Biosynthetic engineering of multi-modular polyketide synthases [electronic resource] : an acyltransferase perspective
Keywords: Chemical Engineering

Polyketide synthases (PKSs) are multi-functional enzymes, which synthesize natural products known as polyketides. These complex molecules have a diverse range of medicinal properties. Biosynthetic engineering of the multi-modular PKSs is attractive due to their modularity and colinearity, which, if understood at the molecular level, could allow for the efficient and predictable regioselective chemical manipulation of polyketides. A minimum combination of a [beta]-ketosynthase (KS), an acyltransferase (AT), and an acyl carrier protein (ACP) is required for the assembly of acyl-CoA precursors into complex polyketide products. This work will focus on AT domains, which are the primary gatekeepers for stepwise incorporation of acyl-CoA building blocks into a growing polyketide chain. In our initial investigations, protein interactions between AT, ACP and flanking AT linkers from a prototypical multimodular 6-deoxyerythronolide B synthase (DEBS) were systematically explored, guided by recent high-resolution structures. Our results indicate that N/C terminal linkers of the modular DEBS AT domain contributed to both efficiency and specificity of transacylation. Representative DEBS AT3 and AT6 domains were also observed to have greater than 10-fold AT specificities for their cognate ACP substrates as compared to other ACPs in the DEBS PKS. In comparison, there is only modest discrimination for its native ACP by the standalone AT from the "AT-less" disorazole synthase (DSZS). These "AT-less" multimodular PKS lack AT domains in their modular assembly, and instead, transacylation is supplied by a trans-acting discrete AT. With its higher transacylation activity for DEBS ACPs compared to their natural ATs (> 40-fold), DSZS AT presents new opportunities for regioselective modification of a polyketide backbone and thus prompting further structural and biochemical investigations. Towards the analysis of DSZS AT, we report crystal structures of trans-acting AT resolved at 1.51 Å, and that of its acetate complex at 1.35 Å resolution. Comprehensive alanine-scanning mutagenesis of its native ACP1 substrate also identified a conserved Asp45 residue on the ACP for AT interactions. This conserved residue is proposed to contribute to the observed AT promiscuity. Supplementing in silico protein docking with these results, a model for DSZS AT and ACP interactions was derived. Working towards high-resolution structural characterization of this interface, we developed a novel strategy for covalently cross-linking and purifying a catalytically relevant DSZS AT-ACP complex. Finally, trans complementation of methylmalonyl CoA specific DEBS modules was also accomplished in vitro with DSZS AT for malonyl CoA incorporation. From our investigations, we have gained new insights into the protein-protein interactions that play a major role in the efficient biosynthesis of structurally complex polyketides. These results also reveal important considerations and opportunities for biosynthetic engineering within the multi-functional assembly lines.