Final Report for the Link energy Fellowship
A sustainable energy future is critical for environmental and strategic reasons. Fossil fuel use has increased greenhouse gas emissions, and continued consumption could adversely change global climate. In addition, the United States must rely on foreign petroleum suppliers, leading to unfavorable trade deficits, instability, and conflict.1 One leading alternative to petroleum used for transportation is ethanol derived from cellulosic biomass.2 A major barrier for biological-based biomass conversion is a cost effective method of releasing sugars from recalcitrant cellulosic biomass by enzymatic hydrolysis. Thermophilic, anaerobic bacteria offer a potential solution, as they produce efficient native hydrolytic enzymes.3 However, all thermophilic bacteria isolated to date convert sugars to organic acids in addition to ethanol, which makes them impractical for cellulose conversion. The anaerobic, saccharolytic, thermophilic bacteria are a class of organisms with unique properties relevant for bioconversion of low cost cellulosic biomass feedstocks. The rate and efficiency of their cell associated enzymes to hydrolyze insoluble cellulose and xylan hold top values in the reported literature. In this regard, they hold a distinct advantage over the microorganisms that currently dominate biotechnological applications, which are unlikely to match the native hydrolytic ability of thermophilic bacteria due to the complexity of engineering highly efficient hydrolytic enzymes and the thermodynamic rate advantage of hydrolysis at higher temperatures. The branched fermentation pathways of these organisms, which produce organic acids in addition to solvents, are the primary obstacles for their use in an industrial process. Other challenges, such as product tolerance and fermentation robustness also need to be addressed, but low product yields above all else preclude their consideration for a commercial process. The establishment of a preliminary genetic system in Thermoanaerobacterium saccharolyticum JW/SL-YS485, a xylanolytic thermophile, opens the possibility for the establishment of this strain as a tractable model organism for anaerobic thermophilic bacteria. T. saccharolyticum also holds applied value due to it’s ability to ferment insoluble xylan and biomass derived sugars. The overall objective of this thesis is to establish high yield ethanol production in this strain through metabolic pathway engineering. The central objective of this project was to demonstrate that a thermophilic bacteria could be engineered to produce ethanol as sole end product. This was undertaken by gene disruption of metabolic pathways leading to acetic and lactic acid. In addition, the central fermentative pathways of T. saccharolyticum were investigated at the enzymatic and genomic level, and an alternative metabolic engineering strategy for high yield ethanol production was attempted by deletion of hydrogenase genes.