Extensive Alanine Scanning of Loop Regions in Ketosynthase Domains Identifies Non-Active Site Mutations with Drastic Effects on Polyketide Biosynthesis

Modular polyketide synthases (PKSs) produce diverse natural products with significant pharmaceutical value, but their protein engineering for drug discovery is hampered by the unpredictable substrate specificity of their ketosynthase (KS) domains. While previous studies have focused on the KS active sites, we conducted extensive alanine scanning of loop regions of a KS domain, most of which are distant from the catalytic center. In vitro screening of 46 point mutants revealed that ∼70% of the mutants retained their activity, whereas ∼25% of the mutants displayed severely reduced activity, and two mutants unexpectedly showed enhanced activity. Interestingly, most mutations with significant impact were located more than 20 Å away from the catalytic center. These findings provide the first clear evidence that KS residues beyond the canonical active site play crucial roles in polyketide biosynthesis. Our results show essential foundational data to understand KS functions, which could be used for developing more effective KS engineering strategies beyond traditional active site modifications.