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Laboratory Screening Protocol to Identify Novel Oleaginous Yeasts

  • Irnayuli R. Sitepu
  • Antonio L. Garay
  • Tomas Cajka
  • Oliver Fiehn
  • Kyria L. Boundy-MillsEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1995)

Abstract

Oleaginous microbes, which contain over 20% intracellular lipid, predominantly triacylglycerols (TG), by dry weight, have been discovered to have high oil content by many different protocols, ranging from simple staining to more complex chromatographic methods. In our laboratory, a methodical process was implemented to identify high oil yeasts, designed to minimize labor while optimizing success in identifying high oil strains among thousands of candidates. First, criteria were developed to select candidate yeast strains for analysis. These included observation of buoyancy of the yeast cell mass in 20% glycerol, and phylogenetic placement near known oleaginous species. A low-labor, semiquantitative Nile red staining protocol was implemented to screen numerous yeast cultures for high oil content in 96-well plates. Then, promising candidates were selected for more quantitative analysis. A more labor-intensive and quantitative gravimetric assay was implemented that gave consistent values for intracellular oil content for a broad range of yeast species. Finally, an LC-MS protocol was utilized to quantify and identify yeast triacylglycerols. This progressive approach was successful in identifying 30 new oleaginous yeast species, out of over 1000 species represented in the Phaff Yeast Culture Collection.

Key words

Oleaginous microbes Yeast Triacylglycerol Nile red Gravimetric Liquid chromatography–mass spectrometry Lipidomics 

Notes

Acknowledgments

L.A. Garay was funded by the National Mexican Council of Science and Technology (CONACYT) Fellowship number 291795. This work, including the efforts of T. Cajka and O. Fiehn, was funded by the National Institutes of Health [grant numbers NIH HL113452 and NIH DK097154], as well as the NIH instrument funding [grant number NIH S10-RR031630].

References

  1. 1.
    Ratledge C (1979) Resources conservation by novel biological processes. Part 1: Grow fats from wastes. Chem Soc Rev 8:283–296CrossRefGoogle Scholar
  2. 2.
    Garay LA, Sitepu IR, Cajka T, Chandra I, Shi S, Lin T, German JB, Fiehn O, Boundy-Mills KL (2016) Eighteen new oleaginous yeast species. J Ind Microbiol Biotechnol 43(7):887–900CrossRefGoogle Scholar
  3. 3.
    Sitepu IR, Sestric R, Ignatia L, Levin D, Bruce German J, Gillies LA, Almada LA, Boundy-Mills KL (2013) Manipulation of culture conditions alters lipid content and fatty acid profiles of a wide variety of known and new oleaginous yeasts species. Bioresour Technol 144:360–369CrossRefGoogle Scholar
  4. 4.
    Ledesma-Amaro R (2015) Microbial oils: a customizable feedstock through metabolic engineering. Eur J Lipid Sci Technol 117(2):141–144CrossRefGoogle Scholar
  5. 5.
    Sitepu I, Garay L, Sestric R, Levin D, Block DE, German J, Boundy-Mills K (2014) Oleaginous yeasts for biodiesel: current and future trends in biology and production. Biotechnol Adv 32(7):1336–1360.  https://doi.org/10.1016/j.biotechadv.2014.08.003 CrossRefPubMedGoogle Scholar
  6. 6.
    Tsouko E, Papanikolaou S, Koutinas A (2016) Production of fuels from microbial oil using oleaginous microorganisms. In: Handbook of biofuels production. Elsevier B.V., Amsterdam, p 201Google Scholar
  7. 7.
    Oguri E, Masaki K, Naganuma T, Iefuji H (2012) Phylogenetic and biochemical characterization of the oil-producing yeast Lipomyces starkeyi. Antonie Van Leeuwenhoek 101(2):359–368CrossRefGoogle Scholar
  8. 8.
    Gujjari P, Suh S-O, Coumes K, Zhou J (2011) Characterization of oleaginous yeasts revealed two novel species: Trichosporon cacaoliposimilis sp. nov. and Trichosporon oleaginousus sp. nov. Mycologia 103(5):1110–1118.  https://doi.org/10.3852/10-403 CrossRefPubMedGoogle Scholar
  9. 9.
    Cajka T, Fiehn O (2014) Comprehensive analysis of lipids in biological systems by liquid chromatography-mass spectrometry. TrAC Trends Anal Chem 61:192–206CrossRefGoogle Scholar
  10. 10.
    Kind T, Liu K-H, Lee DY, DeFelice B, Meissen JK, Fiehn O (2013) LipidBlast in silico tandem mass spectrometry database for lipid identification. Nat Methods 10(8):755–758.  https://doi.org/10.1038/nmeth.2551 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Suutari M, Priha P, Laakso S (1993) Temperature shifts in regulation of lipids accumulated by Lipomyces starkeyi. J Am Oil Chem Soc 70:891–894CrossRefGoogle Scholar
  12. 12.
    Sitepu I, Ignatia L, Franz A, Wong D, Faulina S, Tsui M, Kanti A, Boundy-Mills K (2012) An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species. J Microbiol Methods 91(2):321–328CrossRefGoogle Scholar
  13. 13.
    Lundin H (1950) Fat synthesis by micro-organisms and its possible applications in industry. J Inst Brew 56(1):17–28CrossRefGoogle Scholar
  14. 14.
    Cajka T, Fiehn O (2017) LC–MS-based lipidomics and automated identification of lipids using the LipidBlast in-silico MS/MS library. In: Bhattacharya S (ed) Lipidomics: methods and protocols, Methods in molecular biology, vol 1609. Springer, New York.  https://doi.org/10.1007/978-1-4939-6996-8_14 CrossRefGoogle Scholar
  15. 15.
    Cajka T, Garay LA, Sitepu IR, Boundy-Mills KL, Fiehn O (2016) Multiplatform mass spectrometry-based approach identifies extracellular glycolipids of the yeast Rhodotorula babjevae UCDFST 04-877. J Nat Prod 79(10):2580–2589CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Irnayuli R. Sitepu
    • 1
  • Antonio L. Garay
    • 1
    • 2
  • Tomas Cajka
    • 3
    • 4
  • Oliver Fiehn
    • 3
  • Kyria L. Boundy-Mills
    • 1
    Email author
  1. 1.Phaff Yeast Culture Collection, Department of Food Science and TechnologyUniversity of California, DavisDavisUSA
  2. 2.PepsicoPlanoTXUSA
  3. 3.West Coast Metabolomics CenterUniversity of California, DavisDavisUSA
  4. 4.Department of MetabolomicsInstitute of Physiology CASVidenska 1083Czech Republic

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