Proteomics and characterization of lignocellulosic carbohydrate utilization for Thermoanaerobacterium thermosaccharolyticum reveals two thermophilic Gram-positive polysaccharide utilization loci

Saccharolytic bacteria are of increasing interest due to their importance in human health and the environment. In particular, their abilities to utilize diverse plant polysaccharides make them attractive candidate organisms for production of lignocellulosic biofuels. Thermoanaerobacterium thermosaccharolyticum is a thermophilic saccharolytic anaerobe able to utilize a variety of sugars and oligosaccharides. Here, the genomes and substrate utilization preferences of three strains of T. thermosaccharolyticum were examined in depth and compared using growth assays and untargeted proteomics, focusing on carbohydrate active enzymes (CAZymes) and sugar transporter expression. The analysis revealed the wide range of carbohydrates being utilized by T. thermosaccharolyticum , but there were substantial differences in CAZyme abundances and abilities to grow on polysaccharides. Comparing the global proteomic responses between growth on hexoses and pentoses showed a much higher expression of proteins for hemicellulose-derived sugars. This is consistent with the greater structural complexity of hemicellulose relative to cellulose, the two major components of lignocellulose. Despite being having a minor presence in lignocellulose, arabinose surprisingly drives elevated CAZyme abundance, at levels comparable to those induced by hemicellulose-derived complex carbohydrates. Measuring proteomic responses across carbohydrates and strains enabled the identification of two (Gram-positive) thermophilic polysaccharide utilization loci (gpPULs). These two gpPULs, to our knowledge among the first reported in thermophilic anaerobes, enabled the development of a model describing thermophilic hemicellulose utilization in T. thermosaccharolyticum . This work advances understanding of the role of saccharolytic species in more applied environments like biofuel production as well as in the biosphere, and identifies genes that can extend substrate utilization in other species.