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As the effects of climate change become apparent, metabolic engineers and synthetic biologists are exploring sustainable sources for transportation fuels. The design and engineering of microorganisms to produce gasoline, diesel, and jet fuel compounds from renewable feedstocks can significantly reduce our dependence on fossil fuels as well as lower the emissions of greenhouse gases. Over the past 2 decades, a considerable amount of work has led to the development of microbial strains for the production of advanced fuel compounds from both C5 and C6 sugars. In this work, we combined two strategies—adaptive laboratory evolution and rational metabolic engineering—to improve the yeast Saccharomyces cerevisiae’s ability to utilize d-xylose, a major C5 sugar in biomass, and produce the advanced biofuel isobutanol. Whole genome resequencing of several evolved strains followed by reverse …