Broadbelt & Tyo: Establishing a Clostridia foundry for biosystems design by integrating computational modeling, systems-level analyses, and cell-free engineering technologies

Rapid population growth, a rise in global living standards, economic competitiveness, and climate change concerns have intensified the need for sustainable, low-cost production of biofuels and bioproducts. Industrial biotechnology using microbial cell factories is one of the most attractive approaches for addressing this need, particularly when large-scale chemical synthesis is untenable. Unfortunately, designing, building, and optimizing biosynthetic pathways in cells remains a complex and formidable challenge for many reasons. First, Design-Build-Test (DBT) cycles for optimizing a given biosynthetic pathway can take on the order of weeks to months, resulting in hundreds of person-years of research and development time to bring a new bioproduct to market. Second, most high-throughput platforms for testing engineered organisms focus on Escherichia coli or yeast, which presents researchers a limited set of platform organisms, accessible feedstock and target molecules, and stable operating environments in which to work. Third, a lack of computational tools for pathway prediction and large-scale systems biology data analysis limits integrated design and forward engineering. To address the complex issues above, new tools and engineering approaches are needed to derive interdisciplinary paradigms for biosystems design. Working both in vitro (cell-free) and in vivo, the goal of this proposal is to interweave and advance state-of-the-art computational modeling, genome editing, systems-biology analyses, and cell-free technologies to expand the breadth of platform organisms that meet DOE bioenergy goals. Specifically, we aim to establish the Clostridium Foundry for Biosystems Design (cBioFAB) to enable high-level synthesis of next-generation biofuels and bioproducts from lignocellulosic biomass. cBioFAB will re-conceptualize the way we engineer complex biological systems by linking pathway design, prospecting, validation, and production in an integrated framework that relies on computational modeling, cell-free technologies, and system-level omics data. Our framework will enable end-to-end process monitoring, enhance learning, and accelerate troubleshooting. A key innovation is the use of high-throughput cell-free systems to accelerate design testing by more than an order of magnitude beyond what is feasible today. This unprecedented capability will come from the ability to bypass transformation idiosyncrasies and low-throughput platforms for testing genetic designs as a result of slow anaerobic growth that currently plague engineering efforts in clostridia. When successful, the end goal of this project will be a new integrated framework for biosystems design in clostridia, as well as generalized workflows, processes, and technologies that enable transformative projects across numerous chassis organisms. This outcome will be exemplified through the ability to model, design, and predictably engineer industrially deployed clostridia strains to manufacture a variety of fuels (e.g., butanol) and building block chemicals (e.g., 1,3-butanediol) via existing and de novo pathways at significantly reduced time to market.