Biofuel Research Journal

Biofuel Research Journal

Saccharomyces cerevisiae secretion of recombinant bacteriophage endolysin LysKB317 inhibits Limosilactobacillus fermentum in corn mash fermentation

Document Type : Research Paper

Authors
Shao-Yeh Lu 1
Maulik H. Patel 2
Ronald E. Hector 3
Michael J. Bowman 3
Christopher D. Skory 1
1 U.S. Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, Renewable Product Technology Research Unit, Peoria, IL 61604, USA.
2 Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN, 37830, USA.
3 The U.S. Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, Bioenergy Research Unit, Peoria, IL 61604, USA.
10.18331/BRJ2024.11.4.4
Abstract
This study investigated the secretion of endolysin LysKB317 integrated into the HO locus of Saccharomyces cerevisiae strain NRRL Y-2034 to enable the yeast to simultaneously perform ethanol fermentation and control bacterial contaminants frequently present in ethanol refineries. The cell wall hydrolase gene was expressed using TEF1 and NAT5 promoter and terminator sequences with α-MF secretion signal and an N-terminus poly-histidine tag. LysKB317 was detectable by western blot analysis, which showed a molecular weight slightly larger than the 33 kDa native protein, presumably due to residual amino acids from the α-MF secretion signal peptide or S. cerevisiae glycosylation. Secreted LysKB317 was confirmed to be active using turbidity reduction and cell viability assay. Contaminated corn mash fermentations with yeast secreting LysKB317 demonstrated a significant reduction in bacterial contamination by at least 2-log compared to the contamination controls without LysKB317 expression. Moreover, LysKB317 expression led to a 73% decrease in acetic acid concentration and a 67% decrease in lactic acid levels. Contaminated fermentations with yeast expressing LysKB317 also exhibited a 16% improvement in ethanol production over the contamination controls without LysKB317, with no significant difference observed when compared to yeast-only controls during a 72-h corn mash fermentation. These findings suggest that a yeast endolysin secretion platform holds promise for mitigating bacterial contamination in biorefineries and potentially reducing reliance on antibiotics usage. 

Graphical Abstract

Saccharomyces cerevisiae secretion of recombinant bacteriophage endolysin LysKB317 inhibits Limosilactobacillus fermentum in corn mash fermentation

Highlights

  • HO-locus integrated endolysin in yeast is genetically stable and expressed constitutively.
  • Yeast-secreted endolysin reduces bacterial contamination by more than 2-logs.
  • Secreted endolysin can reduce acetic and lactic acid levels by at least 60% in contaminated fermentation compared to untreated controls.
  • The system improves ethanol production by at least 16% in contaminated fermentations compared to untreated controls.
  • The secretion system provides sustainable and continuous bacterial contaminant control in corn mash fermentation and offers rapid industrial adaptability.

Keywords
Endolysin
Saccharomyces cerevisiae
Yeast secretion
Fuel ethanol
Limosilactobacillus fermentum
Antimicrobial
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Biofuel Research Journal
Volume 11, Issue 4 - Serial Number 4
Autumn 2024
Pages 2243-2255