Recombinant Phage Endolysin LysMP to Control Gram-Positive Bacterial Contaminants in Fuel Ethanol Production

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

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/6/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Background: Antibiotics are commonly utilized to combat microbial contamination in biorefineries used for production of fuel ethanol. Contaminants are predominately lactic acid bacteria (LAB) that are often pervasive in fermentation tanks. These LAB compete for nutrients and produce inhibitory acids that have a negative impact on ethanol-producing yeast, resulting in decreased yields and stuck fermentations. There is increasing interest in developing new antimicrobial alternatives, such as bacteriophage-encoded peptidoglycan hydrolases, called endolysins, to target bacterial contamination. Results: We demonstrate that the endolysin LysMP is an effective method for controlling growth of LAB. The LysMP gene was codon optimized and synthesized based on a prophage sequence discovered in the genome Limosilactobacillus fermentum KGL7. Recombinant enzyme was expressed in E. coli and purified through IMAC for characterization and testing. Optimal activity was in 0.3 to 0.5 M NaCl at pH 6, while maintaining pH stability between pH 4 to 8 and thermostability from 20°C to 40°C, up to 48h. It also retains more than 80% of its activity at 10% ethanol (v/v) for up to 48h. Addition of 1 mM Fe2+ or Ca2+, Mg2+, and Zn2+ at 0.1 mM were shown to enhance lytic activity. Introducing LysMP at 250 µg/mL in model corn mash fermentations effectively mitigated L. fermentum contamination and prevented stuck fermentation. 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. 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). Interestingly, LysMP also demonstrated lytic activity against other non-related Gram-positive bacteria, including Clostridium, Streptococcus, Bacillus, and Listeria spp., suggesting that there could be potential clinical and veterinary applications. Conclusion: LysMP is an effective solution for reducing LAB contamination in biofuel refineries and provides alternative mitigation strategies for supplanting antibiotic usage as a first line defense.