Exploring and exploiting plant biomass degradation by Bacteroidetes
Sammanfattning: Bacteroidetes bacteria have evolved to become excellent biomass degraders. They achieved this by applying carbohydrate-active enzymes (CAZymes) and organizing genes connected to the degradation of specific polysaccharides into discrete gene cassettes, so-called polysaccharide utilization loci (PULs). Consequently, CAZymes and PULs may hold the potential to improve biomass valorization processes in biorefineries and to advance our understanding of human and livestock gut health. CAZymes are extremely diverse in activity and structure, and for some enzyme families only little is known to date. For example, certain carbohydrate esterases (CEs) combine multiple catalytic domains within one protein, resulting in multicatalytic enzyme architectures, and the properties of these have been little explored. In this thesis, I present biochemical data showcasing the existence of intramolecular synergy between the active domains of multicatalytic CEs (BoCE6-CE1). The observed intramolecular synergy facilitated more efficient degradation of xylan-rich biomass compared to non-multicatalytic CEs, giving a possible explanation as to why multicatalytic CEs exist in the genomes of Bacteroidetes species. Well-defined activity profiles of several here characterized CEs support the hypothesis that each catalytic domain fulfills an individual role during concerted plant biomass degradation, explaining why some PULs encode multiple CEs from the same enzyme family. Further, the investigated CEs cleaved xylan decorations and increased the activity of xylanase-mediated biomass degradation up to 20-fold (FjCE6-CE1). During the investigation of the CAZyme repertoire of different species I also identified a remarkably active and promiscuously acting acetyl xylan esterase (DmCE6A), as well as a rare enzyme architecture that may offer new insights into the multitude of interacting enzyme activities necessary to degrade plant biomass (BeCE15A-Rex8A). PULs encode a plethora of CAZymes and have been shown to be vital for the glycan degradation abilities of Bacteroidetes species. However, the investigation of PULs is aggravated by their usually large size, which often limits the scope of genetic studies. In this thesis, I present a new method for the transfer of PULs between Bacteroidetes species, thus expanding the tools available for the identification and characterization of PULs and their components. The PUL transfer was demonstrated for a previously characterized mixed-linkage β-glucan utilization locus and conferred the ability to metabolize mixed-linkage β-glucan to the receptor strain.
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