Global bioethanol production exceeds 110 billion liters annually, yet its expansion remains constrained by the limited range of carbon sources fermentable by Saccharomyces cerevisiae. Glycerol-a major byproduct of biodiesel production-has recently gained attention as both a biomass pretreatment solvent and a fermentable substrate in emerging integrated biorefineries. However, native S. cerevisiae cannot efficiently ferment glycerol, xylose, or acetic acid, and no single engineered strain has previously demonstrated co-fermentation of all these substrates with glucose. In this study, we expanded the metabolic capacity of the previously engineered strain SK-FGG4 (capable of fermenting glycerol and glucose) to enable co-utilization of xylose and acetic acid, generating strain SK2-5. Using CRISPR-based genome editing, we replaced the native ALD6 with a Pichia stipitis xylose assimilation pathway (PsXR, PsXDH), co-expressed with xylulose kinase. Mitochondrial NDE1 and NDE2 were replaced with Salmonella enterica acetylating acetaldehyde dehydrogenase (SeEutE). Overexpression of JEN1 and a mutated ACS1 (L707P) further enhanced acetic acid assimilation, while an additional PsXDH copy improved xylose fermentation efficiency. Under microaerobic conditions, strain SK2-5 achieved over 95% theoretical ethanol conversion efficiency from a mixed substrate of glycerol, xylose, acetic acid, and glucose. To our knowledge, this is the first demonstration of a single S. cerevisiae strain capable of efficiently co-fermenting all four carbon sources. These results establish a flexible metabolic framework for future strain development in glycerol-integrated biorefineries and support coupling with acid-catalyzed glycerolysis and biodiesel-ethanol co-production.

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Journal Article

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Khattab SMR, Katahira M, Watanabe T

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