Functional characterization of a plastidial cytochrome b5-fused Δ4-desaturase from Ostreococcus tauri in higher plants

Marine microalgae are the primary producers of important lipids in oceanic ecosystems. In particular, they sustain the food web with omega-3 very-long-chain polyunsaturated fatty acids (n-3 PUFAs), which play a protective role against various human metabolic disorders and are thus considered highly beneficial to health. Ostreococcus tauri is a marine pico-eukaryote that contains high levels of several n-3 PUFAs, including docosahexaenoic acid (22:6n3; DHA), octadecapentaenoic acid (18:5n3, OPA), and hexadecatetraenoic acid (16:4n3), each with a distinct distribution. While DHA and OPA are restricted to microsomal and plastidial lipids, respectively, 16:4n3 is found in galactolipids as well as in betaine and neutral lipids. The genome of O. tauri contains 14 genes encoding fatty acid desaturases. In this study, we characterized the enzyme encoded by OT_ostta13g01550 (Ot13bDES) as a plastidial cytochrome b5-fused delta-4 desaturase involved in 16:4n3 biosynthesis. Transient heterologous expression of Ot13bDES in Nicotiana benthamiana led to the production of 16:4n3 and 16:3n6, but failed to produce 18:5n3 when Ot13bDES was coexpressed with plastidial Δ6-desaturases, suggesting Ot13bDES has a strict Δ4 regioselectivity. Lipidomic analyses of stable transgenic Arabidopsis lines further showed a nearly 100 % conversion rate of 16:3n3 to 16:4n3 in the best-performing lines, demonstrating that Ot13bDES has a very high catalytic activity. Additionally, 16:4n3 was predominantly localized to monogalactosyldiacylglycerol (MGDG). This study provides the first functional characterization of a plastidial cytochrome b5-fused delta-4 desaturase through heterologous expression in higher plants.