Engineering metazoan fatty acid synthase to control chain length applied in yeast

Metazoan fatty acid (FA) synthases (mFASs) facilitate the de novo synthesis of C16- and C18-FAs through iterative extensions within the FA cycle and hydrolytic release. Here we re-engineer mFAS to fine-tune the interplay between FA extension and FA hydrolytic release for the targeted production of short- and medium-chain fatty acids. Single amino acid exchanges in the ketosynthase domain can redirect FA product profiles from predominantly C8 (G113W) to C8/C10 (G113F) and C12/C14 (G113M). Integration of a thioreductase domain enables the production of medium-chain fatty aldehydes and alcohols. We apply our approach for controlling chain length in FA biosynthesis to the microbial production of C10- and C12-FAs, translate it into a yeast cell factory and achieve C10/C12-FAs titers of 674 mg l^-1 and 67% purity of total free FAs. Our work demonstrates a modular platform for programmable FA synthesis and paves the way toward sustainable bioproduction of valuable oleochemicals.