Acyltransferases in Microbial Triacylglycerol Biosynthesis: Structure, Mechanisms, Regulation, and Engineering

Abstract Triacylglycerols (TAGs) are structurally diverse lipids whose functions are dictated by acyl composition, chain-length distribution, and sn-positional architecture. Microbial TAG production offers a sustainable route to bulk oils and high-value specialty lipids, yet the field has shifted from maximizing total lipid titers to precise control of TAG molecular structure. Acyltransferases (ATs) serve as key enzymatic nodes governing acyl-chain entry, positional incorporation, terminal TAG formation, and lipid droplet biogenesis. This review systematically covers five core AT families (GPAT, AGPAT, DGAT, WS/DGAT, PDAT) across bacteria, yeasts, fungi, and microalgae, highlighting their conserved catalytic motifs, membrane topology, substrate selectivity, and regulatory networks. We discuss engineering strategies including AI-assisted design, enzyme engineering, gene overexpression, metabolic rewiring, fermentation optimization, and multiomics-guided regulation, focusing on producing high-value structured TAGs such as human milk fat substitutes and very-long-chain TAGs. Future advances require synergistic integration of AT mechanisms, acyl-CoA supply, lipid droplet engineering, and scalable bioprocessing.