time seem to lack TAG lipase, which is active even in this darkness.

Example 3 Growth of TAG Lipase Mutant and TAG Accumulation
Two TAG lipase mutants, Obi-LOD68 strain and 5P-LOD18 strain, and their parent strains, Obi strain and 5P strain, were cultured in 1/3 DENSO medium under two light conditions. One is culture under continuous light, and the other is culture under light conditions of 8 hours light period and 16 hours dark period.

  In continuous light culture, no significant difference in growth rate and growth yield was observed between the parent strain and the lipase mutant (FIG. 3A). However, when the amount of accumulated TAG after 17 days was examined, the amount of accumulated TAG of the lipase mutant was much higher than that of the wild-type strain (Table 1).

When the same strain was cultured in 1/3 DENSO medium under light conditions of 8 hours light period and 16 hours dark period, the growth of the Obi strain and the 5P strain stopped at around OD ₇₅₀ of 3. In contrast, the TAG lipase mutant had an OD _{750 exceeding} 3 and continued to grow (FIG. 3B).

As the cell density of the culture increases, the amount of light available to each cell decreases. Probably, in the Obi and 5P strains with an OD ₇₅₀ of 3, the amount of energy obtained during the 8-hour light period is approximately equal to the amount of energy required to maintain the cells throughout the light and dark periods. Was.

  On the other hand, the growth yield of the TAG lipase mutant grown under the same light conditions was higher than the wild type. This suggests that those mutants that cannot use TAG as energy in the dark have either increased energy efficiency gained in photosynthesis, reduced energy to maintain cells, or both. I thought it would be. These changes are unlikely to be directly related to TAG lipase mutations, and alterations in the expression of various metabolic enzymes were signaled by changes in metabolite profiles caused by TAG lipase deficiency It was thought to be something. Thus, the unexpected result was obtained that the TAG lipase mutant was advantageous for growth in poor conditions.

  In the TAG lipase mutant, particularly the Obi-LOD68 strain, the TAG content maintained 44% even under this light condition (Table 1).

  As described above, this study showed that the TAG lipase mutant has better growth and TAG accumulation ability under poor light conditions than the wild type. Therefore, under conditions of short morning, evening or day length, the TAG lipase mutant exhibits higher TAG productivity than the wild type, and can be expected to increase the annual TAG production.

[Obi株、低クロロフィル変異体である5P株、Obi株由来のTAGリパーゼ欠損体であるObi-LOD68株、5P株由来のTAGリパーゼ変異体である5P-LOD18株のそれぞれを、連続光下(L24:D0)又は明期8時間及び暗期16時間の光条件下(L8:D16)で培養し、連続光培養の場合は培養開始後17日、明暗培養の場合は、培養開始後14及び24日目でサンプリングし、細胞内の油脂含有量を測定した]

(Obi strain, low chlorophyll mutant 5P strain, Obi-derived TAG lipase deficient Obi-LOD68 strain, 5P strain derived TAG lipase mutant 5P-LOD18, each under continuous light ( (L24: D0) or 8 hours light period and 16 hours dark period under light conditions (L8: D16), 17 days after the start of culture in the case of continuous light culture, 14 and after the start of culture in the case of light and dark culture. Sampling was performed on day 24, and the fat content in cells was measured.]

[Example 4] Genome analysis of TAG lipase mutant
The genomic nucleotide sequences of three strains, Obi-LOD68, 5P-LOD11, and 5P-LOD18, were determined by Illumina HiSeq 2000, and mutation analysis was performed. In the genome of the Obi-LOD68 strain, mutations involving amino acid sequence changes were detected in 23 genes. In the genomes of the 5P-LOD11 and 5P-LOD18 strains, 10 and 19 gene mutations with amino acid sequence changes were detected, respectively. The gene having a mutation in common among these three independent mutants is a gene named pev02gs0318601 (gene base sequence: SEQ ID NO: 1, mRNA base sequence: SEQ ID NO: 2, amino acid sequence: SEQ ID NO: 3 ), And was highly homologous to Sugar dependent 1 (AtSDP1), a TAG lipase that acts during germination of Arabidopsis seeds. Therefore, the pev02gs0318601 gene was named SDP1.

The Obi-LOD68 strain had a single base substitution at the Splice acceptor site, and had a splice site mutation. In the 5P-LOD11 strain, a deletion of 139 bp including exons and introns was confirmed, and in the 5P-LOD18 strain, nonsense mutation due to base substitution was confirmed. Therefore, the pev02gs0318601 (= SDP1) gene was amplified by PCR from the DNA of the Obi-LOD69 strain having the same phenotype, and the nucleotide sequence was determined using ABI BigDye ^™ Terminator v3.1 Cycle Sequencing Kit and ABI 3730xl sequencer. As a result, it was confirmed that the SDP1 gene of the Obi-LOD69 strain had a missense mutation due to single base substitution.

  In summary, Obi-LOD68 strain, Obi-LOD69 strain, 5P-LOD11 strain, 5P-LOD18 strain, (1) TAG degradation deficiency in the dark period, (2) under continuous light or bright and dark culture conditions It was concluded that the increase in the amount of intracellular TAG accumulation and the improvement of (3) growth under low light conditions were caused by mutation of the SDP1 gene.

FERM BP-10484
FERM BP-22179
FERM BP-22293

Claims (8)

Eukaryotic microalgae mutant with reduced triacylglycerol (TAG) lipase activity of SDP1 protein.Increases intracellular TAG accumulation and increases growth under short-day culture conditions compared to the parent strain. The eukaryotic microalgal variant, wherein the SDP1 protein is an improved protein having an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 and having TAG lipase activity.   The eukaryotic microalgal mutant according to claim 1, wherein the gene encoding the SDP1 protein is disrupted.   The eukaryotic microalgal mutant according to claim 1, wherein the expression of the gene encoding the SDP1 protein is reduced.   2. The eukaryotic microalgal variant according to claim 1, wherein the translation efficiency of the gene encoding the SDP1 protein is reduced.   The eukaryotic microalgal variant according to any one of claims 1 to 4, which belongs to the phylum Chlorophyta.   The eukaryotic microalgae mutant according to claim 5, which belongs to the treboxia algae web.   The eukaryotic microalgal variant according to claim 6, which belongs to the genus Coccomyxa or the genus Pseudococcomyxa.   A TAG production method comprising a step of culturing the eukaryotic microalgal mutant according to any one of claims 1 to 7.

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