Decoding Iterative Chain Elongation: Mechanistic and Structural Insights into <i>Schizochytrium</i> -Sourced Ketosynthase-Chain Length Factor

Long-chain polyunsaturated fatty acids (LC-PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential for health but difficult to obtain sustainably, making engineered microbial production an attractive alternative. LC-PUFAs are produced in certain marine protists via iterative decarboxylative Claisen condensation catalyzed by the ketosynthase-chain length factor (KS_B-CLF). However, the molecular mechanisms governing substrate recognition, chain-length control, and catalytic efficiency of KS_B-CLF remain poorly understood. Here, we employ a combination of computationally guided design, mutagenesis, and in vitro assays to unravel the structural basis of <i>Schizochytrium</i>-KS_B-CLF activity and to enhance LC-PUFA production. Structural analyses identified a conserved catalytic triad (C196-H332-H367) within the KS_B domain. Additionally, key electrostatic residues (K302, R541, R636) were shown to mediate binding with acyl carrier protein (ACP) and acyl-CoA. Rational engineering of hydrophobic substrate channel residues yielded a F235W mutant with 77% and 28% enhanced activity toward C18-CoA and C20-CoA, respectively, attributed to channel elongation and cavity expansion. Engineering <i>Schizochytrium</i> strains with the F235W mutation increased EPA and docosapentaenoic acid (DPA) yields without negatively impacting biomass or lipid production. Together, these findings provide a mechanistic framework for PUFA synthase optimization and mark a significant step toward scalable, sustainable production of nutritionally vital omega-3 fatty acids.