Applied Microbiology and Biotechnology

, Volume 102, Issue 20, pp 8909–8920 | Cite as

Characterization of oil-producing yeast Lipomyces starkeyi on glycerol carbon source based on metabolomics and 13C-labeling

  • Yuki MaruyamaEmail author
  • Yoshihiro Toya
  • Hiroshi Kurokawa
  • Yuka Fukano
  • Atsushi Sato
  • Hiroyasu Umemura
  • Kaoru Yamada
  • Hideaki Iwasaki
  • Norio Tobori
  • Hiroshi Shimizu
Applied microbial and cell physiology


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.


Lipomyces 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.

Ethical approval

This article does not contain any studies with human participants performed by any of the authors.

Supplementary material

253_2018_9261_MOESM1_ESM.pdf (179 kb)
ESM 1 (PDF 179 kb)


  1. Amaretti A, Raimondia S, Leonardia A, Rossia M (2012) Candida Freyschussii: an oleaginous yeast producing lipids from glycerol. Chem Eng Trans 27:139–144. CrossRefGoogle Scholar
  2. An PNT, Yamaguchi M, Fukusaki E (2017) Metabolic profiling of Drosophila melanogaster metamorphosis: a new insight into the central metabolic pathways. Metabolomics 13:29. CrossRefGoogle Scholar
  3. Barnes LD, Kuehn GD, Atkinson DE (1971) Yeast diphosphopyridine nucleotide specific isocitrate dehydrogenase. Purification and some properties. Biochemistry 10:3939–3944. CrossRefPubMedGoogle Scholar
  4. Canelas AB, Ras C, ten Pierick A, van Dam JC, Heijnen JJ, van Gulik WM (2008) Leakage-free rapid quenching technique for yeast metabolomics. Metabolomics 4:226–239. CrossRefGoogle Scholar
  5. Edwards JS, Ibarra RU, Palsson BO (2001) In silico predictions of Escherichia coli metabolic capabilities are consistent with experimental data. Nat Biotechnol 19:125–130. CrossRefPubMedGoogle Scholar
  6. Evans CT, Ratledge C (1985) The role of NAD+: isocitrate dehydrogenase in lipid accumulation by the oleaginous yeast Rhodosporidium toruloides CBS 14. Can J Microbiol 31:845–850. CrossRefGoogle Scholar
  7. Feist AM, Palsson BO (2010) The biomass objective function. Curr Opin Microbiol 13:344–349. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Habe H, Sato S, Fukuoka T, Kitamoto D, Yakushi T, Matsushita K, Sasaki K (2011) Membrane-bound alcohol dehydrogenase is essential for glyceric acid production in Acetobacter tropicalis. J Oleo Sci 60:489–494. CrossRefPubMedGoogle Scholar
  11. 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. CrossRefPubMedGoogle Scholar
  12. 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. CrossRefPubMedGoogle Scholar
  13. 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. CrossRefGoogle Scholar
  14. Ito T, Tanaka M, Shinkawa H, Nakada T, Ano Y, Kurano N, Soga T, Tomita M (2013) Metabolic and morphological changes of an oil accumulating trebouxiophycean alga in nitrogen-deficient conditions. Metabolomics 9:S178–S187. CrossRefGoogle Scholar
  15. Joyce AR, Palsson BO (2007) Toward whole cell modeling and simulation: comprehensive functional genomics through the constraint-based approach. Prog Drug Res 64:265–309. CrossRefPubMedGoogle Scholar
  16. Korenaga H, Naganuma T, Uzuka Y, Tanaka K (1977) Determination of simple defined media for normal growth and lipid production of the yeast Lipomyces starkeyi. Nippon Nōgeikagaku Kaishi 51:449–455. CrossRefGoogle Scholar
  17. Kurokawa H, Fukano Y, Ohki T, Naganuma T (2017) Conversion of glycerol to triacylglycerol by Lipomyces yeast. Oleoscience 17:127–133Google Scholar
  18. Kurtzman CP, Boekhout JW (2011) The yeast, a taxonomic study, Fifth Edition. ElsevierGoogle Scholar
  19. Lin J, Shen H, Tan H, Zhao X, Wu S, Hu C, Zhao ZK (2011) Lipid production by Lipomyces starkeyi cells in glucose solution without auxiliary nutrients. J Biotechnol 152:184–188. CrossRefPubMedGoogle Scholar
  20. Liu L, Zong M, Hu Y, Li N, Lou W, Wu H (2017) Efficient microbial oil production on crude glycerol by Lipomyces starkeyi AS 2.1560 and its kinetics. Process Biochem 58:230–238. CrossRefGoogle Scholar
  21. Matsumoto M, Nojima D, Tanaka T (2016) Development of outdoor mass cultivation for green oil production using marine microalgae. Bioindustry 33:10Google Scholar
  22. Meesters PAEP, Huijberts GNM, Eggink G (1996) High-cell-density cultivation of the lipid accumulating yeast Cryptococcus curvatus using glycerol as a carbon source. Appl Microbiol Biotechnol 45:575–579. CrossRefGoogle Scholar
  23. Nakagawa Y, Shinmia Y, Kosoa S, Tomishige K (2010) Direct hydrogenolysis of glycerol into 1,3-propanediol over rhenium-modified iridium catalyst. J Catal 272:191–194. CrossRefGoogle Scholar
  24. OECD/FAO (2011) OECD-FAO Agricultural Outlook 2011–2020, OECD Publishing and FAO. Accessed 19 Jan 2018
  25. Pavel K, Petr S, Ivana B (2005) WO2005/021476. US Patent. Accessed 19 Jan 2018
  26. Ratledge C (2014) The role of malic enzyme as the provider of NADPH in oleaginous microorganisms: a reappraisal and unsolved problems. Biotechnol Lett 36:1557–1568. CrossRefPubMedGoogle Scholar
  27. Sauer U, Canonaco F, Heri S, Perrenoud A, Fischer E (2004) The soluble and membrane-bound transhydrogenases UdhA and PntAB have divergent functions in NADPH metabolism of Escherichia coli. J Biol Chem 279:6613–6619. CrossRefPubMedGoogle Scholar
  28. Soga T, Igarashi K, Ito C, Mizobuchi K, Zimmermann HP, Tomita M (2009) Metabolomic profiling of anionic metabolites by capillary electrophoresis mass spectrometry. Anal Chem 81:6165–6174. CrossRefPubMedGoogle Scholar
  29. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Sugimoto M, Wong DT, Hirayama A, Soga T, Tomita M (2010) Capillary electrophoresis mass spectrometry-based saliva metabolomics identified oral, breast and pancreatic cancer-specific profiles. Metabolomics 6:78–95. CrossRefPubMedGoogle Scholar
  31. Tang W, Zhang S, Wang Q, Tan H, Zhao ZK (2009) The isocitrate dehydrogenase gene of oleaginous yeast Lipomyces starkeyi is linked to lipid accumulation. Can J Microbiol 55:1062–1069. CrossRefPubMedGoogle Scholar
  32. Tapia VE, Anschau A, Coradini AL, Franco TT, Deckmann AC (2012) Optimization of lipid production by the oleaginous yeast Lipomyces starkeyi by random mutagenesis coupled to cerulenin screening. AMB Express 2:64–71. CrossRefGoogle Scholar
  33. 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. CrossRefPubMedGoogle Scholar
  34. Theodoulou FL, Holdsworth M, Baker A (2006) Peroxisomal ABC transporters. FEBS Lett 580:1139–1155. CrossRefPubMedGoogle Scholar
  35. 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. CrossRefPubMedGoogle Scholar
  36. UN (2015) World population prospects: the 2015 revision. United Nations DESA/Population division. Accessed 19 Jan 2018
  37. van Winden WA, Wittmann C, Heinzle E, Heijnen JJ (2002) Correcting mass isotopomer distributions for naturally occurring isotopes. Biotechnol Bioeng 80:477–479. CrossRefPubMedGoogle Scholar
  38. Varma A, Palsson BO (1994) Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110. Appl Environ Microbiol 60:3724–3731PubMedPubMedCentralGoogle Scholar
  39. Wasylenko TM, Ahn WS, Stephanopoulos G (2015) The oxidative pentose phosphate pathway is the primary source of NADPH for lipid overproduction from glucose in Yarrowia lipolytica. Metab Eng 30:27–39. CrossRefPubMedGoogle Scholar
  40. 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. CrossRefGoogle Scholar
  41. 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
  42. Yang F, Hanna MA, Sun R (2012) Value-added uses for crude glycerol–a byproduct of biodiesel production. Biotechnol Biofuels 5:13. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Yang ZK, Niu YF, Ma YH, Xue J, Zhang MH, Yang WD, Liu JS (2013) Molecular and cellular mechanisms of neutral lipid accumulation in diatom following nitrogen deprivation. Biotechnol Biofuels 6:67. CrossRefPubMedPubMedCentralGoogle Scholar
  44. Yang X, Jin G, Gong Z, Shen H, Bai F, Zhao ZK (2014) Recycling biodiesel-derived glycerol by the oleaginous yeast Rhodosporidium toruloides Y4 through the two-stage lipid production process. Biochem Eng J 91:86–91. CrossRefGoogle Scholar
  45. 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. CrossRefPubMedGoogle Scholar
  46. Zhu Z, Zhang S, Liu H, Shen H, Lin X, Yang F, Zhou YJ, Jin G, Ye M, Zou H, Zhao ZK (2012) A multi-omic map of the lipid-producing yeast Rhodosporidium toruloides. Nat Commun 3:1112. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Yuki Maruyama
    • 1
    Email author
  • Yoshihiro Toya
    • 2
  • Hiroshi Kurokawa
    • 3
  • Yuka Fukano
    • 3
  • Atsushi Sato
    • 1
  • Hiroyasu Umemura
    • 1
  • Kaoru Yamada
    • 1
  • Hideaki Iwasaki
    • 1
  • Norio Tobori
    • 3
  • Hiroshi Shimizu
    • 2
  1. 1.Analytical Technology Research Center, Research and Development HeadquartersLion CorporationTokyoJapan
  2. 2.Department of Bioinformatic Engineering, Graduate School of Information Science and TechnologyOsaka UniversityOsakaJapan
  3. 3.Functional Materials Science Research Laboratories, Research and Development HeadquartersLion CorporationTokyoJapan

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