Characterization of ABC Sugar Transporters for Xylan Utilization in Extremely Thermophilic Anaerocellum bescii

Abstract

Sustainable biofuels can be produced using agricultural feedstocks rich in lignocellulose, such as corn stalks and barley straw—low-cost, renewable feedstocks that are typically discarded. However, lignocellulose is both physically and chemically challenging to degrade. Anaerocellum bescii, a thermophilic bacterium that thrives at ~75°C, is a promising candidate for biorefining processes due to its powerful arsenal of carbohydrate-active enzymes (CAZymes), which can efficiently break down lignocellulose and convert it to ethanol and other biofuels. However, efforts to metabolically engineer A. bescii are hindered by a limited understanding of its ATP-binding cassette (ABC) sugar transport systems, which play a vital role in importing extracellular oligosaccharides into the cell. The Xylan Degradation Locus (XDL) and Conserved Xylan Utilization Locus (CXUL) are involved in xylose utilization and together encode three putative ABC sugar transporters. The XDL includes AxoFGE (Athe_0174–0176) and XloEFG (Athe_0179–0181), with Athe_0174 and Athe_0181 as the associated substrate binding proteins, while the CXUL gene cluster xynUVW (Athe_0614–0616) encodes Athe_0614. By heterologously expressing and purifying these proteins, followed by ligand screening using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC), we identified their hemicellulosic sugar preferences and roles in transport. Additionally, ROSIE docking simulations, contact residue mapping, and sequence alignments with homologous proteins revealed conserved amino acids involved in xylo-oligosaccharide recognition. This work advances our understanding of how A. bescii utilizes diverse hemicellulosic substrates, accelerating its development as a platform for sustainable bioproduction of biofuels and other valuable chemicals.