Novel Endolysin LysMP for Control of Limosilactobacillus and Lactobacillus Bacterial Contamination in Small-Scale Corn Mash Fermentation

Authors: PATEL, MAULIK - Orise Fellow; Lu, Shao; Liu, Siqing; Skory, Christopher - Chris

Submitted to: Biotechnology for Biofuels and Bioproducts
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/18/2023
Publication Date: 9/29/2023
Citation: Patel, M.H., Lu, S.-Y., Liu, S., Skory, C.D. 2023. Novel endolysin LysMP for control of Limosilactobacillus fermentum contamination in small-scale corn mash fermentation. Biotechnology for Biofuels and Bioproducts. 16(1):144. https://doi.org/10.1186/s13068-023-02400-5.
DOI: https://doi.org/10.1186/s13068-023-02400-5

Interpretive Summary: Recurring bacterial contamination in commercial corn-based fuel ethanol facilities can result in expensive plant shutdowns and economic losses. Chemical-based products and antibiotics are often used to control contamination but are often ineffective or are associated concerns on antibiotic resistance. In this research, we developed an effective method to control bacterial contamination with an enzyme, called endolysin, that targets and kills bacteria commonly associated with contamination at fuel ethanol fermentation facilities. This research is important for generating innovative non-antibiotic methods to combat drug-resistant bacterial contamination and to reduce antibiotics usage in fuel ethanol industries. It can restore ethanol production efficiency and reduce production cost associated with bacterial contamination.

Technical Abstract: Traditional bioethanol fermentation industries are not operated under strict sterile conditions and are prone to microbial contamination. Lactic acid bacteria (LAB) are often pervasive in fermentation tanks, competing for nutrients and producing inhibitory acids that have a negative impact on ethanol-producing yeast, resulting in decreased yields and stuck fermentations. Antibiotics are frequently used to combat contamination, but antibiotic stewardship has resulted in a shift to alternative antimicrobials. We demonstrate that endolysin LysMP, a bacteriophage-encoded peptidoglycan hydrolase, is an effective method for controlling growth of LAB. The LysMP gene was synthesized based on the prophage sequence in the genome of Limosilactobacillus fermentum KGL7. Analysis of the recombinant enzyme expressed in E. coli and purified by immobilized metal chelate affinity chromatography (IMAC) showed an optimal activity at pH 6, with stability from pH 4 to pH 8 and from 20°C to 40°C up to 48h. Moreover, it retains more than 80% of its activity at 10% ethanol (v/v) for up to 48h. LysMP can lysis various LAB species. When LysMP was added at 250 µg/mL to yeast corn mash fermentations containing L. fermentum, it reduced bacterial load by at least 4-log fold compared to the untreated controls and prevented stuck fermentation. In comparison, untreated controls with contamination increased from an initial bacterial load of 1.50 x 107 CFU/mL to 2.25 x 109 CFU/mL and 1.89 x 109 CFU/mL after 24h and 48h, respectively. Glucose in the treated samples was fully utilized, while untreated controls with contamination had more than 4%(w/v) remaining at 48h. Furthermore, there was at least a 5-fold reduction in lactic acid (0.085 M untreated contamination controls compared to 0.016 M treated), and a 4-fold reduction in acetic acid (0.027 M untreated contamination controls vs. 0.007 M treated), when LysMP was used to treat contaminated corn mash fermentations. Most importantly, final ethanol yields increased from 6.3%(w/v) contamination controls to 9.3%(w/v) treated, an approximate 50% increase and were comparable to uncontaminated controls 9.3%(w/v). LysMP could be a good alternative to replace antibiotics and to mitigate LAB contamination in biofuel refineries.