SRA

Extremely thermophilic bacteria from the genus Caldicellulosiruptor can degrade various polysaccharide components of plant cell walls. Previous experimental studies identified a variety of carbohydrate-active enzymes in model species C. saccharolyticus and C.bescii, while prior transcriptomic experiments identified their putative carbohydrate uptake transporters. We investigated the mechanisms of transcriptional regulation of carbohydrate utilization genes using a comparative genomics approach applied to fourteen Caldicellulosiruptor species. The reconstruction of carbohydrate utilization regulatory network includes the predicted binding sites for 34 mostly local regulators and point to the regulatory mechanisms controlling expression of genes involved in degradation of plant biomass. The Rex and CggR regulons control the central glycolytic and primary redox reactions. The identified transcription factor binding sites and regulons were validated with transcriptomic and transcription start site data for C. bescii grown on cellulose, cellobiose, glucose, xylan, and xylose. The XylR and XynR regulons control xylan-induced transcriptional response of genes involved in degradation of xylan and xylose utilization. The reconstructed regulons informed the carbohydrate utilization reconstruction analysis and improved functional annotations of 51 transporters and 11 catabolic enzymes. Using gene deletion, we confirmed that the shared ATPase component MsmK is essential for growth on oligo- and polysaccharides but not for the utilization of monosaccharides. By elucidating the carbohydrate utilization framework in C. bescii, strategies for metabolic engineering can be pursued to optimize yields of bio-based fuels and chemicals from lignocellulose. Overall design: Five different growth substrates were used (glucose, cellobiose, crystalline cellulose (Avicel), xylose, birchwood xylan (Sigma), 5 g/L each). Growth experiments were performed in triplicate at 78°C as closed cultures without pH control (400 mL volume, shaken at 150 rpm). Cells were harvested in mid-to-late exponential growth stage (0.5-1.5 × 108 cells mL-1). Cells were pelleted by centrifugation at 6,000 × g for 5 minutes, supernatant was decanted, and cell pellets were frozen in liquid nitrogen.