PhD defence will take place on April 24th at 9:00 in room 401 at TBI
The thesis is entitled: Rational design of multienzymatic assemblies to unravel plant biomass deconstruction.
The presentation will be in English.
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Jury composition :
- Mr. Evangelos TOPAKAS, Reviewer – National Technical University of Athens
- Ms. Elizabeth FICKO-BLEAN, Reviewer – Station Biologique de Roscoff
- Ms. Samantha VERNHETTES, Reviewer – Institut Jean-Pierre Bourgin – Sciences du Végétal
- Mr. Michael O’DONOHUE, Examiner – INSA Toulouse
- Mr. Edward A. BAYER, Examiner – Weizmann Institute of Science
- Ms. Claire DUMON, PhD Supervisor – INSA Toulouse
- Mr. Cédric MONTANIER, Co-supervisor – INSA Toulouse
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Abstract :
To cope with the inherent complexity of plant cell walls and to extract oligosaccharides as carbon source, certain anaerobic microorganisms produce a wide range of glycoside hydrolases organized in a multienzyme complex: the cellulosome. Due to their intrinsic flexibility, determining how the spatial organization within these assemblies influence catalysis remains a major challenge.
It was hypothesized that constraining enzyme positioning would enable direct evaluation of topology-activity relationships in such systems. To address this question, a strategy based on two small proteins, Jo and In, was employed. These proteins spontaneously form a covalent isopeptide bond, creating a rigid scaffold that locks fused enzymes into a defined spatial arrangement.
Three cellulosomal GHs from Acetivibrio thermocellus (AtCel8A, AtCel9R, and AtXyn11A), were selected to generate a library of 12 distinct multienzyme JoIn based complexes. They were successfully produced, purified, and characterized on a range of soluble and lignocellulosic substrates. Small-angle X-ray scattering and mass spectrometry analyses were combined to correlate enzyme topology and inter-enzyme spatial arrangement with soluble oligosaccharide release profiles. Spatial layout dependent differences in catalytic behavior and product distributions were observed, demonstrating that spatial constraints imposed directly influence enzymatic performance. Collectively, these results demonstrate that structure-function relationships can be systematically investigated using the JoIn system, although such relationships are more complex than initially anticipated and cannot be explained solely by enzyme proximity.
In parallel, engineering of the Jo/In pair was performed to expand the number of accessible spatial configurations and better approximate the architectural complexity of designer cellulosomes. Although no fully orthogonal pair was identified, the study provides new insights into the molecular determinants governing Jo/In partner recognition.
Overall, this work expands current understanding of the JoIn platform and, through a multidisciplinary and systematic approach, advances knowledge on the design of synthetic multienzyme assemblies. The results highlight the central role of spatial topology as a key parameter governing multienzyme biocatalysis and establish a framework for rationally engineering programmable enzyme complexes.
