Engineering the Substrate Specificity of a Modular Polyketide Synthase for Installation of Consecutive Non-Natural Extender Units

Abstract

There is significant interest in diversifying the structures of polyketides to create new analogues of these bioactive molecules. This has traditionally been done by focusing on engineering the acyltransferase (AT) domains of polyketide synthases (PKSs) responsible for the incorporation of malonyl-CoA extender units. Non-natural extender units have been utilized by engineered PKSs previously; however, most of the work to date has been accomplished with ATs that are either naturally promiscuous and/or located in terminal modules lacking downstream bottlenecks. These limitations have prevented the engineering of ATs with low native promiscuity and the study of any potential gatekeeping effects by domains downstream of an engineered AT. In an effort to address this gap in PKS engineering knowledge, the substrate preferences of the final two modules of the pikromycin PKS were compared for several non-natural extender units and through active site mutagenesis. This led to engineering of the methylmalonyl-CoA specificity of both modules and inversion of their selectivity to prefer consecutive non-natural derivatives. Analysis of the product distributions of these bimodular reactions revealed unexpected metabolites resulting from gatekeeping by the downstream ketoreductase and ketosynthase domains. Despite these new bottlenecks, AT engineering provided the first full-length polyketide products incorporating two non-natural extender units. Together, this combination of tandem AT engineering and the identification of previously poorly characterized bottlenecks provides a platform for future advancements in the field.

Figure 1.

The pikromycin polyketide synthase and its products. ACP = acyl carrier protein; AT = acyltransferase; DH = dehydratase; ER = enoylreductase; KR = ketoreductase; KS = ketosynthase; KS^Q = ketosynthase-like decarboxylase; TE = thioesterase.

Figure 2.

Wild-type and chimeric module systems designed to probe the specificity of each AT from PikAIII and PikAIV. R1 is the wild-type PikAIII (red) and wild-type PikAIV (purple) with the native PikAIV TE (blue). AT swaps were used to create R2 and R3 from R1. R4 is PikAIII fused to the PikAIV TE. Each Pik AT was swapped into Ery6TE (R6, grey) to generate R7 and R8.

Figure 3.

Models of (A) EryAT6 Y744R and (B) PikAT6 Y753R ATs after undergoing MD simulations. Distances between catalytic residues in the mutant EryAT6 (4.8 Å) are long but catalytically-competent. The same distance in the mutant PikAT6 (8.4 Å) is characteristic of an inactive AT domain.

Scheme 1.

Bimodular extender unit competition assay. The two final Pik modules are incubated with the synthetic pentaketide chain mimic 10 and a mixture of the native extender 8 and an equimolar amount of one of 9a-d in vitro. Products 1, 3a-d, and 4a-d are produced when the PikAIII-extended chain bypasses module 6 and is cyclized by the TE. Products 4a-d bypass the KR domain in PikAIII. Non-reduced products derived from the native extender 8 were not observed. NADPH was produced in situ with an NADPH regeneration system. Product distributions shown are for the wild-type system and are calculated separately for one- and two-extension products. Error was ± 5% of indicated value.

Scheme 2.

Single module extender unit competition assay. The fusion protein PikAIIITE or the final module of the DEBS PKS, Ery6TE, are incubated with the synthetic pentaketide chain mimic 10 and a mixture of the native extender 8 and an equimolar amount of 9a in vitro. NADPH was produced in situ with an NADPH regeneration system. Error was ± 5% of indicated value. Percentages are shown for wild-type systems.