Engineering Caldicellulosiruptor bescii to Analyze Cellulase and Hemicellulase Enzymes for Biofuel Applications

Abstract

The Caldicellulosiruptor genus is known to produce cellulases as well as hemicellulases, with some of them having multiple domains. Caldicellulosiruptor bescii is an anaerobic extreme thermophile in this genus that produces the protein CelA, a multidomain cellulase. Since CelA is a glycoside hydrolase, it allows C. bescii to degrade complex, untreated biomass, making it potentially useful in second generation biofuel production, as it could be used in consolidated bioprocessing, which is a more streamlined version of the current methods for production. Using procedures developed specifically for C. bescii was necessary in ensuring growth of the bacteria as well as in the preparation of competent cells to use for transformations. This study confirms that C. bescii is capable of degrading cellulose with varying levels of crystallinity. Visualizing the entire structure of CelA will be helpful to get a sense of how its domains interact, as currently, the solved crystalline structures of its two catalytic domains have only been captured independently of each other. By transforming the MACB1018 strain of C. bescii with a replicating vector, its production of CelA can be upregulated and production of a histidine tagged version of CelA is enabled, which would allow for the use of fast protein liquid chromatography to purify it as well as subsequent imaging using Cryogenic Electron Microscopy.