Understanding Substrate Specificity of Polyketide Synthase Modules by Generating Hybrid Multimodular Synthases

Abstract

Modular polyketide biosynthesis can be harnessed to generate rationally designed complex natural products through bioengineering. A detailed understanding of the features that govern transfer and processing of polyketide biosynthetic intermediates is crucial to successfully engineer new polyketide pathways. Previous studies have shown that substrate stereochemistry and protein-protein interactions between polyketide synthase modules are both important factors in this process. Here we investigated the substrate tolerance of different polyketide modules and assessed the relative importance of inter-module chain transfer versus chain elongation activity of some of these modules. By constructing a variety of hybrid modular polyketide synthase systems and assaying their ability to generate polyketide products, it was determined that the substrate tolerance of each individual ketosynthase domain is an important parameter for the successful recombination of polyketide synthase modules. Surprisingly, however, failure by a module to process a candidate substrate was not due to its inability to bind to it. Rather, it appeared to result from a blockage in carboncarbon bond formation, suggesting that proper orientation of the initially formed acyl thioester in the ketosynthase active site was important for the enzymecatalyzed decarboxylative condensation reaction.

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases

“…Early studies revealed the importance of ACP-KS interactions (6,7), as well as the utility of intermodular linker interactions (8), in the design of productive chimeric assembly lines. At the same time, the relatively broad, but not unrestricted, substrate tolerance of typical KS domains was also recognized (9). In one particularly thorough study, Menzella et al (10) carried out a large scale assessment of the feasibility of combinatorial biosynthesis by pairwise combinations of 14 modules derived from 8 different PKS clusters, thereby allowing the in vivo characterization of 154 chimeric bimodular PKSs, with the bimodular DEBS derivative (Fig.…”

Acyl‐Chain Elongation Drives Ketosynthase Substrate Selectivity in trans‐Acyltransferase Polyketide Synthases

“…There was no observable elongation for the β-methyl substrate (7), despite initial acylation of the KS. In addition, no elongation was observed with oxazolyl and pyranyl substrates (8) and (9), which are moieties present in various PKS intermediates [17] . Overall, these results suggest that, not only does PsyA KS1 impose selectivity at the elongation step of its catalytic action, but that a) b) the selectivity is in excellent agreement with sequence-based phylogenetic predictions (Figure S14) [13] .…”

Engineering the acyltransferase substrate specificity of assembly line polyketide synthases

“…Most domains in the assembly line receive their substrates from previous catalytic steps and not by diffusive loading; these enzymes therefore may not have been evolutionarily driven to develop strict substrate specificities and could be inherently robust to rearrangement [9]. Indeed, loading AT domains, KS, ACP, KR, DH and ER domains from the 6-deoxyerythronolide B synthase (DEBS) have been shown to accept substrates that vary considerably from their native substrates [10][11][12][13][14][15]. Alternatively, extender AT domains receive their substrates diffusively and usually exhibit strict specificity towards a single a-carboxyacyl-CoA building block [10,14].…”