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

Abstract

Control of bacterial contamination in bioethanol fermentation facilities has traditionally relied on chemical-based products such as hop acids and use of antibiotics. Recent emphasis on antibiotic stewardship has prompted new research into the development of alternative approaches to microbial remediation strategies. We recently described a recombinant peptidoglycan hydrolase, endolysin LysKB317, which inhibited Limosilactobacillus fermentum strains in corn mash fermentation. Here, Saccharomyces cerevisiae EBY100 was used to anchor recombinant LysKB317 using cell surface display with the a-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 a 9.5% decrease in control yeast. Recombinant S. cerevisiae expressing LysKB317 used for small-scale corn mash fermentation, when infected with L. fermentum, could proactively control bacterial infection for 72 h with at least 1-log fold reduction. Analysis of fermentation products showed improved ethanol concentrations from 3.4% to at least 5.9% compared to the 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, respectively. 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.

Novel endolysin LysMP for control of Limosilactobacillus fermentum contamination in small-scale corn mash fermentation

“…Research on endolysins has mostly been focused on human and animal health [ 26 , 27 ], but the use of endolysins to prevent bacterial contamination with fuel ethanol production has been shown to be an effective strategy [ 1 , 16 ]. Furthermore, yeast cell surface display of endolysin [ 39 ] and direct yeast secretion of endolysin [ 40 ] have the potential to improve control of contaminants.…”

“…Divalent metal cations have been shown to improve lytic activity of endolysins such as Listeria targeting endolysin HPL118 and Bacillus targeting endolysin LysB4 [ 47 ]. The LysMP activity in the presence of divalent cations were examined based on metal ion-binding site prediction (MIB2; [ 39 ]) and protein–ligand binding site prediction ( COACH; [ 48 ]). The structural prediction analyses have revealed the potential presence of a zinc finger domain, with various divalent metal ion binding sites distributed across the endolysin (Additional file 1 : Figs.…”

From Saccharomyces cerevisiae to Ethanol: Unlocking the Power of Evolutionary Engineering in Metabolic Engineering Applications

“…For this purpose, S. cerevisiae EBY100 strain was used to anchor a recombinant peptidoglycan hydrolase, the lactobacilli phage endolysin LysKB317, with the a-agglutinin proteins Aga1p-Aga2p. The resulting recombinant S. cerevisiae strain expressing LysKB317 showed 83.8% decrease in bacterial cell counts, improved ethanol production and reduced levels of lactic and acetic acid [72]. Second generation bioethanol processes are known to be more prone to bacterial contamination due to longer pretreatment and fermentation times which allow lactic acid bacteria a longer time for competition with yeast strains [70].…”

Photopolymerization of L-DOPA-Alginate Immobilized Cell Wall Laccase for Textile Dye Decolorization

“…The light of the photon is absorbed by the photoinitiator, where the light energy is transferred into chemical energy; after that, intra-or extra-molecular groups are covalently crosslinked by free radicals [8][9][10]. As stated before, the catalytic and biochemical properties of enzymes can be improved by immobilization, and this action can be eased if the protein is expressed on the cell surface [11][12][13]. Cell-surface display represents the type of expression in which target peptides or proteins are attached to the cell surface of bacteria, yeast, insect, or mammalian cells.…”