engleski

Gas chromatography of fatty acids in transformed yeast upon induced expression of a plant fatty acid elongase (KCS)

The 3-ketoacyl-CoA synthase (KCS ; EC 2.3.1.86) is a fatty acid elongase, which catalyzes the synthesis of several very long chain monounsaturated fatty acids (VLCMFA) in plants. The aim of this study was to clone KCS gene (CgKCS) from plant species Cardamine graeca L. into yeast cells and to analyse expression of the recombinant protein. Coding region of the CgKCS was linked to the galactose-inducible GAL1 promoter in the expression vector pYES2 and introduced into Saccharomyces cerevisiae strain BY4741. After the induced expression, total fatty acid content and acyl composition in transformed yeast was determined by preparing fatty acid methyl esters (FAME) extracted in hexane and analyzed by gas-chromatography (GC) using the 37 Component FAME Mix (Supelco) as an external standard. The conditions used were optimized to allow the detection of VLCFAs of up to C24 in length. Separation of FAMEs was affected by a type of column (HP-5 or DB-FFAP, Agilent)) and temperature program rate. Comparative GC analyses of fatty acid profiles in transformed yeast cells upon CgKCS expression revealed the presence of saturated and monounsaturated VLCFAs that are not normally present in untransformed yeast, with a distinct peak corresponding to nervonic acid 24:1 Δ15 (cis-tetracos-15-enoic acid).

expression; gas-chromatography; gene KCS; nervonic acid; yeast Saccharomyces cerevisiae

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