Bacteriocin-producing Saccharomyces cerevisiae inhibits bacterial contamination in raw starch-to-ethanol fermentations

• Bacterial contaminants in raw starch fermentations reduced ethanol yields. • Lactic acid bacteria (LAB) are the main contaminants in raw sorghum fermentations. • An amylolytic S. cerevisiae strain was engineered to co-produce mundticin • The mundticin-producing strain inhibited > 96% of LAB contaminants. • Fermentations with mundticin-producing yeast increased ethanol yields by > 5% Contaminating lactic acid bacteria (LAB) compete with yeast cells for nutrients during starch-to-ethanol fermentations, affecting yeast cell viability and ethanol yields, with significant economic implications. Antibiotics are typically used to control LAB contaminants in the bioethanol industry; however, the global rise in antibiotic resistance necessitates the use of alternative antimicrobials, such as recombinant bacteriocins. In this study, 31 LAB were isolated from red sorghum flour fermentations, including Enterococcus (80.65%), Pediococcus (16.13%) and Lactococcus (3.22%) species, with varying sensitivities to the bacteriocin, mundticin ST4SA. To counteract LAB during starch fermentation, an industrial amylolytic S. cerevisiae ER T1 strain (derived from Ethanol Red TM ) that produces an α-amylase and glucoamylase, was engineered to co-express a recombinant mundticin ST4SA. The S. cerevisiae T1M18 strain exhibited the greatest antimicrobial activity, inhibiting the growth of all the LAB isolates, except Lactococcus taiwanensis SI24. When inoculated into a sterile red sorghum fermentation spiked with isolate Pediococcus acidilactici SI17, the T1M18 strain reduced bacterial numbers by 99.99% and yielded 13% more ethanol than the ER T1 control strain. In non-sterile fermentations, T1M18 increased the ethanol concentration by 4.27 g/L relative to ER T1. Both fermentations were conducted without antibiotics, highlighting the potential of the recombinant bacteriocin to reduce antibiotic use in the bioethanol industry. This study demonstrated that S. cerevisiae co-expressing recombinant amylases and bacteriocins can simultaneously hydrolyse raw starch and inhibit the growth of LAB contaminants, thereby increasing ethanol yields in consolidated bioprocessing of starch.