Abstract
Lipomyces starkeyi is an oil-producing yeast that can produce triacylglycerol (TAG) from glycerol as a carbon source. The TAG was mainly produced after nitrogen depletion alongside reduced cell proliferation. To obtain clues for enhancing the TAG production, cell metabolism during the TAG-producing phase was characterized by metabolomics with 13C labeling. The turnover analysis showed that the time constants of intermediates from glycerol to pyruvate (Pyr) were large, whereas those of tricarboxylic acid (TCA) cycle intermediates were much smaller than that of Pyr. Surprisingly, the time constants of intermediates in gluconeogenesis and the pentose phosphate (PP) pathway were large, suggesting that a large amount of the uptaken glycerol was metabolized via the PP pathway. To synthesize fatty acids that make up TAG from acetyl-CoA (AcCoA), 14 molecules of nicotinamide adenine dinucleotide phosphate (NADPH) per C16 fatty acid molecule are required. Because the oxidative PP pathway generates NADPH, this pathway would contribute to supply NADPH for fatty acid synthesis. To confirm that the oxidative PP pathway can supply the NADPH required for TAG production, flux analysis was conducted based on the measured specific rates and mass balances. Flux analysis revealed that the NADPH necessary for TAG production was supplied by metabolizing 48.2% of the uptaken glycerol through gluconeogenesis and the PP pathway. This result was consistent with the result of the 13C-labeling experiment. Furthermore, comparison of the actual flux distribution with the ideal flux distribution for TAG production suggested that it is necessary to flow more dihydroxyacetonephosphate (DHAP) through gluconeogenesis to improve TAG yield.
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Acknowledgements
This work was supported by the Japan Science and Technology Agency, Adaptable and Seamless Technology Transfer Program, JST A-STEP through target-driven R&D. We thank Dr. Takafumi Naganuma (Yamanashi University, Kofu, Japan) for providing advice on L. starkeyi culture. We thank Dr. Kenjiro Kami (Human Metabolome Technologies Inc., Tsuruoka, Japan) for providing advice on metabolome data interpretation.
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This study was funded by Adaptable and Seamless Technology Transfer Program through target-driven R&D (A-STEP) from the Japan Science and Technology Agency (JST).
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Maruyama, Y., Toya, Y., Kurokawa, H. et al. Characterization of oil-producing yeast Lipomyces starkeyi on glycerol carbon source based on metabolomics and 13C-labeling. Appl Microbiol Biotechnol 102, 8909–8920 (2018). https://doi.org/10.1007/s00253-018-9261-5
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DOI: https://doi.org/10.1007/s00253-018-9261-5