CAREER: Synthetic Regulatory Systems for Dynamic Metabolic Pathways

非技术描述：工程微生物代谢途径允许从可再生和低成本的原料生产化学品，生物燃料，材料和药品。为了使这项技术具有可扩展性和经济可行性，工程微生物宿主必须能够持续平衡途径代谢以应对高滴度和高产产量的生产。动态代谢调节系统（DMRSs）是对途径酶和代谢物（酶作用的化学物质）的浓度提供动态控制的合成生物成分。这项研究将为DMRSs如何改变代谢途径的动态行为以及DMRSs如何提高途径生产力提供系统和定量的理解。这些知识将使研究人员能够更容易、更有效、更可靠地设计代谢控制系统，提高各种代谢途径的生产率、产量和稳健性，并使微生物生产化学品、药品、生物燃料和材料在经济上更加可行。该项目将为研究动态对自然代谢途径的影响提供有用的工具，揭示自然调节系统的进化意义。这些工具可以进一步扩展到调节各种复杂的合成系统，如化学控制，污染物清洁微生物。该项目还将通过一系列活动，包括“热门话题研讨会”和“实验室研究体验”，为来自当地公立学校的K-12学生和教师提供教育和培训机会，这些教育和培训计划将促进学生对前沿科学的兴趣，并培养研究系统和合成生物学的基本技能。技术描述：提供代谢途径动态控制的合成调控系统（称为DMRSs）是微生物工程中非常强大的工具，用于从复杂的代谢途径生产化学品。先前的概念验证研究表明，DMRSs能够显著提高途径的生产率和产量。然而，由于复杂的蛋白质-代谢物/蛋白质- dna相互作用，构建有效的DMRSs具有挑战性，通常需要测试大量的参数和调控拓扑（例如上调与下调，远程反馈与短程反馈）。为了使DMRSs稳定地工作并适用于广泛的途径，该项目旨在对代谢动力学以及代谢动力学如何影响途径生产力进行系统的了解。结合实验和理论策略，包括生物传感器集成荧光测量、单细胞成像、色谱代谢物定量和动力学模型，该项目将系统地表征代谢动力学、代谢波动和不同调控拓扑下的细胞间代谢变化。

Nontechnical Description: Engineering microbial metabolic pathways allows the production of chemicals, biofuels, materials, and pharmaceuticals from renewable and low-cost feedstocks. For this technology to be scalable and economically viable, engineered microbial hosts must be able to continuously balance the pathway metabolism to cope with high titer and high yield production. Dynamic metabolic regulation systems (DMRSs) are synthetic biological components that provide dynamic control to the concentration of pathway enzymes and metabolites (the chemicals on which enzymes act). This study will provide a systematic and quantitative understanding of how DMRSs change the dynamic behavior of metabolic pathways and how DMRSs improve pathway productivity. The knowledge will enable researchers to design metabolic control systems more easily, effectively, and reliably, improving productivities, yields and robustness for a variety of metabolic pathways and making the microbial production of chemicals, pharmaceuticals, biofuels, and material more economically viable. This project will provide tools that are useful for studying the effect of dynamics on natural metabolic pathways, revealing the evolutionary significance of natural regulatory systems. These tools can be further extended to regulate a variety of complex synthetic systems, such as chemical-controlled, contaminant-cleaning microbes. This project will also provide educational and training opportunities for K-12 students and teachers from local public schools with a high percentage of underrepresented students through a series of activities including "Hot Topic Workshops" and "Lab Research Experience" These educational and training programs will promote students' interests in cutting-edge science and to develop basic skills for research in systems and synthetic biology. Technical Description: Synthetic regulatory systems that provide dynamic control on metabolic pathways (referred to as DMRSs) are very powerful tools in microbial engineering for the production of chemicals from complex metabolic pathways. Previous proof-of-concept studies have demonstrated that DMRSs were able to significantly enhance both pathway productivities and yields. However, due to the complex protein-metabolite/protein-DNA interactions, construction of effective DMRSs is challenging and often needs to test a large number of parameters and regulatory topologies (e.g. upregulation v.s. downregulation, long-range feedback vs. short-range feedback). For DMRSs to work robustly and to be applicable to a broad range of pathways, this project aims to develop a systematic understanding of metabolic dynamics and of how metabolic dynamics affects pathway productivities. Using a combination of experimental and theoretical strategies, including biosensor-enabled ensemble-fluorescence measurement, single cell imaging, chromatographic metabolite quantifications, and kinetic models, this project will systematically characterize metabolic dynamics, metabolic fluctuation, and cell-to-cell metabolic variation under various regulatory topologies.