Genome Engineering a Platform Approach for Biogasoline and Coproducts

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1067730GillIntellectual MeritThere is significant need for fundamental research on next-generation hydrocarbon biofuels that are compatible with the existing fuels infrastructure and meet future greenhouse gas policy requirements. The platform for the proposed research uses engineered microorganisms to produce isopentenol from renewable feedstocks. The isopentenol can then be converted to iso-octane or other co-products using inorganic catalysts. The sustainable microbial production of isopentenol requires a bioprocess that operates at stoichiometric yields, fast rates, and high product concentrations. However, isopentenol flux in current engineered organisms is low, and isopentanol inhibits growth and metabolism at concentrations well below what is required for commercialization.The engineering of complex traits through the simultaneous and specific manipulation of dozens of genes across multiple pathways has remained an area of intense effort at the forefront of metabolic engineering. The size of mutational space encoded by even the smallest microbial genomes is immense. How nature manages to effectively access this space is not completely understood, and prior efforts in this area have not previously been extended far beyond the level of individual genes or pathways. The proposed research will address the challenge of engineering complex traits through the use of a new genome engineering toolkit. Specifically, the genome engineering toolkit uses a new approach for genome engineering that combines advanced DNA synthesis, recombineering, and genomics to potentially improve upon traditional approaches by several orders of magnitude. The objectives of the proposed research are to: 1) increase flux to isopentenol using rational genome engineering of E. coli strains, 2) map isopentenol tolerance mutations at the genome-scale, and, 3) iteratively optimize performance via combinatorial genome engineering. Broader ImpactsThe basic genome-engineering approach developed for this proposed research has potential for widespread application across all of areas of biotechnology, where engineering of complex phenotypes is an important determinant in cost and life cycle analyses. The educational activities focus on a comprehensive plan for involvement of undergraduate students in biofuels research through the Colorado Center for Biorefining and Biofuels (C2B2) and CU-NREL Renewable and Sustainable Energy Institute (RASEI).