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

Authors: Lu, Shao; PATEL, MAULIK - Orise Fellow; Hector, Ronald; Bowman, Michael; Skory, Christopher

Submitted to: Biofuel Research Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/15/2024
Publication Date: 12/1/2024
Citation: Lu, S.Y., Patel, M., Hector, R.E., Bowman, M.J., Skory, C.D. 2024. Saccharomyces cerevisiae secretion of recombinant bacteriophage endolysin LysKB317 inhibits Limosilactobacillus fermentum in corn mash fermentation. Biofuel Research Journal. https://doi.org/10.18331/BRJ2024.11.4.4.
DOI: https://doi.org/10.18331/BRJ2024.11.4.4

Interpretive Summary: Commercial corn-based fuel ethanol facilities rely on yeast to convert agricultural sugars to alcohol but recurring bacterial contamination from the environment and raw materials can inhibit yeast growth and severely impact ethanol productivity. This results in expensive plant shutdowns and economic losses that can cost millions of dollars to a single facility. Antibiotics are often combined with other chemical-based products to help control contamination even though they are often ineffective or are associated with antibiotic resistance concerns. In this research, ARS scientists developed an effective method to control bacterial contamination with a yeast engineered to produce an enzyme, called endolysin, that targets and kills bacteria commonly associated with contamination at biofuel ethanol fermentation facilities. Engineered yeast were able to control introduced bacterial contaminants and fully restore ethanol production in corn mash fermentations. This research demonstrates the effectiveness of this innovative non-antibiotic method to combat drug-resistant bacterial contamination and to reduce antibiotics usage in fuel ethanol industries.

Technical 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 a-MF secretion signal and a N-terminus poly-histidine tag. LysKB317 was detectable by western blot analysis, which showed a MW slightly larger than the 33 kDa native protein, presumably due to S. cerevisiae glycosylation or cleavage amino acids residual from a-MF secretion signal peptide. 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-hour 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.