Oil-and-fat-producing yeast mutant strain that excessively accumulates oil and fat

Abstract

Provided is a mutant strain of a fat-producing yeast having a higher fat accumulation than conventional fat-producing yeasts belonging to the genus Saccharomyces. The mutant strain is a lipomyces starkeyi strain of which the preservation number is NITE BP-03940.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480042634.5 (22) Application Date 2024.08.08 (30) Priority Data 2023-129032 2023.08.08 JP (85) PCT International Application Entering National Phase Date 2025.12.24 (86) PCT International Application Application Data PCT / JP2024 / 028356 2024.08.08 (87) PCT International Application Publication Data WO2025 / 033480 JA 2025.02.13 (83) Biological Preservation Information NITE BP-03940 2023.07.11 (71) Applicant's School / Legal Entity: Teikyo University, Japan (72) Inventor: Yuko Takayama (74) Patent Agency: Beijing Luhao Intellectual Property Agency Co., Ltd., 11002 Patent Attorneys: Qin Li, Shunxing Xie (51) Int.Cl. C12N 1 / 165 (2026.01) C12P 7 / 6463 (2022.01) C12R 1 / 645 (2006.01) (54) Invention Title: Lipid-Producing Yeast Mutant with Excessive Lipid Accumulation (57) Abstract: This invention provides a lipophilic yeast mutant with a higher lipid accumulation than previously known lipophilic yeasts. The mutant is a *Lipophila stearothermii* strain with accession number NITE BP-03940. Claims 1 page, Description 3 pages, Drawings 1 page, CN 121399246 A 2026.01.23 CN 1 21 39 92 46 A 1. A *Saccharomyces davidii* strain, with accession number NITE BP-03940. 2. A method for inducing lipid accumulation, characterized in that the strain described in claim 1 is used. 3. A method for inducing lipid accumulation, comprising: a step of pre-culturing the strain described in claim 1 to the stationary phase; and a step of transplanting the cells obtained in the step into a culture medium for formal culture and performing formal culture. Claims 1 / 1 page 2 CN 121399246 A Lipid-producing yeast mutant strain with excessive lipid accumulation Technical Field

[0001] This invention relates to a lipid-producing yeast mutant strain with excessive lipid accumulation. Background Art

[0002] As an oil-producing yeast, the genus Lipomyces accumulates up to 70% oil per single cell. Its oil composition is close to that of palm oil. Therefore, it is not only used as a biofuel, but is also expected to be used as a substitute oil in food or cosmetics, and research is being rapidly promoted.

[0003] In order to clarify the molecular mechanism of lipid production by lipophilic yeast, the inventors of this application developed a cell biology research method. In this process, a lipid accumulation induction medium that can reduce the cost of the culture medium itself and increase the amount of lipid accumulation in the cells was developed (Patent Document 1).

[0004] However, the requirements for lipid-producing microorganisms and lipid production methods with high lipid yield have not changed and have continued to this day.

[0005] Prior Art Documents Patent Document 1: Japanese Patent Application Publication No. 2021-058103 Summary of the Invention

[0006] (I) Technical Problem to be Solved The technical problem of the present invention is to provide a lipophilic yeast mutant strain with a higher lipid accumulation than previously known lipophilic yeasts, and in particular, to provide a lipophilic yeast mutant strain that shows a higher lipid accumulation under specific culture conditions.

[0007] (II) Technical Solution The aforementioned technical problem can be solved by the following invention.

[0008] [1] A strain of *Staphylococcus sylvatica*, with the accession number NITE BP-03940.

[0009] [2] A method for inducing lipid accumulation, characterized in that the strain described in [1] is used.

[0010] [3] A method for inducing lipid accumulation, comprising: a step of pre-culturing the strain described in [1] to a stable phase; and a step of transplanting the cells obtained in the aforementioned step into a culture medium for formal culture and performing formal culture.

[0011] (III) Beneficial Effects According to the present invention, a lipid-producing yeast mutant strain with a higher lipid accumulation than conventionally known lipid yeasts can be provided. Furthermore, by using the mutant strain of the present invention, a more effective method for inducing lipid accumulation can be provided. Brief Description of the Drawings

[0012] Figure 1: The graph on the left side of Figure 1 shows the amount of triglycerides (TAG) per cell (unit: μg / cell) when cultured under normal culture conditions, and the graph on the right side of Figure 1 shows the amount of triglycerides per cell when cultured under specific culture conditions. The bar charts on the left side of each chart (days 0, 1, 2, 3, and 4 from left to right) represent the control results, while the bar charts on the right side (same as above) represent the results of the lipophilic yeast mutant strain of the present invention. Detailed Embodiments

[0013] (Lipophilic Yeast Mutant Strain of the Present Invention) The lipophilic yeast mutant strain of the present invention is *Saccharomyces davidii* Ls66 strain (accession number NITE BP-03940), which exhibits a higher lipophilic accumulation than previously known lipophilic yeasts and can be used as an effective lipophilic bacterium. The mycological characteristics of the Ls66 strain are as follows: A budding yeast. It proliferates well in YPD medium (composition referred to in the examples described later).It is a diploid cell, which cannot be induced to form spores using the "spore-forming medium for natural yeast" and "spore-forming method for natural yeast" disclosed in the Japanese patent applications (Japanese Patent Application Nos. 2019-182882 and 2021-058102) of the inventor of this application. Furthermore, the "spore-forming medium for natural yeast" consists of water and agar, and the "spore-forming method for natural yeast" includes a step of culturing on an agar medium composed of water and agar.

[0014] (Lipid Accumulation Induction Method of the Invention) The lipid accumulation induction method of the present invention can be implemented according to conventionally known lipid accumulation induction methods, except that it uses the lipid-producing yeast mutant strain of the present invention for culturing.

[0015] For example, in the lipid accumulation induction method of the present invention, a pre-culturing is performed, and the culture is inoculated into a suitable lipid accumulation induction medium, thereby enabling formal culturing. For example, it can be carried out at a culturing temperature of 20~35°C for approximately 1~7 days with shaking.

[0016] Furthermore, by culturing the lipophilic yeast mutant strain of the present invention under specific culture conditions, lipophilic production can be achieved more efficiently.

[0017] For example, for pre-culture, culturing with YPD medium and inoculating with N medium (composition referred to in the examples described below) can produce lipophilic substances. Moreover, in a preferred embodiment of the present invention, by performing pre-culture on YPD medium to the stationary phase, the lipophilic production can be further increased.

[0018] In the present invention, the accumulation of lipophilic substances in the lipophilic yeast can be confirmed and quantified using conventionally known methods. For example, methods include: staining the lipophilic substances with a staining solution such as Oil Red, taking pictures of the yeast under a microscope, and confirming them with the naked eye; and methods for separating and quantifying the lipophilic components present in the cell extract by dissolving or physically destroying the cell wall (e.g., commercially available assay kits (e.g., Triglyceride E Test Wako).

[0019] Examples Hereinafter, the present invention will be specifically described by way of examples, but these examples do not limit the scope of the present invention.

[0020] Example 1: Isolation and Identification of Oil-Producing Yeast Mutant Strains In this example, Ls100 strain (preserved in NBRP), a derivative of the wild-type strain *Saccharomyces davidii* strain NBRC1289 (obtained from NBRP (National BioResource Project)), was used. After ultraviolet treatment, the strain was screened based on the amount of triglyceride accumulation. As described below, even under normal culture conditions, the amount of oil accumulation was greater than that of strains NBRC1289 and Ls100. In addition, under specific culture conditions, an oil-producing yeast mutant strain (named Ls66 strain) with even greater oil accumulation was successfully obtained.

[0021] Example 2: Determination of lipid accumulation in yeast in lipogenic yeast mutant strain. Specification, page 2 / 3, CN 121399246 A. In this example, *Saccharomyces davidii* strain Ls100 was used as a control to determine the time-dependent change in lipid accumulation in *Saccharomyces davidii* strain Ls66 (accession number NITE BP-03940), which is the lipogenic yeast mutant strain of the present invention. The experiment was conducted under normal culture conditions (4 independent cultures; N=4) and the specific culture conditions of the present invention (2 independent cultures; N=2).

[0022] (Normal culture conditions) For normal culture conditions, YPD medium (2% D-glucose, 1% yeast extract, 2% peptone) was used for pre-culture at 26°C, and cells in the logarithmic growth phase were recovered. The cells were washed three times with sterile 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)) at a cell concentration of approximately 3 × 106 cells / mL, and cultured at 26°C. Every other day, 5 × 107 cells were recovered for triglyceride quantification.

[0023] (Specific culture conditions in this invention) For the specific culture conditions in this invention, YPD culture was used for pre-culture at 26°C until a stable state (3 × 108 cells / mL), and the cells were recovered. The cells were washed three times with sterile water, and then transplanted into N medium at a cell concentration of approximately 4 × 106 cells / mL, and cultured at 26°C. Every other day, 5 × 10⁷ cells were recovered for triglyceride quantification.

[0024] The results are shown in Figure 1.The graph on the left of Figure 1 shows the amount of triglycerides (TAG) per cell (unit: μg / cell) under normal culture conditions, and the graph on the right of Figure 1 shows the amount of triglycerides per cell under specific culture conditions. The bar graphs on the left of each graph (at the start of culture, after 1 day, after 2 days, after 3 days, after 4 days) are the control results, and the bar graphs on the right (same as above) are the results of the oil-producing yeast mutant strain of the present invention.

[0025] Even under normal culture conditions (cells on day 0 of the start of culture are in the logarithmic growth phase), the amount of oil accumulated was about twice that of the control Ls100 strain (graph on the left of Figure 1).

[0026] In addition, under specific culture conditions (cells on day 0 of the start of culture are in the stationary phase), the amount of oil accumulated was about 2.5 times that of the control Ls100 strain, confirming a further increase in the amount of oil accumulated (graph on the right of Figure 1).

[0027] It has been confirmed that the oil-producing yeast mutant strain of the present invention, by changing only the strain, can improve the oil yield compared to the Ls100 strain to date. Furthermore, by changing the culture conditions, the oil yield can be further improved.

[0028] Industrial Applicability: The oil-producing yeast mutant strain of the present invention can be used in the field of oil production using yeast.

[0029] Accession Number: Oil-producing yeast strain Ls66 was internationally deposited on July 11, 2023, at the Patent and Microbial Collection Center of the Japan Institute for Product Evaluation and Research (NPMD) (Room 122, 2-5-8, Kamisamaashi, Kisarazu City, Chiba Prefecture 292-0818) with accession number NITE BP-03940. Specification 3 / 3 pages 5 CN 121399246 A Figure 1 Specification Drawing 1 / 1 page 6 CN 121399246 A

Claims

1. A type of oily yeast called *Stelleria stolonifera* The strain has the accession number NITE BP-03940.

2. A method for inducing lipid accumulation, characterized in that, Use the strain described in claim 1.

3. A method for inducing lipid accumulation, comprising: The process of pre-culturing the strain described in claim 1 to the stable phase; The process of transplanting the cells obtained in the above steps into a culture medium for formal culture and then performing formal culture.

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