A novel high-oil-producing strain belonging to the genus Lipomyces, and a method for producing oils and fats using the high-oil-producing strain

Abstract

The present invention aims to obtain a novel mutant strain of yeast belonging to the genus Lipomyces that has superior oil-producing ability compared to known high-oil-producing mutant strains, and to provide a method for producing an oil substitute for palm oil using the novel mutant strain. [Solution] The novel high fat-producing mutant strain according to the present invention is a yeast belonging to the genus Lipomyces, and is a high fat-producing strain deposited with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation under Accession Number NITE BP-04113. This novel high fat-producing mutant strain was obtained by inducing mutation in the mutant N5 strain obtained by UV irradiation of a wild-type strain of Lipomyces starkeyi, CBS1807, and further inducing mutation in the mutant N5 strain by neutron beam irradiation, separating a low-density fraction by Percoll density gradient centrifugation, and repeatedly culturing the resulting strain.

Description

[Technical Field]

[0001] The present invention relates to a novel high fat / oil producing strain belonging to the genus Lipomyces, and a method for producing fat / oil using the high fat / oil producing strain. [Background technology]

[0002] Palm oil is a vegetable oil that is solid at room temperature and is obtained from the fruit of the oil palm. Palm oil is characterized by its high saturated fatty acid content, with palmitic acid and oleic acid accounting for approximately 80% of the fatty acids. In addition to being used as a cooking oil, palm oil is also known to be used as a raw material for margarine, shortening, and soap. Palm oil is also used as a frying oil for snack foods such as instant fried noodles and potato chips.

[0003] Palm oil is the most widely produced vegetable oil in the world, with 62.56 million tons produced worldwide in 2015. Oil palm is a plant that grows in hot and humid tropical regions and is native to West Africa and Central and South America, but currently over 80% of palm oil production occurs in Malaysia and Indonesia. Because oil palm bears fruit throughout the year, the yield per unit area is much higher than other vegetable oil sources, and it can be produced 8 to 10 times more than soybean oil or rapeseed oil. As a result, palm oil is cheaper than other vegetable oils and can be supplied stably, so many countries import it.

[0004] Oil palms can only grow in the hot and humid tropical regions directly under the equator, but these conditions overlap with the distribution of tropical rainforests. As a result, the development of oil palm plantations requires the clearing of tropical rainforests, and large areas of tropical rainforest are lost each year due to clearing. Large-scale forest fires also occur during plantation development. This loss of tropical rainforests puts the rare wildlife that live there at risk of extinction.

[0005] Furthermore, with the rapid expansion of demand for palm oil, workers are being forced into poor working conditions and land development has led to conflicts between local residents and developers. Palm oil is not only a global environmental problem, but also a source of labor and human rights issues.

[0006] For these reasons, in recent years, methods for producing alternative oils to palm oil using microorganisms have been explored. Lipomyces yeast, an oil-producing yeast, is known to produce oils with fatty acid compositions similar to those of palm oil. The oils produced by Lipomyces yeast also have physical properties similar to those of palm oil, and are therefore expected to be alternatives to palm oil from the perspectives of environmental risk, climate change risk, and sustainability.

[0007] In methods for producing alternative oils using oil-producing yeast, because the amount of oil produced by wild-type Lipomyces starkeyi is insufficient, it has also been considered to mutate the wild-type strain to obtain a high-oil-producing strain (high-oil-accumulating strain) that produces a large amount of oil. Non-Patent Documents 1 and 2 disclose that mutations are induced in CBS1807, a wild-type strain of Lipomyces starkeyi, by ethyl methyl sulfonic acid or UV irradiation, and the high-oil-producing strain is repeatedly concentrated by density gradient centrifugation to obtain a concentrated fraction of the high-oil-producing strain.

[0008] Patent Document 1 discloses a method for producing a high oil-producing strain by subjecting an oil-producing yeast belonging to the genus Lipomyces or Rhodosporidium to density gradient centrifugation using a reagent consisting of silicic acid colloid particles coated with polyvinylpyrrolidone. Patent Document 1 also discloses treating the yeast with the mutagen ethyl methanesulfonate (EMS) or treating the yeast with UV light, and then subjecting the yeast to density gradient centrifugation using a reagent consisting of silicic acid colloid particles coated with polyvinylpyrrolidone. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent No. 7082340 [Non-patent literature]

[0010] [Non-Patent Document 1] Yamazaki H, Kobayashi A, Ebina S, et al. (2019). Highly selective isolation and characterization of Lipomyces starkeyi mutants with increased production of triacylglycerol. Applied Microbiology and Biotechnology. 103, 6297-6308. [Non-patent document 2] Hiroaki Takaku et al., Isolation and characterization of Lipomyces starkeyi mutants with greatly increased lipid productivity following UV irradiation, J Biosci Bioeng., 131(6), 613-621, (2021) Summary of the Invention [Problem to be solved by the invention]

[0011] The present inventors induced mutations in the wild-type Lipomyces starkeyi strain CBS1807 by UV irradiation, resulting in the N5 strain, a high-oil-producing strain. The present inventors also induced further mutations in the N5 strain by UV irradiation, resulting in a new high-oil-producing strain, N5-15 (deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation under accession number NITE BP-03796). However, for the industrial production of palm oil substitutes using oleaginous yeast, new strains with higher oil production rates and oil conversion efficiencies than N5-15 are needed. The present invention aims to obtain a high-oil-producing yeast strain belonging to the genus Lipomyces that produces a greater amount of oil (oil accumulation) than N5-15, and to provide a method for producing palm oil substitutes using the high-oil-producing strain. [Means for solving the problem]

[0012] When obtaining the N5-15 strain, the inventors induced mutations in the N5 strain by UV irradiation, but they then investigated whether it would be possible to obtain a new mutant strain that produces more oils and fats than the N5-15 strain by inducing mutations through neutron irradiation, which led to the completion of the present invention.

[0013] Specifically, the present invention provides: The present invention relates to a yeast strain belonging to the genus Lipomyces that is highly oil-producing and has been deposited with the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under accession number NITE BP-04113.

[0014] The high oil-producing strain (Q97 strain, described below), which has been entrusted to the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation under accession number NITE BP-04113, was obtained by inducing mutations in the N5 strain by neutron irradiation, separating the upper layer, which is a low-density fraction, using Percoll density gradient centrifugation, concentrating the high oil-producing strain by repeated cultivation, and then isolating and screening it.

[0015] The present invention also relates to a method for producing fats and oils using a high fat and oil-producing strain of an oleaginous yeast belonging to the genus Lipomyces, which is deposited with the National Institute of Technology and Evaluation Patent Microorganisms Depositary under Accession Number NITE BP-04113. [Effects of the Invention]

[0016] The oil-producing yeast of the present invention can produce palm oil substitutes more efficiently than known oil-producing yeasts belonging to the genus Lipomyces and the N5-15 strain, and as a result, the method for producing oils and fats of the present invention can produce palm oil substitutes at lower cost than conventional production methods using known oil-producing yeasts. [Brief explanation of the drawings]

[0017] [Figure 1] Photographs showing changes in the bacterial cell layer after density gradient centrifugation. WT indicates the wild-type strain, K14 indicates the K14 strain, and UV indicates the wild-type strain treated with UV. [Figure 2] Micrographs of strains N1 to N9 and the control strain are shown. [Figure 3] 1 is a graph showing the cell concentrations of the N1, N3, N5, N8 and control strains during 3 days of culture. [Figure 4] A graph showing the mean particle size measured over time for each strain is shown. [Figure 5] Microscopic photographs of each strain on day 3 of culture are shown. [Figure 6] A graph showing the glucose concentration in the medium over time for each strain is shown. [Figure 7] The graph shows the amount of triglyceride (TAG) per medium measured over time for each strain. [Figure 8] The graph shows the amount of triglyceride (TAG) measured over time per 10 8 cells for each strain. [Figure 9] Photographs showing changes in the bacterial cell layer after density gradient centrifugation. WT indicates the wild-type strain, N5 indicates the N5 strain, and UV indicates the UV-treated N5 strain. [Figure 10] 1 shows a graph measuring the cell concentrations of the N5-1 to N5-16 strains and the control strain on day 3 of culture. [Figure 11] The graph shows the amount of glucose consumed by the N5-1 to N5-16 strains and the control strain on the third day of culture. [Figure 12] 1 shows a graph measuring triglyceride (TAG) per medium of the N5-1 to N5-16 strains and the control strain on day 3 of culture. [Figure 13] 1 shows a graph of the triglyceride (TAG) measured per 10 8 cells of the N5-1 to N5-16 strains and the control strain on day 3 of culture. [Figure 14] 1 is a graph showing the cell concentrations of the N5-2, N5-13, and N5-15 strains and the control strain during 6 days of culture. [Figure 15] 1 is a graph showing the average particle diameters of the N5-2 strain, the N5-13 strain, the N5-15 strain, and the control strain during 6 days of culture. [Figure 16] Microscopic photographs of each strain on day 6 of culture are shown. [Figure 17] 1 is a graph showing the glucose concentrations in the media of the N5-2, N5-13, and N5-15 strains and the control strain during 6 days of culture. [Figure 18] 1 is a graph showing triglycerides (TAG) per medium of the N5-2, N5-13, and N5-15 strains and the control strain during 6 days of culture. [Figure 19] 1 is a graph showing triglycerides (TAG) per 10 8 cells of the N5-2, N5-13, and N5-15 strains and the control strain during 6 days of culture. [Figure 20] 1 is a graph showing hypothetical fat and oil yields of the N5 strain, the N5-15 strain, and the Q1 to Q100 strains (relative values ​​assuming that the hypothetical fat and oil yield of the N5-15 strain is 1). [Figure 21] 1 is a graph showing the TAG amount (per medium) of 10 candidate high oil-producing strains and control strains (N5 strain and N5-15 strain). [Figure 22]1 is a graph plotting the relationship between the relative bacterial concentration (a relative value when the bacterial concentration of the N5-15 strain is set to 1) and the relative particle size (a relative value when the particle size of the N5-15 strain is set to 1). [Figure 23] 1 is a graph showing the amount of TAG per medium of the N5 strain, the N5-15 strain, and three candidate strains. [Figure 24] 1 shows micrographs of the N5 strain, the N5-15 strain, and the Q97 strain at the end of the culture. [Figure 25] 1 is a graph showing the amount of TAG per 10 8 cells of the N5 strain, the N5-15 strain, and the Q97 strain. [Figure 26] 1 is a graph plotting the relationship between the number of days cultured and the bacterial concentration of the N5 strain, the N5-15 strain, and the Q97 strain. DETAILED DESCRIPTION OF THE INVENTION

[0018] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below with reference to the accompanying drawings, but the present invention is not limited to the following description.

[0019] <Identification of mutant strains with high lipid accumulation> 1. Breeding of N5 strain from wild strain Mutations were induced in Lipomyces starkeyi CBS1807 (wild-type strain) by UV irradiation. The upper layer, which is a low-density fraction, was separated by Percoll density gradient centrifugation, and high-oil-producing strains were enriched by repeated cultivation and fractionation, followed by isolation and screening.

[0020] (Mutation induction by UV irradiation) 3 mL of YPD medium (Table 1) was placed in a sterile test tube and Lipomyces starkeyi CBS1807 was inoculated from a stock plate. The final concentrations in Table 1 are in w / v% units, and the same applies to Tables 2 to 6 described below. After culturing at 30°C for 2 days (pre-culture), the culture medium was collected. The collected culture medium was diluted to a cell concentration of 1.0 x 10 7YPD medium was used to adjust the concentration to cells / mL, and 7 mL of this was cultured in an L-shaped test tube for 24 hours (main culture). Next, the entire main culture was transferred to a 15 mL tube and centrifuged at 2270 × g for 5 minutes, and the supernatant was removed.

[0021] [Table 1]

[0022] The precipitated cells were suspended in 7 mL of 1x PBS and centrifuged at 2270 x g for 5 minutes to remove the supernatant, washing the cells. Then, 7 mL of 1x PBS was added to the L-shaped test tube to suspend the cells. The cell concentration was 1.0 x 10 7 The cells were diluted with 1x PBS to give a concentration of 100 cells / mL, and 10 mL of the cell solution was dispensed into sterile dishes.

[0023] Next, the petri dish containing the bacterial solution was placed 25 cm away from the UV tube of a UV irradiation device (Toshiba Lighting & Technology Corporation, model GL15, output 4.9 W), and UV was irradiated for 20 seconds. For example, the UV irradiation energy for 20 seconds was 0.816 (mW / cm 2 ) × 20 (seconds) = 16.32 (mJ / cm2). After UV irradiation, the petri dishes were cultured at 30°C for 2 days. The entire bacterial cell suspension in the petri dish was added to a 15 mL tube and centrifuged at 2270 × g for 5 minutes, after which the supernatant was removed. 1 mL of SG medium (Table 2) was added to the tube to suspend the bacterial cells, and the tube was centrifuged at 2270 × g for 5 minutes, after which the supernatant was removed, to wash the bacterial cells.

[0024] [Table 2]

[0025] 7 mL of SG medium was added to the tube to suspend the cells. The entire volume of the culture medium in the tube was inoculated into a sterile L-shaped test tube and cultured at 30°C and 120 rpm for 3 days (pre-culture). The pre-culture was then cultured until the final cell concentration was 1.25 × 10 6The culture was adjusted to a cell concentration of 1.25 × 10 cells / mL using S5% medium (Table 3), and inoculated into a 200 mL baffled flask at a 75 mL scale. The culture was then cultured at 30°C and 160 rpm for 24 hours (preculture). The preculture solution was adjusted to a cell concentration of 1.25 × 10 cells / mL. 6 The culture medium was adjusted to 100 cells / mL using S5% medium, and 75 mL of the culture medium was inoculated into a 200 mL baffled flask. After that, the culture was incubated at 30°C and 160 rpm for 2 days (main culture).

[0026] [Table 3]

[0027] (Enrichment of high-oil-producing mutant strains by density gradient centrifugation using Percoll reagent) The number of bacteria in this culture was measured using a cell counter, and 1.0 × 10 cells were added to a 15 mL tube. 9 After the main culture, suspensions of Lipomyces starkeyi CBS1807 (wild strain) and K14 strain (Non-Patent Document 1) were similarly collected in tubes.

[0028] The tube was centrifuged (swing rotor, 2270 × g, 5 minutes, room temperature) and the supernatant was removed. 500 μL of sterile 1 × PBS was added to the tube to suspend the cells, and the entire volume was transferred to a 1.5 mL tube. The 1.5 mL tube was then filled up to the 1.0 mark with 1 × PBS.

[0029] Eight milliliters of Percoll reagent (40% concentration) was placed in an ultracentrifuge tube, and the entire bacterial suspension from the 1.5-mL tube was added. After mixing by inverting the ultracentrifuge tube, ultracentrifugation was performed using an ultracentrifuge (70.1Ti rotor, 22,000 rpm, 20 minutes, room temperature, accelerate slow, decline no break). After ultracentrifugation, the bacterial layer formed was observed. Then, 1 mL of the upper layer, which is the low-density fraction, was transferred to a 15-mL tube using a peristaltic pump.

[0030] The cells were washed by adding 5 volumes of sterile 1x PBS to the tube to suspend them, centrifuging at 2270 x g for 5 minutes and removing the supernatant. Next, 1 mL of SG medium was added to the tube to suspend the cells, and centrifuging at 2270 x g for 5 minutes to remove the supernatant. The cells were washed by adding 7 mL of SG medium to the tube to suspend the cells.

[0031] (Bacterial cell culture) The entire volume of the culture medium in the tube was inoculated into a sterile L-shaped test tube and cultured at 30°C and 120 rpm for 3 days (pre-culture). The pre-culture medium was cultured at a final cell concentration of 1.25 × 10 6 The culture was adjusted to a cell concentration of 1.25 × 10 cells / mL using S5% medium, and inoculated into a 200 mL baffled flask at a 75 mL scale. The culture was then cultured at 30 °C and 160 rpm for 24 hours (preculture). The preculture solution was adjusted to a cell concentration of 1.25 × 10 cells / mL. 6 The culture medium was adjusted to 100 cells / mL using S5% medium, and 75 mL of the culture medium was inoculated into a 200 mL baffled flask. After that, the culture was incubated at 30°C and 160 rpm for 2 days (main culture).

[0032] (Enrichment of high-oil-producing mutants by density gradient centrifugation using Percoll reagent: 2nd round) The above-mentioned procedures from (enrichment of the group of high oil-producing mutant strains by density gradient centrifugation using Percoll reagent) to (cell culture) were repeated.

[0033] (Enrichment of high-oil-producing mutants by density gradient centrifugation using Percoll reagent: 3rd round) The above-mentioned procedures from (enrichment of the group of high-oil-producing mutant strains by density gradient centrifugation using Percoll reagent) to (cell culture) were further repeated. However, the main culture in (cell culture) was carried out for 3 days.

[0034] (Enrichment of high-oil-producing mutant strains by density gradient centrifugation using Percoll reagent: 4th round) The above-mentioned procedures from (Enrichment of high-oil-producing mutants by density gradient centrifugation using Percoll reagent) to (Bacterial cell culture) were repeated once more. Then, 1 mL of the main culture medium was added to the tube, and a five-fold volume of sterile 1x PBS was added to suspend the bacterial cells. The cells were then centrifuged at 2270 x g for 5 minutes and the supernatant was removed, thereby washing the bacterial cells. Next, 1 mL of S5% medium was added to the tube to suspend the bacterial cells, and the cells were then centrifuged at 2270 x g for 5 minutes and the supernatant was removed, thereby washing the bacterial cells.

[0035] The enrichment of the low-density cell populations that produced and accumulated high levels of lipids should be observed to shift to the low-density fraction of the bacterial cell layer with increasing concentration cycles, and it was thought that they would be located in a higher layer than the wild-type and K14 strain cell populations. As shown in Figure 1, after the first concentration run, the floating of the bacterial cell layer was not observed in the UV-treated sample compared to the control K14 strain. However, in the UV-treated sample, the bacterial cell layer was observed to be higher than the upper layer of the K14 strain bacterial cell layer by the second concentration run. By the third and fourth concentration runs, the bacterial cell layer became more distinct. This indicates that the UV-treated sample contained more highly lipid-producing cell populations than the K14 strain. Note that in Figure 1, WT represents the wild-type strain. This also applies to the figures described below.

[0036] (Isolation of strains) The cells were suspended in 100 μL of S5% medium and plated onto S5% solid medium (Table 4) and cultured at 30°C for 3 days to isolate colonies. Lipomyces starkeyi produces an acidic polysaccharide composed of mannose, galactose, glucuronic acid, and a small amount of glucose. Polysaccharide-producing cells form viscous colonies. Therefore, strains that readily convert glucose (a carbon source) in the medium to polysaccharides may have reduced lipid production. Therefore, colonies that did not exhibit a glossy appearance, an indicator of polysaccharide production, were transferred to S5% medium and cultured at 30°C for 3 days. From the cultures, those with a glossy appearance, believed to be polysaccharide producers, were removed, yielding nine strains, N1 to N9.

[0037] [Table 4]

[0038] (Primary screening of high-oil-producing strains by microscopic observation) Colonies of nine strains (N1 to N9), a wild-type strain as a negative control, and a K14 strain as a positive control were inoculated into an L-shaped test tube containing 7 mL of SG medium, and cultured at 30°C and 120 rpm for 3 days (pre-culture). The pre-culture was cultured at a final cell concentration of 1.25 × 10 6 The culture was adjusted to a cell concentration of 1.25 × 10 cells / mL using S5% medium, and inoculated into a 200 mL baffled flask at a 75 mL scale. The culture was then cultured at 30 °C and 160 rpm for 24 hours (preculture). The preculture solution was adjusted to a cell concentration of 1.25 × 10 cells / mL. 6 The culture was adjusted to 75 cells / mL using S5% medium and inoculated into a 200 mL baffled flask at a 75 mL scale. Then, the culture was incubated at 30°C and 160 rpm for 3 days (main culture).

[0039] The culture medium on day 3 of the main culture was observed under a microscope, and the size of the fat globules was compared. Secondary screening was performed on strains N1, N3, N5, and N8, which had fat globules of equal or larger size than the positive control strain K14. Figure 2 shows micrographs of strains N1 to N9 and the control strain.

[0040] (Secondary screening of high-oil-producing strains by physicochemical analysis) Colonies of the N1, N3, N5, and N8 strains, as well as the wild-type strain as a negative control and the K14 strain as a positive control, were inoculated into L-shaped test tubes containing 7 mL of SG medium and cultured at 30°C and 120 rpm for 3 days (pre-culture). The pre-culture was then cultured until the final bacterial cell concentration was 1.25 × 10 6 The culture was adjusted to a cell concentration of 1.25 × 10 cells / mL using S5% medium, and inoculated into a 200 mL baffled flask at a 75 mL scale. The culture was then cultured at 30 °C and 160 rpm for 24 hours (preculture). The preculture solution was adjusted to a cell concentration of 1.25 × 10 cells / mL.6 The culture was adjusted to 75 cells / mL using S5% medium and inoculated into a 200 mL baffled flask at a 75 mL scale. Then, the culture was incubated at 30°C and 160 rpm for 3 days (main culture).

[0041] (Measurement of cell number) The cell count (bacterial count) in the medium was determined by diluting the culture medium with 0.75% saline, sonicating it using an ultrasonic homogenizer (SONICS, Vibra cell) (output 20%, on time 5 seconds, off time 5 seconds, 2 cycles), and measuring it using a CDA-1000 (Sysmex). It is also possible to measure the average particle size at the same time.

[0042] (Measurement of glucose concentration in the medium) 1 mL of the culture medium was placed in a 1.5 mL tube, and the glucose concentration was measured using a Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.). Based on the measured values, the amount of glucose consumed (g / L) by each strain during culture was calculated.

[0043] (Measurement of the amount of triglyceride (triacylglycerol: TAG) produced) The culture medium was collected in a 2 mL tube for a Multi-Beads Shocker and centrifuged at 2270 × g for 5 minutes, after which the supernatant was removed. The cells were washed by adding 1 mL of PBS to the tube to suspend them. The cells were then centrifuged at 2270 × g for 5 minutes and the supernatant was removed. The tube was heated at 70 °C for 5 minutes using a heat block to inactivate intracellular lipolytic enzymes. The tube was frozen at 30 °C and the cells were lyophilized using a freeze dryer (EYELA, FDU-1200). 500 μL of PBS and 1 g of 0.5 mm glass beads, previously washed with 0.1 N hydrochloric acid and dried, were added to the tube containing the lyophilized cells. The cells were disrupted using a Multi-Beads Shocker (Yasui Kikai Co., Ltd.) at 2500 rpm for 900 seconds. 500 μL of 1 × PBS was added to the tube and the cells were permeated at 37 °C for 10 minutes using a mixer (Taitec Corporation, Micromixer E-36). This sample was used for TAG measurement. 1 mL of Triglyceride E-Test Wako (Fujifilm Wako Pure Chemical Corporation) coloring solution and 6.7 μL of the TAG measurement sample were added to a 1.5 mL tube and mixed by inversion. The tube was heated at 37°C for 10 minutes using a heat block, with mixing by inversion every 2 minutes. After heating, the absorbance at 600 nm was measured using a similarly heated coloring solution as a control, and the TAG concentration was determined from the calibration curve.

[0044] Figure 3 shows a graph of the cell concentration measured over time for the N1, N3, N5, N8, and control strains during three days of culture. Differences in growth rate were observed from the early stages of culture, with the N1, N3, N5, and N8 strains growing at slower rates than the wild-type strain and resulting in lower final cell concentrations. On day 3 of culture, the cell concentrations of the N1, N3, N5, N8, and N8 strains were 0.90, 0.91, 0.89, and 0.89 times those of the wild-type strain, respectively.

[0045] Figure 4 shows a graph of the average particle size measured over time for the N1, N3, N5, N8, and control strains during three days of culture. On day 4 of culture, the average particle size was approximately 1.05 times larger for the N1 strain, approximately 1.06 times larger for the N3 strain, approximately 1.07 times larger for the N5 strain, and approximately 1.07 times larger for the N8 strain compared to the wild-type strain.

[0046] Figure 5 shows micrographs of each strain on day 3 of culture. As shown in Figure 5, it was confirmed that the N1, N3, N5, and N8 strains had larger fat globules than the wild-type strain, confirming their superior oil and fat production ability.

[0047] Figure 6 shows a graph of the residual glucose level (glucose concentration) in the medium measured over time based on the amount of glucose consumed by each strain. As shown in Figure 6, on the third day of culture, the N1, N3, N5, and N8 strains utilized more glucose in the medium than the wild-type strain.

[0048] Figure 7 shows a graph of the amount of TAG per medium measured over time for each strain. As shown in Figure 7, on day 3 of culture, the amount of TAG per medium was approximately 1.93-fold higher for the N1 strain, approximately 2.03-fold higher for the N3 strain, approximately 2.51-fold higher for the N5 strain, and approximately 1.87-fold higher for the N8 strain compared to the wild-type strain.

[0049] Figure 8 shows the results of 10 cells for each strain. 8 The graph shows the amount of TAG per cell measured over time. 8 On the third day of culture, the amount of TAG per cell was approximately 2.16 times higher in the N1 strain, approximately 2.23 times higher in the N3 strain, approximately 2.83 times higher in the N5 strain, and approximately 2.11 times higher in the N8 strain compared to the wild-type strain.

[0050] 2. Breeding of N5-15 strain from N5 strain The high-oil-producing Lipomyces starkeyi N5 strain (the mutant with the highest TAG production) obtained by the above-mentioned procedure was further mutated by UV irradiation to create a new mutant strain with even higher oil production. Specifically, similar to the method used to breed the N5 strain from the wild-type strain, the upper layer, which is the low-density fraction, was separated by Percoll density gradient centrifugation, and the high-oil-producing strain was concentrated by repeated cultivation and fractionation, after which it was isolated and screened.

[0051] (Mutation induction by UV irradiation) 3 mL of YPD medium was collected in a sterile test tube, and Lipomyces starkeyi N5 strain was inoculated from a stock plate and cultured at 30°C for 2 days (preculture). The collected culture medium was diluted to a cell concentration of 1.0 × 10 7 The YPD medium was used to adjust the concentration to cells / mL, and 7 mL of this was cultured in an L-shaped test tube for 24 hours (main culture). Next, the entire main culture was transferred to a 15 mL tube and centrifuged at 2270 × g for 5 minutes, and the supernatant was removed.

[0052] The precipitated cells were suspended in 7 mL of 1x PBS and centrifuged at 2270 x g for 5 minutes to remove the supernatant, washing the cells. Then, 7 mL of 1x PBS was added to the L-shaped test tube to suspend the cells. The cell concentration was 1.0 x 10 7 The cells were diluted with 1x PBS to give a concentration of 100 cells / mL, and 10 mL of the cell solution was dispensed into sterile dishes.

[0053] Next, the petri dish containing the bacterial cell solution was placed 25 cm away from the UV tube of a UV irradiation device (Toshiba Lighting & Technology Corporation, model GL15, output 4.9 W) and irradiated with UV for 25 seconds. After UV irradiation, the petri dish was cultured at 30°C for 2 days. The entire bacterial cell suspension in the petri dish was added to a 15 mL tube and centrifuged at 2270 × g for 5 minutes, after which the supernatant was removed. 1 mL of SG medium was added to the tube to suspend the bacterial cells, and the cells were washed by centrifuging at 2270 × g for 5 minutes and removing the supernatant.

[0054] 7 mL of SG medium was added to the tube to suspend the cells. The entire volume of the culture medium in the tube was inoculated into a sterile L-shaped test tube and cultured at 30°C and 120 rpm for 3 days. (Pre-preculture) The pre-preculture was added to a final cell concentration of 1.25 x 10 6 The culture was adjusted to a cell concentration of 1.25 × 10 cells / mL using S7% medium (Table 5), and inoculated into a 200 mL baffled flask at a 75 mL scale. The culture was then cultured at 30°C and 160 rpm for 24 hours (preculture). The preculture was adjusted to a cell concentration of 1.25 × 10 cells / mL. 6The culture medium was adjusted to 100 cells / mL using S7% medium, and 75 mL of the culture medium was inoculated into a 200 mL baffled flask. After that, the culture was cultured at 30°C and 160 rpm for 2 days (main culture).

[0055] [Table 5]

[0056] (Enrichment of high-oil-producing mutant strains by density gradient centrifugation using Percoll reagent) The number of bacteria in this culture was measured using a cell counter, and 1.0 × 10 cells were added to a 15 mL tube. 9 After the main culture, suspensions of Lipomyces starkeyi CBS1807 (wild-type strain) and N5 strain were similarly collected in tubes.

[0057] The tube was centrifuged (swing rotor, 2270 × g, 5 minutes, room temperature) and the supernatant was removed. 500 μL of sterile 1 × PBS was added to the tube to suspend the cells, and the entire volume was transferred to a 1.5 mL tube. The 1.5 mL tube was then filled up to the 1.0 mark with 1 × PBS.

[0058] Eight milliliters of Percoll reagent (40% concentration) was placed in an ultracentrifuge tube, and the entire bacterial suspension from the 1.5-mL tube was added. After mixing by inverting the ultracentrifuge tube, ultracentrifugation was performed using an ultracentrifuge (70.1Ti rotor, 22,000 rpm, 20 minutes, room temperature, accelerate slow, decline no break). After ultracentrifugation, the bacterial layer formed was observed. Then, 1 mL of the upper layer, which is the low-density fraction, was transferred to a 15-mL tube using a peristaltic pump.

[0059] The cells were washed by adding 5 volumes of sterile 1x PBS to the tube to suspend them, centrifuging at 2270 x g for 5 minutes and removing the supernatant. Next, 1 mL of SG medium was added to the tube to suspend the cells, and centrifuging at 2270 x g for 5 minutes to remove the supernatant. The cells were washed by adding 7 mL of SG medium to the tube to suspend the cells.

[0060] (Bacterial cell culture) The entire volume of the culture medium in the tube was inoculated into a sterile L-shaped test tube and cultured at 30°C and 120 rpm for 3 days (pre-culture). The pre-culture medium was cultured to a final cell concentration of 1.25 × 10 6 The culture was adjusted to a cell concentration of 1.25 × 10 cells / mL using S7% medium, and inoculated into a 200 mL baffled flask at a 75 mL scale. The culture was then cultured at 30 °C and 160 rpm for 24 hours (preculture). The preculture solution was adjusted to a cell concentration of 1.25 × 10 cells / mL. 6 The culture medium was adjusted to 100 cells / mL using S7% medium, and 75 mL of the culture medium was inoculated into a 200 mL baffled flask. After that, the culture was cultured at 30°C and 160 rpm for 2 days (main culture).

[0061] (Enrichment of high-oil-producing mutants by density gradient centrifugation using Percoll reagent: 2nd round) The above-mentioned procedures from (enrichment of the group of high oil-producing mutant strains by density gradient centrifugation using Percoll reagent) to (cell culture) were repeated.

[0062] (Enrichment of high-oil-producing mutants by density gradient centrifugation using Percoll reagent: 3rd round) The above-mentioned procedures from (Enrichment of high-oil-producing mutant strains by density gradient centrifugation using Percoll reagent) to (Bacterial cell culture) were further repeated. A five-fold volume of sterile 1x PBS was added to the tube to suspend the bacterial cells, which were then centrifuged at 2270 x g for 5 minutes to remove the supernatant, thereby washing the bacterial cells. Next, 1 mL of S7% medium was added to the tube to suspend the bacterial cells, which were then centrifuged at 2270 x g for 5 minutes to remove the supernatant, thereby washing the bacterial cells.

[0063] The enrichment of the low-density cell populations that produced and accumulated high amounts of oil should be observed to shift to the low-density fraction of the bacterial cell layer with increasing enrichment cycles, and it was thought that they would be located in a higher layer than the wild-type and N5 strain cell populations. Figure 9 is a photograph showing the changes in the bacterial cell layer after density gradient centrifugation. As shown in Figure 9, after the first enrichment run, the floating of the bacterial cell layer was not observed in the UV-treated sample compared to the control N5 strain. However, in the UV-treated sample, a bacterial cell layer was observed in a higher position than the upper layer of the N5 strain bacterial cell layer after the second enrichment run. By the third enrichment run, the bacterial cell layer became more distinct. This indicates that the UV-treated sample contained more highly oil-producing cell populations than the N5 strain.

[0064] (Isolation of strains) 100 μL of S7% medium was added to the tube to suspend the bacterial cells, which were then plated on S7% solid medium (Table 6) and cultured at 30°C for 3 days to isolate colonies. Colonies that did not form mucoids were re-cultured on S5% medium and cultured at 30°C for 3 days. Of the re-cultured bacterial cells, shiny ones that were thought to be producing polysaccharides were removed, and 16 strains, N5-1 to N5-16, were obtained.

[0065] [Table 6]

[0066] (Primary screening of high oil-producing strains) Colonies of 16 strains, N5-1 to N5-16, and the wild-type strain and N5 strain as controls were inoculated into L-shaped test tubes containing 7 mL of SG medium and cultured at 30°C and 120 rpm for 3 days (pre-culture). The pre-culture solution was cultured until the final bacterial cell concentration was 1.25 × 10 6 The culture was adjusted to a cell concentration of 1.25 × 10 cells / mL using S5% medium, and inoculated into a 200 mL baffled flask at a 75 mL scale. The culture was then cultured at 30 °C and 160 rpm for 24 hours (preculture). The preculture solution was adjusted to a cell concentration of 1.25 × 10 cells / mL. 6The culture was adjusted to 75 cells / mL using S5% medium and inoculated into a 200 mL baffled flask at a 75 mL scale. Then, the culture was incubated at 30°C and 160 rpm for 3 days (main culture).

[0067] The culture medium was collected on the third day of the main culture, and the cell concentration (number of cells, Figure 10), glucose consumption (Figure 11), TAG production per medium (Figure 12), and cell count per 10 cells were measured in the same manner as in "1. Breeding of the wild-type strain into the N5 strain." 8 The amount of TAG produced per cell was measured (FIG. 13). As shown in FIGS. 10 to 13, the N5-2, N5-13, and N5-15 strains were confirmed to have significantly increased TAG production compared to the N5 strain, so secondary screening was performed on these three strains.

[0068] (Secondary screening of high oil-producing strains) Colonies of the N5-2, N5-13, and N5-15 strains, as well as the wild-type and N5 strains as controls, were inoculated into 50 mL of SG medium in a 200 mL baffled flask and cultured at 30°C and 160 rpm for 3 days (pre-culture). The pre-culture was then cultured to a final cell concentration of 3.0 × 10 6 The culture was adjusted to a cell concentration of 1.2 × 10 cells / mL using S7% medium, and inoculated into a 500 mL baffled flask at a 200 mL scale. The culture was then cultured at 30°C and 160 rpm for 24 hours (preculture). The preculture was adjusted to a cell concentration of 1.2 × 10 cells / mL. 7 The culture medium was adjusted to 100 cells / mL using S7% medium, and 200 mL of the culture medium was inoculated into a 500 mL baffled flask. The culture was then cultured at 30°C and 120 rpm for 6 days (main culture).

[0069] Figure 14 is a graph showing the cell concentrations of the N5-2, N5-13, and N5-15 strains and the control strain during 6 days of culture. Differences in growth rate were observed from the early stages of culture, with the N5-2, N5-13, and N5-15 strains showing slower growth rates and lower final cell concentrations than the N5 strain. On day 3 of culture, the cell concentrations of the N5-2 strain, N5-13 strain, and N5-15 strain were 0.88-fold, 0.90-fold, and 0.79-fold, compared to the N5 strain.

[0070] Figure 15 is a graph showing the average particle diameters of the N5-2, N5-13, and N5-15 strains and the control strain during 6 days of culture. The average particle diameters of the N5-2, N5-13, and N5-15 strains were already larger than that of the wild-type strain on day 0 of culture, with the N5-2 and N5-13 strains reaching their maximum on day 4 of culture and the N5-15 strain continuing to grow until day 6 of culture. On day 4 of culture, the N5-2 strain was approximately 1.05 times larger, the N5-13 strain was approximately 1.06 times larger, and the N5-15 strain was approximately 1.12 times larger than the N5 strain.

[0071] Figure 16 shows micrographs of the wild-type strain (WT), N5 strain, N5-2 strain, N5-13 strain, and N5-15 strain on day 6 of culture. As shown in Figure 16, it was confirmed that the cells of the N5-2 strain, N5-13 strain, and N5-15 strain were larger than those of the N5 strain. Furthermore, the N5-2 strain, N5-13 strain, and N5-15 strain had larger fat globules than the N5 strain, confirming that they have superior oil and fat production ability.

[0072] 17 is a graph showing the glucose concentrations (residual glucose concentrations) in the medium of the N5-2, N5-13, and N5-15 strains and the control strain during 6 days of culture. As shown in Fig. 17, the N5, N5-2, and N5-13 strains had assimilated all of the glucose in the medium by day 6 of culture, but the N5-15 strain had a faster glucose assimilation rate than the other strains and consumed all of the glucose between days 5 and 6 of culture.

[0073] 18 is a graph showing the amount of TAG per medium of the N5-2, N5-13, and N5-15 strains and the control strain during 6 days of culture. As shown in FIG. 18, the amount of TAG per medium was approximately 1.11-fold higher for the N5-2 strain, approximately 1.09-fold higher for the N5-13 strain, and approximately 1.35-fold higher for the N5-15 strain on the 4th day of culture compared to the N5 strain.

[0074] FIG. 19 shows the results of the cell growth of 10 cells of the N5-2, N5-13 and N5-15 strains and the control strain during 6 days of culture. 8 19 is a graph showing the amount of TAG per cell.8 The TAG amount per cell was approximately 1.17 times higher for the N5-2 strain, approximately 1.21 times higher for the N5-13 strain, and approximately 1.82 times higher for the N5-15 strain. The greater the amount of glucose assimilated, the greater the TAG production, and a correlation was observed between glucose consumption and the amount of TAG produced. In other words, the N5-15 strain, which consumed glucose most rapidly, also produced the most TAG. This N5-15 strain has been deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation under accession number NITE BP-03796.

[0075] 3.Breeding of strain Q97 from strain N5 The high-oil-producing strain, Lipomyces starkeyi N5 (the mutant with the highest TAG production among mutants obtained by UV irradiation of the wild-type species), obtained by the above-mentioned procedure, was mutated by neutron irradiation instead of UV irradiation to breed a new mutant with higher oil production than N5-15. Specifically, after neutron irradiation of the N5 strain, the upper layer, which is a low-density fraction, was separated by Percoll density gradient centrifugation, and high-oil-producing strains were concentrated by repeated cultivation and fractionation, after which they were isolated and screened.

[0076] (Mutation induction by neutron irradiation) 50 mL of YPD medium was collected into a 200 mL baffled flask, and Lipomyces starkeyi N5 strain was inoculated from a stock plate and cultured at 30°C for 48 hours (preculture). The collected culture medium was diluted to a cell concentration of 1.0 × 10 7 The YPD medium was used to adjust the cell density to 1.0 × 10 cells / mL, and 50 mL of the culture was cultured in a 200 mL baffled flask for 16 hours (main culture). 7 The cells were adjusted to 1.5 mL / mL using YPD medium, and 500 μL of the mixture was transferred to a 1.5 mL tube and irradiated with neutrons at the neutron source station of the J-PARC Center (3 irradiation zones: 1.81 Gy, 5.43 Gy, and 13.92 Gy).

[0077] (Bacterial cell culture) 50 mL of SG medium was added to a 200 mL baffled flask, and the neutron-irradiated bacterial cells were suspended and cultured at 30°C for 72 hours (pre-preculture). 6 The culture was adjusted to a cell concentration of 1.25 × 10 cells / mL using S7% medium (Table 5), and inoculated into a 200 mL baffled flask at a 50 mL scale. The culture was then cultured at 30°C for 24 hours (preculture). The preculture solution was adjusted to a cell concentration of 1.25 × 10 cells / mL. 6 The culture was adjusted to 100 cells / mL using S7% medium, and 50 mL of the culture was inoculated into a 200 mL baffled flask. Then, the culture was cultured at 30°C for 72 hours (main culture).

[0078] (Enrichment of high-oil-producing mutant strains by density gradient centrifugation using Percoll reagent) The number of bacteria in this culture was measured using a cell counter, and 1.0 × 10 cells were added to a 15 mL tube. 9 The culture medium containing the cells was collected.

[0079] The tube was centrifuged (swing rotor, 2270 × g, 5 minutes, 25 ° C) and the supernatant was removed. 1 mL of sterile 1 × PBS was added to the tube to suspend the bacterial cells, and the entire volume was transferred to a 15 mL tube. It was then centrifuged at 2270 × g, 5 minutes, 25 ° C. The supernatant was removed, and 1 mL of sterile 1 × PBS was added to the tube to suspend the bacterial cells.

[0080] Eight milliliters of Percoll reagent (40% concentration) was placed in an ultracentrifuge tube, and the entire bacterial suspension from the 15-mL tube was added. After mixing by inverting the ultracentrifuge tube, ultracentrifugation was performed using an ultracentrifuge (70.1Ti rotor, 22,000 rpm, 20 minutes, room temperature, accelerate slow, decline no break). After ultracentrifugation, the bacterial layer formed was observed. Then, 2 mL of the upper layer, which is the low-density fraction, was transferred to a 15-mL tube using a peristaltic pump.

[0081] Sterile 1x PBS was added to a 15 mL tube up to the 15 mL mark and the contents were mixed. The tube was centrifuged at 2270 x g for 5 minutes at 25°C, and the supernatant was removed. Next, 5 mL of YPD medium was added to the 15 mL tube to suspend the bacterial cells, which were then inoculated into an L-shaped test tube. Another 5 mL of YPD medium was added to the tube. The culture was then incubated at 30°C for 3-4 days, and when the medium became cloudy, it was made into a glycerol stock.

[0082] (Enrichment of high-oil-producing mutants by density gradient centrifugation using Percoll reagent: 2nd round) For the glycerol stock, the above-mentioned procedures from (pre-preculture) to (glycerol stock) were repeated.

[0083] (Enrichment of high-oil-producing mutants by density gradient centrifugation using Percoll reagent: 3rd round) The glycerol stock obtained after the second Percoll density gradient centrifugation was subjected to the same procedures as described above (pre-preculture) through (ultracentrifugation). After ultracentrifugation, the bacterial cell layer was observed. Then, 2 mL of the upper layer, which was the low-density fraction, was transferred to a 15 mL tube using a peristaltic pump.

[0084] The collected upper layer of cells was suspended in YPD medium and inoculated onto YPD solid medium. From several hundred mutant strains, 100 strains (strains Q1 to Q100) with dull colonies (i.e., colonies that did not form mucoids) were selected and subjected to the screening test described below as candidate strains for high oil production. The glossiness of the colonies is due to exopolysaccharides, which are synthesized from sugars in the medium, just like oils. Therefore, mutant strains with dull colonies can be assumed to produce less exopolysaccharides, not waste sugars in the medium, and have high oil production capacity.

[0085] (Primary screening of candidate high oil-producing strains Q1 to Q100) 100 colonies of strains Q1 to Q100, as well as strains N5 and N5-15 as controls, were inoculated into an L-shaped test tube containing 5 mL of SG medium and cultured at 30°C and 150 rpm for 72 hours (pre-culture). The pre-culture was performed until the final bacterial cell concentration reached 1.25 × 10 6 The culture was adjusted to a cell concentration of 1.25 × 10 cells / mL using S7% medium, and inoculated into a 200 mL baffled flask at a 50 mL scale. The culture was then cultured at 30°C and 150 rpm for 24 hours (preculture). The preculture solution was adjusted to a cell concentration of 1.25 × 10 cells / mL. 6 The culture medium was adjusted to 50 cells / mL using S7% medium, and the cells were inoculated into a 200 mL baffled flask at a 50 mL scale. Then, the cells were cultured at 30°C and 150 rpm for 72 hours (main culture).

[0086] After 72 hours of main cultivation, the culture solution was collected and the cell concentration (number of cells) was measured in the same manner as in "1. Breeding of N5 strain from wild type strain." At the same time, the average particle diameter of each cell was measured and calculated as (4 / 3) x (average particle diameter / 2) 3 The volume of the bacterial cells was calculated according to the formula "×π". Furthermore, the hypothetical oil yield (volume of bacterial cells × number of bacteria) was calculated from the number and volume of bacterial cells of each strain. The hypothetical oil yield is equal to the amount of oil in the medium when the bacterial cells are filled with oil, and was therefore used as the criterion for the primary screening, which is a simple screening method.

[0087] 20 is a graph showing the hypothetical oil yields of the N5 strain, the N5-15 strain, and the Q1 to Q100 strains (relative values, with the hypothetical oil yield of the N5-15 strain defined as 1). Of the 100 strains Q1 to Q100, 27 strains had hypothetical oil yields exceeding 1. Therefore, the top 10 strains with the highest hypothetical oil yields (strain Q46, strain Q60, strain Q70, strain Q71, strain Q85, strain Q89, strain Q94, strain Q96, strain Q97, and strain Q100) were selected, and these strains were subjected to secondary screening.

[0088] (Secondary screening of 10 candidate high oil-producing strains) 50 mL of SG medium was taken into a 200 mL baffled flask, and colonies of strains Q46, Q60, Q70, Q71, Q85, Q89, Q94, Q96, Q97, and Q100, as well as N5 and N5-15 strains as controls, were inoculated and cultured at 30°C for 72 hours (pre-culture). The pre-culture was adjusted to a final cell concentration of 1.25 × 10 6 The culture was adjusted to a cell concentration of 1.25 × 10 cells / mL using S7% medium, and inoculated into a 200 mL baffled flask at a 50 mL scale. The culture was then cultured at 30°C for 24 hours (preculture). The preculture solution was adjusted to a cell concentration of 1.25 × 10 cells / mL. 6 The culture was adjusted to 100 cells / mL using S7% medium, and 50 mL of the culture was inoculated into a 200 mL baffled flask. Then, the culture was cultured at 30°C for 72 hours (main culture).

[0089] After 72 hours of main cultivation, the culture medium was collected and the cell concentration (number of cells), the average particle size of each cell, and the amount of TAG were measured.

[0090] 21 is a graph showing the TAG amounts (TAG amount per medium / relative value when the TAG amount of the N5-15 strain is set to 1) of 10 candidate high-oil-producing strains and control strains (N5 strain and N5-15 strain). The Q89 strain, Q94 strain, and Q97 strain had a TAG amount of 1 or more, while the TAG amount of the Q100 strain was close to 1. The other six candidate strains had TAG amounts of 0.9 or less.

[0091] Figure 22 is a graph plotting the relationship between the relative bacterial concentration (relative value when the bacterial concentration of the N5-15 strain is set to 1) and the relative particle size (relative value when the particle size of the N5-15 strain is set to 1) of the 10 candidate high oil-producing strains and the control strains (strain N5 and strain N5-15). Strains Q89, Q94, and Q97, which had high amounts of TAG per medium, had high relative bacterial concentrations but small relative particle sizes. On the other hand, strain Q100 had the largest relative particle size among the 10 candidate high oil-producing strains. Therefore, of the strains with the highest TAG amounts, two strains with the highest relative bacterial concentrations (strain Q89 and strain Q97) and strain Q100, which had the largest relative particle size, were selected and subjected to tertiary screening.

[0092] (Third screening of three candidate high oil-producing strains) Colonies of the Q89, Q97, and Q100 strains, as well as the N5 and N5-15 strains as controls, were inoculated into 50 mL of SG medium in a 200 mL baffled flask and cultured at 30°C for 72 hours (pre-culture). The pre-culture was then cultured to a final cell concentration of 1.25 × 10 6 The culture was adjusted to a cell concentration of 1.25 × 10 cells / mL using S7% medium, and inoculated into a 200 mL baffled flask at a 50 mL scale. The culture was then cultured at 30°C for 24 hours (preculture). The preculture solution was adjusted to a cell concentration of 1.25 × 10 cells / mL. 6 The culture was adjusted to 100 cells / mL using S7% medium, and inoculated into a 200 mL baffled flask at a 50 mL scale. Then, the culture was cultured at 30°C for 144 hours (main culture).

[0093] After 72 hours of main cultivation, the culture medium was sampled every 24 hours, and the cell concentration (number of cells), average particle size of each cell, TAG amount, and residual glucose amount were measured. The cells were also observed under a microscope.

[0094] 23 is a graph showing the TAG amount (mg / mL) per medium of the three candidate high-oil-producing strains and the control strains (N5 and N5-15) after 96 hours of culture. The Q97 strain had the highest TAG amount per medium, 1.14 times that of the N5-15 strain. After 96 hours of main culture, the TAG amount of the Q89 strain was almost the same as that of the Q97 strain. However, after further main culture, after 120 hours of main culture, the TAG amount of the Q89 strain was lower than that of the Q97 strain and was lower than that of the N5-15 strain. In other words, it was confirmed that the Q97 strain had significantly higher oil-producing ability than the other candidate oil-producing strains when the main culture was continued for 120 hours or more.

[0095] Figure 24 shows micrographs (600x magnification) of the Q97, N5, and N5-15 strains after 96 hours of culture. As shown in Figure 24, the Q97 strain not only exhibits superior cell proliferation ability compared to the N5 strain, but also has a larger bacterial cell diameter, and therefore can be said to possess both high cell proliferation ability and lipid accumulation ability.

[0096] Figure 25 shows the cell growth rate of the Q97 strain and the control strains (N5 strain and N5-15 strain) after 96 hours of culture. 8 1 is a graph showing the amount of TAG per cell. 8 The amount of TAG per cell was 1.25 times that of the N5 strain and 2 times that of the N5-15 strain.

[0097] 26 is a graph showing the changes in bacterial concentration of the Q97 strain and the control strains (N5 strain and N5-15 strain) over a 6-day (144-hour) culture period. The N5 strain had a higher bacterial concentration during the culture period than the N5-15 strain, but the Q97 strain was confirmed to have an even higher bacterial concentration than the N5 strain.

[0098] Generally, there is a trade-off between the oil accumulation ability and cell proliferation ability of oleaginous yeast. In fact, the N5-15 strain exhibited twice the oil accumulation ability compared to the N5 strain, while its cell proliferation ability was slightly less than 60%. However, as shown in Figures 25 and 26, the Q97 strain exhibited superior oil accumulation ability and cell proliferation ability compared to the N5 strain. Furthermore, as shown in Figure 23, the Q97 strain had 1.14 times the amount of TAG per medium as the N5-15 strain, confirming that it had higher oil production ability than the N5-15 strain.

[0099] Analysis of the fatty acid composition of TAG from strains Q89, Q97, and Q100 after 6 days of main cultivation revealed that the fatty acid composition of all the candidate oil-producing strains was almost the same as that of the wild-type strain (WT). In other words, the fatty acid composition of strain Q97 consisted mainly of palmitic acid and oleic acid, confirming that this composition is sufficient to be expected as an alternative to palm oil.

[0100] The Q97 strain, which was confirmed to have the highest oil-producing ability, was deposited at the Patent Microorganisms Deposit Center of the National Institute of Technology and Evaluation (NITE) on May 15, 2024, under accession number NITE BP-04113. [Industrial Applicability]

[0101] The strain of the present invention is useful as a high-oil-producing strain for industrial production of alternative palm oil that can be used as a food ingredient or a cosmetic ingredient, etc. Furthermore, the production method of the present invention is useful for industrial production of alternative palm oil.

Claims

1. This is a yeast strain belonging to the genus Lipomyces, which is a high oil-producing strain deposited at the Patent Microorganisms Deposit Center of the National Institute of Technology and Evaluation under accession number NITE BP-04113.

2. A method for producing fats and oils using a high fat-producing strain of oleaginous yeast belonging to the genus Lipomyces, which is deposited with the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center under Accession Number NITE BP-04113.

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