Utilizing inhibitors and genetic engineering to study the biosynthesis of unsaturated fatty acids and lipids in two thraustochytrid strains

Thraustochytrids are oleaginous heterotrophic marine eukaryotic microbes known to produce large amounts of docosahexaenoic acid (DHA)-containing triacylglycerols (TAGs), and they also synthesize other unsaturated fatty acids like palmitoleic acid (C16:1 n-7) and vaccenic acid (C18:1 n-7), and other terpenoids like squalene. These compounds have great economic value and are in high demand. However, DHA produced from thraustochytrids is still more costly than from fish oil whose availability is limited and unstable. Hence, a deeper understanding of the lipid metabolism and genetic manipulation tools in thraustochytrids is vital to developing high productive strains for profitable biomanufacturing. Here, the results suggest that precursor availability for the Polyketide synthase pathway is not the rate-limiting factor for DHA synthesis. FAS inhibition could enhance DHA-rich triacylglycerol (TAG) production. T66Des9 was then identified as a Δ12-desaturase-like Δ9-desaturase, contributing to the production of C16:1 n-7 and, indirectly, C18:1 n-7. Furthermore, four Acyl-CoA:diacylglycerol acyltransferases (DGAT)-like genes encoded proteins were identified to possess acyl-CoA:sterol acyltransferase (ASAT) activity, contributing to the production of steryl esters. In addition, the expression two of the four genes enhanced squalene production in SR21. The discoveries of the above-mentioned genes pave the way to enhance DHA-rich TAG, C16:1 n-7, C18:1 n-7, and squalene production in thraustochytrids through metabolic engineering, and highlight the enzyme functional diversity.