Gatekeeping Activity of Collinear Ketosynthase Domains Limits Product Diversity for Engineered Type I Polyketide Synthases

Abstract

Engineered type I polyketide synthases (type I PKSs) can enable access to diverse polyketide pharmacophores and generate non-natural natural products. However, the promise of type I PKS engineering remains modestly realized at best. Here, we report that ketosynthase (KS) domains, the key carbon-carbon bond-forming catalysts, control which intermediates are allowed to progress along the PKS assembly lines and which intermediates are excluded. Using bimodular PKSs, we demonstrate that KSs can be exquisitely selective for the upstream polyketide substrate while retaining promiscuity for the extender unit that they incorporate. It is then the downstream KS that acts as a gatekeeper to ensure the fidelity of the extender unit incorporation by the upstream KS. We also demonstrate that these findings are not universally applicable; substrate-tolerant KSs do allow engineered polyketide intermediates to be extended. Our results demonstrate the utility for evaluating the KS-induced bottlenecks to gauge the feasibility of engineering PKS assembly lines.

Figure 1

Modular architecture illustrated for a type I PKS furnishing an α-pyrone product.

Figure 2

Design and evaluation of PKS assembly lines. (A) The Plt P1 PKS. The nonfunctional KR domain is labeled KR0. (B) P2 PKS in which the module-1 AT has been inactivated and FabD is added. Product yields are evaluated relative to the stoichiometry of the 4,5-dichloropyrrolyl-SNAC substrate added to the assay. (C) Cal C1 PKS. (D) The C1 and C2 PKSs. No difference in yield of 2 is observed between C1 and C2 PKSs. (E) Competition experiment between Mal and MeMal extender units leading to products 1–4. (F) Yields of 1–4 produced in the competition assays. (G) Assay design and relative amounts of diketides 5 and 6 produced by the four unimodular PKSs. Time-dependent formation of 5 and 6 by (H) P2 and (I) C2 PKSs.

Figure 3

Expanded competition experiment. (A) Assay design in which four extender units are provided to the P1–P2 and C1–C2 PKSs. Pyrones 7 and 8 collectively represent products in which the PKS module-1 incorporates the cognate extender unit while pyrones 9 and 10 collectively represent molecules in which module-1 incorporates a noncognate extender unit. Product profiles and yields, relative to the 4,5-dichloropyrrolyl-SNAC substrate, for (B) P1 and P2, and (C) C1 and C2 PKSs.

Figure 4

Model of how KS domains maintain fidelity in PKS assembly lines.