Engineering a hypoxia-tolerant Saccharomyces cerevisiae for rapid ethanol production via co-utilization of glucose and acetic acid and redox-enhanced flocculation

Abstract

Enhancing the robustness of microbial cell factories is essential for improving both first- and second-generation bioethanol production. During fermentation, Saccharomyces cerevisiae produces acetic acid as a by-product under certain conditions, which inhibits cellular functions and reduces fermentation efficiency. Additionally, pretreatment of lignocellulosic biomass releases acetic acid, further exacerbating fermentation stress toward the yeast. Hypoxic fermentation, combined with metabolic engineering, offers an alternative strategy to mitigate these challenges. To address this, we used CRISPR-Cas9 gene editing to sequentially delete NADH-dependent glycerol-3-phosphate dehydrogenase 1 (GPD1), cytosolic aldehyde dehydrogenase (ALD6), and mitochondrial external NADH dehydrogenase isoforms (NDE1 and NDE2), while integrating an empty plasmid into the LEU2 locus to generate control strains C1 to C5. Notably, strain C5 (GPD1Δ ALD6Δ NDE1Δ NDE2Δ), exhibited a 150% increase in the fermentation rate compared to strain C1 when fermenting a minimal medium containing 10% glucose and 0.4% acetic acid under hypoxic conditions. To further enhance acetic acid utilization and ethanol production, we integrated a plasmid containing acetylating acetaldehyde dehydrogenase from Salmonella enterica (SeEutE) into the LEU2 locus, generating EutE strains E1 to E5. Strain E5 (GPD1Δ ALD6Δ NDE1Δ NDE2Δ [SeEutE]) exhibited a 200% increase in fermentation rate compared to strain C5, with 75% ethanol-induced flocculation. Strain E5 consumed approximately 25% of the supplemented acetic acid and achieved near-theoretical ethanol yields from the total consumed glucose and acetic acid. Furthermore, strain E5 exhibited a 9% improvement in the fermentation rate under hypoxic conditions compared to hyperoxic conditions. These enhancements together represent an overall improvement of more than 343% compared to the parent strain. Thus, by integrating quadruple deletion (GPD1Δ ALD6Δ NDE1Δ NDE2Δ) with the heterologous expression of SeEutE integration, we introduce a novel strategy to construct a hypoxia and acetate tolerant S. cerevisiae strain. This engineered strain achieves rapid, redox-balanced fermentation and ethanol-induced flocculation, offering a significant advance by overcoming limitations in glucose fermentation rate, redox imbalance, and weak acetate tolerance.