APPLIED MICROBIAL AND CELL PHYSIOLOGY Highly selective isolation and characterization of Lipomyces starkeyi mutants with increased production of triacylglycerol Harutake Yamazaki 1 & Suzuka Kobayashi 1 & Sayaka Ebina 1 & Shiho Abe 1 & Satoshi Ara 1 & Yosuke Shida 2 & Wataru Ogasawara 2 & Katsurou Yaoi 3 & Hideo Araki 4 & Hiroaki Takaku 1 Received: 18 February 2019 /Revised: 6 May 2019 /Accepted: 16 May 2019 /Published online: 5 June 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract The oleaginous yeast Lipomyces starkeyi is an attractive organism for the industrial production of lipids; however, the amount of lipid produced by wild-type L. starkeyi is insufficient. The study aims to obtain L. starkeyi mutants that rapidly accumulate large amounts of triacylglycerol (TAG). Mutagenized yeast cells at the early stages of cultivation were subjected to Percoll density gradient centrifugation; cells with increased production of TAG were expected to be enriched in the resultant upper fraction because of their lower density. Among 120 candidates from the upper fractions, five mutants were isolated that accumulated higher amounts of TAG. Moreover, when omitting cells with mucoid colony morphology, 11 objective mutants from 11 candidates from the upper fraction were effectively (100%) isolated. Of total 16 mutants obtained, detailed characterization of five mutants was performed to reveal that five mutants achieved about 1.5–2.0 times TAG concentration (4.7–6.0 g/L) as compared with the wild-type strain (3.6 g/L) at day 5. Among these five mutants, strain E15 was the best for industrial use because only strain E15 showed significantly higher TAG concentration as well as significantly higher degree of lipid to glucose and biomass to glucose yields than the wild-type strain. Thus, Percoll density gradient centrifugation is an effective method to isolate mutant cells that rapidly accumulate large amounts of TAG. It is expected that by repeating this procedure as part of a yeast-breeding program, L. starkeyi mutants suitable for industrial lipid production can be easily and effectively obtained. Keywords Oleaginous yeast . Lipomyces starkeyi . Triacylglycerol . Percoll . Lipid droplet Introduction Global energy demand is constantly increasing, and excessive use of fossil fuels has caused increases in greenhouse gas emissions. As such, there is an urgent need to reduce emissions of greenhouse gases that cause global warming. Future energy demands compel us to explore the production of biofuels, i.e., clean fuels produced through the harnessing of biological pro- cesses, as a suitable alternative source of energy. For example, biodiesel is already an alternative fuel to petroleum diesel used widely in Europe. However, since biofuels are currently made primarily from edible oils, the production of biofuels is com- petitive to feed and food production, leading to a global food supply imbalance, and requires large areas of land for produc- tion, which may cause deforestation and destruction of the ecosystem (Gui et al. 2008). To overcome this problem, oils produced by microorgan- isms are becoming an attractive alternative to petroleum be- cause their production and use do not affect food supply or require a large amount of space. It has been reported that many microorganisms can accumulate large amounts of lipid per cell (Ratledge 2004; Liang and Jiang 2013). Among these microorganisms, oleaginous yeasts, including Yarrowia * Hiroaki Takaku htakaku@nupals.ac.jp 1 Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Akiha-ku, Niigata 956-8603, Japan 2 Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan 3 Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan 4 Research Institute for Creating the Future, FUJI OIL HOLDINGS INC, 4-3 Kinunodai, Tsukubamirai-shi, Ibaraki 300-2497, Japan Applied Microbiology and Biotechnology (2019) 103:6297–6308 https://doi.org/10.1007/s00253-019-09936-3