Exploring and exploiting plant biomass degradation by Bacteroidetes
Doctoral thesis, 2021
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.
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.
polysaccharide utilization locus
plant biomass degradation
carbohydrate esterases
multidomain enzymes
Bacteroidetes
PUL transfer
carbohydrate-active enzymes
xylan
Opponent: Prof. Birte Svensson, Department of Biotechnology and Biomedicine, Technical University of Denmark, Denmark
Author
Cathleen Kmezik
Chalmers, Biology and Biological Engineering, Industrial Biotechnology
Multimodular fused acetyl–feruloyl esterases from soil and gut Bacteroidetes improve xylanase depolymerization of recalcitrant biomass
Biotechnology for Biofuels,;Vol. 13(2020)
Journal article
A polysaccharide utilization locus from the gut bacterium dysgonomonas mossii encodes functionally distinct carbohydrate esterases
Journal of Biological Chemistry,;Vol. 296(2021)
Journal article
Characterization of a novel multidomain CE15-GH8 enzyme encoded by a polysaccharide utilization locus in the human gut bacterium Bacteroides eggerthii
Scientific Reports,;Vol. 11(2021)
Journal article
Kmezik C, Porter NT, Pope PB, Koropatkin NM, Martens E, Larsbrink J. Enabling metabolism of mixed-linkage β-glucan in Bacteroides thetaiotaomicron by transfer of a polysaccharide utilization locus using a new versatile method
Microorganisms, including bacteria, have adapted over the span of billions of years to inhabit vastly different ecosystems on Earth. Many different types of bacteria exist and species that belong to the phylum Bacteroidetes inhabit many environments, for example soils and the gastrointestinal tracts of animals. In these varying habitats, Bacteroidetes bacteria excel in the deconstruction of recalcitrant biomass, and they do this by producing highly specific and efficient enzymes that act on the carbohydrates present in biomass. These carbohydrate-active enzymes (CAZymes) are sometimes encoded by gene clusters called polysaccharide utilization loci (PULs), which are a special feature of the Bacteroidetes phylum. CAZymes are highly diverse and certain types have been poorly described, including carbohydrate esterases (CEs) with multiple catalytic domains or in other words multicatalytic CEs.
With this thesis, I contribute to the understanding of multicatalytic CEs by demonstrating how their active domains complement each other in a synergistic manner. The biochemical characterization of various PUL encoded CEs also led to the identification of a highly active acetyl xylan esterase and a multicatalytic CE with a novel and rare enzyme architecture. The large size of PULs (often spanning tens of thousands of DNA basepairs) hampers genetic studies. In this thesis, I also present a new genetic tool, the pICKUP method, which allows for the transfer of PULs between Bacteroidetes species thus expanding the genetic toolbox available to conduct PUL research.
Overall, the work presented in this thesis contributes to our understanding of microbial plant biomass degradation and has implications for biorefinery applications and gut health.
With this thesis, I contribute to the understanding of multicatalytic CEs by demonstrating how their active domains complement each other in a synergistic manner. The biochemical characterization of various PUL encoded CEs also led to the identification of a highly active acetyl xylan esterase and a multicatalytic CE with a novel and rare enzyme architecture. The large size of PULs (often spanning tens of thousands of DNA basepairs) hampers genetic studies. In this thesis, I also present a new genetic tool, the pICKUP method, which allows for the transfer of PULs between Bacteroidetes species thus expanding the genetic toolbox available to conduct PUL research.
Overall, the work presented in this thesis contributes to our understanding of microbial plant biomass degradation and has implications for biorefinery applications and gut health.
Subject Categories
Industrial Biotechnology
Biological Sciences
Chemical Sciences
ISBN
978-91-7905-532-5
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4999
Publisher
Chalmers
Opponent: Prof. Birte Svensson, Department of Biotechnology and Biomedicine, Technical University of Denmark, Denmark