Microbial Lipid Production. Methods and Protocols

0508403 - FGÚ 2020 RIV US eng M - Monography Chapter
Sitepu, I. R. - Garay, A. L. - Čajka, Tomáš - Fiehn, O. - Boundy-Mills, K. L.
Laboratory Screening Protocol to Identify Novel Oleaginous Yeasts.
Microbial Lipid Production. Methods and Protocols. New York: Humana Press, 2019 - (Balan, V.), s. 33-50. Methods in Molecular Biology, 1995. ISBN 978-1-4939-9483-0
Institutional support: RVO:67985823
Keywords : oleaginous microbes * yeast * triacylglycerol * nile red * gravimetric * liquid chromatography–mass spectrometry * lipidomics
OECD category: Analytical chemistry

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.
Permanent Link: http://hdl.handle.net/11104/0299320