Glycerol is an eco-friendly solvent enhancing plant-biomass decomposition through the glycell process to bio-based chemicals. Nonetheless, the lack of efficient conversion of glycerol by natural Saccharomyces cerevisiae restrains many biorefineries-scenarios. Here, we outline a comprehensive strategy for generating efficient glycerol fermenting S. cerevisiae via rewriting the oxidation of cytosolic nicotinamide adenine dinucleotide by O2-dependent dynamic shuttle while abolishing glycerol

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... orylation and biosynthesis pathways. By following a vigorous glycerol oxidative pathway, our engineered strain demonstrated a breakthrough in conversion efficiency (CE), reaching up to 0.49g-ethanol/g-glycerol-98% of theoretical conversion-with production rate >1 gL-1h-1 on rich-medium. Interestingly, the glycerol consumption and its fermentation unrepressed during the mixing by glucose until the strain produced >86 g/L of bioethanol with 92.8% of CE. Moreover, fine-tuning of O2 boosted the production rate to >2 gL-1h-1 with 82% of CE. Impressively, the strategy flipped the ancestral yeast even from non-growing on glycerol, on the minimal medium, to a fermenting strain with productivities 0.25-0.5 gL-1h-1 and 84-78% of CE, respectively. Our findings promote utlising glycerol efficiently in several eco-friendly biorefinery approaches.