Rational engineering acyltransferase domain of modular polyketide synthase for expanding substrate specificity

Polyketides have valuable pharmaceutical properties and exhibit a high degree of structural diversity. This diversity can trace back to the polyketide synthesis assembly line, in which the gatekeeper domain ATs strictly control the selection and incorporation of the simple extender units. And thus engineering attempts targeted on ATs have been made to obtain novel polyketide skeletons, which are useful for pharmaceutical and cellular function studies. So far, method of combinatorial biosynthesis has become the most attractive and effective way, particularly through alteration of the acyltransferase (AT) specificity. Herein, upon the complex structures of a broadly selective SpnD-AT, we introduce a structure-directed protocol to manipulate the well-studied EryAT6 to broaden its substrate scope. This protocol can also be generalized to canonical AT domains engineering in the generation of novel and useful chemical probes for dissecting cellular functions.