Microalgal long-chain polyunsaturated fatty acids (LC-PUFAs) have emerged as promising alternatives to depleting fish oils. However, the overproduction of LC-PUFAs in microalgae has remained challenging. Here, we report a sequential metabolic engineering strategy that systematically overcomes the metabolic bottlenecks and overproduces LC-PUFAs. Malonyl CoA-acyl carrier protein transacylase, catalyzing the first committed step in type II fatty acid synthesis, and desaturase 5b, involved in fatty acid desaturation, were coordinately expressed in Phaeodactylum tricornutum. Engineered microalgae hyper-accumulated LC-PUFAs, with arachidonic acid (ARA) and docosahexaenoic acid (DHA) contents of up to 18.98 μg/mg and 9.15 μg/mg (dry weight), respectively. Importantly, eicosapentaenoic acid (EPA) was accumulated up to a highest record of 85.35 μg/mg by metabolic engineering. ARA and EPA were accumulated mainly in triacylglycerides, whereas DHA was found exclusively in phospholipids. Combinatorial expression of these critical enzymes led to the optimal increment of LC-PUFAs without unbalanced metabolic flux and demonstrated the practical feasibility of generating sustainable LC-PUFA production.
Burgeoning demand for long-chain polyunsaturated fatty acids (LC-PUFA) due to their established pharmacological and economic significances along with the declining sources of fish urge to explore the sustainable sources of LC-PUFA. Being the predominant LC-PUFA source for marine fishes at the base of the aquatic food web, microalgae has been hailed as a promising natural source for LC-PUFA. However, the potential of algal systems to overproduce LC-PUFA via metabolic engineering is warranted to meet the ever-increasing demand. In this study, we identified and overexpressed 6-desaturase, the key enzyme involved in fatty acid desaturation and exemplified its potential on elevating PUFA and lipid content in Nannochloropsis oceanica. 6-desaturase overexpression enhanced growth and photosynthetic efficiency. Transgenic cells exhibited a remarkable increase in EPA content and reached up to 62.35 mg/g DW, the highest EPA production in transgenic N. oceanica by expressing a single key enzyme without impeding growth. Total lipid content was significantly increased by 1.7-fold in the transgenic cell than WT. Together, these findings exemplify a potential candidate for LC-PUFA overproduction and also open a new avenue for sustainable production of microalgal PUFAs.
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