S59: Microbial genomics and metabolic engineering of mesophilic and thermophilic anaerobic bacteria

Metabolic engineering of industrially-robust bacteria for conversion of lignocellulosic biomass to fuels and/or value-added co-products requires a comprehensive understanding of the relationships between genome content, gene and gene product expression, enzyme activity levels, carbon and electron flow through metabolic pathways, and end-product synthesis patterns. Genome sequence data is necessary, but by itself is insufficient to provide insight into how growth conditions such as carbon source and loading, pH, and end-product accumulation influence carbon and electron flow to various metabolic end-products. Complete genome sequences are essential for metabolic engineering, but genetic manipulations can have pleiotrophic effects that cannot be predicted a priori without an understanding of how carbon and electron flow are regulated. Comparative bioinformatics analyses of mesophilic and thermophilic, cellulolytic and hemicelluloytic bacteria has revealed three significant problems with genome sequence annotations that can impact metabolic engineering. First, increasing numbers of sequenced genomes are in draft form only, consisting of numerous contigs. Without completion of the genome sequencing, genome annotations will remain incomplete. Second, key metabolic enzyme may be encoded by multiple gene homologues. Metabolic engineering requires precise knowledge of the gene(s) of interest, and it is essential to know which gene encodes the enzyme or enzyme subunit that is active in the pathway of interest. Third, the annotations of many genes are simply incorrect. Finally, we have developed a simple, rapid, broad host-range method of transforming both mesophilic and thermophlic anaerobic bacteria by conjugation, which will greatly facilitate metabolic engineering of C. thermocellum and other gram positive anaerobes.