Discovery of catalytic tools for isoprenoid production

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The isoprenoids are a large and structurally diverse class of natural products with more than 55,000 known compounds. Many isoprenoids have been used as flavors, pharmaceuticals, and industrial polymers. All isoprenoids are currently supplied either by purification from plants or by chemical synthesis involving petrochemicals. However, the yield from natural sources is often inadequate to meet industrial demands, and chemical synthesis is not desirable due to environmental perturbation. To overcome these, the enzymatic production of isoprenoids using biological systems has become an attractive alternative to the traditional purification and chemical synthesis; however, our current knowledge of isoprenoid metabolism remains insufficient to realize the biological supply of isoprenoids. ******The long-term goal of my research program is to supply isoprenoid products by sustainable biological methods, thus replacing the petrochemical-derived products. To advance the knowledge of isoprenoid metabolism, I propose to study two subclasses of isoprenoid natural products - sesquiterpene lactones (SLs) and natural rubber (NR). SLs have been used as building blocks for many pharmaceuticals while NR is an indispensable biopolymer for hundreds of industrial products. Precise mechanisms of SL and NR biosynthesis remain elusive, and integrative approaches combining biochemistry, genetics, and genomics will be used to elucidate the mysteries of SL and NR biosynthesis. ******First, how stereoselective biosynthesis of various SLs is achieved in nature will be studied by isolating and characterizing enzymes from two medicinal plant species. Stereoselective synthesis of chemicals is essential in the pharmaceutical industry, and thus this work will provide biological methods to produce stereoselective chemicals. Second, combinatorial uses of plant enzymes will be attempted in microbes to produce novel SLs that are not present in nature, based on our discovery of one promiscuous enzyme in SL biosynthesis. More than 100 combinations of enzymes will be synthetically made in yeast, and the resulting new SL products will be identified and purified for structural studies. Ultimately, these new SLs will be examined for possible anti-bacterial activities. Third, as we do not fully understand how NR is biosynthesized in plants, in part due to gaps in our knowledge of several protein components in the NR pathway, additional necessary proteins for NR biosynthesis will be identified and characterized from plants using biochemical and genetic approaches. The newly identified protein component(s) can be used to devise alternative bio-systems that can produce NR outside of the tropical para-rubber trees, thereby securing polymer sustainability. Collectively, the proposed research will lay a foundation of knowledge for a sustainable supply of isoprenoid products critically needed in our society.**