Title: Covalent Jo-In complexes for the assembly of artificial cellulosomes
Abstract:
Lignocellulose is the primary structural component of plant cell wall, and Earth’s most abundant renewable organic resource[1,2]. Certain anaerobic bacteria are able to efficiently degrade lignocellulose through cellulosomes, large extracellular multienzyme assemblies with exceptional enzymatic diversity and catalytic synergy[3,4]. These systems have inspired the work of designer cellulosomes (DC) for biomass deconstruction[5,6]. However, DC architectures rely on non-covalent interactions, whose intrinsic flexibility and potential for cross-reactivity can limit the precise control of the fused enzymes[7,8]. This project aims to explore a covalent assembly system, named Jo-In, derived from the Streptococcus pneumoniae RrgA adhesin. This system facilitates the spontaneous formation of an intramolecular isopeptide bond, providing a robust and irreversible linkage[9,10]. This system has been previously validated in-house for in vitro enzyme fusion applications[11]. The project focuses on the identification and characterization of orthogonal Jo-In pairs to enable the modular assembly of multi-component enzymes with enhanced structural stability and defined stoichiometry. To achieve this, we screened multiple Jo-In candidates and evaluated their cross interaction through covalent bonding assays. We further investigated their 3D structure using X-ray crystallography, revealing a large interface within the complex, stabilized by two conserved salt bridges across all studied pairs. Guided by these structural insights, we engineered rational mutations that directly impacted complex formation. Finally, we examined the shape and size of a newly designed Jo-In fusion pair using Small-Angle X-ray Scattering (SAXS) experiments, providing low-resolution envelopes consistent with the expected overall conformation.
