Saccharomyces cerevisiae secretion of recombinant bacteriophage endolysin LysKB317 inhibits Limosilactobacillus fermentum in corn mash fermentation

Abstract

This study investigated the secretion of endolysin LysKB317 integrated into the HO locus of Saccharomyces cerevisiae strain NRRL Y-2034 to enable the yeast to simultaneously perform ethanol fermentation and control bacterial contaminants frequently present in ethanol refineries. The cell wall hydrolase gene was expressed using TEF1 and NAT5 promoter and terminator sequences with α-MF secretion signal and an N-terminus poly-histidine tag. LysKB317 was detectable by western blot analysis, which showed a molecular weight slightly larger than the 33 kDa native protein, presumably due to residual amino acids from the α-MF secretion signal peptide or S. cerevisiae glycosylation. Secreted LysKB317 was confirmed to be active using turbidity reduction and cell viability assay. Contaminated corn mash fermentations with yeast secreting LysKB317 demonstrated a significant reduction in bacterial contamination by at least 2-log compared to the contamination controls without LysKB317 expression. Moreover, LysKB317 expression led to a 73% decrease in acetic acid concentration and a 67% decrease in lactic acid levels. Contaminated fermentations with yeast expressing LysKB317 also exhibited a 16% improvement in ethanol production over the contamination controls without LysKB317, with no significant difference observed when compared to yeast-only controls during a 72-h corn mash fermentation. These findings suggest that a yeast endolysin secretion platform holds promise for mitigating bacterial contamination in biorefineries and potentially reducing reliance on antibiotics usage.