Biochemical and Regulatory Analyses of Xylanolytic Regulons in Caldicellulosiruptor bescii Reveal Genus-Wide Features of Hemicellulose Utilization

species scavenge carbohydrates from runoff containing plant biomass that enters hot springs and from grasses that grow in more moderate parts of thermal features. While only a few species can degrade cellulose, all known species are hemicellulolytic. The most well-characterized species, Caldicellulosiruptor bescii, decentralizes its hemicellulase inventory across five different genomic loci and two isolated genes. Transcriptomic analyses, comparative genomics, and enzymatic characterization were utilized to assign functional roles and determine the relative importance of its six putative endoxylanases (five glycoside hydrolase family 10 [GH10] enzymes and one GH11 enzyme) and two putative exoxylanases (one GH39 and one GH3) in C. bescii. Two genus-wide conserved xylanases, XynA (GH10) and Xyl3A (GH3), had the highest levels of sugar release on oat spelt xylan, were in the top 10% of all genes transcribed by , and were highly induced on xylan compared to cellulose. This indicates that a minimal set of enzymes are used to drive xylan degradation in the genus , complemented by hemicellulolytic inventories that are tuned to specific forms of hemicellulose in available plant biomasses. To this point, synergism studies revealed that the pairing of specific GH family proteins (GH3, -11, and -39) with GH10 proteins released more sugar than mixtures containing five different GH10 proteins. Overall, this work demonstrates the essential requirements for to degrade various forms of xylan and the differences in species genomic inventories that are tuned for survival in unique biotopes with variable lignocellulosic substrates. Microbial deconstruction of lignocellulose for the production of biofuels and chemicals requires the hydrolysis of heterogeneous hemicelluloses to access the microcrystalline cellulose portion. This work extends previous and efforts to characterize hemicellulose utilization by integrating genomic reconstruction, transcriptomic data, operon structures, and biochemical characteristics of key enzymes to understand the deployment and functionality of hemicellulases by the extreme thermophile Caldicellulosiruptor bescii. Furthermore, comparative genomics of the genus revealed both conserved and divergent mechanisms for hemicellulose utilization across the 15 sequenced species, thereby paving the way to connecting functional enzyme characterization with metabolic engineering efforts to enhance lignocellulose conversion.