Glycosyltransferases involved in linking polyketide macrolactones with deoxysugars are key enzymes for combinatorial biosynthesis of novel macrolide anti-infective agents. In this proposal, a new gene shuffling technology, RACHITT, is applied to the desosarninyl transferase (Des VII) from the pikromycin biosynthetic pathway of Streptomyces venezuelae. Des VII is an enzyme with remarkable inherent substrate flexibility, and we expect to expand further its substrate spectrum to include 16-membered ring macrolactones. This technology is expected to provide glycosyltransferase enzymes that can catalyze the biosynthesis of 12-, 14-, and 16-membered ring macrolides. Moreover, we expect to generate a recombinant DesVII that has a higher enzymatic activity toward the 14-membered ring macrolactone narbonolide. This will be a key step in combinatorial biosynthesis of ketolide antibiotics that have a potential to combat multi-drug resistant bacterial pathogens.
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