Characterization of oil-producing yeast Lipomyces starkeyi on glycerol carbon source based on metabolomics and 13C-labeling
- 224 Downloads
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.
KeywordsLipomyces starkeyi Glycerol Triacylglycerol production Metabolomics 13C-labeling experiment Flux analysis
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.
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).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants performed by any of the authors.
- Goncalves EC, Wilkie AC, Kirst M, Rathinasabapathi B (2016) Metabolic regulation of triacylglycerol accumulation in the green algae: identification of potential targets for engineering to improve oil yield. Plant Biotechnol J 14:1649–1660. https://doi.org/10.1111/pbi.12523 CrossRefPubMedPubMedCentralGoogle Scholar
- Habe H, Shimada Y, Yakushi T, Hattori H, Ano Y, Fukuoka T, Kitamoto D, Itagaki M, Watanabe K, Yanagishita H, Matsushita K, Sakaki K (2009) Microbial production of glyceric acid, an organic acid that can be mass produced from glycerol. Appl Environ Microbiol 75:7760–7766. https://doi.org/10.1128/AEM.01535-09 CrossRefPubMedPubMedCentralGoogle Scholar
- Hasunuma T, Harada K, Miyazawa S, Kondo A, Fukusaki E, Miyake C (2010) Metabolic turnover analysis by a combination of in vivo 13C-labelling from 13CO2 and metabolic profiling with CE-MS/MS reveals rate-limiting steps of the C3 photosynthetic pathway in Nicotiana tabacum leaves. J Exp Bot 61:1041–1051. https://doi.org/10.1093/jxb/erp374 CrossRefPubMedGoogle Scholar
- Hirayama A, Kami K, Sugimoto M, Sugawara M, Toki N, Onozuka H, Kinoshita T, Saito N, Ochiai A, Tomita M, Esumi H, Soga T (2009) Quantitative metabolome profiling of colon and stomach cancer microenvironment by capillary electrophoresis time-of-flight mass spectrometry. Cancer Res 69:4918–4925. https://doi.org/10.1158/0008-5472.CAN-08-4806 CrossRefPubMedGoogle Scholar
- Ichihashi K, Yuki D, Kurokawa H, Igarashi A, Yajima T, Fujiwara M, Maeno K, Sekiguchi S, Iwata M, Nishino H (2011) Dynamic analysis of phorbol esters in the manufacturing process of fatty acid methyl esters from Jatropha curcas seed oil. J Am Oil Chem Soc 88:851–861. https://doi.org/10.1007/s11746-010-1741-4 CrossRefGoogle Scholar
- Kurokawa H, Fukano Y, Ohki T, Naganuma T (2017) Conversion of glycerol to triacylglycerol by Lipomyces yeast. Oleoscience 17:127–133Google Scholar
- Kurtzman CP, Boekhout JW (2011) The yeast, a taxonomic study, Fifth Edition. ElsevierGoogle Scholar
- Matsumoto M, Nojima D, Tanaka T (2016) Development of outdoor mass cultivation for green oil production using marine microalgae. Bioindustry 33:10Google Scholar
- OECD/FAO (2011) OECD-FAO Agricultural Outlook 2011–2020, OECD Publishing and FAO. https://doi.org/10.1787/agr_outlook-2011-en. Accessed 19 Jan 2018
- Pavel K, Petr S, Ivana B (2005) WO2005/021476. US Patent. https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2005021476&redirectedID=true. Accessed 19 Jan 2018
- Spier F, Buffon JG, Burkert CA (2015) Bioconversion of raw glycerol generated from the synthesis of biodiesel by different oleaginous yeasts: lipid content and fatty acid profile of biomass. Indian J Microbiol 55:415–422. https://doi.org/10.1007/s12088-015-0533-9 CrossRefPubMedPubMedCentralGoogle Scholar
- Tchakouteu SS, Kalantzi O, Gardeli C, Koutinas AA, Aggelis G, Papanikolaou S (2015) Lipid production by yeasts growing on biodiesel-derived crude glycerol: strain selection and impact of substrate concentration on the fermentation efficiency. J Appl Microbiol 118:911–927. https://doi.org/10.1111/jam.12736 CrossRefPubMedGoogle Scholar
- Tsakona S, Kopsahelis N, Chatzifragkou A, Papanikolaou S, Kookos IK, Koutinas AA (2014) Formulation of fermentation media from flour-rich waste streams for microbial lipid production by Lipomyces starkeyi. J Biotechnol 189:36–45. https://doi.org/10.1016/j.jbiotec.2014.08.011 CrossRefPubMedGoogle Scholar
- UN (2015) World population prospects: the 2015 revision. United Nations DESA/Population division. https://esa.un.org/unpd/wpp/publications/files/key_findings_wpp_2015.pdf. Accessed 19 Jan 2018
- Xu J, Zhao X, Wang W, Du W, Liu D (2012) Microbial conversion of biodiesel byproduct glycerol to triacylglycerols by oleaginous yeast Rhodosporidium toruloides and the individual effect of some impurities on lipid production. Biochem Eng J 65:30–36. https://doi.org/10.1016/j.bej.2012.04.003 CrossRefGoogle Scholar
- Yamauchi H, Mori H, Kobayashi T, Shimizu S (1983) Mass production of lipids by Lipomyces starkeyi in microcomputer-aided fed-batch culture. J Ferment Technol 61:275–281Google Scholar
- Zhang H, Zhang L, Chen H, Chen YQ, Ratledge C, Song Y, Chen W (2013) Regulatory properties of malic enzyme in the oleaginous yeast, Yarrowia lipolytica, and its non-involvement in lipid accumulation. Biotechnol Lett 35:2091–2098. https://doi.org/10.1007/s10529-013-1302-7 CrossRefPubMedGoogle Scholar