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

Northwestern University, Evanston IL

Investigators

Abstract

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.