S160: Exploring microbial resources for useful biocatalysts

Microorganisms produce a wide variety of molecules through well programmed biosynthetic pathways. Their abundant biosynthetic enzymes represent a rich source of useful biocatalysts. This work aimed to discover new biocatalysts from microorganisms that can introduce functional groups to natural products for structural optimization. Streptomyces rimosus is an actinomycete known as the producer of oxytetracycline. We have recently discovered that it is a useful glycosylation biocatalyst. This bacterium can selectively introduce a 4'-deoxy-hex-4'-enopyranosiduronic acid moiety into the substrates such as quercetin and pradimicin analogs to generate novel glycosides. Pochonia chlamydosporia is a filamentous fungus that produces radicicol, an anticancer and antifungal natural product. From the radicicol biosynthetic pathway, we have identified the first fungal flavin-dependent halogenase Rdc2. This enzyme catalyzes the in vitro chlorination of monocillin IV, confirming that it is a post-PKS tailoring enzyme involved in radicicol biosynthesis. A homology model of Rdc2 indicated that the substrate binding site of this enzyme may accommodate a variety of compounds. In vitro studies confirmed that this enzyme has broad substrate specificity towards various aromatic substrates such as dihydroresorcylide, curcumin, isoquinolines and tetracyclines. Additionally, Rdc2 can catalyze dihalogenation to yield corresponding dihalogenated products. Further investigation into its specificity towards halogen donors revealed that this enzyme is also a brominase that can catalyze both mono- and dibromination. Collaborative actions of Rdc2 and another fungal biocatalyst, Beaueria bassiana, led to the synthesis of a novel chlorinated glycoside of dihydroresorcylide. Thus, Rdc2 represents a useful biocatalyst for prepration of halogenated molecules.