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

Abstract

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.