CAREER: Engineering Non-Growth Metabolism for High-Yield Biochemical Production

[1452549]微生物是一种很有前途的催化剂，可以将糖和非食物生物质等可再生资源转化为对我们社会至关重要的燃料和化学品。该方案解决了实现微生物催化剂的一个关键挑战，即提高微生物催化剂生产燃料或化学品的速率和效率。该建议将通过研究限制催化剂生产力的潜在酶调节，大大改善微生物催化剂。虽然许多酶的调控已经在分离中进行了研究，但酶的系统水平，条件依赖性调控已被证明难以阐明，但这种理解对于设计高生产率微生物催化剂至关重要。如果成功，该提案将通过降低柴油、航空燃料和汽油的广泛替代产品的生产成本，以及用于制造塑料、防腐剂、香精和香料以及许多其他消费品的化学品的生产成本，影响美国生物制造业的竞争力。拟议的工作还将通过研究、全球健康与生物技术新课程和可持续发展与全球健康证书课程来培养本科生、硕士和博士水平的学生。该证书将为未来的生物工程师提供对生物技术、具有挑战性的社会问题以及市场分析和风险工具的综合理解。该证书将在目前的试点提案中作为master？年代的程序。该项目将带来更大的STEM教育基础设施，并促进低收入国家未被充分代表的少数民族与STEM学员的互动。这将通过增加科学活动和合作、对具体国家社会挑战的技术经济分析，以及通过培养具有全球思维的工程师，使我们的全球合作伙伴受益。所提出的工作的基本原理是在没有细胞生长的情况下实现高通量代谢，以实现高效的非生长相关生物制造过程。通过优化底物向产品的转化，而不牺牲细胞生长所需的底物消耗，具有快速、非生长产物代谢的发育细胞将消除繁荣生物制造经济的主要障碍。然而，许多生化产物是生长偶联的，一般来说，非生长细胞的代谢率很低。本提案的总体目标是确定在非生长条件下抑制糖酵解代谢的变构调节和翻译后修饰（酶水平调节）。中心假设是酶水平调控主导了固定期的代谢下调。提出的工作将启动两种新的方法来工程化和表征代谢调控，并发展对非生长中心碳代谢调控的系统级理解。该提议将在蛋白质组水平上产生最小的细胞，从而避免基因缺失的问题。该提案还将开发一种快速、有针对性地降解蛋白质的方法，通过降低副产物酶来设计生物转化，使产量接近最佳。该方法将使新的生物学研究成为可能，因为它将有助于制造条件突变体以新的方式扰乱生物系统。第二种方法将根据热力学确定限速酶，从而将工程努力集中在特定的酶上。开发的工作流程将确定反应是接近平衡（非限速）还是远离平衡（限速），适用于广泛的工业相关条件。新的工作流程将用于研究中心碳代谢调节，以获得对有机酸和萜烯生产调节的系统级视角。该职业奖由CBET部门的生物技术和生化工程项目颁发，由分子和细胞生物学部门的系统和合成生物学项目共同资助。

1452549 Tyo, Keith E. Microbes are a promising catalyst to convert renewable resources such as sugars and non-food biomass into fuels and chemicals that are essential to our society. This proposal addresses a key challenge to realizing microbial catalysts, namely increasing the rate and efficiency that a microbial catalyst can produce the fuel or chemical. This proposal will substantially improve microbial catalysts by investigating the underlying enzyme regulation that limits catalyst productivity. While the regulation of many enzymes has been studied in isolation, the systems-level, condition-dependent regulation of enzymes has proved difficult to elucidate, but this understanding will be essential to engineering high productivity microbial catalysts. If successful, this proposal would impact the biomanufacturing competitiveness of the United States by reducing production costs of a wide range of drop-in replacements for diesel, jet fuel, and gasoline, as well as chemicals used to make plastics, preservatives, flavors and fragrances, and many other consumer products. The proposed work will also train students at the undergrad, master and doctoral levels through research, a new course in Global Health and Biotechnology and a certificate program in Sustainability and Global Health. The certificate will provide future biological engineers with an integrated understanding of biotechnology, challenging societal problems, and tools for market analysis and risk. The certificate will be piloted in the current proposal as a master?s program. This program will result in greater STEM educational infrastructure and promote interaction of under-represented minorities in low-income countries with STEM trainees. This will benefit our global partners through increased scientific activity and collaboration, technoeconomic analysis of country-specific societal challenges, as well as our society by training globally minded engineers.The rationale for the proposed work is to enable high flux metabolism in the absence of cell growth for highly productive non-growth-associated biomanufacturing processes. Developing cells with fast, non-growth product metabolism would remove a major barrier to a thriving biomanufacturing economy, by optimizing substrate conversion to product without sacrificing substrate consumption for cell growth. However, many biochemical products are growth-coupled, and in general non-growing cells have low metabolic rates. The overall objective of this proposal is to identify allosteric regulation and post-translational modification (enzyme-level regulation) that represses glycolytic metabolism in non-growth conditions. The central hypothesis is that enzyme-level regulation dominates metabolic downregulation in stationary phase. The proposed work will launch two new methods for engineering and characterizing metabolic regulation and developing a systems-level understanding of non-growth central carbon metabolic regulation. The proposal will generate minimal cells at the proteomic-level, circumventing problems with genetic deletions. The proposal will also develop a method for rapid, targeted degradation of proteins to engineer bioconversions with near optimal yields by knocking down byproduct enzymes. The method will enable new biological studies, as it will be useful for making conditional mutants to perturb biological systems in new ways. The second method will identify rate-limiting enzymes, based on thermodynamics, and thus focus engineering efforts on specific enzymes. The developed workflow will determine if reactions are near equilibrium (non-rate-limiting) or away from equilibrium (rate limiting) for a broad range of industrially relevant conditions. The novel workflow will be deployed to study central carbon metabolic regulation to garner a systems-level perspective on regulation of organic acid and terpene production.This CAREER award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.