Grease-producing yeast mutant strain capable of realizing cell cycle synchronization

Abstract

Provided is a grease-producing yeast mutant strain which can be synchronously stopped in a specific cell cycle at which the grease yield is high by temperature change. The mutant strain is a lipomyces starkeyi strain which is pre-cultured at 23-26 DEG C and then formally cultured at 32-35 DEG C and is synchronously stopped in a G1 phase, or is a lipomyces starkeyi strain which is pre-cultured at 20-23 DEG C and then formally cultured at 30-32 DEG C and is synchronously stopped in a G2 / M phase.

Description

TECHNICAL FIELD

[0001] The present application relates to a mutant strain of oleaginous yeast that can achieve cell cycle synchronization. BACKGROUND

[0002] As oleaginous yeast of the genus Lipomyces, which accumulates up to 70% of oil per single cell, the oil component thereof is close to palm oil, and thus is not only used as biofuel but also expected to be used as a substitute oil for food or cosmetics, and research thereof is rapidly advancing.

[0003] The present inventors found that the amount of oil accumulation increased by 1.4 times by synchronizing at the G1 phase of the cell cycle when advancing to clarify the molecular mechanism of oil production of oleaginous yeast (Non-Patent Literature 1).

[0004] However, the demand for oleaginous microorganisms with high oil production rate and methods of producing oil has continued to date without change.

[0005] Prior Art Documents Non-Patent Literature Non-Patent Literature 1: Morimoto Y. et al., Journal of Cell Science (2022) 135(16), jcs259996 SUMMARY

[0006] (I) Technical Problem to be Solved The technical problem of the present application is to provide a mutant strain of oleaginous yeast that can be synchronized to arrest at a specific cell cycle with high oil production rate by temperature change.

[0007] (II) Technical Solution The aforementioned technical problem can be solved by the present application as follows.

[0008] [1] A mutant strain of Lipomyces starkeyi which is synchronized to arrest at the G1 phase by formal culture at 32 to 35°C after preculture at 23 to 26°C.

[0009] [2] The mutant strain of Lipomyces starkeyi according to [1] which has the accession number NITE BP-03941.

[0010] [3] A mutant strain of Lipomyces starkeyi which is synchronized to arrest at the G2 / M phase by formal culture at 30 to 32°C after preculture at 20 to 23°C.

[0011] [4] The mutant strain of Lipomyces starkeyi according to [3] The strain has the accession number NITE BP-03942 or NITE BP-03943.

[0012] [5] A method for inducing lipid accumulation, characterized in that any one of the strains [1] to [4] is used.

[0013] [6] A method for inducing lipid accumulation, comprising: pre-culturing any one of the strains [1] to [4] at 20 to 26°C, followed by culturing at 30 to 35°C.

[0014] (III) Beneficial Effects According to the present invention, without the need for expensive chemical treatments or special machinery, it is possible to synchronize specific cell cycles that are arrested at high lipid yields simply by changing the temperature. By utilizing this mutant strain, it is possible to achieve an increase in lipid accumulation. Attached Figure Description

[0015] Figure 1 A graph illustrating the results of intracellular triglyceride quantification in the drug-based cell cycle synchronization of *Saccharomyces davidii*, as described in the reference example below.

[0016] Figure 2 Histograms showing the results of intracellular DNA content determination for Ls100, Ls76, Ls63, and Ls93 strains implemented in Example 2 described later, based on FACS (fluorescence-activated cell sorting).

[0017] Figure 3 A graph showing the results of intracellular triglyceride quantification of the Ls100, Ls76, Ls63, and Ls93 strains implemented in Example 3 described later. Detailed Implementation

[0018] (The lipid-accumulating yeast mutant strain of the present invention) The lipid-accumulating yeast mutant strain of this invention is *Saccharomyces davidii*. The mutant strain, after pre-culturing at normal temperature, can be brought to a state of cell cycle synchronization arrest if the temperature during culture is increased to a higher level than usual.

[0019] The first lipid-accumulating yeast mutant strain of the present invention is pre-cultured at 23~26℃, and then the temperature is raised to 32~35℃ for formal culture, thereby synchronizing and arresting at the G1 phase.

[0020] The second lipid-accumulating yeast mutant strain of the present invention is pre-cultured at 20-23°C, and then the temperature is raised to 30-35°C for formal culture, thereby synchronizing and arresting at the G2 / M phase.

[0021] As the first lipid-accumulating yeast mutant of the present application, Ls76 strain (Deposit No. NITE BP-03941) of Lipomyces starkeyi can be exemplified. As the second lipid-accumulating yeast mutant of the present application, Ls63 strain (Deposit No. NITE BP-03942) and Ls93 strain (Deposit No. NITE BP-03943) can be exemplified. These mutants exhibit higher lipid accumulation amount than the previously known Lipomyces genus, and can be used as effective lipid-producing microorganisms. The mycological characteristics of the Ls76 strain, Ls63 strain, and Ls93 strain are budding yeasts, which proliferate well at 23°C, but stop proliferation if the temperature exceeds 30°C.

[0022] (Lipid accumulation induction method of the present application) The lipid accumulation induction method of the present application can be performed according to the previously known lipid accumulation induction method, except that the lipid-accumulating yeast mutant of the present application is used, and the mutant is cultured under conditions capable of synchronously arresting the cell cycle (particularly, G1 phase or G2 / M phase).

[0023] For example, in the lipid accumulation induction method of the present application, preliminary culture is performed, and the culture is inoculated into a suitable lipid accumulation induction medium, whereby the main culture can be performed. The main culture can be performed at a culture temperature of 20 to 35°C, and is performed by shaking culture for about 1 to 7 days.

[0024] In the lipid accumulation induction method using the first lipid-accumulating yeast mutant of the present application, the preliminary culture is performed at 23 to 26°C, and the main culture is performed at 32 to 35°C, whereby G1 phase synchronization arrest can be achieved.

[0025] In the lipid accumulation induction method using the second lipid-accumulating yeast mutant of the present application, the preliminary culture is performed at 20 to 23°C, and the main culture is performed at 30 to 32°C, whereby G2 / M phase synchronization arrest can be achieved.

[0026] The preliminary culture described above can be performed using, for example, YPD medium.

[0027] In the present application, the lipid accumulation in the lipid-accumulating yeast can be confirmed and quantified using the previously known methods. For example, the method in which the lipid is dyed with oil red, and the photograph of the yeast under the microscope is taken and confirmed by the naked eye can be exemplified. The method in which the cell extract is prepared by dissolving or physically destroying the cell wall, and the lipid component present in the extract is separated and quantified can also be exemplified.

[0028] Examples Hereinafter, the present application will be specifically described by examples, but the scope of the present application is not limited by these examples.

[0029] Reference Example: Measurement of the amount of lipid accumulation in each cell cycle synchronization In order to synchronize the cell cycle of Starmerella bombicola , expensive drug treatment or special machines are required. In this reference example, using a wild strain isolated from soil, i.e., Starmerella bombicola NBRC1289 strain (obtained from NBRP (National BioResource Project)), the amount of triacylglycerol (TAG) (unit: mg / 10 8 cells) in Starmerella bombicola after synchronization to each cell cycle was measured.

[0030] (Synchronization arrest at S phase, or synchronization arrest at G2 / M phase) YPD medium (2% D-glucose, 1% yeast extract, 2% polypeptone) was used for preculturing at 26°C, and hydroxyurea (HU) or nocodazole (Noc) was added, and after 3.5 hours of culture, the cells were recovered. As described later, hydroxyurea can synchronize arrest at S phase, and nocodazole can synchronize arrest at G2 / M phase. The recovered cells were washed 3 times with sterilized water, and then transplanted into N medium (0.3% sucrose, 0.025% KH2PO4, 0.066% MgSO4·7H2O, trace element solution (FeCl3·6H2O 1.7 mg / L, MnSO4·6H2O 0.51 mg / L, ZnSO4·7H2O 4.5 mg / L)) so that the cell concentration became about 3 x 10 6 cells / mL, and cultured at 26°C for 18 hours. 5 x 10 7 cells were recovered, and triacylglycerol was quantified.

[0031] (Synchronization arrest at G1 phase) YPD medium was used for preculturing at 26°C, and nocodazole (Noc) was added, and after 3 hours of culture, the cells were recovered. After washing 3 times with sterilized water, it was transferred to YPD medium and cultured for 30 minutes, and then rapamycin (Rapa) was added and further cultured for 2.5 hours. As described later, rapamycin can synchronize arrest at G1 phase. It was washed 3 times with sterilized water, and transplanted into N medium so that the cell concentration became about 3 x 10 6 cells / mL, and cultured at 26°C for 18 hours. 5 x 10 7 cells were recovered, and triacylglycerol was quantified.

[0032] The results are shown in Figure 1 .

[0033] Figure 1 , Asy refers to N medium, HU refers to hydroxyurea, Noc refers to nocodazole, and Rapa refers to rapamycin.

[0034] Cells with hydroxyurea (HU) were synchronized to arrest at S phase, cells with nocodazole (Noc) were synchronized to arrest at G2 / M phase, and cells with rapamycin (Rapa) were synchronized to arrest at G1 phase.

[0035] Figure 1 Error bars (mean ± standard deviation) were calculated from independent cell culture [n = 16, 10, 14, 20 (number of experiments from left in the graph)]. Figure 1 "p" in means P < 0.001 (compared with N medium; Dunnett's multiple comparison test).

[0036] It was confirmed that the accumulation of triacylglycerol was the highest (about 1.4 times) when synchronized to arrest at G1 phase.

[0037] Example 1: Isolation and identification of oleaginous yeast mutant strains In this example, LslOO strain (deposited in NBRP) derived from Starmerella bombicola NBRC 1289 strain was subjected to UV treatment, and the colonies were transplanted to YPD agar medium and cultured under high temperature conditions (30°C and 32°C). It was confirmed whether the cell strain that grew poorly under high temperature conditions would still exhibit poor growth under high temperature conditions again. The cell strain that grew poorly under high temperature conditions was cultured under high temperature conditions, and whether it would be synchronized to arrest at a specific cell cycle was used as an index to perform screening by FACS analysis, and as a result, oleaginous yeast mutant strains that were synchronized to arrest at a cell cycle under high temperature conditions were successfully obtained. More specifically, one strain of cells that were synchronized to arrest at G1 phase (designated as Ls76 strain) and two strains of cells that were synchronized to arrest at G2 / M phase (designated as Ls63 strain and Ls93 strain) were obtained. In addition, about 100 strains that grew poorly under high temperature conditions were obtained from about 1700 colonies subjected to UV irradiation, and finally, three strains that were synchronized to arrest at a cell cycle were obtained.

[0038] Example 2: Synchronization of cell cycle arrest of oleaginous yeast mutant strains (Ls76 strain that was synchronized to arrest at G1 phase) YPD was used for pre-culture at 26°C, and after the temperature was raised to 32°C and cultured for 3.5 hours, the cells were recovered. After washing with sterilized water three times, the cells were transplanted to N medium so that the cell concentration would be about 3 x 10 6 cells / mL, and the culture was started at 26°C or 32°C. Every other day, 5 x 10 7 cells were recovered, and the intracellular DNA content and triacylglycerol were quantified.

[0039] (Ls63 strain and Ls93 strain synchronized and arrested at G2 / M phase) Using YPD culture, pre-culture was performed at 23°C, the temperature was raised to 30°C and cultured for 3.5 hours, and then the cells were recovered. The cells were washed with sterilized water three times, and then transplanted to N medium at a cell concentration of about 3 x 10 6 7 The cells were recovered at intervals of one day, and the intracellular DNA content and the triacylglycerol content were quantified.

[0040] The results of the intracellular DNA content measured by FACS (fluorescence-activated cell sorting) analysis are shown in Figure 2 .

[0041] In each column, the lower half indicates the DNA content at the start of 32°C culture when the culture temperature was changed from 26°C to 32°C, and the upper half indicates the DNA content 3.5 hours after the start of the 32°C culture.

[0042] For example, in the upper half of the LslOO strain, the left peak indicates the state before the start of DNA replication, and the right peak indicates the state after the end of DNA synthesis (i.e., 2-fold in terms of DNA content).

[0043] The obtained mutant strains exhibit the following properties: Ls76 strain (Deposit No. NITE BP-03941): After culture at 26°C, culture at 32°C for 3.5 hours, thereby synchronized and arrested at the G1 phase.

[0044] Ls63 strain (Deposit No. NITE BP-03942): After culture at 23°C, culture at 30°C for 3.5 hours, thereby synchronized and arrested at the G2 / M (1) phase.

[0045] Ls93 strain (Deposit No. NITE BP-03943): After culture at 23°C, culture at 30°C for 3.5 hours, thereby synchronized and arrested at the G2 / M (2) phase.

[0046] The G2 / M arrested cells Ls63 strain and Ls93 strain can start the cell cycle again by returning to low temperature (reversible strain), and have a higher value in cell biology.

[0047] Example 3: Time-dependent change in the amount of intracellular oil accumulated in the oleaginous yeast mutant strain ​In this embodiment, mutant strains of *Saccharomyces davidii* Ls76, Ls63, and Ls93 were used. By changing the culture medium and the culture temperature to a high temperature, the changes in intracellular DNA content and the amount of triglycerides accumulated in the mutant strains over time were observed at the G1 phase or G2 / M phase when cell proliferation was stopped.

[0048] For strain Ls76, pre-culture was performed using YPD culture base at 26°C, then the temperature was raised to 32°C and cultured for 3.5 hours before cell harvesting. The cells were washed three times with sterile water and then cultured at a concentration of approximately 3 × 10⁻⁶ cells / mL. 6 Cells were transplanted into N medium at a rate of 100 cells / mL and cultured at 26°C or 32°C. After 1 day and 4 days of culture, 5 × 10⁵ cells were recovered. 7 We analyzed individual cells and quantified intracellular DNA content and triglyceride levels.

[0049] For strains Ls63 and Ls93, pre-culture was performed using YPD culture base at 23°C, then the temperature was raised to 30°C and cultured for 3.5 hours before cell harvesting. The cells were washed three times with sterile water and then cultured at a concentration of approximately 3 × 10⁻⁶ cells / mL. 6 Cells were transplanted into N medium at a rate of 100 cells / mL and cultured at 23°C or 30°C. After 1 day and 4 days of culture, 5 × 10⁵ cells were recovered. 7 We analyzed individual cells and quantified intracellular DNA content and triglyceride levels.

[0050] The same experiment was conducted using the parent strain of the aforementioned temperature-sensitive mutant strain, namely strain Ls100, as a control.

[0051] Intracellular triglyceride levels in Ls100, Ls76, Ls63, and Ls93 strains after 1 day and 4 days of culture were shown in [the figures]. Figure 3 The Ls63 strain was found to have extremely significant oil accumulation.

[0052] The oil-producing yeast mutant strain of the present invention does not require expensive chemical treatments or special machinery; it can synchronize cell cycles that are arrested at a high oil yield simply by changing the temperature. By utilizing this mutant strain, an increase in oil accumulation can be achieved.

[0053] Industrial applicability The oil-producing yeast mutant strain of the present invention can be used in the field of oil production using yeast.

[0054] Collection Number Starch's oil yeast The Ls76 strain was internationally deposited on July 11, 2023, at the National Institute of Technology and Evaluation Patent Microorganisms Depository (NPMD) (2-5-8 Kamitsuma-Kamase, Kimitsu-shi, Chiba-ken, 292-0818, Japan) under Accession No. NITE BP-03941.

[0055] L. starkeyi The Ls63 strain was internationally deposited on July 11, 2023, at the National Institute of Technology and Evaluation Patent Microorganisms Depository (NPMD) (2-5-8 Kamitsuma-Kamase, Kimitsu-shi, Chiba-ken, 292-0818, Japan) under Accession No. NITE BP-03942.

[0056] L. starkeyi The Ls93 strain was internationally deposited on July 11, 2023, at the National Institute of Technology and Evaluation Patent Microorganisms Depository (NPMD) (2-5-8 Kamitsuma-Kamase, Kimitsu-shi, Chiba-ken, 292-0818, Japan) under Accession No. NITE BP-03943.

Claims

1. A strain of the yeast Lipomyces starkeyi, which is synchronized to arrest in the G1 phase by formal cultivation at 32 to 35°C after precultivation at 23 to 26°C.

1. A strain of the yeast Lipomyces starkeyi, which is synchronized to arrest in the G1 phase by formal cultivation at 32 to 35°C after precultivation at 23 to 26°C.

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

3. A strain of the yeast Lipomyces starkeyi, which is synchronized to arrest at the G2 / M phase by formal cultivation at 30 to 32°C after precultivation at 20 to 23°C.

3. A strain of the yeast Lipomyces starkeyi, which is synchronized to arrest at the G2 / M phase by formal cultivation at 30 to 32°C after precultivation at 20 to 23°C.

4. The L. starkeyi of claim 3 Strain having accession number NITE BP-03942 or NITE BP-03943.

5. A method for inducing lipid accumulation, characterized by, The strain according to any one of claims 1 to 4 is used.

6. A method for inducing lipid accumulation, comprising: The strain according to any one of claims 1 to 4 is pre-cultured at 20 to 26°C, and then cultured at 30 to 35°C.

Images