Novel Recombinant Endolysin LysMYM94 against Dry-Grind Corn Mash Bioethanol Fermentation Contaminant Limosilactobacillus fermentum.

Bacterial contamination by lactic acid bacteria particularly Limosilactobacillus fermentum poses a significant challenge to commercial bioethanol fermentation facilities, resulting in reduced productivity and yield. Here we characterize a novel bacteriophage endolysin, LysMYM94 isolated from a L. fermentum prophage, that selectively targets lactic acid bacteria contaminants isolated from bioethanol fermentation plants. Recombinant endolysin LysMYM94, a 35.9 kDa endolysin, consists of an N-acetylmuramidase GH25-like enzymatically active domain and a SH3b-like cell-binding domain. The endolysin exhibited lytic activity against 17 strains when tested against a bacterial panel of 24 strains. The enzyme maintained lytic activity over a temperature range of 4℃ to 60℃ with highest retained lytic activity observed at 50℃ for 72 h, with broad lytic activity between pH 3.5 to 8.0 and maximal activity near pH at 6.0, and stable in ethanol concentration up to 10% v/v up to 72 h in 300 mM NaCl. In contaminated corn mash fermentation, LysMYM94 at 1 µM was able to decrease bacterial load by at least a 2.5-log fold change while reducing acetic acid and lactic acid levels. Ethanol production for treated samples was comparable to the level of uncontaminated control. These findings demonstrate that LysMYM94 is an effective alternative to antibiotics for mitigating lactic acid bacteria contamination. The robust antimicrobial properties of this enzyme can prevent stalled fermentation and restore S. cerevisiae ethanol production under industrially relevant fermentation conditions.