S1: Engineering fermentative yeasts to express bactericidal enzymes to inhibit lactobacilli contamination in fuel ethanol fermentations

Lactic acid bacteria (LAB) are a pervasive contaminant in any ethanol fermentation system. In fuel ethanol fermentation, Lactobacillus species have been linked to a decrease in fermentation efficiency and ‘stuck’ fermentations. Bacteriophage derived peptidoglycan hydrolases (endolysins) can serve as biopesticides and kill problematic bacteria. We predict that by engineering a Saccharomyces cerevisiae yeast strain to express/secrete endolysins during the fermentation process we can inhibit lactobacilli contamination. We have synthesized and purified multiple recombinant endolysins. The endolysins have shown bactericidal activity towards highly problematic strains of L. fermentum, L. gasseri, L. brevis and L. casei in zymogram analysis and turbidity reduction assays. These endolysins achieved optimal specific activities under conditions common in fuel ethanol fermentations (pH 5.5 and up to 5% ethanol). Native endolysin domains are modular, typically consisting of one or two lytic domains and a single cell wall binding domain. In the belief that few bacteria can resist three unique bactericidal activities, we have constructed fusion enzymes harboring three different acting lytic domains coupled to a cell wall binding domain. This triple-acting fusion enzyme provides a single molecule that we predict will be expressed by an engineered yeast strain, and will be both highly bactericidal and refractory to bacterial resistance development. S. cerevisiae expressing triple-acting fusion enzymes are expected to increase ethanol yields while reducing ethanol production losses and facility down-time for cleaning.