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New tools for probing polyketide biosynthesis
The need for more drugs to curb the increasing rise in antibiotic resistant infections has seen an increase in research to discover more antibiotic scaffolds. Polyketides are natural products that can be manipulated to yield a structurally diverse library for drug discovery programs. Modular polyketide synthases (PKSs) are nano-factories that biosynthesize polyketides in an assembly line fashion making use of two sets of active sites in each module; one set for increasing polyketide chain length and another set for fixing the oxidation state of the β-carbons. Although many strategies exist for the introduction of diversity into polyketide structure, these strategies often result in the production of unnatural polyketides with reduced yields. The primary factors responsible for this observed reduction in chemical output are substrate selectivity of downstream enzymes and disruption of protein-protein interactions. One strategy, domain inactivation via active site mutagenesis, however, maintains the three dimensional architecture of the PKS and is therefore a much more viable strategy. This strategy is best suited for a PKS in which all or almost all of the modules possess the full complement of tailoring domains. To this end, the genes for the fluvirucin B1 synthase have been cloned and characterized. The fluvirucin B1 PKS has five modules, four of which are fatty acid synthase-like and hence represent an ideal system for the domain inactivation strategy. A simple assay to examine the activity of the tailoring domains has also been designed. This assay differs from traditional methods in that it doesn't require a chain elongation step. To do this, we have designed an electrophilic β-lactam that reacts specifically with the phosphopantetheine group of holo-ACPs to yield β-keto acyl ACPs which serve as the substrate for the tailoring domain enzymes. A mechanism based fluorescence transfer assay which is very simple, inexpensive and robust has also been developed to investigate the substrate tolerance of KS domains. Together, these new tools will be valuable in providing insights into the mechanism of polyketide biosynthesis and go a long way in aiding engineering efforts towards the production of polyketides with unnatural structures.
Borketey, Sheringham Lawrence, "New tools for probing polyketide biosynthesis" (2013). Doctoral Dissertations Available from Proquest. AAI3603058.