Metabolic engineering of fatty acid biosynthesis in Chlorella vulgaris using an endogenous omega-3 fatty acid desaturase gene with its promoter

This study demonstrated the overexpression of omega-3 fatty acid desaturase (ω-3 FAD) gene under the control of its endogenous promoter and to elucidate its effect on fatty acid biosynthesis pathway in transgenic Chlorella vulgaris. Two stable transgenic lines (Ch-TL1 and Ch-TL2) were selected and cultured under nitrate-replete and nitrate-deficient BBM culture conditions, respectively to determine the growth, biomass production, total oil content, fatty acid composition and the expression level of four fatty acid biosynthetic genes of the transgenic C. vulgaris. Results revealed that both transgenic lines and wild-type displayed similar growth patterns. Higher biomass production and total oil content were recorded for both transgenic lines that were cultured under nitrate-deficient condition. The ω-3 FAD gene expression was consistently up-regulated in Ch-TL2 in all culture conditions which led to higher α-linolenic acid (C18:3n3) content. Similarly, the expression of β-ketoacyl ACP synthase I (KAS I), stearoyl-ACP desaturase (SAD) and omega-6 desaturase (ω-6 FAD) genes were also up-regulated in Ch-TL2. However, the accumulation of palmitic (C16:0), stearic (C18:0), oleic (C18:1) and linoleic (C18:2) acids were found to be differentially regulated at either transcriptional or post-transcriptional levels. PCR-base genome walking of Ch-TL2 genomic DNA successfully elucidated the integration sites and flanking regions of the inserted vector cassette. These findings provide valuable insight that could pave way for further genetic improvement of the C. vulgaris for various downstream applications.