Author Summary The yeast Saccharomyces cerevisiae is being genetically engineered to produce renewable biofuels from sustainable plant material. Efficient biofuel production from plant material requires conversion of the complex suite of sugars found in plant material, including the five-carbon sugar xylose. Because it does not efficiently metabolize xylose, S. cerevisiae has been engineered with a minimal set of genes that should overcome this problem; however, additional genetic changes are required for optimal fermentative conversion of xylose into biofuel. Despite extensive knowledge of the regulatory networks controlling glucose metabolism, less is known about the regulation of xylose metabolism and how to rewire these networks for effective biofuel production. Here we report genetic mutations that enabled the conversion of xylose into bioethanol by a previously ineffective yeast strain. By comparing altered protein and metabolite abundance within yeast cells containing these mutations, we determined that the mutations synergistically alter metabolic pathways to improve the rate of xylose conversion. One change in a gene with well-characterized aerobic mitochondrial functions was found to play an unexpected role in anaerobic conversion of xylose into ethanol. The results of this work will allow others to rapidly generate yeast strains for the conversion of xylose into biofuels and other products.
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