Doctor of Philosophy in Chemistry (PhD)
Fundamental and applied carbohydrate enzymology
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Xyloglucan (XyG) is a ubiquitous plant heteropolysaccharide representing up to one-quarter of the total carbohydrate content of terrestrial plant cell walls. Given its structural complexity, XyG requires a consortium of backbone-cleaving endo-xyloglucanases and sidechain-cleaving exo-glycosidases for complete saccharification. The Gram-negative soil saprophyte Cellvibrio japonicus is a treasure trove for carbohydrate active enzymes (CAZymes) due to its robust capacity to degrade different plant polysaccharides. The XyG utilization machinery in C. japonicus is incompletely understood, despite recent characterization of associated sidechain-cleaving exo-glycosidases. I present here my attempts to identify and functionally characterize the endo-xyloglucanase(s) and the XyG-specific β-1,4 exo-glucosidase catalyzing the first and final steps, respectively, in the XyG saccharification pathway in C. japonicus. Bioinformatic analysis identified one Glycoside Hydrolase Family 74 (CjGH74), three GH5_4 (CjGH5D, CjGH5E and CjGH5F) and three GH9 (CjGH9A, CjGH9B and CjGH9C) candidates with putative endo-xyloglucanase activity. Biochemical and structural analyses that involved CjGH74 and the three CjGH5_4 enzymes clearly demonstrated the exquisite specificity of the four enzymes towards XyG. Scrutiny of the modular architecture of the CjGH5_4 enzyme, CjGH5F, identified the module of unknown function X181. Affinity gel electrophoresis and isothermal titration calorimetry identified the X181 module as a member of a new CBM family that exclusively binds galactose-containing polysaccharides including XyG and galactomannans, congruent with the displayed endo-xyloglucanase activity of the pendent catalytic domain. Surprisingly, reverse genetic analysis in C. japonicus displayed the lack of growth perturbation on XyG upon the deletion of the four specific endo-xyloglucanases, suggesting the presence of other enzyme(s) with potential endo-xyloglucanase activity. Biochemical characterization of the GH9 enzyme CjGH9B revealed its high catalytic efficiency towards mixed linkage β-glucan and its weak side-activity against xyloglucan, which might be sufficient to rescue the quadruple deletion mutant. Bioinformatic analysis identified the four CjGH3 enzymes Bgl3A, Bgl3B, Bgl3C, and Bgl3D as potential targets for the XyG-specific exo-β-glucosidase in C. japonicus. Comprehensive genetic and biochemical approaches interestingly revealed the fundamental contribution of Bgl3D in XyG utilization in C. japonicus. Together, these data shed light on the initial and final steps of xyloglucan saccharification in C. japonicus and identify useful enzymes for selective biomass deconstruction.
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