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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVING BIOCHEMICAL PROCESSES FOR THE PRODUCTION OF SUSTAINABLE FUELS AND CHEMICALS

Location: Renewable Product Technology Research Unit

Title: Expression of bacteriophage endolysins in Saccharomyces cerevisiae

Authors
item Khatibi, Piyum
item Roach, Dwayne
item Hughes, Stephen
item Donovan, David
item Bischoff, Kenneth

Submitted to: American Society for Microbiology General Meeting
Publication Type: Abstract Only
Publication Acceptance Date: May 21, 2013
Publication Date: N/A

Technical Abstract: One of the challenges facing the fuel ethanol industry is the management of bacterial contamination during fermentation. Species of Lactobacillus are the predominant contaminants that reduce ethanol yields and cause “stuck” fermentations, decreasing the profitability of biofuel production with expensive antibiotic treatments and disinfecting costs. Moreover, ethanol fermentations treated with antibiotics may promote 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 and improved mitigation strategies for bacterial contamination. In this study, bacteriophage lytic enzymes (endolysins) were expressed in Saccharomyces cerevisiae and tested for their ability to kill different Lactobacillus species. The specific objectives of this work were to (1) express genes encoding bacteriophage endolysins in S. cerevisiae, (2) test the yeast-expressed lytic enzymes for activity against species of Lactobacillus, and (3) apply the recombinant enzymes to reduce lactobacilli contamination in experimentally infected fermentations. The streptococcal phage LambdaSA2 endolysin and the Lactobacillus bacteriophage endolysin LysA were previously shown to have strong lytic activity against Lactobacillus when expressed in E.coli. When expressed in yeast, however, the LambdaSA2 enzyme was insoluble, while LysA was both soluble and active. The recombinant LysA killed strains of L. fermentum, L. brevis, and L. mucosae, all isolated as contaminants from commercial ethanol facilities. LysA was also effective at reducing lactobacilli concentrations from 5 log(CFU/ml) to less than 2 log(CFU/ml) in a series of experimentally infected fermentations. Implementing novel and antibiotic free strategies to kill bacteria contaminating fermentations will enable more cost effective fuel ethanol production and will impact producers and consumers across the farm-food-fork continuum.

Last Modified: 12/22/2014
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