Bacterial contamination control in corn mash fermentation using recombinant endolysin secretion by Saccharomyces cerevisiae

Authors: Lu, Shao; PATEL, MAULIK - Orise Fellow; Hector, Ronald; Bowman, Michael; Skory, Christopher

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Abstract Only
Publication Acceptance Date: 8/4/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: In 2023, the United States produced 15.6 billion gallons of bioethanol, representing 53% of global production. Bioethanol fermentation is often susceptible to contamination with lactic acid bacteria (LAB) that compete for nutrient resources, result in the accumulation of yeast inhibitory byproducts, such as acetic acid and lactic acid, and reduce ethanol productivity of yeast. Current bacterial mitigation methods include extensive facility cleaning or various chemical treatments, such as hop acids, chlorine dioxide, and antibiotics. However, these treatments often require fermentation facility shutdowns for extended application periods, are minimally effective, or may potentially contribute to the emergence of antibiotic-resistant bacteria. To combat LAB infection, we genetically modified a previously described bacteriophage endolysin gene, LysKB317, with yeast codon optimization, an alpha mating factor secretion signal (MF-alpha), a TEF1 protomer and NAT5 terminator. We integrated this modified gene into the yeast strain Saccharomyces cerevisiae NRRL Y-2034 at the HO gene site. Confirmation of endolysin expression and secretion was achieved through western blot analysis of the spent media. Lytic activity against Limosilactobacillus fermentum was shown by bacterial cell viability assay and turbidity reduction assays. L. fermentum was selected because it has previously been shown to drastically inhibit bioethanol productivity. In corn mash fermentations, yeast LysKB317 secretion significantly reduced bacterial load by >2-log CFU/mL and decreased acetic acid and lactic acid concentration level by 73% (p < 0.0001) and 67% (p < 0.0001), respectively, over a 72-hour fermentation. Fermentations with modified yeast produced 16.7% (p< 0.0001) more ethanol than the unmodified yeast strains when challenged with L. fermentum contamination (9.6% and 8.0% w/v, respectively). Additionally, glucose utilization in the presence of bacterial contamination was significantly improved with modified yeast compared to unmodified yeast with only 0.31% w/v remaining, compared to the 2.6% w/v in the unmodified yeast control sample. This further represents an 88.2% improvement in glucose utilization (p < 0.0001) at the end of 72-hour fermentation. Overall, this yeast-mediated endolysin secretion technology presents a proactive and continuous solution to bacterial contamination in bioethanol production, offering an effective and economical solution to mitigating the risk associated with bacterial contamination while minimize antibiotics use.