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

Abstract

To provide an oil-and-fat-producing yeast mutant strain having a higher oil and fat accumulation level than that of a conventionally known genus Lipomyces.SOLUTION: The mutant strain is a Lipomyces starkeyi bacterial strain with Accession Number NITE BP-03940.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an oleaginous yeast mutant that overaccumulates oleaginous substances. [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 inventor has developed cell biological research techniques to elucidate the molecular mechanism of lipid production in Lipomyces, and in the process developed an lipid accumulation-inducing medium that can reduce the cost of the medium itself and increase the amount of lipid accumulated in the cells (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] [Patent documents]

[0003] [Patent Document 1] Patent Publication No. 2021-058103 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide an oleaginous yeast mutant that accumulates a greater amount of oil than known Lipomyces species, in particular, an oleaginous yeast mutant that accumulates a greater amount of oil under specific culture conditions. [Means for solving the problem]

[0005] The above problems can be solved by the present invention described below. [1] Lipomyces starkeyi (accession number NITE BP-03940) Lipomycesstarkeyi ) strain. [2] A method for inducing fat accumulation, characterized by using the strain of [1]. [3] Pre-culturing the strain of [1] to stationary phase; A step of transplanting the cells obtained in the previous step into a medium for main culture and carrying out main culture. A method for inducing fat accumulation comprising the steps of: Effect of the Invention

[0006] According to the present invention, it is possible to provide an oleaginous yeast mutant strain that accumulates more oils and fats than the conventionally known Lipomyces yeast strains. Furthermore, by using the mutant strain of the present invention, it is possible to provide a more efficient method for inducing accumulation of oils and fats. [Brief description of the drawings]

[0007]

Figure 1

[0008] (Oleaf-accumulating yeast mutant of the present invention) The lipid-accumulating yeast mutant of the present invention is Lipomyces starchii ( Lipomyces starkeyi ) Ls66 strain (accession number NITE BP-03940), which accumulates more fats and oils than the conventionally known Lipomyces species, and can be used as an efficient fat-and-oil producing bacterium. The mycological properties of the Ls66 strain are as follows: It is a budding yeast. It grows well in YPD medium (see the examples below for the composition). It is a diploid cell, and spore formation cannot be induced by the "natural yeast spore formation medium" and "natural yeast spore formation method" disclosed in the patent application (Japanese Patent Application No. 2019-182882, Japanese Patent Publication No. 2021-058102) in which the present inventor is the inventor. The "natural yeast spore formation medium" is composed of water and agar, and the "natural yeast spore formation method" includes a process of culturing on an agar medium composed of water and agar.

[0009] (Method of inducing fat accumulation of the present invention) The method for inducing fat / oil accumulation of the present invention can be carried out in accordance with a conventionally known method for inducing fat / oil accumulation, except that the fat / oil-producing yeast mutant of the present invention is used for cultivation. 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, for example, 20 to 35°C, and the culture may be shaken for about 1 to 7 days.

[0010] Furthermore, by culturing the fat-and-oil producing yeast mutant of the present invention under specific culture conditions, fats and oils can be produced more efficiently. For example, fats and oils can be produced by culturing the bacteria in a pre-culture in a YPD medium and inoculating the bacteria into an N medium (see the Examples below for the composition). In addition, in a preferred embodiment of the present invention, fats and oils can be produced by culturing the bacteria in a YPD medium until the bacteria reach the stationary phase.

[0011] 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. For example, the fats and oils can be stained with a stain such as Oil Red, and then photographed under a microscope to visually confirm the amount of fats and oils, or the fats and oils can be separated and quantified by dissolving or physically destroying the cell walls to prepare a cell extract and then isolating and quantifying the fats and oils present in the extract (for example, a commercially available measurement kit (e.g., Triglyceride E-Test Wako (Wako))). EXAMPLES

[0012] The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited thereto.

[0013] Example 1: Isolation and identification of oleaginous yeast mutants In this example, a wild-type strain, Lipomyces starchii ( Lipomyces starkeyi ) The Ls100 strain (already deposited at NBRP), a derivative of the NBRC1289 strain (obtained from NBRP (National BioResource Project)), was used and subjected to ultraviolet light treatment, followed by screening using the amount of triacylglycerol accumulated as an indicator. As will be described later, even under normal culture conditions, the strain accumulated more lipids than the NBRC1289 strain and the Ls100 strain, and furthermore, under specific culture conditions, an oleaginous yeast mutant strain (named Ls66 strain) that accumulated even more lipids was successfully obtained.

[0014] Example 2: Measurement of the amount of fat accumulated in yeast in a mutant strain of fat-producing yeast In this example, as a control, Lipomyces starchii ( Lipomyces starkeyi The lipid-producing yeast mutant of the present invention, Lipomyces starkeyi Ls66 strain (accession number NITE BP-03940), was used to measure the time-dependent change in lipid accumulation in yeast. The experiment was performed under normal culture conditions (independent culture performed four times; N=4) and the specific culture conditions of the present invention (independent culture performed two times; N=2).

[0015] (Normal culture conditions) For the normal culture conditions, YPD medium (2% D-glucose, 1% yeast extract, 2% hypopeptone) was used for pre-culture at 26°C, and the cells in the logarithmic growth phase were harvested. After washing three times with sterilized water, the cells were cultured in N medium (0.3% sucrose, 0.025% KH 2 PO 4 , 0.066% MgSO 4 7H 2 O, Trace element solution (FeCl 3 6H 2O 1.7 mg / L, MnSO 4 6H 2 O 0.51 mg / L, ZnSO 4 7H 2 O 4.5mg / L) with a bacterial 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. 7 Cells were harvested and triacylglycerol was quantified.

[0016] (Specific Culture Conditions in the Present Invention) In the specific culture conditions of the present invention, the bacteria are pre-cultured at 26°C using YPD medium, and then cultured until the bacteria reach a stationary state (3x10 8 The cells were collected and washed three times with sterile water, and then added to N medium at a cell concentration of approximately 4x10 6 The cells were then transferred to the cells at a concentration of 5x10 cells / mL and cultured at 26°C. 7 The cells were harvested and triacylglycerol was quantified.

[0017] The results are shown in Figure 1. The graph on the left side of Figure 1 shows the amount of triacylglycerol (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 triacylglycerol per cell when cultured under specific culture conditions. The bars on the left side of each graph (from the left, 0 days (at the start of culture), 1 day, 2 days, 3 days, and 4 days) show the results of the control, and the bars on the right side (same as above) show the results of the oil-producing yeast mutant strain of the present invention.

[0018] Even under normal culture conditions (cells were in the logarithmic growth phase on day 0 of culture), the amount of lipids accumulated was approximately twice as much as that of the control Ls100 strain (graph on the left side of Figure 1). Furthermore, under certain culture conditions (cells in stationary phase on day 0 of culture) the amount of lipid accumulated was approximately 2.5 times that of the control Ls100 strain, and a further increase in lipid accumulation was observed (graph on the right side of Figure 1). It has been confirmed that the fat-producing yeast mutant of the present invention can increase fat production efficiency compared to the conventional Ls100 strain simply by changing the strain, and that the fat production efficiency can be further increased by changing the culture conditions. [Industrial Applicability]

[0019] 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]

[0020] Lipomyces Starkey ( Lipomyces starkeyi The Ls66 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-03940.

Claims

1. A Lipomyces starkeyi strain having the accession number NITE BP-03940.

2. A method for inducing lipid accumulation, comprising using the strain according to claim 1.

3. Pre-cultivating the strain according to claim 1 to stationary phase; A step of transplanting the cells obtained in the previous step into a medium for main culture and carrying out main culture. A method for inducing fat accumulation comprising the steps of:

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