Strategic Acyl Carrier Protein Engineering Enables Functional Type II Polyketide Synthase Reconstitution In Vitro

Microbial polyketides represent a structurally diverse class of secondary metabolites with medicinally relevant properties. Aromatic polyketides are produced by type II polyketide synthase (PKS) systems, each minimally composed of a ketosynthase-chain length factor (KS-CLF) and a phosphopantetheinylated acyl carrier protein (-ACP). Although type II PKSs are found throughout the bacterial kingdom, and despite their importance to strategic bioengineering, type II PKSs have not been well-studied . In cases where the KS-CLF can be accessed via heterologous expression, often the cognate ACPs are not activatable by the broad specificity surfactin-producing phosphopantetheinyl transferase (PPTase) Sfp and, conversely, in systems where the ACP can be activated by Sfp, the corresponding KS-CLF is typically not readily obtained. Here, we report the high-yield heterologous expression of both cyanobacterial sp. PCC 7428 minimal type II PKS (gloPKS) components in , which allowed us to study this minimal type II PKS . Initially, neither the cognate PPTase nor Sfp converted gloACP to its active state. However, by examining sequence differences between Sfp-compatible and -incompatible ACPs, we identified two conserved residues in gloACP that, when mutated, enabled high-yield phosphopantetheinylation of gloACP by Sfp. Using analogous mutations, other previously Sfp-incompatible type II PKS ACPs from different bacterial phyla were also rendered activatable by Sfp. This demonstrates the generalizability of our approach and breaks down a longstanding barrier to type II PKS studies and the exploration of complex biosynthetic pathways.