P73: Bacteriophage endolysins expressed in yeast kill strains of Lactobacillus that contaminate fermentations

One of the challenges facing the fuel ethanol industry is the management of bacterial contamination during fermentation.  Species of Lactobacillus are the predominant contaminants that reduce ethanol yields and cause “stuck” fermentations, decreasing the profitability of biofuel production with expensive antibiotic treatments and disinfecting costs.  Moreover, ethanol fermentations treated with antibiotics may promote the emergence of drug-resistant bacterial strains, and antibiotic residues in distillers’ dried grains with solubles (DDGS) are a concern for the feed and food industries.  This underscores the need for new and improved mitigation strategies for bacterial contamination.  In this study, bacteriophage lytic enzymes (endolysins) were expressed in Saccharomyces cerevisiae and tested for their ability to kill different Lactobacillus species.  The specific objectives of this work were to (1) express genes encoding bacteriophage endolysins in S. cerevisiae, (2) test the yeast-expressed lytic enzymes for activity against species of Lactobacillus, and (3) apply the enzymes in experimentally infected fermentations.  The streptococcal phage LambdaSA2 endolysin and the Lactobacillus bacteriophage endolysins LysA and LysA2 were previously shown to have strong lytic activity against Lactobacillus when expressed in E.coli.  When expressed in yeast, however, the LambdaSA2 enzyme was insoluble, while LysA and LysA2 were both soluble and active.  The recombinant enzymes killed strains of Lactobacillus that were isolated as contaminants from commercial ethanol facilities.  Yeast-expressed LysA reduced lactobacilli concentrations in experimentally infected fermentations.  Implementing novel and antibiotic free strategies to kill bacteria contaminating fermentations will enable more cost effective fuel ethanol production and will impact producers and consumers across the farm-food-fork continuum.