Medium-Chain Triglyceride Production in Nannochloropsis via a Fatty Acid Chain Length Discriminating Mechanism

Depending on their fatty acid chain length, triacylglycerols (TAGs) have distinct applications; thus, a feedstock with a genetically designed chain length is desirable to maximize process efficiency and product versatility. Here, ex vivo, in vitro, and in vivo profiling of the large set of type-2 diacylglycerol acyltransferases (NoDGAT2s) in the industrial oleaginous microalga Nannochloropsis oceanica revealed two endoplasmic reticulum-localized enzymes that can assemble medium-chain fatty acids (MCFAs) with 8-12 carbons into TAGs. Specifically, NoDGAT2D serves as a generalist that assembles C8-C18 fatty acids (FAs) into TAG, whereas NoDGAT2H is a specialist that incorporates only MCFAs into TAG. Based on such specialization, stacking of NoDGAT2D with MCFA- or diacylglycerol (DAG)-supplying enzymes or regulators, including rationally engineering Cuphea palustris acyl-ACP thioesterase, Cocos nucifera lysophosphatidic acid acyltransferase and Arabidopsis thaliana WRINKLED1, elevated the medium-chain triglyceride (MCT) share in total TAG 66-fold and MCT productivity 64.8-fold at the peak phase of oil production. Such functional specialization of NoDGAT2s in the chain length of substrates and products reveals a dimension of control in the cellular TAG profile, which can be exploited for producing designer oils in microalgae.