Microbial type II polyketides serve as powerful medicinally relevant agents. These molecules are biosynthesized by polyketide synthases (PKSs) comprised of a core ketosynthase-chain length factor (KS-CLF) and phosphopantetheinylated acyl carrier protein (holo-ACP). While engineering type II PKSs holds potential to unlock sustainable access to diverse bioactive molecules, the inability to obtain cognate type II KS-CLFs andholo-ACPs forin vitrostudies represents a longstanding barrier. Herein, we share how the sequence and structural analysis of theGloeocapsa sp.PCC 7428 ACP allowed us to tune to a requisite weak yet specific interaction with a phosphopantetheinyl transferase to afford theholo-ACP. This, coupled with our ability to heterologously express the cognate KS-CLF in high quantities, unlocked access to polyketide products viain vitromultienzyme assembly. We hope this work inspires future studies of type II PKSs that have previously evaded heterologous expression or have yet to be explored.Abstract Figure