Stereochemical Dissection of the Strobilurin PKS Reveals the Complex Biosynthetic Logic of Iterative <i>EZE</i> Triene Construction

Type I Iterative polyketide synthases (PKS) use a limited set of catalytic extension and β-processing domains to create complex polyketides. A remarkable case is that of the strobilurin PKS where a single dehydratase (DH) domain creates an EZE triene over three dehydration cycles. Here we dissect the strobilurin PKS and assay catalytic domains individually and in combination with stereo-defined synthetic substrates in vitro, to reveal the complex and varying selectivities of methylation, ketoreduction and dehydration that lead to this remarkable result. At the diketide stage all stereoselectivities are consistent with those known for other related systems, giving an E product. But at the triketide stage, 2R-methylation is selectively achieved, that is followed by rapid keto-reduction to give an unusual 3-L-alcohol. In-turn, this is eliminated to give the unusual Z-alkene. These selectivities are flexible and change in response to the structure of the substrate at every stage. This uncovers the complete and intricate regio- and stereo-selectivities of a highly reducing iterative Type I PKS for the first time, and highlights the important differences to the well-studied cis-AT modular PKS β-processing enzymes that appear to have inflexible selectivities.