A renaissance in the field of modular polyketide synthases has begun. New tools and paradigms are enabling deeper insights into the architectures and activities of these enzymatic assembly lines and are facilitating our long-term goal of applying the synthetic power of modular polyketide synthases to the development and production of new medicines. Using the updated definition of the “module”, our lab has engineered diverse tri- /tetra-/pentaketide synthases that are functional both in vivo and in vitro. While these short assembly lines are uncommon in nature, they are ideal for our structural and functional studies. In Specific Aim 1 we propose to plunge-freeze these assembly lines as they are synthesizing their polyketide products and investigate them by cryo-electron microscopy. Since this approach has enabled us to capture high-resolution, dynamic information of the priming ketosynthase and acyltransferase of a model triketide synthase, we will apply it to synthases that contain other regions of interest. One objective is to learn how the ketoreductase, dehydratase, and enoylreductase processing enzymes are oriented relative to one another and the neighboring ketosynthase+acyltransferase didomains to understand how acyl carrier protein domains move between these enzymes during the extension and processing of polyketide intermediates. Thus, we will investigate at least thirteen engineered tri-/tetraketide synthases and two natural synthases functionally validated in our lab that contain different sets of these processing enzymes. In Specific Aim 2 we propose to elucidate interactions between processed polyketide intermediates and the ketosynthases that gatekeep for them. We have strong hypotheses for how sets of substrate tunnel residues interact with intermediates closest to the reactive thioester to ensure they are properly modified by upstream processing enzymes. Thus, we will appropriately mutate the gatekeeping residues of ketosynthases in less active model synthases as well as model synthases with inactivated upstream processing enzymes and determine whether their productivities improve as predicted. Since our data indicate that polyketide intermediates rigidify the ketosynthase dimer and dimeric ketosynthase+acyltransferase didomains can be readily identified in cryo-electron microscopy studies, we will also perform electron microscopy on stalled synthases to solve structures of polyketide-bound ketosynthase+acyltransferase dimers. In Specific Aim 3 we propose to determine key domain-domain interfaces. We have evidence that interfaces between processing enzymes and downstream KSs drive the ordered self- assembly of synthase polypeptides more than the small interface observed between the C- and N-terminal docking domains and seek structures of representative complexes. We also aim to determine how acyl carrier protein domains dock with ketosynthases during the transacylation reaction. If we are successful in these projects, it will great...