From functional metagenomics to unique synthetic expression strategies in iron-reducing bacteria.

Abstract

Cellulose and hemicellulose are renewable sources of fermentable sugars. The use of fermentable sugars for the production of alternative energy sources (i.e. ethanol, butanol, etc.) is an attractive solution to alleviate the shortage and high prices of petroleum. Cellulases and hemicellulases are the two groups of glycosyl hydrolases responsible for breaking down the polysaccharide component of biomass into their respective sugar moieties. The enzymatic hydrolysis of cellulose and hemicellulose has relied on enzymes originally produced by culturable organisms. This thesis describes the use of metagenomics coupled to high-throughput screening techniques to identify glycosyl hydrolases originally encoded by uncultured organisms. The findings of this thesis include the identification and biochemical characterization of a unique endoglucanase. Besides catalyzing the hydrolysis of soluble and insoluble cellulosic substrates, this endoglucanase exhibited a domain architecture that has not been previously reported in the literature. This thesis also describes two different strategies to engineer the surface of (Fe+3)-reducing bacteria. These expression systems are a valuable tool for studying the cellular respiration of Geobacter and Shewanella. Furthermore, they have practical applications in the area of whole-cell biocatalysis in microbial fuel cells. The first strategy involved using an autodisplay system to engineer the cell envelope of Geobacter and Shewanella. The autodisplay system translocated a functional β-galactosidase enzyme to the cell envelope of G. sulfurreducens and S. oneidensis. Furthermore, this system proved to be an effective tool for catalyzing reactions in electrochemical cells using biofilms of G. sulfurreducens cells. The second strategy exploited the use of in-frame fusions with the c-type cytochrome OmcZ to translocate a recombinant protein to the outer membrane and extracellular matrix of Geobacter sulfurreducens. This is the first time that the c-type cytochrome OmcZ has been used to engineer biofilms of Geobacter sulfurreducens.