Metabolic engineering of Crambe abyssinica for producing high erucic acid oil
Sammanfattning: Vegetable oils are renewable and more environmentally friendly than fossil oils, and thus are good alternatives to fossil oils for industrial applications. Erucic acid (C22:1Δ13) is a valuable feedstock in the chemical industry. The current plant sources for erucic acid production are high erucic acid rapeseed (HEAR) and mustard. However, these crops readily outcross with food quality rapeseed, causing food oil safety concerns. Crambe abyssinica (crambe) is a dedicated non-food industrial oil crop, accumulating 22:1 to up to 60% of its seed oil, mainly in triacylglycerol (TAG). Further increasing the 22:1 content in crambe oil would reduce its downstream processing costs for purification or, if levels above 90% can be achieved, enable its direct application in the chemical industry. This thesis focused on producing ultra-high erucic acid in crambe through genetic engineering. By modifying three genes (BnFAE1, CaFAD2-RNAi and LdLPAAT) involved in the biosynthesis of 22:1, genetically modified (GM) crambe lines that accumulate up to 73% of erucic acid in seed oil were developed. For re-transformation of the best erucic line to further increase the 22:1 level, a hygromycin based selection marker system was established. To study the phosphatidylcholine (PC) involvement in the accumulation of 22:1 in crambe, candidate genes encoding three enzymes (LPCAT, PDCT and PDAT) were cloned from wild type (WT) crambe. Several RNAi expression vectors with these genes were transformed into WT crambe. Significant changes in fatty acid composition were observed in the transgenic lines but not an increase in 22:1 level. The functions of these three enzymes in the 22:1 accumulation were discussed. By in vitro and in vivo investigation of enzymatic activities in GM and WT crambe, a bottleneck to higher 22:1 accumulating at early stages of seed development in GM crambe was pointed out. Comparative transcriptomic analysis of GM and WT crambe was carried out to identify key genes and to further elucidate the molecular mechanisms involved in 22:1 biosynthesis and TAG accumulation.
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