Engineered reversal of the β-oxidation cycle in clostridia for the synthesis of fuels and chemicals

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 with limited sets of platform organisms, accessible feedstock and target molecules, and stable operating environments in which to work. Third, a focus on linear heterologous pathways limits co-development of multiple products and integrated design. With support from the Department of Energy, we will address these challenges in a new interdisciplinary venture to provide tools and engineering strategies that enable high-level synthesis of next-generation biofuels and bioproducts from lignocellulosic biomass in clostridia. Specifically, we aim to develop a new technology platform for engineering reversal of the β-oxidation cycle in clostridia for the synthesis of fuels and chemicals. Products include medium-chain alcohols (e.g., decanol), medium chain diols (e.g., butanediol or hexanediol), and medium-chain dicarboxylic acids (e.g., adipic acid). 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, omics measurements, systems-biology analyses, and cell-free technologies to expand the set of platform organisms that meet DOE bioenergy goals. Our work is expected to be transformative in several fields, as we will (i) reconceive how we engineer complex biological systems by linking pathway design, prospecting, and validation in a new integrated framework, (ii) develop a new technology platform for engineering reversal of the β-oxidation cycle in clostridia for the synthesis of fuels and chemicals, and (iii) open new paths for synthesis of next-generation biofuels and bioproducts from lignocellulosic biomass. Looking forward, the project will expand the scope of biomanufacturing practice and become a key driver of global innovation and economic growth.