Metabolic engineering of a cellulolytic thermophile for renewable production of biofuels
Willams-Rhaesa, Amanda Michele
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Sustainable generation of liquid transportation fuels is critical to mitigate the environmental impacts of fossil fuels. Lignocellulosic biomass is an attractive renewable resource but the natural recalcitrance of plant materials to enzymatic and microbial deconstruction must first be overcome. Costly pretreatment processing of the biomass and the addition of exogenous cellulase enzymes to release sugars are necessary. Microbes then utilize the sugars in standard industrial ethanol production. Consolidated bioprocessing (CBP) has the potential to decrease the costs associated with the use of lignocellulosic feedstocks by combining biomass deconstruction and microbial conversion of cellulose and hemicellulose into ethanol all in one fermentation vessel. Cellulolytic thermophiles have been explored for use in this technology as their high growth temperatures facilitate breakdown of the plant material, and decrease the risk of contamination of fermentation vessels. Caldicellulosiruptor bescii (Topt 78°C) is the most thermophilic cellulolytic bacterium known and can utilize plant biomass without pretreatment. The recent development of a genetic system in this organism has greatly increased its potential utility for CBP. The goal of this research was to engineer C. bescii for maximum ethanol production. First, the limitations of growing wild-type C. bescii were explored on high concentrations of both a model substrate, crystalline cellulose (Avicel), and the real-world substrate, switchgrass. This study revealed that substrate utilization was limited by nitrogen availability and the production of >150 mM organic acids. Generation of a neutral product, such as ethanol, would alleviate this second limitation. Second, improvements were made to the genetics system, including the development of a more stable genetic background and also of a native xylose-inducible promoter, thereby expanding the genetic toolkit for this organism. Third, the more stable background was used for heterologous expression of a cytoplasmic bifunctional alcohol dehydrogenase with and without a reduced ferredoxin NAD oxidoreductase. This six-subunit membrane-bound enzyme provides redox balance for ethanol production and allowed for the generation of a maximum of 75 mM ethanol from cellulose at 60°C. This is the highest ethanol production by C. bescii to date. These advancements bode well for the future use of C. bescii as a platform organism for biotechnology.