RUI: A platform technology for elucidating design rules of the Epoxomicin synthase, enabling biosynthesis of complex anti-tumor molecules

具有生物活性的天然产品（药品、保健品）通常很难用化学方法复制，因为它们是由一系列的反应产生的，这些反应是在不断生长的分子中一点一点地添加不同的亚基。因此，它们主要是从植物材料中收获的，通常产量很低。该项目将研究一种在没有细胞的情况下制造较大药物分子亚基的策略，然后将这些亚基结合在一起产生最终产品。如果成功，这将大大提高大分子药物生产的效率，也可以提高识别更有效药物分子的速度。研究工作将主要由本科生推动，他们将参与所有拟议的实验和广泛传播结果。本科生还将通过一个已建立的“边做边学”实验室，以及通过将SciTrek项目扩展到当地学校，为K-12课程提供探究式学习模块。目前生物制造以前难以处理的天然产物的方法在按需生物合成新的天然产物的能力方面受到限制。该项目的主要目标是实现和促进按需生物合成天然产物及其衍生物。具体来说，一种阐明环氧霉素天然产物生物合成机制基础的新方法将通过以下方式发展：1)“解剖”合成环氧霉素的非核糖体肽-聚酮合成酶（NRP-PKS）巨酶，2)鉴定底物特异性的分子决定因素，以及3)设计环氧霉素生物合成机制以生成衍生物。这些代谢工程将在无细胞环境中进行；该平台的开放性将提供直接访问代谢反应条件，以实现比传统代谢工程平台更快的设计-构建-测试周期。潜在的贡献包括：A)了解NRP-PK巨酶生物合成复杂天然产物的生化原理；B)一种平台技术，使复杂天然产品的工程和生物制造成为可能。通过这项工作取得的进展将直接应用于无数其他NRP-PKS巨型酶的表征和工程。此外，这项工作将为培养本科生进行生物技术研究和向他们的社区传播科学奠定基础。

Bio-active natural products (pharmaceuticals, nutraceuticals) are generally difficult to replicate chemically, because they result from a sequence of reactions that add, piece by piece, different subunits to the growing molecule. As a result, they are harvested primarily from plant material, usually with low yield of product. This project will investigate a strategy that manufactures subunits of the larger drug molecule in the absence of cells, then brings the subunits together to create the final product. If successful, this could dramatically improve the efficiency of large molecule drug production, and could also increase the speed of identification of more effective drug molecules. Research efforts will be driven primarily by undergraduate students who will participate in all proposed experiments and broad dissemination of results. Undergraduate students will also lead inquiry-based learning modules for K-12 outreach through an established "Learn by Doing Lab" and through expansion of the SciTrek program into local schools. Current approaches to the biomanufacturing of previously intractable natural products are limited in their capacity to biosynthesize new natural products of interest on-demand. The primary objective of this project is to enable and facilitate on-demand biosynthesis of natural products and their derivatives. Specifically, a novel approach to elucidating the mechanistic underpinnings of epoxomicin natural product biosynthesis machinery will be developed by 1) "dissecting" the non-ribosomal peptide - polyketide synthase (NRP-PKS) mega-enzyme that synthesizes epoxomicin, 2) identifying the molecular determinants of substrate specificity, and 3) engineering the epoxomicin biosynthesis machinery for the generation of derivatives. These metabolic engineering efforts will be pursued in a cell-free environment; the open nature of this platform will provide direct access to metabolic reaction conditions for design-build-test cycles that are more rapid than traditional platforms for metabolic engineering. Potential contributions include A) knowledge of biochemical principles by which NRP-PK mega-enzymes biosynthesize complex natural products; and B) a platform technology that enables engineering and bio-manufacturing of complex natural products on-demand. Advances made through this work will apply directly to characterization and engineering of a myriad of other NRP-PKS mega-enzymes. In addition, this work will provide a foundation for training undergraduate students in conducting biotechnological research and communicating science to their community.