Engineeringthe Substrate Specificity of a ModularPolyketide Synthase for Installation of Consecutive Non-Natural ExtenderUnits

There is significant interest in diversifying the structures of polyketidesto create new analogues of these bioactive molecules. This has traditionally been done by focusing on engineering the acyltransferase(AT) domains of polyketide synthases(PKSs) responsible for the incorporation of malonyl-CoAextender units. Non-natural extender units have been utilized by engineered PKSs previously; however, most of the work to date has been accomplished with ATs that are either naturally promiscuousand/or located in terminal modules lacking downstream bottlenecks. These limitations have prevented the engineering of ATs with low native promiscuityand the study of any potential gatekeepingeffects by domains downstream of an engineered AT. In an effort to address this gap in PKS engineering knowledge, the substrate preferences of the final two modules of the pikromycinPKS were compared for several non-natural extender units and through active site mutagenesis. This led to engineering of the methylmalonyl-CoAs...