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

Lactic acid bacteria (LAB), such as Limosilactobacillus fermentum are persistent contaminants in bioethanol fermentation facilities and inhibit Saccharomyces cerevisiae, reducing ethanol yields. Rising resistance to antibiotics such as virginiamycin makes contamination control increasingly difficult. Recombinant endolysins such as LysKB317 provide a promising alternative. To improve LysKB317 performance, five variants were engineered by duplicating and rearranging its enzymatically active (EAD) and cell binding (CBD) domains. These constructs were compared to the wild-type endolysin. Differential scanning calorimetry (DSC) and bacterial lytic assays demonstrated that domain shuffling altered bacteriolytic activity and thermostability. The wild-type LysKB317 exhibited the highest initial lytic activity (1.16 × 10^-4 OD/s‧µM), whereas LysKB317_EADx3-CBD displayed comparable bacteriolytic kinetics (7.99 × 10^-5 OD/s‧µM) and LysKB317_CBD-EAD-CBD (ΔCp = 24.66 kJ/mol‧K) exhibited the greatest thermostability. Despite reduced activity, both variants effectively control LAB contaminants and prevented stalled corn mash fermentation. Domain shuffling demonstrated effective bacterial control strategy for generating novel endolysins for biocontrol.