Biology subjects

Maiti, M. K.

Publications and source records attributed to Maiti, M. K..

2 recordsLinked to original sources

bioRxiv2025

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

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.

plant biology↗

bioRxiv2024

Knockout of fatty acid elongase1 homeoalleles in amphidiploid Brassica juncea leads to undetectable erucic acid in seed oil

Indian mustard (Brassica juncea L.) is a major oilseed crop with significant economic and nutritional importance within the Indian subcontinent. While its seed oil offers valuable dietary benefits, including a balanced ratio of human essential fatty acids, the traditional high oil-yielding varieties contain an elevated level of erucic acid (EA, C22:1) that is associated with adverse health effects. Therefore, developing low erucic acid (LEA) mustard cultivars is crucial for broader utilization and consumer safety. In this study, we employed CRISPR/Cas9 genome editing tools to disrupt the fatty acid elongase1 (FAE1) gene that encodes a key enzyme in EA biosynthesis in two high erucic acid (HEA) B. juncea cultivars, PCR7 and JD6. Targeted knockout (KO) of BjFAE1 homeoalleles (BjFAE1.1 and BjFAE1.2) in this amphidiploid plant using CRISPR/Cas9 constructs, each carrying two guide RNAs led to monoallelic and biallelic mutations. Biallelic KO lines showed a near-complete elimination of EA (<0.5% in PCR7, undetectable in JD6) with a significant increase in nutritionally beneficial oleic acid ([~]30% in PCR7, [~]35% in JD6), while the content of essential fatty acids also increased significantly, suggesting rerouting of carbon flux from EA biosynthesis. Importantly, these LEA lines retained key agronomic traits like plant seed yield and oil content, matching the productivity of the control elite cultivars. Our findings underscore the effectiveness of CRISPR/Cas9 technology for editing B. juncea genome, producing LEA seed oil lines with improved nutritional quality and thus expanding the applications of this important oilseed crop.

plant biology↗