Insights into azalomycin F assembly-line contribute to evolution-guided polyketide synthase engineering and identification of intermodular recognition

Abstract Exploring interactions among modules in modular polyketide synthase (PKS) is very important for understanding the overall biosynthetic process and for engineering PKS assembly-lines. Herein, we show that intermodule recognition between the enoylreductase domain ER1/2 inside module 1/2 and the ketosynthase domain KS3 inside module 3 is required for the cross-module enoylreduction in azalomycin F (AZL) biosynthesis. We also show that KS4 of module 4 acts as a gatekeeper facilitating cross-module enoylreduction. Additionally, evidence is provided that module 3 and module 6 in the AZL PKS are evolutionarily homologous, which make evolution-oriented PKS engineering possible. These results reveal an unprecedented intermodule recognition furthering understanding of the mechanism of the PKS assembly-line, thus providing new insights into PKS engineering. It also reveals that gene duplication/conversion and subsequent combinations may be a neofunctionalization process in modular PKS assembly-lines, hence providing a new case for supporting investigation of modular PKS evolution.