High oil production strain belonging to the genus lipomyces and oil production method using the high oil production strain

Description

1 / 31 “HIGH OIL-YIELDING STRAIN BELONGING TO THE GENUS LIPOMYCES AND OIL PRODUCTION METHOD USING THE HIGH OIL-YIELDING STRAIN” Technical Field

[001] The present invention relates to a novel high-oil-producing strain belonging to the genus Lipomyces and to a method of producing oil using the high-oil-producing strain. Antecedent Technique

[002] Palm oil is a vegetable oil derived from the fruit of the oil palm and is solid at room temperature. Palm oil is characterized by containing a high level of saturated fatty acids, with palmitic acid and oleic acid constituting approximately 80% of the total fatty acids. Palm oil is known to be used not only as an edible oil, but also as a raw material for margarine, vegetable shortening, or soaps. Palm oil is also used as a frying oil for fried instant noodles or snacks, such as potato chips.

[003] Palm oil is the most widely produced vegetable oil in the world. In 2015, a total of 6256 * 104 tons of palm oil were produced worldwide. The palm tree grows in tropical areas with hot, humid climates and is native to West Africa and Latin America. Currently, Malaysia and Indonesia account for 80% or more of palm oil production. Because the palm tree bears fruit year-round, its production is far superior to that of other vegetable oil sources, and palm oil can be produced in quantities 8 to 10 times greater than soybean oil or rapeseed oil. Therefore, palm oil is inexpensive compared to other vegetable oils and can be supplied stably, which is why many countries import it. Petition 870250087009, dated 09 / 25 / 2025, p. 45 / 100 2 / 31

[004] Palm oil grows exclusively in equatorial tropical regions with hot and humid climates. These regions offer suitable growing conditions for the species, but they coincide with areas where tropical forests are distributed. Thus, the development of a palm oil plantation inevitably involves the deforestation of a tropical forest, and many tropical forests are deforested and lost every year. Massive forest fires have also occurred during the development of palm oil plantations. Due to this loss of tropical forests, rare species of wildlife that inhabit them are now on the verge of extinction.

[005] Furthermore, the rapid increase in demand for palm oil has led to precarious working conditions for workers or has generated conflicts between local residents and developers over land development. In this way, palm oil has caused labor and human rights problems, as well as global environmental problems.

[006] In this context, methods for producing an alternative to palm oil using microorganisms have been sought in recent years. Oleaginous yeasts belonging to the genus Lipomyces are known to produce oils with fatty acid compositions similar to those of palm oil. Furthermore, the oils produced by Lipomyces yeasts resemble palm oil in physical properties and show promise as an alternative to palm oil in terms of environmental risk, climate change risk, and sustainability.

[007] In a method of producing an alternative oil using wild-type Lipomyces starkeyi as an oil-producing yeast, the amount of oil produced by the yeast is insufficient. To solve this problem, attempts were made to mutate the wild-type strain and thus obtain a strain with high oil production (high accumulation strain). Petition 870250087009, dated 09 / 25 / 2025, page 46 / 100 3 / 31 of oil) that produces large quantities of oil. Non-Patented Literature 1 or 2 describes the induction of mutations in CBS1807, which is a wild-type strain of Lipomyces starkeyi, by means of ethyl methanesulfonate or UV irradiation and repeated concentration of the high-oil-producing mutant strain by density gradient centrifugation to obtain a concentrated fraction of the high-oil-producing strain.

[008] Patent Literature 1 describes a method for producing a high-oil-producing strain using an oleaginous yeast belonging to the genus Lipomyces or Rhodosporidium and subjecting the oleaginous yeast to density gradient centrifugation using a reagent composed of colloidal silicate particles coated with polyvinylpyrrolidone. Patent Literature 1 further describes treating the yeast using ethyl methanesulfonate or UV as a mutagenic agent and then subjecting the treated yeast to density gradient centrifugation using a reagent composed of colloidal silicate particles coated with polyvinylpyrrolidone. List of Citations Patent Literature

[009] PTL 1: Japanese Patent No. 7082340 Non-Patent Literature

[0010] NPL 1: Yamazaki H, Kobayashi A, Ebina S, et al (2019). Highly selective isolation and characterization of Lipomyces starkeyi mutants with increased triacylglycerol production. Applied Microbiology and Biotechnology. 103, 6297-6308. NPL 2: Hiroaki Takaku et al., Isolation and characterization of Lipomyces starkeyi mutants with significantly increased lipid productivity after UV irradiation, J Biosci Bioeng., 131(6), 613-621, (2021) Petition 870250087009, dated 09 / 25 / 2025, page 47 / 100 4 / 31 Summary of the Invention Technical Problem

[0011] The present inventors have successfully obtained the N5 strain as a high-oil-producing strain by inducing mutations in Lipomyces Starkeyi CBS1807, a wild-type strain, through UV irradiation. However, the industrial use of the N5 strain is disadvantageous in terms of oil production rate and oil conversion efficiency. An objective of the present invention is to obtain a high-oil-producing yeast strain belonging to the genus Lipomyces, the high-oil-producing strain being capable of producing (accumulating) greater quantities of oil than the N5 strain, and to provide a method for producing an alternative to palm oil using the high-oil-producing strain. Solution to the Problem

[0012] The present inventors focused on the potential of the method used to obtain the N5 strain as a high oil-producing strain from Lipomyces Starkeyi CBS1807, a wild-type strain, and investigated whether applying this method, using the N5 strain as the parent strain, could result in a new mutant strain with higher oil productivity. As a result, the inventors concluded the present invention.

[0013] Specifically, the present invention relates to a high oil-producing yeast strain belonging to the genus Lipomyces, the high oil-producing strain being deposited with the National Institute of Technology and Evaluation, Patent Microorganisms Depositary, under accession number NITE BP-03796.

[0014] The high oil-producing strain (N5-15 strain described later), deposited with the National Institute of Technology and Evaluation, Patent Microorganisms Depositary, under accession number NITE BP-03796, is obtained as follows: the N5 strain is irradiated Petition 870250087009, dated 09 / 25 / 2025, page 48 / 100 5 / 31 with UV to induce mutations; the resulting mutant strain is subjected to Percoll density gradient centrifugation, after which the top layer is collected as a low-density fraction and the cells from this fraction are cultured; the fractionation and culture steps are repeated to concentrate the high-oil-producing strain; and the strain is then isolated and evaluated.

[0015] The present invention also relates to a method for producing oil, the method including the use of a high oil-producing oleaginous yeast strain belonging to the genus Lipomyces, the high oil-producing strain being deposited with the National Institute of Technology and Evaluation, Patent Microorganisms Depositary, under accession number NITE BP-03796. Advantageous Effects of the Invention

[0016] The oil-producing yeast of the present invention can produce an alternative to palm oil more efficiently than conventional oil-producing yeasts belonging to the genus Lipomyces. Consequently, the oil production method of the present invention can produce an alternative to palm oil at a lower cost than conventional production methods using known oil-producing yeasts. Brief Description of the Drawings

[0017] FIG. 1 shows images illustrating how cell layers change after density gradient centrifugation. “WT” denotes a wild-type strain, “K14” denotes the K14 strain, and “UV” denotes UV-treated cells from the wild-type strain. FIG. 2 shows microscopic images of strains N1 to N9 and control strains. FIG. 3 is a graph showing the cell concentrations of strains N1, N3, N5, and N8 and the control strains over the three days of culture. Petition 870250087009, dated 09 / 25 / 2025, page 49 / 100 6 / 31 FIG. 4 is a graph showing the average cell size measured over time for the different strains. FIG. 5 shows microscopic images of the different strains captured on the third day of culture. FIG. 6 is a graph showing the glucose concentration in the medium measured over time for the different strains. FIG. 7 is a graph showing the triglyceride (TAG) content per volume of medium measured over time for the different strains. FIG. 8 is a graph showing the triglyceride (TAG) content normalized to cell count, measured over time for the different strains. FIG. 9 shows images illustrating how cell layers change after density gradient centrifugation. “WT” denotes a wild-type strain, “N5” denotes the N5 strain, and “UV” denotes N5 strain cells treated with UV. FIG. 10 is a graph showing the cell concentration measured on the third day of culture for strains N5-1 to N5-16. FIG. 11 is a graph showing the glucose consumption measured on the third day of culture for strains N5-1 to N5-16 and the control strains. FIG. 12 is a graph showing the triglyceride (TAG) content per volume of medium measured on the third day of culture for strains N5-1 to N5-16 and the control strains. FIG. 13 is a graph showing the triglyceride (TAG) content normalized to cell count, measured on the third day of culture for strains N5-1 to N5-16. FIG. 14 is a graph showing the cell concentrations of strains N5-2, N5-13 and N5-15 and the control strains over the six days of culture. Petition 870250087009, dated 09 / 25 / 2025, page 50 / 100 7 / 31 FIG. 15 is a graph showing the average cell sizes of strains N5-2, N5-13, and N5-15, and of the control strains over the six days of culture. FIG. 16 shows microscopic images of the various strains captured on the sixth day of culture. FIG. 17 is a graph showing the glucose concentration in the medium measured over the six days of culture for strains N5-2, N5-13 and N5-15 and the control strains. FIG. 18 is a graph showing the triglyceride (TAG) content per volume of medium measured over the six days of culture for strains N5-2, N5-13 and N5-15 and the control strains. FIG. 19 is a graph showing the triglyceride (TAG) content normalized to cell count, measured over the six days of culture for strains N5-2, N5-13 and N5-15 and the control strains. Description of the Modalities

[0018] The embodiments of the present invention will be described below with reference to the drawings. The present invention is not limited to the description below.

[0019] Acquisition of mutant strains with high oil accumulation 1. Development of the N5 strain from the wild-type strain Lipomyces starkeyi CBS1807 (wild-type strain), a yeast belonging to the genus Lipomyces, was irradiated with UV to induce mutations. The resulting mutant strain was subjected to Percoll density gradient centrifugation, after which the top layer was collected as a low-density fraction and the cells from this fraction were cultured. The fractionation and culture steps were repeated to concentrate the high-oil-producing strain, and the strain was isolated and evaluated. Petition 870250087009, dated 09 / 25 / 2025, page 51 / 100 8 / 31

[0020] Induction of mutations by UV irradiation A 3 mL volume of YPD medium (Table 1) was added to a sterile test tube and inoculated with the Lipomyces starkeyi CBS1807 strain from a stock plate. The unit of the final concentration shown in Table 1 is % w / v, and the same applies to Tables 2 to 6 referenced later. The strain cells were cultured at 30°C for two days (pre-culture), after which the culture fluid was collected. The collected culture fluid was adjusted to a cell concentration of 1.0 χ¹⁰⁷ cells / mL using YPD medium, and 7 mL of the culture fluid were transferred to an L-shaped test tube, in which the cells were cultured for 24 hours (main culture). Subsequently, all the main culture fluid was transferred to a 15 mL test tube and centrifuged at 2270 χ¹⁰ g for five minutes, after which the supernatant was removed.

[0021] Table 1 Final concentration: Yeast extract 1%, Hypopeptide 2%, D-(+)-Glucose 2%

[0022] The precipitated cells were washed by suspending them in 1 x PBS mL, centrifuging the suspension at 2270 χ g for five minutes, and then removing the supernatant. Next, 7 mL of 1 x PBS were added to an L-shaped test tube, and the cells were suspended in the PBS. The suspension was diluted to a cell concentration of 1.0 χ 107 cells / mL using 1 χ PBS, and 10 mL of the cell suspension were dispensed into a sterile Petri dish.

[0023] Next, the Petri dish containing the cell suspension was placed at a distance of 25 cm from the UV tube of a UV irradiator (TOSHIBA Lighting & Technology Corporation, model GL15, power Petition 870250087009, dated 09 / 25 / 2025, page 52 / 100 9 / 31 = 4.9 W), and the cell suspension was irradiated with UV for 20 seconds. The energy of the UV irradiation is calculated, for example, as 0.816 (mW / cm2) x 20 (sec) = 16.32 (mJ / cm2) in the case of 20 seconds of irradiation. After UV irradiation, the cells in the Petri dish were cultured at 30°C for two days. The entire cell suspension in the Petri dish was added to a 15 mL tube and centrifuged at 2270 xg for five minutes, after which the supernatant was removed. The cells were washed by adding 1 mL of SG medium (Table 2) to the tube to resuspend them, centrifuging the suspension at 2270 xg for five minutes, and removing the supernatant.

[0024] Table 2 Final concentration (NH4)2SO4 0.5% KH2PO4 0.1% NaCl 0.01% Yeast extract 0.1% Glycerol 2% MgSO4^7H2O 0.05% CaCl2^7H2O 0.01%

[0025] 7 mL of SG medium were added to the tube to suspend the cells. All the culture fluid in the tube was used to inoculate a sterile L-shaped test tube, in which the cells were cultured at 30°C and 120 rpm for three days (initial culture). The initial culture fluid was adjusted to a final cell concentration of 1.25 x 10⁶ cells / mL using 5% S medium (Table 3), and a 200 mL deflector flask was inoculated with the culture fluid to a 75 mL scale. The cells were then cultured in the flask at 30°C and 160 rpm for 24 hours (pre-culture). The pre-culture fluid was adjusted to a final cell concentration of 1.25 x 10⁶ cells / mL using 5% S medium, and a 200 mL deflector flask was inoculated with the Petition 870250087009, dated 09 / 25 / 2025, page 53 / 100 10 / 31 culture fluid on a 75 mL scale. The cells were then cultured in the flask at 30°C and 160 rpm for two days (main culture).

[0026] Table 3 Final concentration (NH4)2SO4 0.5% KH2PO4 0.1% NaCl 0.01% Yeast extract 0.1% D-(+)- Glucose 5% MgSO4^7H2O 0.05% CaCl2^7H2O 0.01%

[0027] Concentration of mutant strains with high oil production by density gradient centrifugation using Percoll's reagent

[0028] The number of cells in the main culture fluid was determined using a cell counter, and a volume containing 1.0 x 109 cells was transferred to a 15 mL tube. A suspension of Lipomyces starkeyi CBS1807 (wild-type strain) subjected to main culture and a suspension of the K14 strain (Unpatented Literature 1) subjected to main culture were also added to the tubes.

[0029] Each tube was centrifuged (oscillating rotor, 2270 xg, five minutes, room temperature) and the supernatant was removed. 500 μL of sterile 1 x PBS was added to the tube to suspend the cells, and the entire suspension was transferred to a 1.5 mL tube. Then, 1 x PBS was added to bring the suspension volume to the 1.0 mL mark on the 1.5 mL tube.

[0030] An 8 mL volume of Percoll's reagent (concentration = 40%) was added to an ultracentrifuge tube, for which to Petition 870250087009, dated 09 / 25 / 2025, p. 54 / 100 11 / 31 of the cell suspension was transferred from the 1.5 mL tube. The ultracentrifuge tube was gently inverted to mix its contents, and the mixture was subjected to ultracentrifugation using an ultracentrifuge (rotor 70.1Ti, 22000 rpm, 20 minutes, room temperature, ACEL SLOW, DECEL NO BREAK). After ultracentrifugation, the formed cell layer was observed. Subsequently, 1 mL of the formed upper layer, as a low-density fraction, was transferred to a 15 mL tube using a peristaltic pump.

[0031] The cells were washed as follows: 1 x sterile PBS, the volume of which was five times that of the transferred top layer, was added to the tube to suspend the cells, and the suspension was centrifuged at 2270 χ g for five minutes, after which the supernatant was removed. The cells were subsequently washed by adding 1 mL of SG medium to the tube to suspend them, centrifuging the suspension at 2270 χ g for five minutes and removing the supernatant. 7 mL of SG medium were added to the tube to suspend the cells.

[0032] Cell culture

[0033] All the culture fluid contained in the tube was used to inoculate a sterile L-shaped test tube, in which the cells were cultured at 30°C and 120 rpm for three days (initial culture). The initial culture fluid was adjusted to a final cell concentration of 1.25 χ 10⁶ cells / mL using 5% S medium, and a 200 mL deflector flask was inoculated with the culture fluid to a 75 mL scale. The cells were then cultured in the flask at 30°C and 160 rpm for 24 hours (pre-culture). The pre-culture fluid was adjusted to a final cell concentration of 1.25 χ 10⁶ cells / mL using 5% S medium, and a 200 mL deflector flask was inoculated with the culture fluid to a 75 mL scale. The cells were then cultured in the flask at 30°C and 160 rpm for two days (main culture). Petition 870250087009, dated 09 / 25 / 2025, page 55 / 100 12 / 31

[0034] Concentration of mutant strains with high oil production by density gradient centrifugation using Percoll's reagent: second round

[0035] The procedures described in [Concentration of mutant strains with high oil production by density gradient centrifugation using Percoll's reagent] through [Cell culture] were repeated.

[0036] Concentration of mutant strains with high oil production by density gradient centrifugation using Percoll's reagent: third round

[0037] The procedures described in [Concentration of mutant strains with high oil production by density gradient centrifugation using Percoll's reagent] up to [Cell culture] were subsequently repeated. The main culture in [Cell culture] was carried out for three days.

[0038] Concentration of mutant strains with high oil production by density gradient centrifugation using Percoll's reagent: fourth round

[0039] The procedures described in [Concentration of mutant strains with high oil production by density gradient centrifugation using Percoll's reagent] up to [Cell culture] were repeated once more. Then, 1 mL of the main culture fluid was added to a tube, and the cells were washed by adding 1 x sterile PBS, the volume of which was five times greater than that of the main culture fluid, to suspend the cells, centrifuging the suspension at 2270 χ g for five minutes and removing the supernatant. Then, the cells were washed again by adding 1 mL of 5% S medium to the tube to suspend the cells, centrifuging the suspension at 2270 χ g for five minutes and removing the supernatant. Petition 870250087009, dated 09 / 25 / 2025, page 56 / 100 13 / 31

[0040] When low-density cells that have produced and accumulated large amounts of oil are repeatedly concentrated, it is believed that the cell layer will gradually shift to a low-density fraction. As a result, the cell layer is expected to appear in a higher position than that of the wild-type strain or the K14 strain after repeated rounds of concentration. After the first concentration process, as shown in FIG. 1, the cell layer of the UV-treated sample was not observed above the cell layer of the K14 strain used as a control. After the second round of concentration, the cell layer of the UV-treated sample was found in a higher position than the upper portion of the cell layer of the K14 strain. The third and fourth rounds of concentration made the cell layer of the UV-treated sample clearly identifiable.This indicates that the cells with high oil production were more concentrated in the UV-treated sample than in the K14 strain. In FIG. 1, “WT” denotes the wild-type strain. The same applies to the other figures referenced later.

[0041] Isolation of strains

[0042] The isolation of the colonies was carried out as follows: 100 μL of 5% S medium were added to the tube to suspend the cells, and then the suspension was seeded onto 5% solid S medium (Table 4), in which the cells were cultured at 30°C for three days. It was found that Lipomyces starkeyi produces an acidic polysaccharide containing mannose, galactose, glucuronic acid, and a small amount of glucose as constituent sugars, and that the cells producing this polysaccharide form a mucoid colony. Thus, strains prone to converting glucose (carbon source) taken from the medium into the polysaccharide should have low oil productivity. With this in mind, colonies that did not show any of the Petition 870250087009, dated 09 / 25 / 2025, page 57 / 100 14 / 31 cells with a bright appearance indicative of polysaccharide production were separated from the plate and re-seeded in S5% medium, in which the cells were cultured at 30°C for three days. Among the cells of the re-seeded colonies, those with a bright appearance and believed to have produced the polysaccharide were removed to obtain nine strains N1 to N9.

[0043] Table 4 Final concentration (NH4)2SO4 0.5% KH2PO4 0.1% NaCl 0.01% Yeast extract 0.1% D-(+)- Glucose 5% MgSO4^7H2O 0.05% CaCl2^7H2O 0.01% Agar 2%

[0044] Primary evaluation of strains with high oil production by microscopic observation

[0045] L-shaped test tubes containing 7 mL of SG medium were inoculated with colonies of the nine strains N1 to N9, the wild-type strain serving as a negative control and the K14 strain serving as a positive control, and the strain cells were cultured at 30°C and 120 rpm for three days (initial culture). Each initial culture fluid was adjusted to a final cell concentration of 1.25 χ 10⁶ cells / mL using 5% S medium, and a 200 mL deflector flask was inoculated with the culture fluid on a 75 mL scale. The cells were then cultured in the flask at 30°C and 160 rpm for 24 hours (pre-culture). The pre-culture fluid was adjusted to a final cell concentration of 1.25 χ¹⁰⁶ cells / mL using 5% S medium, and a 200 mL baffle flask was inoculated with the culture fluid. Petition 870250087009, dated 09 / 25 / 2025, page 58 / 100 15 / 31 on a 75 mL scale. The cells were then cultured in the flask at 30°C and 160 rpm for three days (main culture).

[0046] On the third day of the primary culture, the culture fluids were observed under a microscope to compare the size of the fat globules among them. Strains N1, N3, N5, and N8 presented fat globules as large as or larger than those of strain K14, used as a positive control. These strains N1, N3, N5, and N8 were subjected to secondary evaluation. FIG. 2 shows microscopic images of strains N1 to N9 and the control strains.

[0047] Secondary evaluation of high oil-producing strains through physical and chemical analyses

[0048] L-shaped test tubes containing 7 mL of SG medium were inoculated with colonies of strains N1, N3, N5, and N8, with the wild-type strain serving as a negative control and strain K14 as a positive control. Cells from the strains were cultured at 30°C and 120 rpm for three days (initial culture). Each initial culture fluid was adjusted to a final cell concentration of 1.25 χ¹⁰⁶ cells / mL using 5% S medium, and a 200 mL deflector flask was inoculated with the culture fluid on a 75 mL scale. Cells were then cultured in the flask at 30°C and 160 rpm for 24 hours (pre-culture). The pre-culture fluid was adjusted to a final cell concentration of 1.25 χ¹⁰⁶ cells / mL using 5% S medium, and a 200 mL baffle flask was inoculated with the culture fluid to a 75 mL scale. The cells were then cultured in the flask at 30°C and 160 rpm for three days (main culture).

[0049] Cell count measurement

[0050] The cell count (number of cells) in the medium was measured as follows: the main culture fluid was diluted with a 0.75% saline solution, then ultrasonically treated (production = 20%, on time = 5 seconds, off time = 5 Petition 870250087009, dated 09 / 25 / 2025, page 59 / 100 16 / 31 seconds, two cycles) using an ultrasonic homogenizer (Vibra cell from SONICS) and subjected to cell count measurement using CDA-1000 (Sysmex). The average cell size can be measured simultaneously.

[0051] Measurement of glucose concentration in the medium

[0052] A 1 mL volume of the main culture fluid was transferred to a 1.5 mL tube, and the glucose concentration in the main culture fluid was measured using the Wako Glucose CII-Test (Wako Pure Chemical Industries, Ltd.). The glucose consumption (g / L) of each mutant strain during culture was calculated based on the measured glucose concentration.

[0053] Measurement of the amount of triglycerides produced

[0054] A volume of the main culture fluid was transferred to a 2 mL Multi-beads Shocker tube and centrifuged at 2270 χ g for five minutes, after which the supernatant was removed. The cells were washed by adding 1 mL of PBS to the tube to suspend them, centrifuging the suspension at 2270 χ g for five minutes, and removing the supernatant. The tube was heated using a heating block at 70°C for five minutes to deactivate intracellular lipase. The tube was frozen, and the cells were lyophilized using a freeze dryer (EYELA FDU-1200). To the tube containing the lyophilized cells, 500 μL of PBS and 1 g of 0.5 mm glass beads, which had been washed with 0.1 N hydrochloric acid and previously dried, were added. The cells were then broken (2500 rpm, 900 seconds) using a Multi-bead Shocker (Yasui Kikai Corporation).A volume of 500 μL of 1 χ PBS was added to the tube, and the tube was shaken using a mixer (Taitec Corporation Micromixer E-36) at 37°C for 10 minutes. The sample thus obtained was used as a sample for triglyceride quantification. To a 1.5 mL tube, 1 mL of the chromogenic reagent Triglyceride was added. Petition 870250087009, dated 09 / 25 / 2025, page 60 / 100 17 / 31 E-Test Wako (FUJIFILM Wako Pure Chemical Corporation) and 6.7 pL of the sample for triglyceride quantification were used, and the contents of the tube were mixed by gentle inversion. The tube was heated using a heating block at 37°C for 10 minutes, while the tube was gently inverted every two minutes. After heating, the absorbance of the sample was measured at 600 nm. The chromogenic reagent, heated in the same manner as the sample, was used as a control in the measurement, and the triglyceride concentration in the sample was determined from the calibration curve.

[0055] FIG. 3 is a graph showing the cell concentration measured over time for strains N1, N3, N5, and N8 and the control strains during the three days of culture. The difference in growth rate was observed as early as the initial stage of culture, and the growth rate and final cell concentration were lower for strains N1, N3, N5, and N8 than for the wild-type strain. On the third day of culture, the cell concentration of strain N1 was 0.90 times higher than that of the wild-type strain, the cell concentration of strain N3 was 0.91 times higher than that of the wild-type strain, the cell concentration of strain N5 was 0.89 times higher than that of the wild-type strain, and the cell concentration of strain N8 was 0.89 times higher than that of the wild-type strain.

[0056] FIG. 4 is a graph showing the average cell size measured over time for strains N1, N3, N5, and N8 and the control strains during the three days of culture. On the fourth day of culture, the average cell size of strain N1 was approximately 1.05 times larger than that of the wild-type strain, the average cell size of strain N3 was approximately 1.06 times larger than that of the wild-type strain, the average cell size of strain N5 was approximately 1.07 times larger than that of the wild-type strain, and the average cell size of strain N8 was approximately 1.07 times larger than that of the wild-type strain. Petition 870250087009, dated 09 / 25 / 2025, p. 61 / 100 18 / 31

[0057] FIG. 5 shows microscopic images of the various strains captured on the third day of culture. As seen in FIG. 5, strains N1, N3, N5, and N8 showed larger fat globules than the wild-type strain. This demonstrates that strains N1, N3, N5, and N8 are superior in oil productivity.

[0058] FIG. 6 is a graph showing the remaining glucose content (glucose concentration) in the medium, measured over time for the various strains. As seen in FIG. 6, on the third day of cultivation, it was found that strains N1, N3, N5 and N8 assimilated a greater amount of glucose from the medium than the wild-type strain.

[0059] FIG. 7 is a graph showing the triglyceride (TAG) content per volume of medium measured over time for the various strains. As seen in FIG. 7, on the third day of culture, the triglyceride content per volume of medium for strain N1 was approximately 1.93 times higher than that of the wild-type strain, the triglyceride content per volume of medium for strain N3 was approximately 2.03 times higher than that of the wild-type strain, the triglyceride content per volume of medium for strain N5 was approximately 2.51 times higher than that of the wild-type strain, and the triglyceride content per volume of medium for strain N8 was approximately 1.87 times higher than that of the wild-type strain.

[0060] FIG. 8 is a graph showing the normalized triglyceride (TAG) content relative to cell count, measured over time for the different strains. As observed in FIG. 8, on the third day of culture, the normalized triglyceride content relative to cell count for strain N1 was approximately 2.16 times higher than that of the wild-type strain, the normalized triglyceride content relative to cell count for strain N3 was approximately 2.23 times higher than that of the wild-type strain, the normalized triglyceride content relative to cell count for strain N5 was approximately 2.83 times higher than that of the wild-type strain, and the triglyceride content Petition 870250087009, dated 09 / 25 / 2025, page 62 / 100 The normalized 19 / 31 cell count for the N8 strain was approximately 2.11 times higher than that of the wild-type strain.

[0061] 2. Development of the N5-15 strain from the N5 strain

[0062] The Lipomyces starkeyi N5 strain, obtained as a high-oil-producing strain by the procedures described above (the mutant strain that produced the highest amount of triglycerides), was subsequently irradiated with UV to induce mutations and thus develop a new mutant strain as another high-oil-producing strain. The method employed was the same as that used for the development of the N5 strain from the wild-type strain. Specifically, the mutant strain was subjected to Percoll density gradient centrifugation, after which the top layer was collected as a low-density fraction and the cells of this fraction were cultured. The fractionation and culture steps were repeated to concentrate the high-oil-producing strain, and the strain was isolated and evaluated.

[0063] Induction of mutations by UV irradiation

[0064] A 3 mL volume of YPD medium was added to a sterile test tube and inoculated with the Lipomyces starkeyi N5 strain from a stock plate. The strain cells were cultured at 30°C for two days (pre-culture). The culture fluid was collected and then adjusted to a cell concentration of 1.0 χ¹⁰⁷ cells / mL using YPD medium, and 7 mL of the culture fluid were transferred to an L-shaped test tube in which the cells were cultured for 24 hours (main culture). Subsequently, all the main culture fluid was transferred to a 15 mL test tube and centrifuged at 2270 χ¹⁰ g for five minutes, after which the supernatant was removed.

[0065] The precipitated cells were washed by suspending them in 1 mL of PBS, centrifuging the suspension at 2270 χ g for five minutes, and then removing the supernatant. Then, 7 mL Petition 870250087009, dated 09 / 25 / 2025, page 63 / 100 20 / 31 of 1 x PBS were added to an L-shaped test tube and the cells were suspended in the PBS. The suspension was diluted to a cell concentration of 1.0 χ¹⁰⁷ cells / mL using 1 x PBS, and 10 mL of the cell suspension were dispensed into a sterile Petri dish.

[0066] Next, the Petri dish containing the cell suspension was placed 25 cm away from the UV tube of a UV irradiator (TOSHIBA Lighting & Technology Corporation, model GL15, power = 4.9 W), and the cell suspension was irradiated with UV for 25 seconds. After UV irradiation, the cells in the Petri dish were cultured at 30°C for two days. The entire cell suspension in the Petri dish was added to a 15 mL tube and centrifuged at 2270 χ g for five minutes, after which the supernatant was removed. The cells were washed by adding 1 mL of SG medium to the tube to resuspend them, centrifuging the suspension at 2270 χ g for five minutes, and removing the supernatant.

[0067] 7 mL of SG medium were added to the tube to suspend the cells. All the culture fluid contained in the tube was used to inoculate a sterile L-shaped test tube, in which the cells were cultured at 30°C and 120 rpm for three days (initial culture). The initial culture fluid was adjusted to a final cell concentration of 1.25 χ 106 cells / mL, using 7% S medium (Table 5), and a 200 mL deflector flask was inoculated with the culture fluid to a 75 mL scale. The cells were then cultured in the flask at 30°C and 160 rpm for 24 hours (pre-culture). The pre-culture fluid was adjusted to a final cell concentration of 1.25 χ 10⁶ cells / mL using 7% S medium, and a 200 mL deflector flask was inoculated with the culture fluid to a 75 mL mark. The cells were then cultured in the flask at 30°C and 160 rpm for two days (main culture). Petition 870250087009, dated 09 / 25 / 2025, page 64 / 100 21 / 31

[0068] Table 5 Final concentration (NH4)2SO4 0.5% KH2PO4 0.1% NaCl 0.01% Yeast extract 0.1% D-(+)- Glucose 7% MgSO4^7H2O 0.05% CaCl2^7H2O 0.01%

[0069] Concentration of mutant strains with high oil production by density gradient centrifugation using Percoll's reagent

[0070] The number of cells in the main culture fluid was determined using a cell counter, and a volume containing 1.0 x 109 cells was transferred to a 15 mL tube. A suspension of Lipomyces starkeyi CBS1807 (wild-type strain) subjected to main culture and a suspension of the N5 strain subjected to main culture were similarly added to the tubes.

[0071] Each tube was centrifuged (oscillating rotor, 2270 xg, 5 minutes, room temperature) and the supernatant was removed. 500 μL of 1 x sterile PBS was added to the tube to suspend the cells, and the entire suspension was transferred to a 1.5 mL tube. Then, 1 x PBS was added to bring the suspension volume to the 1.0 mL mark on the 1.5 mL tube.

[0072] An 8 mL volume of Percoll's reagent (concentration = 40%) was added to an ultracentrifuge tube, into which the entire cell suspension was transferred from the 1.5 mL tube. The ultracentrifuge tube was gently inverted to mix its contents, and the mixture was subjected to ultracentrifugation using an ultracentrifuge (rotor 70.1Ti, 22000 rpm, 20 minutes, room temperature, Petition 870250087009, dated 09 / 25 / 2025, p. 65 / 100 22 / 31 (Slow acceleration, no break). After ultracentrifugation, the formed cell layer was observed. Subsequently, 1 mL of the upper layer formed as a low-density fraction was transferred to a 15 mL tube using a peristaltic pump.

[0073] The cells were washed as follows: 1 x sterile PBS 1, the volume of which was five times greater than that of the transferred top layer, was added to the tube to suspend the cells, and the suspension was centrifuged at 2270 xg for five minutes, after which the supernatant was removed. The cells were washed again by adding 1 mL of SG medium to the tube to suspend them, centrifuging the suspension at 2270 xg for five minutes and removing the supernatant. 7 mL of SG medium were added to the tube to suspend the cells.

[0074] Cell culture

[0075] All the culture fluid in the tube was used to inoculate a sterile L-shaped test tube, in which cells were cultured at 30°C and 120 rpm for three days (initial culture). The initial culture fluid was adjusted to a final cell concentration of 1.25 x 10⁶ cells / mL using 7% S medium, and a 200 mL deflector flask was inoculated with the culture fluid to a 75 mL scale. Cells were then cultured in the flask at 30°C and 160 rpm for 24 hours (pre-culture). The pre-culture fluid was adjusted to a final cell concentration of 1.25 x 10⁶ cells / mL using 7% S medium, and a 200 mL deflector flask was inoculated with the culture fluid to a 75 mL scale. The cells were then cultured in the flask at 30°C and 160 rpm for two days (main culture).

[0076] Concentration of mutant strains with high oil production by density gradient centrifugation using Percoll's reagent: second round Petition 870250087009, dated 09 / 25 / 2025, page 66 / 100 23 / 31

[0077] The procedures described in [Concentration of mutant strains with high oil production by density gradient centrifugation using Percoll's reagent] through [Cell culture] were repeated.

[0078] Concentration of mutant strains with high oil production by density gradient centrifugation using Percoll's reagent: third round

[0079] The procedures described in [Concentration of mutant strains with high oil production by density gradient centrifugation using Percoll's reagent] up to [Cell culture] were repeated. The cells in the tube were washed by adding 1 x sterile PBS, the volume of which was five times greater than that of the main culture fluid, to suspend the cells, centrifuging the suspension at 2270 * g for five minutes and removing the supernatant. Then, the cells were washed again by adding 1 mL of 7% S medium to the tube to suspend the cells, centrifuging the suspension at 2270 * g for five minutes and removing the supernatant.

[0080] When low-density cells that have produced and accumulated large amounts of oil are repeatedly concentrated, it is believed that the cell layer will gradually shift to a low-density fraction. As a result, the cell layer is expected to appear in a higher position than that of the wild-type strain or the N5 strain after repeated rounds of concentration. FIG. 9 shows images illustrating how the cell layers change after density gradient centrifugation. After the first concentration process, as shown in FIG. 9, the cell layer of the UV-treated sample was not observed above the cell layer of the N5 strain used as a control. After the second round of concentration, the cell layer of the UV-treated sample was found in a higher position than the upper portion of the cell layer. Petition 870250087009, dated 09 / 25 / 2025, page 67 / 100 24 / 31 cell layer of the N5 strain. The third round of concentration made the cell layer of the UV-treated sample clearly identifiable. This indicates that the cells with high oil production were more concentrated in the UV-treated sample than in the N5 strain.

[0081] Isolation of strains

[0082] The isolation of the colonies was carried out as follows: 100 μL of 7% S medium were added to the tube to suspend the cells, and then the suspension was seeded onto 7% solid S medium (Table 6), in which the cells were cultured at 30°C for three days. Non-mucoid colonies were re-spread onto 5% S medium, in which the cells were cultured at 30°C for three days. Among the cells of the re-spread colonies, those with a shiny appearance and believed to have produced a polysaccharide were removed to obtain 16 strains N5-1 to N5-16.

[0083] Table 6 Final concentration (NH4)2SO4 0.5% KH2PO4 0.1% NaCl 0.01% Yeast extract 0.1% D-(+)-Glucose 7% MgSO4^7H2O 0.05% CaCl2^7H2O 0.01% Agar 2%

[0084] Primary evaluation of strains with high oil production

[0085] L-shaped test tubes containing 7 mL of SG medium were inoculated with colonies of the 16 strains N5-1 to N5-16, with colonies of the wild-type strain and the N5 strain serving as controls. The strain cells were cultured at 30°C and 120 rpm for three Petition 870250087009, dated 09 / 25 / 2025, page 68 / 100 25 / 31 days (initial culture). Each initial culture fluid was adjusted to a final cell concentration of 1.25 χ¹⁰⁶ cells / mL using 5% S medium, and a 200 mL deflector flask was inoculated with the culture fluid to a 75 mL scale. Cells were then cultured in the flask at 30°C and 160 rpm for 24 hours (pre-culture). The pre-culture fluid was adjusted to a final cell concentration of 1.25 χ¹⁰⁶ cells / mL using 5% S medium, and a 200 mL deflector flask was inoculated with the culture fluid to a 75 mL scale. Cells were then cultured in the flask at 30°C and 160 rpm for three days (main culture).

[0086] A volume of each culture fluid was collected on the third day of the primary culture and analyzed for cell concentration (number of cells, FIG. 10), glucose consumption (FIG. 11), amount of triglycerides produced per volume of medium (FIG. 12), and amount of triglycerides produced normalized to the cell count (FIG. 13) in the same way as in 1. Development of the N5 strain from the wild-type strain. As seen in FIGS. 10 to 13, it was observed that the N5-2, N5-13, and N5-15 strains produced a significantly higher amount of triglycerides than the N5 strain. Thus, these three strains were subjected to secondary evaluation.

[0087] Secondary evaluation of strains with high oil production

[0088] A 50 mL volume of SG medium was added to individual 200 mL baffle flasks, which were inoculated with colonies of strains N5-2, N5-13, N5-15 and colonies of the wild type strain and strain N5, serving as controls. The cells in the flasks were cultured at 30°C and 160 rpm for three days (initial culture). Each initial culture fluid was adjusted to a final cell concentration of 3.0 χ¹⁰⁶ cells / mL using 7% S medium, and a 500 mL baffle flask was inoculated with the culture fluid in a Petition 870250087009, dated 09 / 25 / 2025, page 69 / 100 26 / 31 200 mL. The cells were then cultured in the flask at 30°C and 160 rpm for 24 hours (pre-culture). The pre-culture fluid was adjusted to a cell concentration of 1.2 χ 107 cells / mL using 7% S medium, and a 500 mL deflector flask was inoculated with the culture fluid on a 200 mL scale. The cells were then cultured in the flask at 30°C and 120 rpm for six days (main culture).

[0089] FIG. 14 is a graph showing the cell concentration measured over the six days of culture for strains N5-2, N5-13, and N5-15 and the control strains. The difference in growth rate was observed early in the culture, and the growth rate and final cell concentration were lower for strains N5-2, N5-13, and N5-15 than for strain N5. On the third day of culture, the cell concentration of strain N5-2 was 0.88 times higher than that of strain N5, the cell concentration of strain N5-13 was 0.90 times higher than that of strain N5, and the cell concentration of strain N5-15 was 0.79 times higher than that of strain N5.

[0090] FIG. 15 is a graph showing the average cell size measured over the six days of culture for strains N5-2, N5-13, and N5-15 and the control stains. For all strains N5-2, N5-13, and N5-15, the average cell size was already larger than that of the wild-type strain on day 0 of culture. Cells of strains N5-2 and N5-13 reached their maximum size on the fourth day of culture, and cells of strain N5-15 continued to grow until the sixth day. On the fourth day of culture, cells of strain N5-2 were approximately 1.05 times larger than those of strain N5, cells of strain N5-13 were approximately 1.06 times larger than those of strain N5, and cells of strain N5-15 were approximately 1.12 times larger than those of strain N5.

[0091] FIG. 16 shows microscopic images of the wild-type strain (WT) and strains N5, N5-2, N5-13 and N5-15 captured on the sixth Petition 870250087009, dated 09 / 25 / 2025, p. 70 / 100 27 / 31 day of culture. As seen in FIG. 16, it was found that the cells of strains N5-2, N5-13 and N5-15 were larger than those of strain N5. In addition, it was found that strains N5-2, N5-13 and N5-15 had larger fat globules than strain N5. This demonstrates that strains N5-2, N5-13 and N5-15 are superior in oil productivity.

[0092] FIG. 17 is a graph showing the glucose concentration (remaining glucose concentration) in the medium, measured over the six days of culture for strains N5-2, N5-13 and N5-15 and the control strains. As seen in FIG. 17, strains N5, N5-2 and N5-13 completely assimilated the glucose from the medium on the sixth day of culture. The N5-15 strain assimilated glucose more rapidly than the other strains and depleted it between the fifth and sixth day.

[0093] FIG. 18 is a graph showing the triglyceride (TAG) content per volume of medium measured over six days of culture for strains N5-2, N5-13 and N5-15 and the control strains. As seen in FIG. 18, on the fourth day of culture, the triglyceride content per volume of medium for strain N5-2 was approximately 1.11 times that of strain N5, the triglyceride content per volume of medium for strain N5-13 was approximately 1.09 times that of strain N5, and the triglyceride content per volume of medium for strain N5-15 was approximately 1.35 times that of strain N5.

[0094] FIG. 19 is a graph showing the cell count-normalized triglyceride (TAG) content measured over six days of culture for strains N5-2, N5-13, and N5-15 and control strains. As seen in FIG. 19, the cell count-normalized triglyceride content for strain N5-2 was approximately 1.17 times higher than that of strain N5, the cell count-normalized triglyceride content for strain N5-13 was approximately 1.21 times higher than that of strain N5, and the cell count-normalized triglyceride content for strain N5-15 was approximately 1.82 times higher than that of strain N5. The more glucose was assimilated, the more triglycerides were... Petition 870250087009, dated 09 / 25 / 2025, page 71 / 100 28 / 31 ram produced; this indicates a correlation between glucose consumption and triglyceride production. In other words, the N5-15 strain, which showed the fastest glucose consumption, produced triglycerides at the highest rate.

[0095] Fatty acid composition analysis

[0096] The fatty acid composition was analyzed in the fatty acids produced by the cells of the five strains: N5-2, N5-13 and N515, the wild type strain and N5.

[0097] Cell culture

[0098] A 50 mL volume of SG medium was added to individual 200 mL baffle flasks, which were inoculated with colonies of strains N5-2, N5-13, N5-15, the wild-type strain, and strain N5. The cells in the flasks were cultured at 30°C and 160 rpm for three days (initial culture). Each initial culture fluid was adjusted to a final cell concentration of 3.0 χ¹⁰⁶ cells / mL using 7% S medium, and a 500 mL baffle flask was inoculated with the culture fluid on a 200 mL scale. The cells were then cultured in the flask at 30°C and 120 rpm for 24 hours (pre-culture). The pre-culture fluid was adjusted to a cell concentration of 1.2 χ¹⁰⁷ cells / mL using 7% S medium, and a 500 mL baffle flask was inoculated with the culture fluid on a 200 mL scale. The cells were then cultured in the flask at 30°C and 120 rpm for four days (main culture).

[0099] Samples for fatty acid composition analysis

[00100] A sample for triglyceride quantification was prepared from each main culture fluid in the same manner as in “Measurement of the amount of triglycerides produced” of “1. Development of the N5 strain from a wild-type strain” described above. A Multi-bead Shocker tube was loaded with 125 μL of chloroform, 250 μL of methanol, and 100 μL of the sample for quantification. Petition 870250087009, dated 09 / 25 / 2025, page 72 / 100 29 / 31 triglyceride concentration, 0.4 g of glass beads were added (No. 0.40 from Sansho Kenmazai Co., Ltd.). The cells were then disrupted with the Multi-beads Shocker (2500 rpm, 4°C, 900 seconds).

[00101] To the tube, 125 μL of chloroform and 125 μL of sterile purified water were added, and then the cells were again disrupted with the Multi-beads Shocker (2500 rpm, 4°C, 300 seconds). The disrupted cell suspension in the tube was shaken with a vortex mixer and transferred to a new 2 mL tube. 200 pL of chloroform, 200 pL of methanol, and 180 pL of sterile purified water were added to the test tube, and the contents were shaken with a vortex mixer.

[00102] The tube was centrifuged at 4°C and 10000 χ g for five minutes, after which the organic layer formed as the bottom layer was separated and transferred to a new 2 mL tube. The solvent was distilled from the tube using nitrogen gas, and the fatty acids in the tube were converted to methyl esters using a fatty acid methylation kit for triglycerides (Nacalai Tesque, Inc.). The esterified methyl sample was processed into a sample for fatty acid composition analysis according to the kit's instructions for use. The sample for fatty acid composition analysis was subjected to fatty acid composition analysis using gas chromatography under the conditions listed in Table 7.

[00103] Table 7 Gas chromatography conditions GC-2014 system, Gas Chromatography manufactured by Shimadzu Corporation. DB-23 column, manufactured by Aglient Technologies. Length = 60 m, internal diameter = 0.25 mm, film thickness = 0.15 µm. Petition 870250087009, dated 09 / 25 / 2025, page 73 / 100 30 / 31 Injection volume 1μL Injection method Split injection, 10:1 Temperature on the day of injection 250°C Pressure on the day of injection 150 KPa Oven 50°C (1 min), heating to 175°C at 25°C / min, heating to 230°C at 4°C / min Carrier gas Helium, linear velocity = 22.1 cm / s Column flow rate 1.11 mL / min Purge flow rate 3.0 mL / min Total flow rate 15.2 mL / min Detector FID, 280°C Detector gas Hydrogen, air

[00104] Table 8 shows the results of the fatty acid composition analysis of the triglycerides produced by the five strains. The fatty acid composition analysis was performed on the fourth day of cultivation. For strain N5, the palmitic acid content was slightly higher and the stearic acid content slightly lower than for the wild-type strain (WT). The fatty acid composition determined for each of the N5-2, N5-13, and N5-15 strains developed from the mutant strain N5 was substantially the same as that determined for strain N5. The fatty acid composition determined for each of these mutant strains consisted mainly of palmitic acid and oleic acid, and an oil with this composition is a promising candidate to serve as an alternative to palm oil. Table 8 Fatty acids (%) WT N5 N5-2 N5-13 N5-15 Palm oil C14:0 Myristic acid 0.65 0.08 0.08 0.08 0.64 1.10 C16:0 Palmitic acid 35.89 40.51 41.65 41.89 39.59 44.00 Petition 870250087009, dated 09 / 25 / 2025, p. 74 / 100 31 / 31 C16:1 Palmitoleic acid 3.63 3.60 4.16 4.21 3.76 0.20 C18:0 Stearic acid 5.50 3.93 2.88 3.18 3.76 4.40 C18:In9c Oleic acid 46.74 47.25 46.32 45.84 46.84 39.20 C18:2n6c Linoleic acid 3.37 2.38 2.63 2.53 3.25 9.70 Other fatty acids 4.22 2.24 2.28 2.27 2.17 1.40

[00105] The N5-15 strain, which demonstrated the highest oil productivity, was deposited on December 14, 2022, with the National Institute of Technology and Evaluation, Patent Microorganisms Depositary, under accession number NITE BP-03796.

[00106] Industrial Applicability

[00107] The present invention is useful in an industrial method for producing an alternative to palm oil that can be used in applications such as edible oils and cosmetic ingredients. Petition 870250087009, dated 09 / 25 / 2025, p. 75 / 100

Claims

1 / 1 CLAIMS 1. High oil-producing strain of yeast belonging to the genus Lipomyces, characterized in that the high oil-producing strain is deposited with the National Institute of Technology and Evaluation, Patent Microorganisms Depositary, under accession number NITE BP-03796.

2. A method for producing an oil, characterized in that it comprises the use of a high-oil-producing strain of oleaginous yeast belonging to the genus Lipomyces, the high-oil-producing strain being deposited with the National Institute of Technology and Evaluation, Patent Microorganisms Depositary, under accession number NITE BP-03796. Petition 870250087009, dated 09 / 25 / 2025, p. 42 / 100

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