Investigation of Novel Cellulose-Binding TÃPirin Proteins in Caldicellulosiruptor Species

The genus Caldicellulosiruptor represents a group of extremely thermophilic bacteria with potential for production of biofuels and chemicals. Traditional methods of producing biofuels incorporate chemical and thermal pretreatment of plant biomass, followed by enzymatic degradation, to produce fermentable sugars. The use of Caldicellulosiruptor could consolidate these processes into a single step, requiring no biomass pretreatment. The ability of Caldicellulosiruptor to bind to cellulosic biomass is due in part to novel proteins known as tāpirins. Once bound, Caldicellulosiruptor solubilizes the recalcitrant cellulosic biomass, an important step in the degradation of sugars needed for processing. In order to study these unique proteins, tāpirins from several different species were expressed in Escherichia coli. These proteins were then purified via immobilized metal ion affinity chromatography (IMAC), tested for their ability to bind different substrates, and prepared for crystallography. The C-terminal domains of two tāpirins, Calkro_0844 and Calhy_0908, have been successfully crystallized. These two proteins, despite their low homology, bear significant structural resemblance. To determine the specific role of tāpirins and pili in binding to cellulose and complex biomasses, a series of genetic knockouts are also being performed in Caldicellulosiruptor bescii. Overall, tāpirins and pili likely play an important part in the ability of Caldicellulosiruptor to bind and degrade cellulosic biomass, which is crucial in the consolidated bioprocessing approach to biofuel production and chemical synthesis.