Polyketides are among the most diverse and complex natural products. The antibacterial, antifungal, anticancer, antiparasitic, and immunosuppressive properties of polyketides have made them invaluable pharmaceuticals and drug precursors. A subset of these compounds is synthesized by multi-enzyme complexes known as Type I Assembly-Line Polyketide Synthases (T1ALPKSs) which consist of repeating modules composed of multiple domains. A number of investigators have attempted to modify T1ALPKSs to generate new products in a process known as “Combinatorial Biosynthesis.” However, these engineered T1ALPKSs are often inactive or have greatly attenuated catalytic activity. General strategies to improve the overall activities of engineered T1ALPKSs are underdeveloped. And, although modules and domains T1ALPKSs catalyze reactions relevant to synthetic chemistry, few components of T1ALPKS have been expressed or applied as catalysts for chemical synthesis. In-vitro experiments with terminal T1ALPKS domains known as thioesterases address both of these deficiencies. Previous studies indicate that thioesterases catalyze the macrocyclization or hydrolysis of polyketide intermediates as either standalone catalysts or as part of larger multi-enzyme systems.<br/>Herein, we report 1) the expression of 35 thioesterases, 2) the semi-synthesis of new polyketide substrates from narbonolide, 3) the quantification of the products of reactions between these substrates and thioesterases, 4) and insights into the reactivity and selectivity of thioesterase domains. We observed that thioesterases from related pathways react with our substrates to generate products in similar proportions, and we identified general trends for thioesterases from each pathway. Thioesterases that catalyze the rapid cyclization of a linear heptaketide substrate with exceptionally high total turnover numbers and selectivities were identified. Some of the thioesterases we evaluated catalyze the hydrolysis of macrolactones, and synthetic applications of these enzymes were explored. Finally, we expressed thioesterase variants containing active site serine-to-cysteine substitutions, reacted them with our substrates, and quantified the resulting products. This work provides new insights into the catalytic properties of thioesterases and lays the foundation for the application of thioesterases to chemical synthesis.

Insights gained from thioesterase-catalyzed macrocyclization of linear semi-synthetic substrates

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