Recombinant bacteriophage LysKB317 endolysin mitigates Lactobacillus infection of corn mash fermentations

Authors: Lu, Shao; Bischoff, Kenneth; Rich, Joseph; Liu, Siqing; Skory, Christopher - Chris

Submitted to: Biotechnology for Biofuels and Bioproducts
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/27/2020
Publication Date: 9/8/2020
Citation: Lu, S.Y., Bischoff, K.M., Rich, J.O., Liu, S., Skory, C.D. 2020. Recombinant bacteriophage LysKB317 endolysin mitigates Lactobacillus infection of corn mash fermentations. Biotechnology for Biofuels. 13. Article 157. https://doi.org/10.1186/s13068-020-01795-9.
DOI: https://doi.org/10.1186/s13068-020-01795-9

Interpretive Summary: Microbial contamination in commercial corn-based fuel ethanol fermentation facilities is a common problem that results in decreased ethanol production efficiency and loss of profit. A number of antimicrobial agents, including antibiotics, are used to control this contamination, but they are often ineffective or are associated with antibiotic resistance concerns. In this research, we developed an effective method to control bacterial contamination without the use of antibiotics. An enzyme, called endolysin, found in a virus that infects bacteria was shown to kill several bacteria species commonly found in commercial fuel ethanol fermentation facilities. We further demonstrated that this enzyme is tolerant of the environmental conditions frequently found in these facilities. This research is important in generating new ways to combat bacterial contamination in fuel ethanol industries and to restore ethanol production through none antibiotic approach. It also provides researchers new tools and methods to kill bacteria that are drug-resistant.

Technical Abstract: Commercial ethanol fermentation facilities traditionally rely on antibiotics for bacterial contamination control. Here we demonstrate an alternative approach to treat contamination using a novel peptidoglycan hydrolase (LysKB317) isolated from a bacteriophage, EcoSau. This endolysin was specially selected against Lactobacillus strains that were isolated as contaminants from a fuel ethanol plant. The LysKB317 gene was recombinantly expressed in Escherichia coli as a 33 kDa purified enzyme. In turbidity reduction assays, the recombinant enzyme was subjected to a panel of 32 bacterial strains and was active against 28 bacterial strains representing 1 species of Acetobacter, 8 species of Lactobacillus, 1 species of Pediococcus, 3 species of Streptococcus, and 1 species of Weissella. The activity of LysKB317 was optimal around pH 6, but it has broad activity and stability from pH 4.5 – 7.5 up to at least 48 h. Maximum activity was observed at 50°C up to at least 72 h. In addition, LysKB317 was stable in 30% ethanol up to at least 72 h. In experimentally infected corn mash fermentations, 1 µM endolysin reduced bacterial load by 4-log fold change, while 0.01 µM reduced bacteria by 2-log fold change. Concentration of fermentation products (ethanol, residual glucose, lactic acid, and acetic acids) for infected cultures treated with = 0.01 µM LysKB317 were similar to uncontaminated controls. These data demonstrated that exogenously added LysKB317 endolysin is functional in conditions typically found in fuel ethanol fermentations tanks and may be developed as an alternative to antibiotics for contamination control during fuel ethanol fermentations.