Oil-and-fat-producing yeast mutant strain capable of cell cycle synchronization

Abstract

To provide an oil-and-fat-producing yeast mutant strain that can be stopped synchronously with temperature change, within a specific cell cycle having high oil-and-fat production efficiency due to temperature change.SOLUTION: The mutant strain is a Lipomyces starkeyi bacterial strain that, as a consequence of undergoing main culture at 32 to 35°C after pre-culture at 23 to 26°C, is stopped synchronously with G1 phase, or a Lipomyces starkeyi bacterial strain that, as a consequence of undergoing main culture at 30 to 32°C after pre-culture at 20 to 23°C, is stopped synchronously with G2 / M phase.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an oleaginous yeast mutant capable of synchronizing the cell cycle. [Background technology]

[0002] The oleaginous yeast species Lipomyces is capable of accumulating up to 70% oleaginous matter per cell. Because the composition of this oleaginous matter is similar to that of palm oil, it is expected that it will be used not only as a biofuel but also as an alternative oleaginous matter for food and cosmetics, and research into this oleaginous yeast is currently progressing at a rapid pace. The present inventors have attempted to elucidate the molecular mechanism of lipid production in Lipomyces and have found that synchronization at the G1 phase of the cell cycle increases lipid accumulation by 1.4 times (Non-Patent Document 1). However, there remains a continuing demand for oil-producing microorganisms and methods for producing oils with high oil production efficiency. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Morimoto Y. et al., Journal of Cell Science (2022) 135 (16), jcs259996 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide an oil-producing yeast mutant that can be synchronously arrested at a specific cell cycle that has high oil-producing efficiency by changing the temperature. [Means for solving the problem]

[0005] The above problems can be solved by the present invention described below. [1] Lipomyces starchii, which is synchronously arrested in the G1 phase by pre-culturing at 23-26°C and then culturing at 32-35°C.Lipomyces starkeyi ) strain. [2] Lipomyces starkeyi ( Lipomyces starkeyi ) strain. [3] Lipomyces starchii, which is synchronously arrested in the G2 / M phase by pre-culturing at 20-23°C and then culturing at 30-32°C. Lipomyces starkeyi ) strain. [4] The Lipomyces starchii strain of [3], whose accession number is NITE BP-03942 or NITE BP-03943. Lipomyces starkeyi ) strain. [5] A method for inducing fat accumulation, comprising using any one of the strains described in [1] to [4]. [6] A method for inducing fat accumulation, comprising the steps of pre-culturing any one of the strains described in [1] to [4] at 20 to 26°C, and then culturing it at 30 to 35°C. Effect of the Invention

[0006] According to the present invention, it is possible to synchronize and arrest a specific cell cycle that has high efficiency for producing fats and oils by simply changing the temperature, without the need for expensive chemical treatments or special equipment, and by utilizing this mutant strain, it is possible to achieve increased accumulation of fats and oils. [Brief description of the drawings]

[0007]

Figure 1

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Figure 3

[0008] (Oleaf-accumulating yeast mutant of the present invention) The lipid-accumulating yeast mutant of the present invention is Lipomyces starchii ( Lipomyces starkeyi ) mutant, and can be synchronously arrested at a specific cell cycle by raising the culture temperature higher than normal after preliminary incubation at normal temperature. The first lipid-accumulating yeast mutant strain of the present invention can be synchronously arrested in the G1 phase by pre-culturing at 23 to 26° C., followed by main culturing at an elevated temperature of 32 to 35° C. The second lipid-accumulating yeast mutant strain of the present invention can be synchronously arrested in the G2 / M phase by pre-culturing at 20 to 23°C, followed by main culturing at an elevated temperature of 30 to 35°C.

[0009] The first oleaginous yeast mutant of the present invention may be Lipomyces starkeyi Ls76 strain (Accession No. NITE BP-03941). The second oleaginous yeast mutant of the present invention may be Ls63 strain (Accession No. NITE BP-03942) and Ls93 strain (Accession No. NITE BP-03943). These mutants accumulate more oil than the conventionally known Lipomyces genus and can be used as efficient oil-producing bacteria. The mycological properties of the Ls76, Ls63, and Ls93 strains are budding yeasts, which grow well at 23°C but stop growing at temperatures above 30°C.

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

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

[0012] In the present invention, the accumulation of fats and oils in the oil-producing yeast can be confirmed and quantified by a conventionally known method, such as a method of staining the fats and oils with a staining solution such as oil red, taking a picture 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 fats and oils present in the extract. EXAMPLES

[0013] The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited thereto.

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

[0015] (S-phase arrest or G2 / M-phase arrest) The cells were pre-cultured at 26°C using YPD medium (2% D-glucose, 1% yeast extract, 2% hypopeptone), and then hydroxyurea (HU) or nocodazole (Noc) was added and cultured for 3.5 hours, after which the cells were harvested. As described below, hydroxyurea can arrest the cells in the S phase, and nocodazole can arrest the cells in the G2 / M phase. The harvested cells were washed three times with sterile water and then cultured in N medium (0.3% sucrose, 0.025% KH2PO4, 0.066% MgSO4·7H2O, trace element solution (FeCl3·6H2O 1.7mg / L, MnSO4·6H2O 0.51mg / L, ZnSO4·7H2O 4.5mg / L)) at a cell concentration of approximately 3x10 6 The cells were then transferred to a concentration of 5x10 cells / mL and cultured at 26°C for 18 hours. 7 Cells were harvested and triacylglycerol was quantified.

[0016] (Synchronization stopped at G1) 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 harvested. 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 synchronously arrest the cells in the G1 phase. The cells were washed three times with sterile water, and the cell concentration was adjusted to approximately 3x10 6 The cells were then transferred to a concentration of 5x10 cells / mL and cultured at 26°C for 18 hours. 7 Cells were harvested and triacylglycerol was quantified.

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

[0018] Example 1: Isolation and identification of oleaginous yeast mutants In this example, the Ls100 strain (already deposited with NBRP) derived from the Lipomyces Starkey NBRC1289 strain was used, and after ultraviolet treatment, colonies were transferred to YPD agar medium and cultured under high temperature conditions (30°C and 32°C). The cell strains that grew poorly under high temperature conditions were again examined to see whether they showed poor growth under high temperature conditions. The strains that grew poorly under high temperature conditions were cultured under high temperature conditions, and screened by FACS analysis using whether they stopped at a specific cell cycle as an indicator, and an oil-producing yeast mutant strain that stopped the cell cycle under high temperature conditions was successfully obtained. More specifically, one cell strain (named Ls76 strain) that synchronously stopped at the G1 phase and two cell strains (named Ls63 strain and Ls93 strain) that synchronously stopped at the G2 / M phase were obtained. In addition, about 100 strains that grew poorly under high temperature conditions were obtained from about 1700 colonies that emerged after UV irradiation, and finally, three strains that synchronously stopped the cell cycle were obtained.

[0019] Example 2: Cell cycle cessation in oleaginous yeast mutants (Ls76 strain arrested in G1 phase) The cells were pre-cultured at 26°C in YPD medium, and then the temperature was raised to 32°C for 3.5 hours, after which the cells were harvested. After washing three times with sterilized water, the cells were added to N medium at a cell concentration of approximately 3x10 6 The cells were then transferred to the cells at a concentration of 5x10 cells / mL and cultured at 26°C or 32°C. 7The cells were harvested and the intracellular DNA content and triacylglycerol were quantified.

[0020] (Ls63 and Ls93 strains arrested in G2 / M phase) The cells were pre-cultured at 23°C in YPD medium, and then the temperature was raised to 30°C for 3.5 hours, after which the cells were harvested. After washing three times with sterilized water, the cells were added to N medium at a cell concentration of approximately 3x10 6 The cells were then transferred to the cells at a concentration of 5x10 cells / mL and cultured at 26°C or 30°C. 7 The cells were harvested and the intracellular DNA content and triacylglycerol were quantified.

[0021] The results of measuring the intracellular DNA content by fluorescence-activated cell sorting (FACS) analysis are shown in FIG. In each column, the lower row indicates the DNA content at the start of 32°C culture after changing from 26°C culture to 32°C, and the upper row indicates the DNA content 3.5 hours after the start of the 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 began, and the right peak represents the state after DNA synthesis was completed (i.e., DNA content doubled).

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

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

[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 added to N medium at a cell concentration of approximately 3x10 6 The cells were then transferred to the cells at a concentration of 5x10 cells / mL and cultured at 26°C or 32°C. After 1 and 4 days of culture, the cells were transferred to the cells at a concentration of 5x10 cells / mL and cultured at 26°C or 32°C. 7 The cells were harvested 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 3x10 6 The cells were then transferred to the cells at a concentration of 5x10 cells / mL and culture was started at 23°C or 30°C. After 1 and 4 days of culture, 7 The cells were harvested 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 that has high oil production efficiency by simply changing the temperature, without the need for expensive chemical treatments or special equipment, and by utilizing this mutant strain, it is possible to achieve increased accumulation of oil. [Industrial Applicability]

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

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

Claims

1. A Lipomyces starkeyi strain which undergoes synchronous arrest in the G1 phase by primary culture at 32-35°C after preliminary culture at 23-26°C.

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

3. A Lipomyces starkeyi strain which undergoes synchronous arrest in the G2 / M phase by primary culture 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 fat and oil accumulation, comprising using the strain according to any one of claims 1 to 4.

6. A step of pre-culturing the strain according to any one of claims 1 to 4 at 20 to 26°C, followed by culturing at 30 to 35°C. A method for inducing fat accumulation comprising the steps of:

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