Heterologous expression in Saccharomyces and Chlamydomonas reveals host-dependent activity of Brassica juncea fatty acid elongase1 isozymes

Abstract

The fatty acid elongase1 (FAE1) genes of tetraploid Brassica juncea are the key determinant of high erucic acid (EA, C22:1) accumulation in its seed oil. While our previous work demonstrated near-zero EA content in mustard oil via CRISPR/Cas9 knockout of the two homeoalleles, BjFAE1.1 and BjFAE1.2; the contributory function of each isozymes towards EA biosynthesis remains elusive. This study investigated the heterologous expression of BjFAE1.1 and BjFAE1.2 from high EA B. juncea cultivar JD6 in two metabolically distinct eukaryotic microbial hosts: the green microalga Chlamydomonas reinhardtii and the budding yeast Saccharomyces cerevisiae. Despite confirmed protein expression, neither BjFAE1 isozyme produced detectable C20:1 or C22:1 very-long-chain fatty acids (VLCFAs) in transgenic lines of C. reinhardtii. In contrast, expression in S. cerevisiae resulted in significant de novo biosynthesis of VLCFAs, C20:1 ([~]9-11%) and C22:1 ([~]17-19%), confirming their enzymatic activity as functional {beta}-ketoacyl-CoA synthase. Substrate feeding experiments in yeast further validated their capability to elongate oleoyl-CoA (C18:1-CoA) to erucoyl-CoA (C22:1-CoA) via eicosenoyl-CoA (C20:1-CoA), with BjFAE1.1 showing slightly higher activity, as indicated by the enhanced VLCFAs accumulation. These findings highlight the critical influence of the heterologous hosts cellular environment on the enzyme functionality of plant genes involved in lipid metabolism, underscoring challenges for VLCFA production in microalgal platform.