Physiological study of polyunsaturated fatty acid production and the role of delta -6 desaturase in the marine microalga Glossomastix chrysoplasta.

摘要

In this work, we describe characterization of the microalga Glossomastix chrysoplasta, an eicosapentaenoic acid (EPA) producer in the Pinguiophyceae class, Chromophyte division. In addition to fermentation optimization to maximize algal growth and its EPA production, we isolated a delta-6 desaturase gene in G. chrysoplasta and confirmed its function in S. cerevisiae. Delta-6 fatty acid desaturase is a potential rate-limiting step of EPA production, and its isolation would enable molecular characterization concurrently with optimization of fatty acid production conditions.;Glossomastix chrysoplasta was found to produce EPA as 30% of total amount of fatty acids, and up to 22 mg EPA per g dry weight. Up to 72% of the EPA was produced as glycolipid, a component of structural lipids. In combination with increased EPA fraction with increasing growth rate, EPA is thought to be produced as a structural lipid. The optimized growth conditions for continuous culture were found as follows: greater than 500 mumol photons/m2-s light intensity, pH 7.20 to 7.45, and 18 to 20°C temperature. Macronutrient studies indicated the limiting nutrient to be bicarbonate or dissolved carbon dioxide, and this result agrees with the increase in EPA production with decreasing pH.;Isolation and cloning of a Delta6 fatty acid desaturase from G. chrysoplasta was performed in this study using RT-PCR with degenerate primers designed from conserved histidine motifs and 5' and 3' RACE. The isolated protein, GcDES6, was found to contain features characterizing membrane-bound Delta6 desaturase including membrane-spanning regions separating conserved histidine boxes and N-terminal cytochrome b5 fusion. Heterologous expression in S. cerevisiae was used to confirm Delta6 regioselectivity and function of GcDES6. Both o3(18:3Delta9,12,15) and o6(18:2Delta9,12) precursors can be utilized by GcDES6 in vivo with similar desaturase activity. One intron site was found in the cytochrome b5 fusion region of GcDES6. Although the conservation of intron-exon junctions has been found for several desaturases in human and in C. elegans , comparison among the introns in GcDES6 and other delta-6 desaturases has not yet shown any strong similarities.