Functionalization of Plant Oil-Derived Fatty Acids into Di- and Trihydroxy Fatty Acids by Using a Linoleate Diol Synthase as a Key Enzyme

Hydroxy fatty acids (HFAs) are multifunctional lipids exhibiting potent physiological activities via G protein-coupled receptors like GPR120, which modulates fat taste perception, metabolism, and immunity. Here, we explored the biotransformation of plant oil-derived fatty acids into HFAs using linoleate diol synthases (LDSs), fusion enzymes with dioxygenase (DOX) and hydroperoxide isomerase (HPI) domains. For instance, the natural 12-hydroxyoctadec-9Z-enoic acid (ricinoleic acid, 12-HOME) and 13-hydroxyoctadec-9Z-enoic acid prepared from linoleic acid were transformed into novel 8,12- and 8,13-dihydroxyoctadec-9Z-enoic acids (DiHOMEs) as well as their corresponding trihydroxy derivatives: 7,8,12-/8,11,12- and 7,8,13-/5,8,13-trihydroxyoctadec-9Z-enoic acids, respectively, by the LDS from Glomerella cingulata (Gc-LDS). The recombinant Escherichia coli expressing Gc-LDS efficiently produced 9.4 mM 8S,12R-DiHOME from 10 mM 12-HOME (187 U/g dry cells) under reducing conditions. Notably, 8S,12R-DiHOME elicited a potent GPR120-mediated Ca²⁺ response (EC₅₀ = 0.43 μM), likely via enhanced hydrogen bonding with key receptor residues. This study contributes to the LDS-based biotransformation for developing functional HFAs.