Investigation of bacterial transport for improved uptake of hemicellulose-derived sugars and oligosaccharides

Plant lignocellulosic biomass can be a source of fermentable sugars for the production of second generation biofuels and provide a viable alternative to fossil fuels. The hemicellulotic component of many bioenergy grasses, including Miscanthus, is comprised of arabinoglucuronoxylans. As xylans constitute the third most abundant biopolymer, we set out to investigate the uptake of its sugars and oligosaccharides, ie. L-arabinofuranose and xylobiose, by bacteria. Using a phylogenetic, phylogenomic and ‘in silico’ analyses of Galacto/Arabinofuranose SBP from E. coli (ie. GafAEc) we detected bacterial orthologs which could show ‘bias’ towards arabinose binding. The chosen SBPs for further characterisation were GafASw and GafASm from Shewanella sp. ANA-3 and Sinorhizobium meliloti 1021, respectively. Biochemical characterisation showed that GafASm bound L-arabinose 5 times higher affinity than GafAEc, and D-fucose changed the thermal stability of the protein at similar levels to D-galactose. The enhanced affinity for both L-arabinose and D-fucose might suggest that GafSm system is involved in chemotaxis towards root mucilage. Strikingly, GafASw shows different ligand specificity to GafAEc. Structural chareacterisation of GafASw discovered the molecular determinants in the binding pocket proximity which led to the discrepancy. Lastly, we created Transport Deletion (TD) mutants of E. coli which are unable to grow on L-arabinose (ie. TDara), D-xylose (ie. TDxyl) and D-glucuronic acid (ie. TDglcA). The strains recovered their growth when their respective secondary (MFS) were ectopically expressed, thus verifying their applicability as tools for detection of useful bacterial transporters