Periodic Reporting for period 1 - EvoXUL (Evolution of Xylan Utilization Loci 's enzymes for the design of enzymatic assemblies)
Okres sprawozdawczy: 2021-01-11 do 2023-01-10
Lignocellulosic biomass (LCB) is a renewable and inexhaustible carbon source on Earth and its valorisation will drive
sustainable circular bioeconomy. Microorganisms involved in LCB deconstruction produce a huge repertoire of carbohydrate
active enzymes (CAZymes) in order to utilize LCB as carbon source. Specifically, Bacteroides, encode fine-tuned gene
clusters dedicated to polysaccharide metabolism called Polysaccharide Utilization Loci (PUL). The host team recently
discovered a xylan PUL from termite gut whose enzymes showed promising activity on different LCB. In EvoXUL project an
original co-evolution strategy will be deployed to simultaneously engineer XUL’s enzymes towards wheat bran and wheat
straw hydrolysis. In addition, a combination of enzyme assemblies will be created thanks to the Bio Molecular Welding Jo-In
system to further maximize the synergy between the catalysts and to unravel the impact of enzyme spatial organization with
the goal of developing more efficient enzyme cocktails for bioeconomy. The fellow and members of the French National
Institute for Applied Sciences of Toulouse (INSA Toulouse) will establish a successful collaboration plan on the basis of their
respective backgrounds in hydrolases involved in biomass valorisation (biochemistry, applied enzymology, bioprocess
engineering) and protein evolution, engineering, and functional characterization of glycoside hydrolases, respectively. The
acquired knowledge in structure and synergism of modular enzymes will provide rules for tailor made enzyme assemblies for
future industrial applications related with lignocellulosic feedstock biorefinery. The project will provide an opportunity for the
fellow to come back to science and the in depth training in molecular biology, protein engineering and structure-function of
carbohydrate modifying enzymes will allow him to become an independent researcher.
sustainable circular bioeconomy. Microorganisms involved in LCB deconstruction produce a huge repertoire of carbohydrate
active enzymes (CAZymes) in order to utilize LCB as carbon source. Specifically, Bacteroides, encode fine-tuned gene
clusters dedicated to polysaccharide metabolism called Polysaccharide Utilization Loci (PUL). The host team recently
discovered a xylan PUL from termite gut whose enzymes showed promising activity on different LCB. In EvoXUL project an
original co-evolution strategy will be deployed to simultaneously engineer XUL’s enzymes towards wheat bran and wheat
straw hydrolysis. In addition, a combination of enzyme assemblies will be created thanks to the Bio Molecular Welding Jo-In
system to further maximize the synergy between the catalysts and to unravel the impact of enzyme spatial organization with
the goal of developing more efficient enzyme cocktails for bioeconomy. The fellow and members of the French National
Institute for Applied Sciences of Toulouse (INSA Toulouse) will establish a successful collaboration plan on the basis of their
respective backgrounds in hydrolases involved in biomass valorisation (biochemistry, applied enzymology, bioprocess
engineering) and protein evolution, engineering, and functional characterization of glycoside hydrolases, respectively. The
acquired knowledge in structure and synergism of modular enzymes will provide rules for tailor made enzyme assemblies for
future industrial applications related with lignocellulosic feedstock biorefinery. The project will provide an opportunity for the
fellow to come back to science and the in depth training in molecular biology, protein engineering and structure-function of
carbohydrate modifying enzymes will allow him to become an independent researcher.
Research on lignocellulosic biomass (LCB) has been continuously in progress due to its characteristic abundance and renewability. It is an environmentally friendly source of energy and added valued compounds settling the basis for a circular bioeconomy. However, its complete exploitation is mired due to its complex chemical and structural composition forcing microorganisms to deploy diverse strategies based on the action of a wide array of enzymatic activities to achieve its complete hydrolysis. One intriguing example produced by some anaerobic cellulolytic bacteria is the cellulosome, a multienzyme complex that is highly efficient for LCB deconstruction. This relies on substrate targeting and enzyme proximity allowing substrate channelling and synergistic action (Artzi et al., 2017) ). Therefore suggesting that the spatial organisation of the enzymes in this nanomachines plays an important role in catalysis. However, cellulosome efficiency is mainly guided by its flexibility (Andrade Pinheiro et al., 2012), making difficult to investigate the effect of enzymatic spatial proximity during catalysis.
Recently, Enjalbert et al. (2020) circumvented this issue by locking the spatial topology between two hemicellulases using the Jo-In biomolecular welding system (Bonnet et al., 2017); based on two proteins, Jo and In, which are able to form a covalent bond spontaneously, creating a post translational rigid linker.
In this work, we investigated the spatial proximity effect of two hemicellulases from a bacteroides xylan utilization loci (XUL) belonging to termite’s microbiote . This XUL encodes several hemicellulases constituting an interesting model to explore enzyme synergism and spatial organization of related carbohydrate active enzymes. Exploiting the antiparallel association between Jo and In, we achieved the construction, production in vivo, and initial characterization of two different chimeric enzyme spatial organisation called C4 and C12 using a GH11 xylanase and a GH43 xylosidase.
The complexes were produced in E. coli and purified after two chromatographic steps yielding around 35 and 31.5 mg/L for C4 and C12, respectively. The specific activities of the pure complexes against p-nitrophenyl-β-D-xylopyranoside, wheat arabinoxylan (WAX) and wheat bran have been assayed and compared to an equimolar amount of wild type enzymes free in solution. The results show how effectively there is a clear effect of the complex structure on its capacity to hydrolase the substrate, being the activity of C4 approximately 1.5-fold higher than the one for C12 on wheat bran. In parallel, the complexes were analysed by cryoelectron microscopy to gain information on structural organisation, this work provided initial conditions for freezing prior to cryoEM.
The results have been presented in the Lignobiotech research conference, in Vancouver August 2022, and spatial organisation study were used to publish a review in a special issue on carbohydrate modifying enzymes in Essays in biochemistry.
Recently, Enjalbert et al. (2020) circumvented this issue by locking the spatial topology between two hemicellulases using the Jo-In biomolecular welding system (Bonnet et al., 2017); based on two proteins, Jo and In, which are able to form a covalent bond spontaneously, creating a post translational rigid linker.
In this work, we investigated the spatial proximity effect of two hemicellulases from a bacteroides xylan utilization loci (XUL) belonging to termite’s microbiote . This XUL encodes several hemicellulases constituting an interesting model to explore enzyme synergism and spatial organization of related carbohydrate active enzymes. Exploiting the antiparallel association between Jo and In, we achieved the construction, production in vivo, and initial characterization of two different chimeric enzyme spatial organisation called C4 and C12 using a GH11 xylanase and a GH43 xylosidase.
The complexes were produced in E. coli and purified after two chromatographic steps yielding around 35 and 31.5 mg/L for C4 and C12, respectively. The specific activities of the pure complexes against p-nitrophenyl-β-D-xylopyranoside, wheat arabinoxylan (WAX) and wheat bran have been assayed and compared to an equimolar amount of wild type enzymes free in solution. The results show how effectively there is a clear effect of the complex structure on its capacity to hydrolase the substrate, being the activity of C4 approximately 1.5-fold higher than the one for C12 on wheat bran. In parallel, the complexes were analysed by cryoelectron microscopy to gain information on structural organisation, this work provided initial conditions for freezing prior to cryoEM.
The results have been presented in the Lignobiotech research conference, in Vancouver August 2022, and spatial organisation study were used to publish a review in a special issue on carbohydrate modifying enzymes in Essays in biochemistry.
The work carried out in the EvoXUL project could lay the foundations, in the more or less near future, for obtaining new custom-formulated enzymatic cocktails for the degradation of recalcitrant polysaccharides of the plant cell wall. Thus, in a context of circular bioeconomy, agroforestry waste could be a substrate for these new enzymatic cocktails and its degradation would allow obtaining biomolecules that would be used as building blocks for the production of cleaner and more environmentally friendly fuels, new renewable materials for construction, textile industry, ... precursors of compounds of pharmaceutical interest. Undoubtedly, further development of the technology and procedures established during the EvoXUL project for the production of these new enzyme cocktails could attract the interest of established companies in the sector. In addition, the knowledge generated in the project, and is the one that has yet to be obtained, lays the foundations for knowing how the spatial organization in multiprotein complexes affects their function, which could have clear repercussions in fields other than biotechnology such as biomedicine, only we have to think that inside the cell most of the chemical reactions are produced not by isolated enzymes but by protein complexes that can be more or less transitory.
Overall, the work developed in the EvoXUL project contributes towards European policy objectives, specifically its scope is inside of the Sustainable Development Goals of the United Nations: goal 7 (affordable and clean energy) and goal 12 (responsible consumption and production).
Overall, the work developed in the EvoXUL project contributes towards European policy objectives, specifically its scope is inside of the Sustainable Development Goals of the United Nations: goal 7 (affordable and clean energy) and goal 12 (responsible consumption and production).