Controlling Megasynthetase Module–Module Interactions through β-Hairpin Docking Domain Engineering

Bacteria manufacture a diversity of natural products with pharmaceutical value, many from modular polyketide synthase (PKS), nonribosomal peptide synthetase (NRPS), or hybrid pathways. In these pathways, each module extends a biosynthetic intermediate by an acyl unit (PKS) or amino acid (NRPS), employing a carrier domain (CP) to deliver the pathway intermediate to successive active sites and to the subsequent module. Docking domains (DD) at polypeptide termini ensure pathway fidelity by specific noncovalent association of sequential modules. The vatiamide biosynthetic gene cluster encodes a rare trifurcated pathway, enabled by a short linear motif (SLiM) at the C-terminus of VatM that docks with identical β-hairpin domains (βHDs) at the N-termini of VatN, VatQ, and VatS. Taking inspiration from Nature, we examined the utility of DDs for engineering by quantitating affinity and catalytic throughput in the Vat system and an unrelated SLiM-βHD dock from the carmabin pathway. The SLiM-βHD dock was the sole determinant of affinity of natural and engineered module partners (<i>K</i>_d ∼ 1 μM). The effectiveness of engineered DDs was evaluated relative to natural partners and docks. DD affinity was predictive of catalytic success in most, but not all, of the dozen cases tested. Thus, while the DD determines affinity and selectivity, other factors also affect catalytic throughput when a DD is engineered into a non-native environment. This study enhances our understanding of the interactions that enforce PKS/NRPS pathway fidelity and highlights the challenges of engineering these systems to diversify the repertoire of natural products.