A funnel-type pipeline for accelerating discovery of tailored designer xylanosomes for efficient arabinoxylan...

Designer cellulosomes (DCs) are synthetic multi-enzyme complexes composed of a scaffold and docked enzymes, engineered for efficient lignocellulose degradation. However, designing these systems is challenged by an expansive design space, low protein yields, instability, and aggregation. To overcome these significant hurdles, we developed a novel, funnel-type methodological pipeline for the parallel construction, production, and analysis of designer xylanosomes (DXs), a subset of DCs targeting hemicellulose's xylan fraction. This streamlined approach leverages VersaTile, a combinatorial DNA assembly technique, to generate an unprecedented number of modular proteins. We demonstrate the efficient construction of 96 bicatalytic dockerin-enzyme (DE) fusions. Parallel high-throughput assays, including Enzyme-Linked ImmunoSorbent Assay (ELISA) and DNA Sequencer-Assisted Fluorophore-Assisted Carbohydrate Electrophoresis (DSA-FACE), enabled rapid analysis of DE binding efficiencies and substrate preferences, facilitating selection of optimal components for downstream processing. Subsequently, we assembled 23 unique DXs and systematically evaluated their assembly capacity and hydrolytic efficiency. Our findings reveal that DX hydrolytic efficiencies are highly dependent on composition and architecture, yielding either specific arabinoxylan-oligosaccharide (AXOS) mixtures or more complete degradation to monomers. This diversity allows for tailored outcomes, leading to the identification of three superior DX variants. This innovative pipeline significantly accelerates the discovery and optimization of highly efficient multi-enzyme complexes, paving the way for more effective biomass valorization processes.