Strategic combination of different promoters in lactose metabolisation and host chassis selection for high bioethanol titres from dairy wastes

The yeast Saccharomyces cerevisiae is widely used in biotechnological processes but is unable to metabolise lactose, the main component of cheese whey. Here, we engineered industrial S. cerevisiae strains for bioethanol fermentation from lactose. Heterologous expression of LAC12 and LAC4 from Kluyveromyces lactis , encoding for a lactose permease and a β-galactosidase, respectively, under different promoter combinations ( TEF1p / PGK1p ), generated the strains E1 ( TEF1p → LAC4 / PGK1p → LAC12 ) and E2 ( PGK1p → LAC4 / TEF1p → LAC12 ). In synthetic media, E1 exhibits faster lactose consumption up to 150 g/L of lactose, while E2 exhibits improved lactose metabolisation ability at 200 g/L of lactose with a yield near 100%. These results were validated in whey where the strain E2 produced 92.2 g/L of ethanol from 200 g/L lactose, the highest titre reported in the literature from whey. This study provides valuable insight on how the promoter choice influences multi-enzymatic step pathways and the importance of selecting an appropriate host for high-titre processes. • LAC4 and LAC12 genes were expressed under combinations of PGK1/TEF1 promoters. • TEF1p induces higher β-galactosidase activity than PGK1p in the presence of lactose. • The different expression balance between the two genes led to different outcomes. • 92.2 g/L ethanol was attained from cheese whey powder with one of the combinations.