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 FY12, ARS scientists made significant progress toward this objective, as demonstrated by the following activities. A search of publicly available genome databases identified four genes encoding putative antibacterial enzymes. These genes were synthesized and expressed in bacteria to produce recombinant enzymes. The enzymes possessed antibacterial activity against several Lactobacillus strains that are known to contaminate commercial ethanol fermentations. Initial yeast expression experiments have shown successful recombinant enzyme production. 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.