Mutagenesis of Emiliania huxleyi aimed at identifying genes and proteins involved in alkenone metabolic pathways

Alkenones are unique very long chain polyunsaturated fatty acids, characterized by their unusual length (C38-40), spacing and trans configuration of their double bonds. They are produced by a select group of haptophytes, predominantly the coccolithophorid, Emiliania huxleyi. Aimed at identifying genes and proteins involved in alkenone metabolic pathways, we developed mutagenesis and lipid screening tools for the calcifying (PLY 217) and non-calcifying (CCMP 1516) strains of E. huxleyi. Optimal mutagen dose for ultraviolet irradiation was determined at 1 and 3 J/m2, and methyl methanesulfonate exposure 0.4 and 0.6% (v/v), for PLY217 and CCMP1516, respectively. Using these optimal doses, wild type CCMP1516 was mutagenized and mutants in lipid metobolism were selected on plates supplemented with known inhibitors of proteins within the lipid biosynthesis pathways. Twenty-six surviving colonies were picked from the inhibitor plates and characterized by their cell size, lipid content and non-polar lipid profiles using a lipophilic dye, nile red, gas chromatography and mass spectrometry. Six mutant colonies were created with clearly an altered alkenone pathway. In this work, we successfully demonstrated that UV and MMS can be utilized to produce lipid altered mutants in the coccolithophore Emiliania huxleyi.