Biosynthesis of fungal polyketide natural products: Structural and biochemical studies toward understanding programming and substrate specificity

Polyketides are a large and diverse family of natural products encompassing some of the most effective and valuable pharmaceuticals of all time, as well as some of the deadliest toxins. These fascinating molecules are biosynthesized by some of nature’s largest and most complex enzymes, the polyketide synthases. Because of their diverse biological activities, polyketide-based natural products are an attractive target for biosynthetic engineering. In order to effectively engineer polyketide synthases to produce novel compounds, we must understand the native enzymology of these molecular machines. Since they consist of only a single set of catalytic domains, the fungal, iterative polyketide synthases are particularly challenging to decipher and engineer, relative to the assembly-line-like modular polyketide synthases. In this work, biochemical and structural studies are performed to improve understanding of iterative polyketide synthase enzymatic programming. By performing a screen of thirty unnatural starter units, the substrate specificity of the iterative polyketide synthase PksA was evaluated. Characterization of enzymatic derailment products allowed for interrogation of the tolerance of specific catalytic domains to the unnatural substrates. Through mechanism-based crosslinking of a closely related iterative polyketide synthase, CTB1, the structure of a polyketide synthase locked in a loading conformation was observed by cryo-electron microscopy. This first-ever multidomain structure of a non-reducing polyketide synthase may help to guide future engineering efforts, by beginning to establish what conformational changes polyketide synthases undergo during biosynthesis and how programming is achieved across multiple catalytic domains