PROJECT SUMMARY Microorganisms produce structurally diverse molecules, many of which have been successfully repurposed as pharmaceutical agents. These molecules are manufactured by multi-enzyme assemblies, which rely on acyl carrier proteins (ACPs) to modify and transfer chemical intermediates to a team of enzymatic partners. Strategic redesign of natural enzyme assemblies presents an exciting possible route to produce new antibiotics, but the success of any redesign approach hinges on a thorough understanding of what leads to chemically productive ACP-enzyme interactions. The goal of this study is to gain a molecular-level understanding of how ACPs interact with their molecular cargo and enzymatic partners. In the previous funding period, our lab developed new spectrophotometric methodologies that enabled us to unveil the fast and transient interactions between ACPs and their molecular cargo, as well as between ACPs and two enzymatic partners: a ketosynthase (KS) and dehydratase (DH). These studies led to 7 papers with 40 Haverford College undergraduate students earning co-authorship. We now seek to leverage these major advancements to understand the complex interplay between ACP sequence and molecular cargo identity in directing the phenomenon called “chain sequestration,” which is thought to play a critical role in directing biocatalysis. We will also study how chain sequestration effects ACP-KS binding affinity and obtain ACP-KS crosslinked structures for subsequent molecular-level structural characterization. Results from these studies will guide the future biosynthesis of novel small molecules with potential pharmaceutical activity. The work will be executed in the context of independent undergraduate research projects and course-based undergraduate research experiences (CUREs), thereby exposing ~60 undergraduate students to advanced research at the chemistry-biology interface.