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Laboratory Screening Protocol to Identify Novel Oleaginous Yeasts
IR. Sitepu, AL. Garay, T. Cajka, O. Fiehn, KL. Boundy-Mills,
Language English Country United States
Document type Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't
- MeSH
- Staining and Labeling methods MeSH
- Chromatography, Liquid methods MeSH
- Fluorescent Dyes analysis MeSH
- Microscopy, Fluorescence methods MeSH
- Mass Spectrometry methods MeSH
- Yeasts chemistry MeSH
- Lipidomics methods MeSH
- Lipids analysis MeSH
- Oxazines analysis MeSH
- Triglycerides analysis MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
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.
References provided by Crossref.org
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