Transcriptomic and Ultrastructural Insights into Ethanol Hyperproduction and Flocculation in Engineered <i>Saccharomyces cerevisiae</i> under Glucose–Acetic Acid Co-Fermentation

Acetic acid stress and hypoxia are major constraints in lignocellulosic ethanol production by Saccharomyces cerevisiae. A previously engineered strain (E5) carrying sequential deletions of GPD1, ALD6, NDE1, and NDE2, together with heterologous expression of the acetylating acetaldehyde dehydrogenase SeEutE from Salmonella enterica, exhibited markedly enhanced ethanol productivity, hypoxia tolerance, and ethanol-dependent flocculation during glucose–acetic acid co-fermentation. To explore the molecular and structural basis of these traits, comparative transcriptomic and ultrastructural analyses were conducted for strain E5, its intermediate predecessor E4, and the parental strain C1. Strain E5 displayed broad transcriptional remodeling characterized by reduced expression of respiratory and amino-acid biosynthetic pathways and increased expression of glycolytic, membrane-associated, and cell-wall-related genes. Transmission electron microscopy revealed cell enlargement, vacuolar expansion, lipid droplet formation, and pronounced restructuring of the cell envelope. Transcriptomic profiling further revealed elevated expression of subtelomeric gene families, including FLO, PAU, and DAN, associated with flocculation and hypoxic adaptation. This study provides a systems-level analysis integrating transcriptomic and ultrastructural observations to examine how redox pathway engineering is associated with coordinated metabolic, membrane, and cell-wall remodeling supporting ethanol hyperproduction and tolerance to acetic acid and hypoxic stress, offering insights for future metabolic engineering strategies.