A highly conserved ABC transporter mediates cello-oligosaccharide uptake in the extremely thermophilic, lignocellulolytic bacterium <i>Anaerocellum bescii</i> (f. <i>Caldicellulosiruptor bescii</i> )

Cellulose deconstruction and utilization are foundational to renewable biofuel and biochemical production. <i>Anaerocellum bescii</i> (formerly <i>Caldicellulosiruptor bescii</i>) is an extremely thermophilic cellulolytic bacterium, notable for its multi-domain cellulases and hemicellulases that efficiently degrade lignocellulosic biomass. However, the mechanisms by which <i>A. bescii</i> 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 (<i>athe_0595-0598</i>) 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 <i>A. bescii</i> strain HTAB187, which grew poorly on cellobiose and did not grow on cellulose. Comparison of growth with a <i>msmK</i> 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 <i>A. bescii</i> transports cello-oligosaccharides released from cellulose, providing mechanistic insight relevant to consolidated bioprocessing applications.IMPORTANCE<i>Anaerocellum bescii</i> 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 <i>A. bescii</i> 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 <i>A. bescii</i> expands opportunities for its metabolic engineering and furthers our understanding of its carbohydrate utilization systems.