Enrichment of Long-Chain Polyunsaturated Fatty Acids by Coordinated Expression of Multiple Metabolic Nodes in the Oleaginous Microalga Phaeodactylum tricornutum

Abstract

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.

Potentiation of concurrent expression of lipogenic genes by novel strong promoters in the oleaginous microalga Phaeodactylum tricornutum

“…Compared to the D5b overexpressing microalgae previously generated in our lab (Wang et al, 2017a), both lines pPtMD-1 and pPtMD-2 displayed no significant change in fatty acid composition but the overall fatty acid content showed a significant increase.…”

“…Reasoning the crucial regulatory role of Pt667 and Pt207 on successfully driving the GUS gene, we attempted to investigate the efficacy of these promoters on governing the expression of lipogenic genes such as ME (Xue et al, ; Xue et al, ) and PtD5b (Peng et al, ; X. Wang, Liu, et al, ). Subsequently, two expression cassettes, Pt667‐ME and Pt202‐PtD5b were fused together and cloned into the expression vector (Figure a), the consequent recombinant expression construct designated pPtMD (pPt667‐ME‐Pt202‐D5b) was employed to algal transformation.…”

“…As shown in Figure d, NADPH enzymatic activity was found to be significantly increased, which in turn elevated total lipid content. PtD5b catalyzes the desaturation of eicosatetraenoic acid (ETA) to form EPA, a fatty acid of significant health and economic significance (Peng et al, ; L. W. Wang, He, et al, ). Congruently, EPA content was found to be elevated (Figure d).…”

“…Reasoning the crucial regulatory role of Pt667 and Pt207 on successfully driving the GUS gene, we attempted to investigate the efficacy of these promoters on governing the expression of lipogenic genes such as ME (Xue et al, 2015;Xue et al, 2016) and PtD5b (Peng et al, 2014;X. Wang, Liu, et al, 2017a).…”

“…Oleaginous microalgae hold enormous potential for sustainable production of biofuels and a wide array of high‐value products, such as proteins, polysaccharides, carotenoids, and polyunsaturated fatty acids (PUFAs ; Kwon et al, ; X. Wang, Liu, et al, ), due to their advantageous features such as photosynthesis, higher growth rate, oleaginicity and nonencroachment of arable land (Zhang, Li, Agrawal, & San, ). The eukaryotic microalga Phaeodactylum tricornutum is considered an attractive candidate for the production of a wide range commodity components (Kikutani et al, ; Xue et al, ).…”