Acyltransferase (AT) domains of polyketide synthases (PKSs) select extender units for incorporation into polyketides and dictate large portions of the structures of clinically relevant natural products. Accordingly, there is significant interest in engineering the substrate specificity of PKS ATs in order to site-selectively manipulate polyketide structure. However, previous attempts to engineer ATs have yielded mutant PKSs with relaxed extender unit specificity, rather than an inversion of selectivity from one substrate to another. Here, by directly screening the extender unit selectivity of mutants from active site saturation libraries of an AT from the prototypical PKS, 6-deoxyerythronolide B synthase, a set of single amino acid substitutions was discovered that dramatically impact the selectivity of the PKS with only modest reductions of product yields. One particular substitution (Tyr189Arg) inverted the selectivity of the wild-type PKS from its natural substrate toward a non-natural alkynyl-modified extender unit while maintaining more than twice the activity of the wild-type PKS with its natural substrate. The strategy and mutations described herein form a platform for combinatorial biosynthesis of site-selectively modified polyketide analogues that are modified with non-natural and non-native chemical functionality.

Probing and interrogating protein interactions that involve acyl carrier proteins (ACP’s) in fatty acid synthases and polyketide synthases are critical to understanding the molecular basis for the programmed assembly of complex natural products. Here, we have used unnatural amino acid mutagenesis to site specifically install photo-cross-linking functionality into acyl carrier proteins from diverse systems and the ketosynthase FabF from the Escherichia coli type II fatty acid synthase. Subsequently, a photo-cross-linking assay was employed to systematically probe the ability of FabF to interact with a broad panel of ACP’s, illustrating the expected orthogonality of ACP:FabF interactions and the role of charged residues in helix II of the ACP. In addition, FabF residues involved in the binding interaction with the cognate carrier protein were identified via surface scanning mutagenesis and photo-cross-linking. Furthermore, the ability to install the photo-cross-linking amino acid at virtually any position allowed interrogation of the role that carrier protein acylation plays in determining the binding interface with FabF. A conserved carrier protein motif that includes the phosphopantetheinylation site was also shown to play an integral role in maintenance of the AcpP:FabF binding interaction. Our results provide unprecedented insight into the molecular details that describe the AcpP:FabF binding interface and demonstrate that unnatural amino acid based photo-cross-linking is a powerful tool for probing and interrogating protein interactions in complex biosynthetic systems.