Co-Expression of a ..beta..-d-Xylosidase from Thermotoga maritima and a Family 10 Xylanase from Acidothermus cellulolyticus Significantly Improves the Xylan Degrading Activity of the Caldicellulosiruptor bescii Exoproteome

Caldicellulosiruptor species are hyperthermophilic, Gram-positive anaerobes and the most thermophilic cellulolytic bacteria so far described. They have been engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. Xylooligomers, such as xylobiose and xylotriose, that result from the breakdown of plant biomass more strongly inhibit cellulase activity than do glucose or cellobiose. High concentrations of xylobiose and xylotriose are present in C. bescii fermentations after 90 h of incubation, and removal or breakdown of these types of xylooligomers is crucial to achieving high conversion of plant biomass to product. In previous studies, the addition of exogenous β-D-xylosidase substantially improved the performance of glucanases and xylanases in vitro. β-D-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and β-D-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular β-D-xylosidase was identified in the C. bescii genome. Here, we report that the coexpression of a thermal stable β-D-xylosidase from Thermotoga maritima and a xylanase from Acidothermus cellulolyticus in a C. bescii strain containing the A. cellulolyticus E1 endoglucanase significantly increased the activity of the exoproteome as well as growth on xylan substrates. The combination of these enzymes also resulted in increased growth on crystalline cellulose in the presence of exogenous xylan.