Saccharomyces cerevisiae expressing bacteriophage endolysins reduce Lactobacillus contamination during fermentation

Background: One of the challenges facing the fuel ethanol industry is the management of bacterial contamination during fermentation. Lactobacillus species are the predominant contaminants that decrease the profitability of biofuel production by reducing ethanol yields and causing stuck fermentations, which incur additional economic losses via expensive antibiotic treatments and disinfection costs. The current use of antibiotic treatments has led to 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, non-antibiotic, eco-friendly mitigation strategies for bacterial contamination. The specific objectives of this work were to (1) express genes encoding bacteriophage lytic enzymes (endolysins) in Saccharomyces cerevisiae, (2) assess the lytic activity of the yeast-expressed enzymes against different species of Lactobacillus that commonly contaminate fuel ethanol fermentations, and (3) test the ability of yeast expressing lytic enzymes to reduce Lactobacillus fermentum during fermentation. Implementing antibiotic-free strategies to reduce fermentation contaminants will enable more cost-effective fuel ethanol production and will impact both producers and consumers in the farm-to-fork continuum. Results: Two genes encoding the lytic enzymes LysA and LysA2 were individually expressed in S. cerevisiae on multi-copy plasmids under the control of a galactose-inducible promoter. The enzymes purified from yeast were lytic against Lactobacillus isolates collected from fermentors at a commercial dry grind ethanol facility including Lactobacillus fermentum, Lactobacillus brevis, and Lactobacillus mucosae. Reductions of L. fermentum in experimentally infected fermentations with yeast expressing LysA or LysA2 ranged from 0.5 log(10) colony-forming units per mL (CFU/mL) to 1.8 log(10) (CFU/mL) over 72 h and fermentations treated with transformed yeast lysate showed reductions that ranged from 0.9 log(10) (CFU/mL) to 3.3 log(10) (CFU/mL). Likewise, lactic acid and acetic acid levels were reduced in all experimentally infected fermentations containing transformed yeast (harboring endolysin expressing plasmids) relative to the corresponding fermentations with untransformed yeast. Conclusions: This study demonstrates the feasibility of using yeast expressing bacteriophage endolysins to reduce L. fermentum contamination during fuel ethanol fermentations.