To engineer modular polyketide synthases that reliably yield stereocomplex products, a detailed understanding of how their enzymatic domains collectively operate is essential. How their enoylreductase (ER) domains function to install either d-or l-oriented alkyl substituents remains largely mysterious. In this study, we investigated the stereoselectivity of ERs and how they work with other domains within tetraketide-heptaketide synthase platforms constructed from components of the pikromycin and rapamycin synthases. We first determined how ER and acyl carrier protein (ACP) domains associate by mutagenically probing the consensus solution predicted by AlphaFold using an in vitro tetraketide synthase platform. Since ERs were found to contact residues conserved in ACPs from diverse module types, we tested whether ERs operate when inserted into modules that only contain ketoreductase and dehydratase domains in several in vivo platforms. ER-inserted modules were observed to generate both unsaturated and saturated intermediates. Increased production of the saturated intermediate was realized when the downstream KS was also swapped for a KS from an ER-containing module. To access products with desired methyl group orientations, it was necessary to consider how the methyl groups interact with the KS of the module that installed them, as well as with the KS of the downstream module. Additionally, we interconverted d- and l-type ERs by swapping a 14-residue loop and propose the molecular basis for how ERs set stereocenters. This research provides new strategies for controlling the orientations of alkyl substituents in complex polyketides and enables access to stereochemical libraries of polyketide drug leads.
Zhang et al. (Wed,) studied this question.