Abstract
Cellulose deconstruction and utilization are foundational to renewable biofuel and biochemical production. Anaerocellum bescii (formerly Caldicellulosiruptor bescii) is an extremely thermophilic cellulolytic bacterium, notable for its multi-domain cellulases and hemicellulases that efficiently degrade lignocellulosic biomass. However, the mechanisms by which A. bescii transports cello-oligosaccharides released during cellulose degradation into the cell for catabolism remain unclear. Among its 23 ATP-binding cassette (ABC) sugar transporters, we identified a conserved ABC transporter locus (athe_0595-0598) encoding two extracellular binding proteins: Athe_0597 and Athe_0598. Biophysical analyses using differential scanning calorimetry and isothermal titration calorimetry revealed that Athe_0597 binds cello-oligosaccharides of varying lengths (G2-G5), while Athe_0598 is specific to cellobiose (G2). Ligand docking simulations supported these findings and shed light on the subsite configuration of these substrate-binding proteins (SBPs). To assess its physiological importance, we genetically deleted this transporter locus in A. bescii strain HTAB187, which grew poorly on cellobiose and did not grow on cellulose. Comparison of growth with a msmK deletion strain that cannot consume oligosaccharides showed that HTAB187 retains growth on non-cello-oligosaccharides and monosaccharides. Taken together, these results integrate biophysical characterization, structural modeling, and genetic perturbation to elucidate how A. bescii transports cello-oligosaccharides released from cellulose, providing mechanistic insight relevant to consolidated bioprocessing applications.IMPORTANCEAnaerocellum bescii is the most thermophilic lignocellulolytic bacterium known and holds potential for bioprocessing lignocellulosic biomass into renewable fuels. Its diverse ATP-binding cassette (ABC) sugar transporters make it a valuable model for studying thermophilic sugar uptake. Here, we identify a single ABC transporter with two substrate-binding proteins (Athe_0597 and Athe_0598) responsible for cello-oligosaccharide uptake. Genetic deletion of this transporter locus impaired growth on cellobiose and eliminated growth on cellulose. This is the first genetic manipulation in A. bescii to modulate transport of a specific sugar. We also characterize the substrate specificity of the extracytoplasmic binding proteins associated with the locus. One binds various cellodextrins (G2-G5), while the other specifically binds cellobiose (G2). Molecular modeling depicts how each oligosaccharide is docked within the binding pocket of these proteins. Understanding the mechanism of cello-oligosaccharide uptake by A. bescii expands opportunities for its metabolic engineering and furthers our understanding of its carbohydrate utilization systems.