Controlled spatial prOximity for iNnovating enzymatic Cascades to Enhance biomass deconstRucTiOn – Concerto

Today, the increase of the atmospheric CO2 originates from the utilisation of carbon fossil in our daily life. In addition to the development of alternative energy sources (solar, wind, nuclear), plant biomass is one of the main options to replace fuel for transportation sector. Thus, plant biomass is projected to play an important role in this European bioeconomy strategy. The second-generation biofuels relies on a cheap and abundant non-food material: the lignocellulose (LC). LC consists of a complex network of cellulose, hemicelluloses, lignin and proteins that cross-link with each other and is highly recalcitrant to chemical or biological degradation. However, this chemical complexity offers a vast potential in the development of biorefinery for renewable and sustainable molecules and materials for our daily lives. In Nature, lignocellulolytic microorganisms are able to metabolize and recycle plant-derived organic carbon. They achieve this by using complex arsenals of cell wall-degrading enzymes. Most of these enzymes display a modular architecture, composed of catalytic and non-catalytic modules. Some anaerobic bacterium produce a self-assembling multienzymatic complex anchored to the outer membrane that couple enzymes with complementary activities. Previous work has evidenced that spatial proximity is a key to the remarkable efficiency of the cellulosome but it is difficult to evaluate the effect of the distance and active site orientation on enzymatic synergism mainly because of the high flexibility of the cellulosome. Thus, controlling such spatial organization is of high importance to master enzymatic synergism and increase the efficiency yield of PCW deconstruction. To reach this goal, an original approach is required. CONCERTO propose to use the BioMolecular Welding tool, composed of two small proteins Jo and In that are able to spontaneously create an intramolecular isopeptidic bond. Once linked to each other, Jo-In is a rigid complex of around 6 nm wide, displaying available N- and C-terminus for fusion. Furthermore, the anti-parallel organization of Jo-In offers the ability to create chimeras and modulate the relative spatial organization of linked protein domains. However this technology is limited because there is only one pair of Jo-In, and no natural complementary pair exists. This limitation prevents from the development of more complex assemblies that are required to degrade LC. Therefore, in CONCERTO, we propose to tackle this limitation by developing new pair of Jo and In in order to create larger organization of multi-modular enzymes. Thanks to new pairs of Jo and In, original enzymatic complexes will be designed in CONCERTO and will be deeply investigating, on one hand by solving the structure of theses complexes in solution using SAXS analysis and on another hand by carefully characterising the product profile generated by such enzymatic complexes thanks to the development of an analytical strategy that does not exists today. We hypothesize that various spatial organization will alter the product profile of the hydrolysis of complex plant cell wall substrates, helping us to understand the catalytic activity/spatial organization/product profile relationship within nanomachine and paved the way toward the control of biomass deconstruction.