JP6362162B2 - Oil and fat manufacturing method - Google Patents

Description

  The present invention relates to a method for producing microalgae, cultures, and fats and oils.

  In recent years, fuel production using microalgae has been proposed as an alternative fuel for fossil fuels. Since microalgae have a high light collecting ability and carbonic acid fixing ability, fuel production using the microalgae is excellent in terms of high productivity per unit area.

  As a novel microalgae that produces hydrocarbons that can be used as an alternative fuel for fossil fuels, Pseudocollistis strains have been proposed (see, for example, Patent Document 1). Pseudocollistis strains can accumulate hydrocarbons of about 30% of the dry alga body weight, have a high growth rate, and can be cultured under acidic conditions that can be cultivated in open-air systems without contamination by other organisms ( Since the optimum pH is 3 to 4), the hydrocarbon production method using the Pseudocollistis strain is a technique expected to be put to practical use.

Japanese Patent No. 4748154

However, in order to carry out fuel production using microalgae on a commercial basis, there is a problem in terms of cost, and further improvements are required.
This invention is made | formed in view of the said situation, Comprising: The microalga which improved productivity of fats and oils, the culture formed by culture | cultivating this microalgae, and the manufacturing method of fats and oils are provided.

  As a result of diligent research to solve the above problems, the present inventors have found that the productivity of fats and oils can be improved by inhibiting the enzyme activity of TOR (Target of Rapamycin) in microalgae, and the present invention has been completed. I let you.

  That is, this invention provides the manufacturing method of the micro algae, culture, and fats and oils which have the following characteristics.

(1) A microalgae characterized in that the enzyme activity of TOR is reduced or inactivated.
(2) The microalgae according to (1) above, which has a structural gene sequence encoding FKBP12 and a promoter sequence and terminator sequence for expressing the structural gene in the chromosome or as an extrachromosomal gene.
(3) The microalgae according to (2), wherein the structural gene sequence encoding FKBP12 is a sequence derived from an organism species sensitive to rapamycin.
(4) The microalgae according to any one of (1) to (3), wherein the enzyme activity of the TOR is reduced or inactivated by a TOR inhibitor.
(5) A culture obtained by culturing the microalgae according to any one of (1) to (4).
(6) The culture according to (5), further comprising a TOR inhibitor.
(7) A method for producing fats and oils comprising: step A1 for culturing microalgae, and step B1 for adding a TOR inhibitor to a culture solution obtained by culturing the microalgae after step A1. .
(8) The microalgae have a structural gene sequence encoding FKBP12 and a promoter sequence and terminator sequence for expressing the structural gene in the chromosome or as an extrachromosomal gene. Of oil and fat.
(9) The method for producing fats and oils according to (8), wherein the structural gene sequence encoding FKBP12 is a sequence derived from a biological species sensitive to rapamycin.
(10) A method for producing fats and oils, comprising the step A2 of culturing microalgae in which the enzyme activity of TOR is reduced or inactivated.

  ADVANTAGE OF THE INVENTION According to this invention, the micro algae which improved the productivity of fats and oils, the culture formed by culture | cultivating this micro algae, and the manufacturing method of fats and oils can be provided.

It is a result of the growth inhibitory ability evaluation by rapamycin addition. It is a figure which shows the expression vector used for the Example. It is a result of the growth inhibitory ability evaluation by rapamycin addition. It is a result of the growth inhibitory ability evaluation by rapamycin addition. It is a result of the growth inhibitory ability evaluation by rapamycin addition. It is a result of fats and oils accumulation ability evaluation by rapamycin addition. It is a result of fats and oils accumulation ability evaluation by rapamycin addition. It is a result of fats and oils accumulation ability evaluation by rapamycin addition. It is a result of the growth inhibitory ability evaluation by rapamycin addition. It is a result of fats and oils accumulation ability evaluation by rapamycin addition. It is a result of the growth inhibitory ability evaluation by TOR inhibitor addition. It is a result of fats and oils accumulation ability evaluation by TOR inhibitor addition. It is a result of the growth inhibitory ability evaluation by TOR inhibitor addition. It is a result of fats and oils accumulation ability evaluation by TOR inhibitor addition. It is a result of the growth inhibitory ability evaluation by TOR inhibitor addition. It is a result of fats and oils accumulation ability evaluation by TOR inhibitor addition. It is a result of the growth inhibitory ability evaluation by TOR inhibitor addition. It is a result of fats and oils accumulation ability evaluation by TOR inhibitor addition. It is a result of fats and oils accumulation ability evaluation by TOR inhibitor addition. It is a result of fats and oils accumulation ability evaluation by TOR inhibitor addition. It is a result of fats and oils accumulation ability evaluation by TOR inhibitor addition. It is a result of fats and oils accumulation ability evaluation by TOR inhibitor addition. It is a result of fats and oils accumulation ability evaluation by TOR inhibitor addition.

<Microalgae>
The microalgae of the present invention is characterized in that the enzyme activity of TOR is reduced or inactivated.
The microalgae used in the present invention is not particularly limited, and almost all algae can be used. Examples of such algae include Pseudochoristis ellipsoida (hereinafter also referred to as P. ellipsisidea), Botryococcus branii, and Cyanidiosero melodi (C. ), Chlamydomonas reinhardtii (hereinafter also referred to as C. reinhardtii) and the like.
Examples of the microalgae strain used in the present invention include Pseudochoristis ellipsisida N1 strain and Pseudochoristis ellipsisida Obi strain.

TOR (Target of Rapamycin) is a serine threonine kinase involved in intracellular signal transduction. TOR has been identified as a target protein for the antibiotic rapamycin and integrates intracellular and extracellular environmental information such as insulin, other growth factors, nutrition / energy status, and redox status, and through transcription, translation, etc. It is thought to play a central role in regulating size, division and survival.
As described later in Examples, the present inventors have found that fat and oil accumulation can be improved by inhibiting the enzyme activity of TOR in microalgae.

As a method for reducing or inactivating the enzyme activity of TOR, a method of substituting or deleting at least a part of the base of chromosomal DNA of microalgae encoding TOR, an insertion sequence is incorporated into the chromosomal DNA, and a frame shift is caused. Examples thereof include a method, a method of allowing a dominant negative type of TOR to be transiently or stably expressed in microalgae, and the like.
Moreover, the method of adding the enzyme activity inhibitor of TOR to the culture solution of a micro algae is also mentioned. As an enzyme activity inhibitor of TOR, in addition to the above-mentioned rapamycin, ATP competitive inhibitor Torin1 (Theoreen CC, et.al., J. Biol. Chem. (2009) Vol. 20, pp. 8023-32. AZD8055 (see Chresta CM, et.al., Cancer Res. (2010) Vol.70, pp.288-98.) And the like.
By these methods, the enzyme activity of TOR can be reduced or inactivated. From the viewpoint of easy operation, it is preferable that the enzyme activity of TOR is reduced or inactivated by a TOR inhibitor.

Examples of a method for deleting chromosomal DNA of microalgae include a method using homologous recombination. The method using homologous recombination is a drug resistance that cannot be autonomously replicated in the microalgae by linking a selectable marker between the gene fragment to be deleted on the chromosomal DNA of the microalgae or a DNA fragment existing on both outer sides of the DNA. Examples include a method using a plasmid for homologous recombination linked to a plasmid DNA having a gene or the like.
Another example is a method of amplifying a DNA fragment in which a selection marker is linked between a gene to be deleted on a chromosomal DNA of microalgae or a DNA fragment existing on both outer sides of the DNA by a PCR method. After introducing the DNA fragment linearized by treating the plasmid for homologous recombination with an appropriate restriction enzyme or the DNA fragment amplified by the PCR method into microalgae by a conventional method, drug resistance and auxotrophy are indicated. Thus, a transformed strain in which the plasmid for homologous recombination is incorporated on the chromosomal DNA by homologous recombination is selected.

  Another method for deleting chromosomal DNA of microalgae is a method of randomly inserting a gene with a transposon and selecting a strain in which the target gene has been inserted from the group into which the gene has been inserted. It is done. Another example is a method of obtaining a gene-deficient strain in the same manner as a transposon by introducing a foreign gene having a selectable marker into a cell and randomly inserting it into the genome (non-homologous recombination). It is done.

  Examples of methods for introducing genes into microalgae include particle bombardment, electroporation, microinjection, polyethylene glycol, lipofection, adsorption, infection, and protoplast fusion. Specifically, a method of introducing the recombination plasmid into microalgae during the exponential growth phase is preferable. In addition, a method of pretreatment with an enzyme having protease activity before gene introduction into a host cell by electroporation can also be mentioned.

  Hereinafter, preferred embodiments of the microalgae of the present invention will be described, but these embodiments are described as examples for better understanding of the gist of the invention, and the present invention is limited unless otherwise specified. Not what you want.

[First embodiment]
Examples of host microalgae used in this embodiment include C.I. Examples include rapamycin-insensitive ones such as melolae. C. melolae is a single-cell red algae that inhabits one of the most extreme environments for eukaryotes, such as hot and strongly acidic hot springs. In addition, C.I. In the melorae, the base sequence of the entire genome has been determined, and it has only a few introns. Furthermore, it is considered as a model organism for plant research because it can knock out nuclear genes and has low gene duplication.

The microalgae of this embodiment has a structural gene sequence encoding FKBP12 and a promoter sequence and terminator sequence for expressing the structural gene in the chromosome or as an extrachromosomal gene.
FKBP12 is a protein belonging to peptidylprolyl cis-trans isomerase. Rapamycin binds to FKBP12 to form a complex that inhibits the enzymatic activity of TOR by binding to TOR to form a triple complex. C. used for the microalgae of this embodiment. melolae is insensitive to rapamycin, but becomes highly sensitive to rapamycin by highly expressing FKBP12.

The structural gene encoding FKBP12 is not particularly limited, and may be a human-derived gene or a yeast-derived gene, and a Saccharomyces cerevisiae-derived gene is preferable. Here, the structural gene encoding FKBP12 may be cDNA or genomi cDNA.
Further, the structural gene sequence encoding FKBP12 is preferably a sequence derived from a biological species sensitive to rapamycin.

Since the homology between the amino acid sequences of yeast FKBP12 and human FKBP12 (identity of amino acid sequences) is 50 to 60%, the structural gene encoding FKBP12 used in this embodiment is the following (a) to ( It is preferable that the DNA comprises any one of d) and encodes a protein that forms a complex with rapamycin and inhibits the enzyme activity of TOR.
(A) DNA comprising the base sequence represented by SEQ ID NO: 1,
(B) a DNA comprising a base sequence in which one to several bases have been deleted, substituted or added in the base sequence represented by SEQ ID NO: 1;
(C) a nucleotide sequence having a homology (identity of nucleotide sequences) of 30% or more, preferably 50% or more, more preferably 70% or more, and even more preferably 90% or more with the nucleotide sequence represented by SEQ ID NO: 1. DNA consisting of, or
(D) DNA comprising a base sequence capable of hybridizing under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 1.

Furthermore, the structural gene encoding FKBP12 used in this embodiment is more preferably a gene encoding the following protein (e) or (f).
(E) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (f) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (e), and rapamycin A protein that forms a complex and inhibits the enzymatic activity of TOR

Examples of the promoter sequence and terminator sequence include tubulin, actin, rubisco, cauliflower mosaic virus (CaMV) 35S, Agrobacterium NOS, and the like.
In the microalgae of the present embodiment, examples of the method for expressing FKBP12 include a method for transiently overexpressing and a method for stably overexpressing, and handling is prepared in [Oil and fat production method] described later. Therefore, a method of stably overexpressing is preferable.

  Since the microalgae of this embodiment express FKBP12, it is sensitive to rapamycin. And by adding rapamycin to a culture solution, the enzyme activity of TOR in a micro algae falls or deactivates, and fats and oils accumulate in the micro algae of this embodiment.

[Second Embodiment]
Examples of the microalgae used in the present embodiment include C.I. Examples include those sensitive to rapamycin such as reinhardtii. By adding rapamycin to the culture solution, the enzyme activity of TOR in the microalgae is reduced or inactivated, and fats and oils accumulate in the microalgae of this embodiment.
Furthermore, in addition to rapamycin, an ATP competitive inhibitor can be used. ATP competitive inhibitors targeting human TOR (mammalian target of rapamycin; mTOR) have been researched and developed as anticancer agents, and those having potent inhibitory activity against mTOR and selectivity against other kinases have been developed. ing. Such ATP competitive inhibitors include Torin1, AZD8055, and the like. By using these ATP competitive inhibitors, the TOR activity of microalgae in the present embodiment is strongly and selectively suppressed, so that fats and oils accumulate efficiently.

[Third embodiment]
Examples of microalgae used in this embodiment include P.I. ellipsisidea and p. ellipsisideN1 strain and P. aeruginosa. The ellipsiside Obi strain is preferred. P. Elipsoidea is excellent in the production of fats and oils because of its high growth rate (see Japanese Patent No. 4748154).
Similar to the second embodiment, an ATP competitive inhibitor can also be used in this embodiment. According to this embodiment, fats and oils can be efficiently accumulated by strongly and selectively inhibiting TOR using microalgae excellent in the production of fats and oils.

<Culture>
The culture of the present invention is obtained by culturing the above-mentioned microalgae of the present invention. In the culture, genetic manipulation is not applied to the microalgae, and the TOR activity is reduced or inactivated. If not, it is preferable to further contain a TOR inhibitor.

<Oil and fat manufacturing method>
Hereinafter, preferred embodiments of the method for producing hydrocarbons of the present invention will be described, but these embodiments are described as examples for better understanding of the gist of the invention, and unless otherwise specified, It is not intended to limit the invention.

[First embodiment]
The manufacturing method of fats and oils of this embodiment has process A1 which culture | cultivates a micro algae, and process B1 which adds a TOR inhibitor to the culture solution formed by culture | cultivating the said micro algae after said process A1.
Although it does not specifically limit as a micro algae of the culture | cultivation object in this embodiment, A rapamycin sensitive thing is preferable.

First, in step A1, microalgae are cultured. Examples of the medium for culturing microalgae include known freshwater microalgae media and various microalgae media containing various nutrient salts, trace metal salts, vitamins, and the like. Further, an agar plate medium prepared based on a known medium for freshwater microalgae can also be used.
Examples of nutrient salts include nitrogen sources such as NaNO ₃ , KNO ₃ , NH ₄ Cl, (NH ₄ ) ₂ SO ₄ and urea; and phosphorus sources such as K ₂ HPO ₄ , KH ₂ PO ₄ and sodium glycerophosphate.
Examples of trace metals include iron, magnesium, manganese, calcium, and zinc, and examples of vitamins include vitamin B1 and vitamin B12.
Further, in step A1, it is preferable to stir together with the supply of carbon dioxide under aeration conditions as the culture conditions for the microalgae. At that time, it is preferable to carry out stationary culture, shaking, or air-aeration culture by irradiation with a fluorescent lamp for 12 hours, light irradiation with a light / dark cycle such as a 12-hour dark condition, or continuous light irradiation. Moreover, it is preferable to culture | cultivate, adding about 1 to 5% of carbon dioxide in the air from a viewpoint of growth promotion.
Further, there is no particular limitation as long as it does not adversely affect the growth of microalgae. In melolae, the pH of the culture solution is preferably 2-3, and the culture temperature is preferably 42 ° C .; In ellipsiside, the pH of the culture solution is preferably 3 to 4, and the culture temperature is preferably 25 ° C .; In reinhardtii, the pH of the culture solution is preferably 5.5 to 8.5, and the culture temperature is preferably 25 ° C.

Next, in step B1, a TOR inhibitor is added to a culture solution obtained by culturing microalgae. The TOR inhibitor used is not particularly limited, and may be rapamycin or an ATP competitive inhibitor. The amount of addition of the TOR inhibitor is not particularly limited, but is preferably such that the growth inhibition is caused by 10% to 70%, more preferably 20% to 60%, more preferably 50%. A degree of addition is particularly preferred.
Through step B1, fats and oils are accumulated in the microalgae.

When cultured under the above conditions, oil and fat can be collected in about 6 to 8 days from the start of culture.
According to this embodiment, fats and oils can be manufactured with high productivity.
Furthermore, according to this embodiment, since fats and oils can be manufactured even if it does not culture | cultivate on nitrogen deficient conditions, the culture | cultivation process on nitrogen deficient conditions required in the conventional fats and oils manufacturing method can be skipped. Therefore, the method for producing fats and oils of the present embodiment is excellent in terms of cost.

  The produced fats and oils can be collected from cultured algal bodies. A method of crushing cells by a general method such as a French press or a homogenizer and then extracting with an organic solvent such as n-hexane, or collecting cells on a filter such as glass fiber and drying them, followed by an organic solvent, etc. The method of extracting by is mentioned. Moreover, the method of collect | recovering cells by centrifugation, freeze-drying and pulverizing, and extracting with the organic solvent from the powder is also mentioned. The desired oil and fat can be obtained by evaporating the solvent after extraction under reduced pressure or normal pressure, or by heating or normal temperature.

  Oils and fats that accumulate in microalgae are compounds in which fatty acids are ester-bonded to glycerol. As such fats and oils, acylglycerides are preferable, and triacylglycerides are more preferable. For example, these fats and oils can be methyl esterified with methanol and converted into a fuel of the same quality as conventional diesel fuel (Biodiesel fuel). .

[Second Embodiment]
The method for producing fats and oils of the present embodiment uses a microalgae having a structural gene sequence encoding FKBP12 and a promoter sequence and terminator sequence for expressing the structural gene in the chromosome or as an extrachromosomal gene. Is the method. In the present embodiment, the structural gene sequence encoding FKBP12 is preferably a sequence derived from a biological species that is sensitive to rapamycin.
Examples of host microalgae used in this embodiment include C.I. Examples include rapamycin-insensitive ones such as melolae. As described in the first embodiment of microalgae, by expressing FKBP12, the rapamycin-insensitive microalgae is sensitive to rapamycin. And by adding rapamycin to a culture solution, the enzyme activity of TOR in a micro algae falls or deactivates, and fats and oils accumulate in a micro algae.
About another structure, since it is the same as that of the manufacturing method of the fats and oils of 1st Embodiment, the description is abbreviate | omitted.

[Third embodiment]
The manufacturing method of the fats and oils of this embodiment has process A2 which culture | cultivates the micro algae in which the enzyme activity of TOR has fallen or is deactivated.
In the present embodiment, a mutant in which at least a part of the base of chromosomal DNA of microalga encoding TOR is substituted or deleted; an insertion sequence is incorporated into chromosomal DNA of microalga encoding TOR to cause frame shift It is preferable to use a transformant in which a dominant negative type of TOR is transiently or stably overexpressed in microalgae.
Further, a transformant in which an RNAi-inducible nucleic acid capable of suppressing the expression of a TOR gene at the mRNA level is expressed in microalgae is also included. The RNAi-inducible nucleic acid means a nucleic acid that can induce RNA interference by being introduced into microalgal cells. RNA interference refers to the effect that RNA containing a base sequence complementary to mRNA (or a partial sequence thereof) suppresses the expression of the mRNA.
The mRNA targeted by the RNAi-inducing nucleic acid may be a coding region or a non-coding region.
In addition, examples of the RNAi-inducible nucleic acid include siRNA and miRNA. Examples of vectors that are introduced into microalgal cells and cause RNAi as well as siRNA include shRNA (short hairpin RNA / small hairpin RNA) expression vectors.
Further, from the viewpoint that inhibition of TOR activity may cause growth inhibition of microalgae, it is preferable to conditionally induce the expression of the RNAi-inducible nucleic acid described above to regulate the activity of TOR.
About another structure, since it is the same as that of the manufacturing method of the fats and oils of 1st Embodiment, the description is abbreviate | omitted.
According to this embodiment, fats and oils can be manufactured only by culture | cultivating the said micro algae mutant or a transformant, without adding a TOR inhibitor. Therefore, the method for producing fats and oils of the present embodiment is further excellent in terms of cost.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

Example 1
[Inhibition of growth by rapamycin]
Approximately 1.0 × 10 ⁷ cells / mL of C.I. melolae was added, rapamycin was added to final concentrations of 0 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, and 10 μM, and cultured at 40 ° C. for 60 hours. A photograph of each well is shown in FIG.
As shown in FIG. 1, even when 10 μM of rapaicin is used, there is no difference in green density from the 0 μM well. It was confirmed that merolae was insensitive to rapamycin.
For the composition of the medium used for the culture, see Ohnuma M, et. , Plant Cell Physiol. (2008) Vol. 49, pp. 117-120.

[Preparation of expression vector]
C. at the 5 ′ end of FKBP12 (ScFKBP12) from Saccharomyces cerevisiae. A fragment containing the promoter region of the melolae APCC (CMO250C; http://merolae.biol.su-tokyo.ac.jp/) gene and the APCC gene terminator region at the 3 ′ end was transferred by PCR. Amplified. In addition, a 3 × FLAG tag sequence was added between ScFKBP12 and the terminator region of the APCC gene. Used for indication of ScFKBP12 expression in melolae cells. When such a fragment was amplified by the PCR method, a sequence recognized by the restriction enzyme EcoRI was artificially added to the 5 ′ end and 3 ′ end of the DNA, and the fragment was digested with EcoRI. On the other hand, pKFURACm-Gs (see Immura S, et.al., Plant Cell Physiol. (2010) Vol. 51, pp. 707-717.) Was digested with restriction enzyme EcoRI and treated with alkaline phosphatase. After this treatment, it was subjected to agarose electrophoresis, and the vector pKFURACm-Gs fragment and the above-mentioned fragment containing the ScFKBP12 gene were excised from the agarose gel, ligated, and then introduced into Escherichia coli DH5α (manufactured by Toyobo Co., Ltd.) for transformation. A vector was prepared from the obtained transformant, and the target expression vector pKF-ScFKBP12 (see FIG. 2) was obtained. The target vector was confirmed from the construction of a restriction enzyme map and the decoding of the base sequence using a capillary sequencer.

[Preparation of a high expression strain of ScFKBP12]
The pKF-ScFKBP12 and its parent vector pKFURACm-Gs were obtained from Imamara S, et. , Plant Cell Physiol. (2010) Vol. 51, pp. 707-717. It was introduced into melolae M4 strain and transformed. Thereafter, transformants were selected using uracil non-requirement as an index, and the F12 strain, which is a high expression strain of ScFKBP12, and the C12 strain, which is a control strain, were obtained.

[Inhibition of growth by rapamycin]
About 1.0 × 10 ⁷ cells / mL of F12 or C12, which is a high expression strain of ScFKBP12, is added to each well of a 24-well plate, and rapamycin is adjusted so that the final concentrations are 0 nM, 10 nM, 50 nM, 100 nM, 500 nM, and 1000 nM. Was added and cultured at 40 ° C. for 60 hours. The photograph of each well and the cell turbidity of the high expression strain of ScFKBP12 are shown in FIG. Further, Rapamycin was added to the high expression strain of ScFKBP12 so as to have a final concentration of 500 nM, followed by culturing, and the turbidity and cell number of the culture solution were measured over time. The results of cell growth curves observed with two indexes of “cell turbidity” and “cell number” are shown in FIG. 4 and FIG.
As shown in FIG. 3, in the ScFKBP12-expressing strain, a growth inhibitory effect was observed at 10 nM of rapaisin, and a complete growth inhibitory effect was observed at 500 nM, while the growth of the C12 strain was not inhibited. From this, it was confirmed that the high expression strain of ScFKBP12 is sensitive to rapamycin.
As shown in FIG. 4 and FIG. 5, in the absence of rapamycin, the ScFKBP12 high-expressing strain showed similar growth efficiency compared to the C12 strain, but it was confirmed that the growth was suppressed in the presence of rapamycin. It was done.

[Evaluation of oil production capacity]
A rapamycin having a final concentration of 500 nM was added to the high expression strain of ScFKBP12 and cultured, and accumulation of fats and oils in the high expression strain of ScFKBP12 was measured over time. The result of dyeing fats and oils with BODIPY is shown in the lower part of FIG. 6, an autofluorescence photograph of chloroplast is shown in the middle part of FIG. 6, and a bright field image of the high expression strain of ScFKBP12 is shown in the upper part of FIG. Similarly, FIG. 7 shows the accumulation of fats and oils after addition of DMSO to the high expression strain of ScFKBP12.
As shown in the lower part of FIG. 6, accumulation of fats and oils was observed after 24 hours from the addition of rapamycin. In particular, it was confirmed that the accumulation of fats and oils after 48 hours was more remarkable than when DMSO was added as a control in the lower part of FIG.

Further, the amount of triacylglyceride (TAG) produced 2 days after addition of rapamycin in the high expression strain of ScFKBP12 was quantified. Specifically, cells 2 days after the addition of rapamycin were collected and stored at -80 degrees. When used, the cells were dried with a freeze dryer for about 16 hours and subjected to lipid extraction. Extraction of total lipids was performed based on the Bligh & Dyer method (Blight EG, et.al., Can. J. Biochem. Physiol. (1959) Vol. 37, pp. 911-917.).
The dried product was suspended in water, chloroform and methanol (1: 2 (vol / vol)), and the total lipid was extracted. Chloroform and 1% KCl were added to the extract, suspended and centrifuged. After centrifugation, it was separated into two layers, and the lower organic solvent layer containing the total lipid was recovered. Separation of TAG from total lipids was performed by thin layer chromatography. The developing solvent composition at that time was hexane: diethyl ether: acetic acid = 160: 40: 4 (vol / vol). A TAG spot was scraped from the plate after separation, and the TAG was subjected to methanolysis treatment using 15: 0 fatty acid (pentadecanic acid) as an internal standard sample. Specifically, 100 μL of a 1 mM 15: 0 hexane solution and 500 μL of a 5% hydrogen chloride methanol solution were added to silica gel powder containing TAG in a glass test tube with a screw cap, followed by treatment at 85 ° C. for 1 hour. After the methanolysis treatment, the fatty acid methyl ester was recovered with hexane, dried with nitrogen gas, dissolved with 60 μL of hexane, 3 μL of which was subjected to gas chromatography for quantification.

  The results are shown in FIG. As shown in FIG. 8, it was confirmed that the amount of accumulated TAG increased by adding rapamycin and culturing for 2 days as compared with the case of adding DMSO alone and culturing for 2 days as a control.

<< Example 2 >>
[Inhibition of growth by rapamycin]
Approximately 3.0 × 10 ⁶ cells / mL of C.I. Reinhardtii was added, rapamycin was added to a final concentration of 0 nM, 100 nM, 200 nM, 400 nM, 600 nM, and 1000 nM, and cultured at 25 ° C. for 40 hours. The photograph and turbidity of each well are shown in FIG.
For the composition of the medium used for the culture, see Gorman DS, et.al. , Proc. Natl. Acad. Sci. USA, (1965) Vol. 54, pp. 1665-1669.
As shown in FIG. Since cell proliferation of reinhardtii is suppressed, C.I. Reinhardtii was confirmed to be sensitive to rapamycin.

[Evaluation of oil production capacity]
C. C. reinhardtii was added with 400 nM rapamycin and cultured at 25 ° C. for 40 hours. The accumulation of fats and oils in reinhardtii was measured. The result of dyeing fats and oils with BODIPY is shown in FIG.
As shown in FIG. 10, accumulation of fats and oils was observed by adding 400 nM rapamycin.

[Inhibition of growth by ATP competitive inhibitor 1]
Approximately 3.0 × 10 ⁶ cells / mL of C.I. Reinhardtii is added, and Torin1 (Thoren CC, et.al., J. Biol. Chem. (2009) Vol. 20, pp. 8023-32. is adjusted to a final concentration of 0 nM, 1 nM, 10 nM, 100 nM, 1000 nM, and 10000 nM. And the mixture was cultured at 25 ° C. for 100 hours. The photograph and turbidity of each well are shown in FIG.
As shown in FIG. Since cell proliferation of reinhardtii is suppressed, C.I. It was confirmed that reinhardtii is sensitive to Torin1, which is an ATP competitive inhibitor of TOR.

[Evaluation of oil production capacity]
C. Reinhardtii was added with Torin 1 having a final concentration of 1000 nM and cultured at 25 ° C. for 100 hours. The accumulation of fats and oils in reinhardtii was measured. The result of dyeing fats and oils with BODIPY is shown in FIG.
As shown in FIG. 12, in two different visual fields, accumulation of fats and oils was observed by adding a final concentration of 1000 nMTorin1.

[Inhibition of growth by ATP competitive inhibitor 2]
Approximately 3.0 × 10 ⁶ cells / mL of C.I. Reinhardtii is added, and AZD8055 (Chresta CM, et.al., Cancer Res. (2010) Vol. 70, pp. 288-98.) is used so that the final concentration is 0 nM, 1 nM, 10 nM, 100 nM, 1000 nM, and 10000 nM. In addition, the cells were cultured at 25 ° C. for 48 hours. The photograph and turbidity of each well are shown in FIG.
As shown in FIG. 13, C.D. Since cell proliferation of reinhardtii is suppressed, C.I. It was confirmed that reinhardtii is sensitive to AZD8055, which is an ATP competitive inhibitor of TOR.

[Evaluation of oil production capacity]
C. Reinhardtii was added with AZD8055 at a final concentration of 100 nM and cultured at 25 ° C. for 48 hours. The accumulation of fats and oils in reinhardtii was measured. The result of dyeing fats and oils with BODIPY is shown in FIG.
As shown in FIG. 14, accumulation of fats and oils was observed by adding 100 nMAZD8055 in two different visual fields.

Example 3
[Inhibition of growth by ATP competitive inhibitor 1]
Approximately 5.0 × 10 ⁶ cells / mL of P.I. ellipsiside was added, and Torin1 was added to a final concentration of 0 nM, 1 nM, 10 nM, 100 nM, 1000 nM, and 10000 nM, and cultured at 25 ° C. for 48 hours. The photograph and turbidity of each well are shown in FIG.
For the composition of the medium used for the culture, Imamura S, et al. J. Gen. Appl. Microbiol., (2012) Vol. 58, pp. The description of 1-10 was followed.
As shown in FIG. Since cell growth of ellipsisidea is suppressed, It was confirmed that ellipsisidea is sensitive to Torin1, which is an ATP competitive inhibitor of TOR.

[Evaluation of oil production capacity]
P. E. lipsoidea was added with Torin 1 to a final concentration of 10000 nM and cultured at 25 ° C. for 48 hours. The accumulation of fats and oils in ellipsiside was measured. The result of dyeing fats and oils with BODIPY is shown in FIG.
As shown in FIG. 16, accumulation of fats and oils was observed by adding 10,000 nM Torin1.

[Inhibition of growth by ATP competitive inhibitor 2]
Approximately 5.0 × 10 ⁶ cells / mL of P.I. ellipsiside was added, AZD8055 was added to a final concentration of 0 nM, 1 nM, 10 nM, 100 nM, 1000 nM, and 10000 nM, and cultured at 25 ° C. for 48 hours. The photograph and turbidity of each well are shown in FIG.
As shown in FIG. 17, P.P. Since cell growth of ellipsisidea is suppressed, It was confirmed that ellipsisidea is sensitive to AZD8055 which is an ATP competitive inhibitor of TOR.

[Evaluation of oil production capacity]
P. E. lipsoidea was added with 1000 nM AZD8055 and cultured at 25 ° C. for 48 hours. The accumulation of fats and oils in ellipsiside was measured. The result of dyeing fats and oils with BODIPY is shown in FIG.
As shown in FIG. 18, accumulation of fats and oils was observed by adding AZD8055 to a final concentration of 1000 nM.

Example 4
[Evaluation of oil production capacity]
Four test tubes containing 50 ml of medium (see Gorman DS, et.al., Proc. Natl. Acad. Sci. USA, (1965) Vol. 54, pp. 1665-1669.) Are prepared. Were designated as test tube 1, test tube 2, test tube 3 and test tube 4. In each medium, C.I. After each reinhardtii was added, the culture was started by stirring at 25 ° C. with CO ₂ supply under aeration conditions while applying light with a fluorescent lamp. When the logarithmic growth phase was reached, DMSO, final concentration of 1.0 μM rapamycin, final concentration of 1.0 μM Torin1, or final concentration of 0.2 μM AZD8055 was added and cultured at 25 ° C. for 24 hours. The production amount of TAG in Reinhardtii was quantified by the same method as the quantification method by gas chromatography described in Example 1.
The results are shown in FIG. As shown in FIG. 19, it was confirmed that the production amount of TAG was increased by adding TOR inhibitor and culturing for 1 day as compared with the case of adding DMSO alone and culturing for 1 day as a control.

Example 5
[Evaluation of oil production capacity]
Four 200 mL flat flasks were prepared, and flask 1, flask 2, flask 3, and flask 4, respectively. Nitrogen-sufficient culture medium (Imamura S, et.al., J. Gen. Appl. Microbiol., (2012) Vol. 58, pp. 1-10.) Is put in each flask. After adding ellipsiside to each medium, the culture was started by stirring at 25 ° C. with CO ₂ supply under aeration conditions while illuminating with a fluorescent lamp. After 4 days of culture, the culture medium in flask 1 was replaced with a new nitrogen-sufficient medium, and the medium in flask 2 was replaced with a nitrogen-deficient medium (a medium with a composition in which NaNO ₃ having a composition of nitrogen-sufficient medium was replaced with equimolar NaCl). Then, DMSO was added to the medium of flask 3, AZD8055 having a final concentration of 1 μM was added to the medium of flask 4, and further cultured.
The culture medium of flask 1 to flask 4 after 4 days of culture was sampled over time, centrifuged, and the supernatant was removed. The precipitate of ellipsiside was collected. The precipitate obtained from the culture media of flask 1 to flask 4 was freeze-dried, and the dry weight of the dried product was measured.
After drying the precipitate, the TAG weight ratio per dry weight was measured using a TD-NMR (Time Domain Nuclear Magnetic Resonance, time domain nuclear magnetic resonance) apparatus for oil analysis. Olive oil was used as a standard sample. The results are shown in FIG. As shown in FIG. 20, the AZ weight ratio per dry weight was significantly greater under the AZD8055 addition condition than under the nitrogen deficiency condition.

FIG. 21 shows the result of calculating the TAG weight per liter of the culture solution, FIG. 22 shows the result of calculating the TAG weight per unit cell number, and FIG. 23 shows the result of calculating the TAG productivity on the lab scale. Shown in Here, the TAG productivity at the lab scale indicates a value obtained by dividing the TAG weight per 1 L of the culture solution by the total number of culture days.
As shown in FIGS. 21 to 23, it was confirmed that the AZD8055 addition condition was larger than the nitrogen deficiency condition in any calculation method.

Claims (3)

A method for producing fats and oils, comprising: step A1 for culturing microalgae, and step B1 for adding AZD8055 to a culture solution obtained by culturing the microalgae after step A1. The oil and fat production according to claim 1 , wherein the microalgae has a structural gene sequence encoding FKBP12 and a promoter sequence and a terminator sequence for expressing the structural gene in a chromosome or as an extrachromosomal gene. Method. The method for producing fats and oils according to claim 2 , wherein the structural gene sequence encoding FKBP12 is a sequence derived from a biological species sensitive to rapamycin.