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.

Starter Unit Flexibility for Engineered Product Synthesis by the Nonreducing Polyketide Synthase PksA

“…This finding was in complete accord with similar comparisons of intact vs . domain combinations determined recently, and confirms the validity of the deconstruction method for the purposes of the experiments described herein(19). …”

Aflatoxin and deconstruction of type I, iterative polyketide synthase function

“…From a matrix of six NR-PKSs enzyme components we have found that ACPs are substantially interchangeable; maintaining SAT–KS pairs is beneficial to overall synthetic efficiency; chain length is largely, but not exclusively, determined by the KS with occasional participation by the PT; PT active sites are unexpectedly accommodating with respect to the chain lengths of the polyketide intermediates presented to them, yet the regiochemistry of their cyclizations is tightly controlled; and, as noted above, the TE is crucial to success. Enzyme-directed cyclizations must outpace spontaneous reaction and TE-mediated editing, 52, 53 but “rules” have emerged from these studies where rapid combinatorial experiments can be used to quickly survey potential heterodomain combinations for assembly into single PKS chimeras for more efficient synthesis. 53 …”

“…Enzyme-directed cyclizations must outpace spontaneous reaction and TE-mediated editing, 52, 53 but “rules” have emerged from these studies where rapid combinatorial experiments can be used to quickly survey potential heterodomain combinations for assembly into single PKS chimeras for more efficient synthesis. 53 …”

Use of a biosynthetic intermediate to explore the chemical diversity of pseudo-natural fungal polyketides

“…The present results have therefore verified the success of this study for accessing novel chemical space. A number of methods based on PKS engineering have been developed and have produced diverse unnatural polyketide molecules [37][38][39][40][41][42] . These methods are undoubtedly powerful enough to generate analogues of structurally complex metabolites, although they do have some weakness regarding the simultaneous expansion of molecular frameworks.…”