Domain shuffling of endolysin LysKB317 to control Limosilactobacillus fermentum in corn mash fermentation

Authors: Lu, Shao; Wu, Zhengliang; Martinez, Moses; PATEL, MAULIK - Orise Fellow; Skory, Christopher

Submitted to: Biotechnology Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/18/2026
Publication Date: 7/19/2026
Citation: Lu, S.Y., Wu, Z.L., Martinez, M.Y., Patel, M.H., Skory, C.D. 2026. Domain shuffling of endolysin LysKB317 to control Limosilactobacillus fermentum in corn mash fermentation. Biotechnology Reports. https://doi.org/10.1016/j.btre.2026.e00973.
DOI: https://doi.org/10.1016/j.btre.2026.e00973

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: Lactic acid bacteria (LAB), such as Limosilactobacillus fermentum are challenging bacterial contaminants found in bioethanol fermentation facilities. These contaminants can interfere with fermentation and cellular growth of Saccharomyces cerevisiae, resulting in poor fermentation productivity. LAB strains associated with bioethanol refineries have been shown to develop resistance against commonly used antibiotics, such as virginiamycin, which exacerbate contamination control challenges. Recent developments in antibiotic alternatives utilizing recombinant endolysins, such as LysKB317 have been demonstrated to be an effective bacterial mitigation strategy. To further enhance the efficacy of recombinant endolysin, five enzyme variants were developed by duplicating and rearranging the enzymatically active domains (EAD) and cell binding domains (CBD) of LysKB317. These variant constructs were systematically evaluated in comparison to the wild-type LysKB317. A differential scanning calorimeter (DSC) and bacterial lytic assay demonstrated that increasing tandem repeats of EAD, improved the lytic activity of the endolysin but at a cost of reduced thermostability (-0.05% to -13.09%) and thermal transition point of the enzymes compared to the wild type. Irrespective of reduction in thermostability, lytic enzymes with the best and worst lytic rate (LysKB317EADx3-CBD and LysKB317CBD-EAD-CBD respectively) were still successful in treating contamination and preventing stalled fermentation in the corn mash fermentation model when compared to an untreated infection control for 72 h. Our results demonstrated that the domain shuffling strategy can enhance efficiency in generating novel endolysins to combat bacterial contamination.