Saccharomyces cerevisiae surface display of endolysin LysKB317 for control of bacterial contamination in corn ethanol fermentations

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

Submitted to: Frontiers in Bioengineering and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/20/2023
Publication Date: 4/6/2023
Citation: Lu, S.Y., Liu, S., Patel, M., Glenzinski, K.M., Skory, C.D. 2023. Saccharomyces cerevisiae surface display of endolysin LysKB317 for control of bacterial contamination in corn ethanol fermentations. Frontiers in Bioengineering and Biotechnology. 11. Article 1162720. https://doi.org/10.3389/fbioe.2023.1162720.
DOI: https://doi.org/10.3389/fbioe.2023.1162720

Interpretive Summary: Commercial corn-based fuel ethanol facilities rely on yeast to convert agricultural sugars to alcohol, but recurring bacterial contamination can result in expensive plant shutdowns and economic losses. Antibiotics are often used with other chemical-based products to control contamination even though they are often ineffective or are associated with antibiotic resistance concerns. In this research, we developed an effective method to control bacterial contamination with a yeast engineered to produce 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: Control of bacterial contamination in bioethanol fermentation facilities has traditionally relied on chemical-based products such as hop acids and the use of antibiotics. Recent emphasis on antibiotic stewardship has prompted new research into development of alternative approaches to microbial remediation strategies. We recently described a recombinant peptidoglycan hydrolase, endolysin LysKB317, that inhibited Lactobacillus strains in corn mash fermentation. Here, Saccharomyces cerevisiae EBY100 was used to anchor recombinant LysKB317 using cell surface display with the alpha-agglutinin proteins Aga1p-Aga2p. Immunostaining and confocal fluorescence were used for localization of the extracellular interface of the cells. Yeast surface expressed endolysin demonstrated an 83.8% decrease in bacterial cell counts compared to 9.5% decrease in control yeast. Recombinant S. cerevisiae expressing LysKB317 used for small-scale corn mash fermentation, when infected with Limosilactobacillus fermentum, showed the ability to proactively control bacterial infection for 72 hours with at least 1-log fold reduction. HPLC analysis of fermentation products showed improved ethanol concentration from 3.4% to at least 5.9% compared to infection-only control and reduced levels of lactic and acetic acid from 34.7 mM to 13.8 mM and 25.5 mM to 18.1 mM in that order. In an optimized yeast surface display system, proactive treatment of bacterial contaminants by endolysin LysKB317 can improve fermentation efficiency in the presence of L. fermentum contamination.