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bioRxiv · 10.1101/2025.02.26.640372

A Highly Conserved ABC Transporter Mediates Cello-Oligosaccharide Uptake in the Extremely Thermophilic, Lignocellulolytic Bacterium Anaerocellum (f. Caldicellulosiruptor) bescii

Tjo, H.·Jiang, V.·Calvo, A.·Joseph, J. A.·Conway, J. M.
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Abstract

Cellulose deconstruction and utilization are key to unlocking renewable fuel and chemical production. Anaerocellum bescii (formerly Caldicellulosiruptor bescii) is an extremely thermophilic cellulolytic bacterium, among the most effective at degrading lignocellulosic biomass due to its arsenal of multi-domain cellulases and hemicellulases. However, little is known about how it transports the assorted sugars released from lignocellulose degradation into the cell for catabolism. Among its twenty-three ATP-Binding Cassette (ABC) sugar transporters, the mechanism for uptake of cello-oligosaccharides released from cellulose degradation remains unclear. Here, we identify an ABC transporter locus (Athe_0595 -- 0598), highly conserved in the genus with two extracellular binding proteins, Athe_0597 and Athe_0598. Biophysical analyses, including Differential Scanning Calorimetry (DSC) and Isothermal Titration Calorimetry (ITC), reveal that Athe_0597, binds cello-oligosaccharides of varying lengths (G2-5), while Athe_0598 is specific to cellobiose (G2). Computational modeling of ligand docking supports these findings and sheds light on the subsite configuration of the substrate binding proteins. To assess its physiological importance, we genetically deleted this transporter locus in A. bescii strain HTAB187, which does not grow on cellulose and grows poorly on cellobiose. Comparison of growth with a msmK deletion strain that cannot consume oligosaccharides shows that HTAB187 can grow on non-cello-oligosaccharides (e.g. maltose) or monosaccharides. Taken together, this study integrates biophysical characterization, structural modeling, and genetic perturbation to elucidate how A. bescii transports cello-oligosaccharides release from cellulose, opening doors for its future use in applied bioprocessing contexts. ImportanceAnaerocellum bescii is the most thermophilic cellulolytic 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 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 substrate binding proteins associated with the locus; one binds various cellodextrins (G2-5), while the other specifically binds cellobiose (G2). Computational modeling reveals how each sugar docks 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 thermophilic sugar transport.

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Tjo, H., Jiang, V., Calvo, A., Joseph, J. A., Conway, J. M.. 2025-02-26. A Highly Conserved ABC Transporter Mediates Cello-Oligosaccharide Uptake in the Extremely Thermophilic, Lignocellulolytic Bacterium Anaerocellum (f. Caldicellulosiruptor) bescii. https://doi.org/10.1101/2025.02.26.640372

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