Novel recombinant endolysin LysMYM94 against dry-grind corn mash bioethanol fermentation contaminant Limosilactobacillus fermentum

Authors: Lu, Shao; Wu, Zhengliang; Skory, Christopher

Submitted to: Probiotics and Antimicrobial Proteins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/15/2026
Publication Date: 6/27/2026
Citation: Lu, S.Y., Wu, Z.L., Skory, C.D. 2026. Novel recombinant endolysin LysMYM94 against dry-grind corn mash bioethanol fermentation contaminant Limosilactobacillus fermentum. Probiotics and Antimicrobial Proteins. https://doi.org/10.1007/s12602-026-11112-9.
DOI: https://doi.org/10.1007/s12602-026-11112-9

Interpretive Summary: The United States is the world leading bioethanol producer, generating approximately 16.2 billion gallons in 2024–about half of the global supply. Bioethanol is critical to U.S. farmers by creating a substantial consistent market for crops like corn, sugar beet and sugarcane. However, a persistent challenge for ethanol producers has been bacterial contamination, which significantly slows down production and can even lead to failed fermentation batches. To combat these bacteria, chemical-based products and antibiotics are commonly used. Unfortunately, these methods are often ineffective and may contribute to the growing problem of antibiotic resistance in bacteria. In this research, ARS scientists developed an antimicrobial enzyme that specifically targets and destroys the troublesome bacteria that plague ethanol production. This innovative approach efficiently controls bacterial contamination, thereby allowing optimal ethanol production and ensuring reduced production costs. This discovery will further strengthen the U.S. ethanol industry, which processes roughly 5.5 billion bushels of corn, adds over $50 billion to the economy, supports over 300,000 jobs and provides stable energy security.

Technical Abstract: 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 target LAB contaminant isolated from bioethanol fermentation plants. Recombinant endolysin LysMYM94 of 35.9 kDa 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’s lytic activity ranges were determined between 4' to 60' with optimal for 72h at 50', pH 3.5 to pH 8 with optimal pH at 6 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 bacteria load by at least 2.5-log fold change while reducing acetic acid and lactic acid. Ethanol production for treated samples was comparable to level of uncontaminated control. These findings demonstrate that LysMYM94 is an effective antibiotic alternative capable of mitigating lactic acid bacteria contamination. The robust antimicrobial properties of this enzyme can prevent stalled fermentation and restore S. cerevisiae ethanol production under industrial relevant fermentation conditions.