Yeast mutant strain that produces oil / fat and allows cell cycle synchronization

Abstract

Provided is an oil / fat producing yeast mutant strain that can be arrested for synchronization, due to temperature change, within a specific cell cycle phase during which highly efficient oil / fat production is realized.　The mutant strain is a Lipomyces starkeyi (Lipomyces starkeyi) strain that, as a consequence of undergoing main culture at 32-35°C after pre-culture at 23-26°C, is arrested for synchronization within G1 phase, or a Lipomyces starkeyi strain that, as a consequence of undergoing main culture at 30-32°C after pre-culture at 20-23°C, is arrested for synchronization within G2 / M phase.

Description

An oleaginous yeast mutant capable of synchronizing the cell cycle

[0001] The present invention relates to an oil-producing yeast mutant capable of synchronizing the cell cycle.

[0002] The oleaginous yeast species of the genus Lipomyces accumulates as much as 70% of lipids per cell. Because the lipid composition is similar to that of palm oil, Lipomyces is expected to be used not only as a biofuel but also as an alternative lipid for foods and cosmetics, and research into this species is progressing rapidly. The present inventors have been working to elucidate the molecular mechanism of lipid production in Lipomyces and have found that synchronization in the G1 phase of the cell cycle increases lipid accumulation by 1.4-fold (Non-Patent Document 1). However, there remains a continuing need for lipid-producing microorganisms and lipid production methods with high lipid production efficiency.

[0003] Morimoto Y. et al., Journal of Cell Science (2022) 135 (16), jcs259996

[0004] An object of the present invention is to provide an oil-producing yeast mutant that can be synchronously arrested in a specific cell cycle with high oil-producing efficiency by temperature change.

[0005] The above-mentioned problems can be solved by the present invention as follows. [1] A Lipomyces starkeyi strain that undergoes synchronous arrest at the G1 phase by pre-culture at 23 to 26°C followed by main culture at 32 to 35°C. [2] The Lipomyces starkeyi strain of [1], having accession number NITE BP-03941. [3] A Lipomyces starkeyi strain that undergoes synchronous arrest at the G2 / M phase by pre-culture at 20 to 23°C followed by main culture at 30 to 32°C. [4] The Lipomyces starkeyi strain of [3], having accession number NITE BP-03942 or NITE BP-03943. [5] A method for inducing lipid accumulation, characterized by using any of the strains of [1] to [4]. [6] A method for inducing fat accumulation, comprising the steps of pre-culturing any one of the strains [1] to [4] at 20 to 26°C, and then culturing it at 30 to 35°C.

[0006] According to the present invention, it is possible to synchronize and arrest a specific cell cycle with high oil production efficiency simply by changing the temperature, without the need for expensive chemical treatments or special equipment, and by using this mutant strain, it is possible to achieve increased oil accumulation.

[0007] 1 is a graph showing the results of intracellular triacylglycerol quantification in the cell cycle synchronization of Lipomyces starkeyi by drug treatment performed in the Reference Example described below. 2 is a histogram showing the results of measuring the intracellular DNA content by FACS analysis of the Ls100 strain, Ls76 strain, Ls63 strain, and Ls93 strain performed in Example 2 described below. 3 is a graph showing the results of intracellular triacylglycerol quantification of the Ls100 strain, Ls76 strain, Ls63 strain, and Ls93 strain performed in Example 3 described below.

[0008] (Oil-Accumulating Yeast Mutant of the Present Invention) The oil-accumulating yeast mutant of the present invention is a mutant of Lipomyces starkeyi, and can be synchronously arrested at a specific cell cycle by pre-culturing at a normal temperature and then raising the temperature during culture higher than normal. The first oil-accumulating yeast mutant of the present invention can be synchronously arrested at the G1 phase by pre-culturing at 23-26°C, followed by raising the temperature to 32-35°C and then performing main culture. The second oil-accumulating yeast mutant of the present invention can be synchronously arrested at the G2 / M phase by pre-culturing at 20-23°C, followed by raising the temperature to 30-35°C and then performing main culture.

[0009] An example of the first oleaginous yeast mutant strain of the present invention is the Lipomyces starchii Ls76 strain (accession number NITE BP-03941). An example of the second oleaginous yeast mutant strain of the present invention is the Ls63 strain (accession number NITE BP-03942) and the Ls93 strain (accession number NITE BP-03943). These mutant strains accumulate higher amounts of oil than previously known Lipomyces species and can be used as efficient oil-producing bacteria. The Ls76, Ls63, and Ls93 strains are mycologically budding yeasts that grow well at 23°C but stop growing at temperatures above 30°C.

[0010] (Method for inducing fat accumulation of the present invention) The method for inducing fat accumulation of the present invention can be carried out in accordance with a conventionally known method for inducing fat accumulation, except that the fat-producing yeast mutant of the present invention is used and the mutant is cultured under conditions that allow synchronous arrest at the desired cell cycle (particularly the G1 phase or the G2 / M phase). For example, in the method for inducing fat accumulation of the present invention, a preliminary culture is carried out in advance, and the resulting culture is inoculated into an appropriate fat accumulation induction medium to carry out the main culture. The culture temperature can be 20 to 35°C, and the culture is performed with shaking for about 1 to 7 days.

[0011] In the first method for inducing oil accumulation using an oil-accumulating yeast mutant strain of the present invention, synchronization arrest can be achieved at the G1 phase by carrying out preliminary culture at 23 to 26° C. and then main culture at 32 to 35° C. In the second method for inducing oil accumulation using an oil-accumulating yeast mutant strain of the present invention, synchronization arrest can be achieved at the G2 / M phase by carrying out preliminary culture at 20 to 23° C. and then main culture at 30 to 32° C. These preliminary cultures can be carried out using, for example, YPD medium.

[0012] In the present invention, the accumulation of fats and oils in the fat-producing yeast can be confirmed and quantified by a conventionally known method, such as a method of staining the fats and oils with a stain such as oil red, taking a photograph of the yeast under a microscope, and visually confirming the amount, or a method of preparing a cell extract by dissolving or physically destroying the cell wall, and separating and quantifying the fat and oil components present in the extract.

[0013] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0014] Reference Example: Measurement of lipid accumulation amount by synchronizing each cell cycle Synchronizing the cell cycle of Lipomyces starkeyi required expensive chemical treatments and specialized equipment. In this reference example, Lipomyces starkeyi NBRC1289 strain (obtained from NBRP (National BioResource Project)), a wild-type strain isolated from soil, was used to measure the amount of triacylglycerol (TAG) (unit: mg / 10 ８ cells) were measured.

[0015] (Synchronization arrest at S phase or G2 / M phase) Cells were pre-cultured at 26°C using YPD medium (2% D-glucose, 1% yeast extract, 2% hypopolypeptone), and hydroxyurea (HU) or nocodazole (Noc) was added. After culturing for 3.5 hours, the cells were harvested. As described below, hydroxyurea can induce synchronization arrest at S phase, and nocodazole can induce synchronization arrest at G2 / M phase. The harvested cells were washed three times with sterile water and then transferred to N medium (0.3% sucrose, 0.025% KH２ P.O. ４ , 0.066% MgSO ４ ・7H ２ O, Trace element solution (FeCl ３ ・6H ２ O 1.7mg / L, MnSO ４ ・6H ２ O 0.51mg / L, ZnSO ４ ・7H ２ The bacterial cell concentration was adjusted to approximately 3 x 10 ６ The cells were then inoculated at a density of 5 x 10 cells / mL and cultured at 26°C for 18 hours. ７ Cells were collected and triacylglycerol was quantified.

[0016] (Synchronization arrest at G1 phase) The cells were pre-cultured at 26°C using YPD medium, nocodazole (Noc) was added, and the cells were cultured for 3 hours, after which they were collected. After washing three times with sterile water, the cells were transferred to YPD medium and cultured for 30 minutes, after which rapamycin (Rapa) was added and the cells were cultured for an additional 2.5 hours. As described below, rapamycin can cause synchronization arrest at G1 phase. After washing three times with sterile water, the cells were cultured in N medium at a cell concentration of approximately 3 x 10 ６ The cells were then inoculated at a density of 5 x 10 cells / mL and cultured at 26°C for 18 hours. ７ Cells were collected and triacylglycerol was quantified.

[0017] The results are shown in Figure 1. In Figure 1, Asy means N medium, HU means hydroxyurea, Noc means nocodazole, and Rapa means rapamycin. Cells treated with hydroxyurea (HU) were synchronized and arrested in the S phase, cells treated with nocodazole (Noc) were synchronized and arrested in the G2 / M phase, and cells treated with rapamycin (Rapa) were synchronized and arrested in the G1 phase. Error bars (mean ± standard deviation) in Figure 1 were calculated from independent cell cultures [n = 16, 10, 14, and 20 (number of experiments from the left of the graph)]. In Figure 1, "**" indicates P < 0.001 (compared to N medium; Dunnett's multiple comparison test). The highest amount of triacylglycerol accumulation was confirmed when cells were synchronized and arrested in the G1 phase (approximately 1.4-fold).

[0018] Example 1: Isolation and Identification of an Oil-Producing Yeast Mutant In this example, the Ls100 strain (deposited at NBRP), derived from the Lipomyces starkey NBRC1289 strain, was used. After UV treatment, colonies were transferred to YPD agar medium and cultured under high-temperature conditions (30°C and 32°C). Cell strains that showed poor growth under high-temperature conditions were again examined to see if they also showed poor growth under high-temperature conditions. Strains that showed poor growth under high-temperature conditions were cultured under high-temperature conditions and screened by FACS analysis to determine whether they arrested at a specific cell cycle. An oil-producing yeast mutant that arrested at high temperatures was successfully obtained. More specifically, one cell strain (named Ls76 strain) that synchronously arrested at the G1 phase and two cell strains (named Ls63 strain and Ls93 strain) that synchronously arrested at the G2 / M phase were obtained. Of the approximately 1,700 colonies that emerged after UV irradiation, approximately 100 strains that were unable to grow under high temperature conditions were obtained, and ultimately, three strains that showed synchronous cell cycle arrest were obtained.

[0019] Example 2: Synchronized arrest of cell cycle in an oil-producing yeast mutant strain (Ls76 strain with synchronized arrest at G1 phase) The cells were pre-cultured at 26°C in YPD medium, and the temperature was raised to 32°C for 3.5 hours, after which the cells were collected. After washing three times with sterilized water, the cells were cultured in N medium at a cell concentration of approximately 3 x 10 ６ The cells were seeded at a density of 5 × 10 cells / mL and culture was initiated at 26°C or 32°C. ７ The cells were collected and the intracellular DNA content and triacylglycerol were quantified.

[0020] (Ls63 strain and Ls93 strain that undergo synchronous arrest at G2 / M phase) The strains were pre-cultured in YPD medium at 23°C, then the temperature was raised to 30°C and cultured for 3.5 hours, after which the cells were harvested. After washing three times with sterilized water, the cells were cultured in N medium at a cell concentration of approximately 3 x 10 ６ The cells were inoculated at a density of 5 × 10 cells / mL and culture was initiated at 26°C or 30°C. ７ The cells were collected and the intracellular DNA content and triacylglycerol were quantified.

[0021] The results of measuring intracellular DNA content by FACS (fluorescence-activated cell sorting) analysis are shown in Figure 2. In each column, the lower row shows the DNA content at the start of 32°C culture after changing from 26°C culture to 32°C culture, and the upper row shows the DNA content 3.5 hours after the start of 32°C culture. For example, of the two peaks in the upper row of the Ls100 strain, the left peak represents the state before DNA replication begins, and the right peak represents the state after DNA synthesis is completed (i.e., DNA content doubled).

[0022] The obtained mutant strains exhibited the following properties: Ls76 strain (Accession No. NITE BP-03941): After culturing at 26°C, it was possible to synchronously arrest the cells in the G1 phase by culturing at 32°C for 3.5 hours. Ls63 strain (Accession No. NITE BP-03942): After culturing at 23°C, it was possible to synchronously arrest the cells in the G2 / M(1) phase by culturing at 30°C for 3.5 hours. Ls93 strain (Accession No. NITE BP-03943): After culturing at 23°C, it was possible to synchronously arrest the cells in the G2 / M(2) phase by culturing at 30°C for 3.5 hours. The G2 / M-arrested cells Ls63 and Ls93 strains can restart the cell cycle by returning to low temperature (reversible strains), making them even more valuable in cell biology.

[0023] Example 3: Changes over time in the amount of fat and oil accumulated in yeast mutants producing fat and oil-producing yeast In this example, mutant Lipomyces starchii strains Ls76, Ls63, and Ls93 were used, and the changes over time in the intracellular DNA content and the amount of triacylglycerol accumulated in the mutants were observed in a state where cell growth was stopped at the G1 phase or G2 / M by changing the medium and changing the culture temperature to a high temperature.

[0024] The Ls76 strain was pre-cultured in YPD medium at 26°C, then the temperature was raised to 32°C and cultured for 3.5 hours, after which the cells were harvested. After washing three times with sterilized water, the cells were cultured in N medium at a cell concentration of approximately 3 x 10 ６ The cells were seeded at a density of 5 × 10 cells / mL and culture was initiated at 26°C or 32°C. After 1 and 4 days of culture, 5 × 10 cells were seeded at a density of 5 × 10 cells / mL. ７The cells were collected and the intracellular DNA content and triacylglycerol were quantified.

[0025] The Ls63 and Ls93 strains were pre-cultured in YPD medium at 23°C, then the temperature was raised to 30°C and cultured for 3.5 hours, after which the cells were harvested. After washing three times with sterilized water, the cells were cultured in N medium at a cell concentration of approximately 3 x 10 ６ The cells were seeded at a density of 5 × 10 cells / mL and culture was initiated at 23°C or 30°C. After 1 and 4 days of culture, 5 × 10 cells were seeded at a density of 5 × 10 cells / mL. ７ The cells were collected and the intracellular DNA content and triacylglycerol were quantified.

[0026] Similar experiments were carried out using the Ls100 strain, which is the parent strain of these temperature-sensitive mutants, as a control.

[0027] The intracellular triacylglycerol amounts of the Ls100, Ls76, Ls63, and Ls93 strains after 1 and 4 days of culture are shown in Figure 3. Extremely significant accumulation of lipids was confirmed in the Ls63 strain.

[0028] According to the oil-producing yeast mutant strain of the present invention, it is possible to synchronize and arrest a specific cell cycle with high oil production efficiency simply by changing the temperature, without the need for expensive chemical treatments or special equipment, and by using this mutant strain, it is possible to achieve increased oil accumulation.

[0029] The fat-and-oil-producing yeast mutant strain of the present invention can be used in the field of fat-and-oil production using yeast.

[0030] The Lipomyces starkeyi Ls76 strain was deposited internationally on July 11, 2023 at the National Institute of Technology and Evaluation (NPMD) Patent Microorganisms Depositary (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Room 122) under the accession number NITE BP-03941. The Lipomyces starkeyi Ls63 strain was deposited internationally on July 11, 2023 at the National Institute of Technology and Evaluation (NPMD) Patent Microorganisms Depositary (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Room 122) under the accession number NITE BP-03942. Lipomyces starkeyi Ls93 strain was deposited internationally on July 11, 2023 at the National Institute of Technology and Evaluation, National Patent Microorganism Depositary (NPMD) (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Room 122) under the accession number NITE BP-03943.

Claims

1. A Lipomyces starkeyi strain that undergoes synchronous arrest in the G1 phase by pre-cultivation at 23-26°C followed by main cultivation at 32-35°C.

2. The Lipomyces starkeyi strain according to claim 1, having the accession number NITE BP-03941.

3. A Lipomyces starkeyi strain which undergoes synchronous arrest in the G2 / M phase when cultured at 30-32°C after preliminary culture at 20-23°C.

4. The Lipomyces starkeyi strain according to claim 3, having an accession number of NITE BP-03942 or NITE BP-03943.

5. A method for inducing accumulation of fats and oils, comprising using the strain according to any one of claims 1 to 4.

6. A method for inducing accumulation of fats and oils, comprising the steps of pre-culturing the strain according to any one of claims 1 to 4 at 20 to 26°C, and then culturing it at 30 to 35°C.

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