Final Report Summary - CELLUFUEL (Designer Cellulosomes by Single Molecule Cut & Paste) Cellulosomes are extracellular organelles, which enable certain bacteria to degrade lignocellulose. This project investigated the molecular architecture of these unique multi-enzyme devices. We discovered unknown molecular mechanisms for their mechanostability and developed novel technologies towards building man-made designer cellulosomes.In their different native ambients cellulosomes have to provide multiple functions. They anchor the bacteria to the cellulose fibrils through specialized carbohydrate binding modules and they orchestrate the ensemble of digestive enzymes where they have to ensure close proximity of the enzymes to the substrate and at the same time provide enough flexibility to allow enzymatic turnover. The mechanical properties of cellulosomes are therefore of utmost importance for their proper function and for the design of man-made cellulosomes for renewable energies. Their stability at elevated temperatures and their robustness against ambient changes makes them ideal building blocks for the design of novel supramolecular functions e.g. production of biofuel from wood residues or waste.We thus spent a substantial share of our research efforts towards elucidating the bio-mechanics of these molecular assemblies and their protein-constituents. Using AFM-based single molecule force spectroscopy (SMFS) we discovered extreme mechanostabilty in the non-covalent bonds between cohesins and dockerins, reaching force limits beyond 800 pN, values, which up to then had only be reached by covalent bonds. We implemented a new analysis mode in steered MD-simulations and were for the first time able to trace the force propagation pathways in the molecular complexes. With this combination we discovered a "force detour" in the binding interface as the molecular origin of their exceptional properties. This new hybrid of in-vitro and in-silico SMFS provided also a detailed picture of the mechanostability of the cohesin scaffolding at the level of the individual domains and culminated in the discovery that the stability of a particular native domain can be artificially enhanced threefold by the exchange of a single amino acid! Motivated by the need to extend the force range of our SMC&P handles we probed the stability limits of protein-protein interactions in other bacterial strains. We discovered extreme mechano-stability, where the interaction forces between the proteins is only limited by the strength of the covalent bonds of their anchorage. Values of more than 2.5 nN were found in the adhesion complexes of several staphylococcus strains making these ideally suited as all-protein handles for our SMC&P surface assembly. For the latter we developed a microfluidics device that allowed us to produce in an in-vitro expression a library of to 640 different spots within a few hours. This one-step process from the gene to the covalently attached protein product in a chip format proved to be ideally suited for the investigation of large numbers of different constituents and cellulytic enzyme libraries, which the different strains of bacteria provide. For the quantification of the cellulytic activity of our enzyme assemblies we developed novel assay formats based on a highly sensitive fluorescent readout. In the first assay we used the intrinsic fluorescence of the cellulose and in the second approach we employed a radical byproduct of the hydrolysis for the polymerization of a hydrogel with embedded fluorescent labels. In an alternative strategy we employed the AFM to image the local cellulytic degradation of extremely flat cellulose samples from Valonia ventricosa. As an alternative to surface assembled designer cellulosomes we also developed mini scaffoldings for the combination of various cellulytic enzymes based on site specific mild click chemistry. For this purpose, we developed several scaffolding components containing non-natural amino acids in chosen positions.