Type I modular polyketide synthases (PKSs) are a family of enzymes capable of synthesizing a diverse class of natural products, which form the basis for approximately one-third of pharmaceuticals. The biochemical features of these systems include the extension and processing of polyketide chains in a stepwise, stereospecific manner, organized by a series of modules divided into distinct catalytic domains. Taking advantage of these enzymes as biocatalysts would be a powerful addition to the synthetic chemistry toolbox that enables a more sustainable and environmentally responsible approach to molecular diversification. Previous work has indicated that the primary hurdle to utilizing PKS modules to create diverse macrolactones hinges on the selectivity of the thioesterase (TE) domain. We recently demonstrated the ability to form 12-membered macrocycles utilizing an amide hexaketide in conjunction with an engineered TE S148C mutant from the pikromycin biosynthetic pathway. However, these 12-membered rings were formed in significantly attenuated yields in comparison with the native substrate, mostly due to linear hydrolytic byproducts. Thus, we embarked on a directed evolution campaign to identify TE variants with enhanced selectivity for the desired 12-membered rings. After the first round of evolution, five single amino acid mutant TEs were identified with improved yields. Combinations of beneficial mutations produced a TE mutant with three-fold enhanced activity compared to the original enzyme, and further iterative rounds of evolution are underway. This project focuses on merging synthetic chemistry and protein engineering to expand the use of PKS TEs (in conjunction with their upstream modules) as biocatalysts to produce structurally complex polyketide macrocycles. Our study gives critical insights into the amino acid residues that impart improved selectivity and yield with these substrates, and in the pursuit of combinatorial strategies to produce synthetically challenging molecules for drug discovery applications.

Enhancing diversification of macrolactones by directed evolution of PKS cyclase (TE) domains

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ACS Fall 2022

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