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

RUI：一种阐明 Epoxomicin 合酶设计规则的平台技术，可实现复杂抗肿瘤分子的生物合成

• 批准号: 1708919
• 负责人: Katharine Watts
• 金额: $ 28.61万
• 依托单位: California Polytechnic State University Foundation
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1708919&HistoricalAwards=false
• 关键词: RUI; platform; technology; elucidating; design

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.

具有生物活性的天然产物（药物、营养品）通常很难通过化学方法复制，因为它们是由一系列反应产生的，这些反应将不同的亚基一点一点地添加到生长的分子中。因此，它们主要从植物材料收获，通常产品产量低。该项目将研究一种策略，在没有细胞的情况下制造较大药物分子的亚基，然后将亚基聚集在一起以产生最终产品。如果成功，这将大大提高大分子药物生产的效率，并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驱动，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”和将SciTrek计划扩展到当地学校，为K-12外展领导基于探究的学习模块。目前的方法，以前棘手的天然产品的生物制造是有限的，在其能力，生物合成新的天然产品的需求。该项目的主要目标是实现和促进天然产品及其衍生物的按需生物合成。具体地，将通过以下方式开发阐明环氧霉素天然产物生物合成机制的机制基础的新方法：1）“解剖”合成环氧霉素的非核糖体肽-聚酮合酶（NRP-PKS）巨酶，2）鉴定底物特异性的分子决定因素，和3）工程化环氧霉素生物合成机制以产生衍生物。这些代谢工程工作将在无细胞环境中进行;该平台的开放性将为设计-构建-测试循环提供直接进入代谢反应条件的途径，比传统的代谢工程平台更快。潜在的贡献包括A）NRP-PK巨型酶生物合成复杂天然产物的生物化学原理的知识;和B）能够按需工程和生物制造复杂天然产物的平台技术。通过这项工作取得的进展将直接适用于无数其他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科学传播到他们的社区奠定基础。

项目成果

Activation of Energy Metabolism through Growth Media Reformulation Enables a 24-Hour Workflow for Cell-Free Expression

• DOI: 10.1021/acssynbio.0c00283
• 发表时间: 2020-10-16
• 期刊: ACS SYNTHETIC BIOLOGY
• 影响因子: 4.7
• 作者: Levine, Max Z.;So, Byungcheol;Oza, Javin P.
• 通讯作者: Oza, Javin P.

The Genetic Code Kit: An Open-Source Cell-Free Platform for Biochemical and Biotechnology Education

遗传密码套件：用于生物化学和生物技术教育的开源无细胞平台

• DOI: 10.3389/fbioe.2020.00941
• 发表时间: 2020
• 期刊: Frontiers in Bioengineering and Biotechnology
• 影响因子: 5.7
• 作者: Williams, Layne C.;Gregorio, Nicole E.;So, Byungcheol;Kao, Wesley Y.;Kiste, Alan L.;Patel, Pratish A.;Watts, Katharine R.;Oza, Javin P.
• 通讯作者: Oza, Javin P.

Unlocking Applications of Cell-Free Biotechnology through Enhanced Shelf Life and Productivity of E. coli Extracts

• DOI: 10.1021/acssynbio.9b00433
• 发表时间: 2020-04-17
• 期刊: ACS SYNTHETIC BIOLOGY
• 影响因子: 4.7
• 作者: Gregorio, Nicole E.;Kao, Wesley Y.;Oza, Javin P.
• 通讯作者: Oza, Javin P.