Saccharomyces cerevisiae expressing bacteriophage endolysins reduce Lactobacillus contamination during fermentation

Abstract

Background: One of the challenges facing the fuel ethanol industry is the management of bacterial contamination during fermentation. Lactobacillus species are the predominant contaminants that decrease the profitability of biofuel production by reducing ethanol yields and causing "stuck" fermentations, which incur additional economic losses via expensive antibiotic treatments and disinfection costs. The current use of antibiotic treatments has led to the emergence of drug-resistant bacterial strains, and antibiotic residues in distillers dried grains with solubles (DDGS) are a concern for the feed and food industries. This underscores the need for new, non-antibiotic, eco-friendly mitigation strategies for bacterial contamination. The specific objectives of this work were to (1) express genes encoding bacteriophage lytic enzymes (endolysins) in Saccharomyces cerevisiae, (2) assess the lytic activity of the yeast-expressed enzymes against different species of Lactobacillus that commonly contaminate fuel ethanol fermentations, and (3) test the ability of yeast expressing lytic enzymes to reduce Lactobacillus fermentum during fermentation. Implementing antibiotic-free strategies to reduce fermentation contaminants will enable more cost-effective fuel ethanol production and will impact both producers and consumers in the farm-to-fork continuum. Results: Two genes encoding the lytic enzymes LysA and LysA2 were individually expressed in S. cerevisiae on multi-copy plasmids under the control of a galactose-inducible promoter. The enzymes purified from yeast were lytic against Lactobacillus isolates collected from fermentors at a commercial dry grind ethanol facility including Lactobacillus fermentum, Lactobacillus brevis, and Lactobacillus mucosae. Reductions of L. fermentum in experimentally infected fermentations with yeast expressing LysA or LysA2 ranged from 0.5 log 10 colony-forming units per mL (CFU/mL) to 1.8 log 10 (CFU/mL) over 72 h and fermentations treated with transformed yeast lysate showed reductions that ranged from 0.9 log 10 (CFU/mL) to 3.3 log 10 (CFU/mL). Likewise, lactic acid and acetic acid levels were reduced in all experimentally infected fermentations containing transformed yeast (harboring endolysin expressing plasmids) relative to the corresponding fermentations with untransformed yeast.

Controlling microbial contamination during hydrolysis of AFEX-pretreated corn stover and switchgrass: effects on hydrolysate composition, microbial response and fermentation

“…Elimination of contamination in our case is particularly crucial as the hydrolysate being produced is used for comparative microbial research and needs to have consistent characteristics and quality. A variety of methods can be used to control microbial contamination during hydrolysis and fermentation including (1) tailoring the levels of pretreatment degradation products to control contamination while limiting negative impacts on the fermentative organism [ 33 ]; (2) autoclaving the pretreated biomass prior to enzymatic hydrolysis [ 12 , 32 ]; (3) adding antibiotics to the hydrolysis and/or fermentation [ 29 , 30 ]; (4) pasteurizing the fermenters and their contents mid-way through the process [ 29 ]; (5) spiking the hydrolysis/fermentation with high concentrations of ethanol to inhibit growth of contaminating organisms [ 29 , 34 , 35 ]; and (6) expressing bacteriophage lytic enzymes (endolysins) in the fermentative organism [ 36 ]. Of these methods, only autoclaving the pretreated biomass and adding antibiotics are suitable for enzymatic hydrolysis, and have been demonstrated to consistently control contamination [ 12 , 29 , 32 , 37 ].…”

Recombinant Bacteriophage LysKB317 Endolysin Mitigates Lactobacillus Infection of Corn Mash Fermentations

“…SDS-PAGE gels were stained using LabSafe GEL Blue (G-Biosciences) and washed in deionized (DI) water for 1 hr at room temperature. Additional de-staining incubation was done with gels submerged in de-staining buffer (50 mM Tris-HCl, 1% Triton X-114, pH 5.5) at room temperature with gentle swirling overnight or until translucent bands is clearly visible as described [50].…”

Anti-Contamination Strategies for Yeast Fermentations

“…For example, the endolysin LysA2 from a Lactobacillus casei bacteriophage can reduce the growth of a wide spectrum of LAB [110]. The antimicrobial potential of LysA2 has been tested and used for the reduction of contamination problems in fuel ethanol production [108,111]. Like bacteriocins, the production cost of the endolysin protein is significant.…”