Switching Metazoan Fatty Acid Synthase Between Reducing and Non-reducing Elongation Mode via Programming of the Ketoreductase Domain

Polyketides constitute a large class of natural products with important biological activities and applications such as antibiotics, antitumor agents, pesticides, and pigments. Their biosynthesis is catalyzed by polyketide synthases (PKSs) which are multi-domain enzymes evolutionarily related to fatty acid synthases (FASs). Despite their close homology in structure and the chemistry they perform, FASs and PKSs differ fundamentally in their catalytic programming: FASs run fully reducing elongation reactions to yield saturated fatty acids, while iterative PKSs execute reductions just in selected cycles, generating complex oxidized compounds. In this study, we aimed at engineering the metazoan FAS in its KR domain to switch from fully reducing to a non-reducing mode during chain elongation. Guided by recent insights into KR programming, we incorporated a helix into metazoan FAS, which is found in KRs from iterative PKSs and type II FASs with chain length programming. These FAS variants initially catalyze complete fatty acid cycles but lose the ability of {beta}-keto reduction in later elongation rounds, producing intermediates that spontaneously cyclize to pyrone products. Finally, our study provides valuable insight into the mechanism of KR catalysis identifying another amino acid next to the active tyrosine which is capable for intermediate protonation.