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Abstract
The plant cell wall provides a renewable reservoir of energy-rich molecules that can be converted by microbes into useful products. Converting lignocellulosic biomass to fuels would improve environmental impacts of transportation fuels and provide an additional, renewable source of energy. Cellulolytic thermophiles express and secrete an arsenal of enzymes that degrade complex plant cell wall polysaccharides, releasing sugars the bacteria can metabolize. To be able to harness the power of these organisms, tools must be developed for introduction of DNA, gene expression and gene deletion. This work describes the development of genetic tools for Gram-positive, anaerobic, cellulolytic thermophiles Caldicellulosiruptor bescii, Caldicellulosiruptor hydrothermalis and Clostridium thermocellum to enable studies of plant cell wall degradation and to facilitate metabolic engineering. A thermophilic plasmid replicon from C. bescii was introduced into the two other species to test its maintenance and use for gene expression. Gene deletions in C. hydrothermalis and C. thermocellum were made with the aim of improving genetic manipulation. Deleting a restriction enzyme gene, chyI, in C. hydrothermalis increased plasmid transformation efficiency. In an attempt to overcome undesired DNA recombination in C. thermocellum, a recA deletion showed that this gene is required for replication of the native C. bescii thermophilic plasmid. To expand the abilities for gene expression in cellulolytic thermophiles, a thermophilic -glucosidase was developed as a reporter gene and used to characterize three promoters for gene expression, one of which was regulated by maltose. These tools are important advances in the development of genetic systems in non-model cellulolytic thermophiles. They will impact not only our understanding and engineering of these particular organisms, but also for developing genetic systems in new non-model organisms that produce novel enzymes, biochemical reactions and pathways.