Systematic domain swaps of iterative, nonreducing polyketide synthases provide a mechanistic understanding and rationale for catalytic reprogramming

Abstract

Iterative, nonreducing polyketide synthases (NR-PKSs) are multidomain enzymes responsible for the construction of the core architecture of aromatic polyketide natural products in fungi. Engineering these enzymes for the production of non-native metabolites has been a long-standing goal. We conducted a systematic survey of in vitro "domain swapped" NR-PKSs using an enzyme deconstruction approach. The NR-PKSs were dissected into mono- to multidomain fragments and recombined as noncognate pairs in vitro, reconstituting enzymatic activity. The enzymes used in this study produce aromatic polyketides that are representative of the four main chemical features set by the individual NR-PKS: starter unit selection, chain-length control, cyclization register control, and product release mechanism. We found that boundary conditions limit successful chemistry, which are dependent on a set of underlying enzymatic mechanisms. Crucial for successful redirection of catalysis, the rate of productive chemistry must outpace the rate of spontaneous derailment and thioesterase-mediated editing. Additionally, all of the domains in a noncognate system must interact efficiently if chemical redirection is to proceed. These observations refine and further substantiate current understanding of the mechanisms governing NR-PKS catalysis.

Figure 1

Core domain architecture of NR-PKS with highlighted enzyme bound intermediates and products of TE-directed Claisen cyclization or spontaneous O–C bond closure.

Figure 2

Product analysis of combinatorial reactions with Pks4 SAT-KS-MAT. A) Proposed structures for products of chemical redirection. B) Pks4 control reactions. C) Combinatorial reactions containing PT and ACP_n for the given parent PKS. D) Combinatorial reactions containing PT, ACP₂, and TE for the given parent PKS.

Figure 3

UV–vis spectra and high-resolution mass spectra (HRMS) for YWA1 (5) core containing molecules. Product 11 converts to pre-bikaverin (2).

Figure 4

UV–vis spectra and HRMS for naphthopyrone containing products nor-toralactone (4) and norpyrone (19) are presented with those for likely naphthopyrone 13 or naphthopyrone 14.

Figure 5

UV–vis spectra and HRMS for atrochrysone core containing molecules are presented for comparison. Spectral data are consistent for literature values for atrochrysone.

Figure 6

Product analysis of combinatorial reactions with Pks1 SAT-KS-MAT. (A) Proposed structures for products of chemical redirection. (B) Pks1 control reaction. Combinatorial reactions containing (C) PT and ACP_n and (D) PT, ACP₂, and TE for the given parent PKS.

Scheme 1. Possible Intermediates of the Pks4 PT and TE Domains

Figure 7

Product analysis of combinatorial reactions with PksA SAT-KS-MAT. (A) Proposed structures for products of chemical redirection. (B) PksA control reaction. (C) Combinatorial reactions containing PT and ACP_n for the given parent PKS. (D) Combinatorial reactions containing PT, ACP₂, and TE for the given parent PKS.

Figure 8

UV–vis spectra and HRMS are presented for pannorin (23) and hex-pannorin (22).

Figure 9

Combinatorial reactions containing PksA SAT-KS-MAT with the addition of (A) PksA PT, ACP, TE; (B) CTB1 PT, ACP₂, TE; and (C) CTB1 PT, PksA ACP, CTB1 TE.

Figure 10

Reactions of (A) deconstructed Pks1 (SAT-KS-MAT + PT + ACP₂ + TE) and (B) intact Pks1.

Figure 11

Formations of (A) YWA1 (5) and nor-rubrofusarin (32) by (B) 10 μM each CTB1 SAT-KS-MAT and Pks1 PT, ACP₂, and TE; and (C) 1 μM M4P6.