Discovery of catalytic tools for isoprenoid production

异戊二烯类化合物是一大类结构多样的天然产物，有55,000多种已知化合物。许多异戊二烯类化合物已被用作香料、医药和工业聚合物。目前，所有异戊二烯类化合物都是通过工厂提纯或涉及石化产品的化学合成来供应的。然而，来自自然资源的产量往往不足以满足工业需求，而且由于环境干扰，化学合成并不可取。为了克服这些问题，利用生物系统酶法生产类异戊二烯已经成为一种有吸引力的替代传统的提纯和化学合成的方法；然而，我们目前对类异戊二烯代谢的了解仍然不足以实现类异戊二烯的生物供应。 我的研究计划的长期目标是通过可持续的生物方法提供类异戊二烯产品，从而取代石化衍生产品。为了加深对类异戊二烯代谢的了解，我建议研究两类天然产物-倍半萜内酯(SLS)和天然橡胶(NR)。SLS已被用作许多药物的基础材料，而天然橡胶是数百种工业产品不可或缺的生物聚合物。SL和NR生物合成的确切机制仍然不清楚，结合生物化学、遗传学和基因组学的综合方法将被用来解释SL和NR生物合成的奥秘。 首先，将通过从两种药用植物中分离和鉴定酶来研究各种SLS在自然界中如何实现立体选择性的生物合成。立体选择性合成化学物质在制药工业中是必不可少的，因此这项工作将为生产立体选择性化学物质提供生物方法。其次，基于我们在SL生物合成中发现的一种混杂酶，我们将尝试在微生物中组合使用植物酶来产生自然界中不存在的新的SLS。将在酵母中合成100多种酶组合，并对由此产生的新SL产品进行鉴定和纯化，以进行结构研究。最终，将对这些新的SLS进行可能的抗菌活性检查。第三，由于我们还不完全了解NR在植物中是如何生物合成的，部分原因是我们对NR途径中的几种蛋白质成分的了解不足，因此将使用生化和遗传方法从植物中鉴定和表征NR生物合成所需的额外蛋白质。新发现的蛋白质成分(S)可用于设计替代生物系统，在热带副橡胶树外产生天然橡胶，从而确保聚合物的可持续性。总而言之，拟议的研究将为可持续供应我们社会迫切需要的类异戊二烯产品奠定知识基础。

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.