1a.Objectives (from AD-416)
1. Characterize the spectrum of activity of phage endolysins against our collection of bacterial contaminants from fuel ethanol plants.
2. Mutatgenesis/directed evolution to broaden the spectrum of activity of endolysins.
3. Clone endolysin genes for functional expression in a cellulosic microbial catalyst.
4. Demonstrate efficacy of endolysins in ethanol fermentations.
1b.Approach (from AD-416)
Fuel ethanol is not produced under aseptic conditions, and acute bacterial infections occur unpredictably leading to "stuck" fermentations. We propose developing a cellulosic yeast ethanologen that secretes a bactericidal lytic enzyme with specificity against contaminating lactic acid bacteria. Purified endolysins maintain their near-species specificity and can lyse both biofilm and senescent forms of Gram positive bacteria when exposed externally. The expression of these enzymes in yeast will be accomplished via a proprietary methodology that avoids inactivation due to aberrant glycosylation, a major pitfall of heterologous yeast expression systems.
Bacterial contamination in commercial fuel ethanol plants lowers the yield of ethanol and increases production costs. The contaminating bacteria compete with the yeast for sugar and nutrients, and they produce acids that can inhibit the fermenting yeast. The objective of this research is to develop strains of yeast that express antibacterial proteins to inhibit the competing bacteria during fuel ethanol production. In FY10, progress was made toward identifying candidate genes that express putative antibacterial enzymes. The products of these genes will be characterized for antibacterial activities against bacterial strains that contaminate commercial fuel ethanol facilities. This work has potential to improve the economics of cellulosic biofuel production by:. 1)increasing ethanol yield through elimination of competing bacteria;. 2)reducing production costs by avoiding large scale antibiotic use; and. 3)decreasing environmental impact by replacing broad range antibiotics with biodegradable, enzyme-based antibacterials. Activities for this project were coordinated by the ADODR via emails and telephone interactions between cooperating scientists at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, Illinois, and the USDA-ARS Animal Biosciences and Biotechnology Lab, Beltsville, Maryland.