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

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 carbon-carbon bond formation, suggesting that proper orientation of the initially formed acyl thioester in the ketosynthase active site was important for the enzyme-catalyzed decarboxylative condensation reaction. 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 carbon-carbon bond formation, suggesting that proper orientation of the initially formed acyl thioester in the ketosynthase active site was important for the enzyme-catalyzed decarboxylative condensation reaction. Polyketides are a large family of structurally diverse and complex natural products produced by bacteria and fungi. Many of these compounds, such as the important clinical agents erythromycin (1Cortes J. Haydock S.F. Roberts G.A. Bevitt D.J. Leadlay P.F. Nature. 1990; 348: 176-178Crossref PubMed PubMed PubMed PubMed and J. PubMed J. J. PubMed are by modular polyketide polyketide ketosynthase polyketide ketosynthase the condensation of to to these diverse and complex PubMed J. PubMed polyketide biosynthetic are of large polyketide synthase each to active the for the and of a the polyketide chain are to a module module the acyl chain from the module its ketosynthase and the the acyl domain the is the acyl chain in a and the is by and the module transfer the acyl chain to the module for processing PubMed J. PubMed modular of these biosynthetic Nature. PubMed for the large of and in the polyketide of the and of modules in a biosynthetic the of each module for in the and of the of modular that can be harnessed to generate rationally designed complex through J. J. Leadlay P.F. PubMed PubMed PubMed rationally modular it is to features govern processing of the polyketide Previous studies PubMed PubMed J. PubMed PubMed have shown that the transfer of the polyketide is by each chain transfer between modules. of the ketosynthase for the stereochemistry of the acyl chain J. PubMed J. PubMed J. PubMed is important in module and of these inter-module and interactions from of the synthase J. PubMed J. PubMed the of these we have the ability of modules from different to complex a of modules from the erythromycin (1Cortes J. Haydock S.F. Roberts G.A. Bevitt D.J. Leadlay P.F. 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