Self-Assembling Protein Scaffold System for Easy in Vitro Coimmobilization of Biocatalytic Cascade Enzymes

Biocatalytic cascades represent an attractive approach for the synthesis of valuable chemicals. To be competitive with chemical synthesis, cascade reactions need to be efficient, robust, self-sufficient, and ideally performed as one-pot in vitro reactions. Immobilization of enzymes has the potential to improve enzyme stability and increase reaction efficiency. However, coimmobilization of multiple different enzymes on the same solid surface is difficult, requiring optimized chemistry for each catalyst. To address this challenge, we developed an easy-to-adapt, genetically programmable and self-assembling protein scaffolding system for the simple immobilization of biocatalytic cascades. We adopted the self-assembling properties of the bacterial microcompartment protein EutM from Salmonella enterica to engineer scaffolds for covalent linkage with biocatalysts using SpyTag–SpyCatcher covalent bond formation. Our results show that our scaffolding system can be readily isolated from Escherichia coli, self-assembles, and remains stable in vitro under a range of conditions relevant for biocatalysis. Furthermore, cargo proteins spontaneously covalently attach to the protein scaffolds in vitro. As initial proof-of-concept, we coimmobilize a dual enzyme cascade for chiral amine synthesis and show that scaffolding of the cascade reduces the time required to reach final conversions, compared to the free enzyme system. Detailed analyses suggest that this may result from stabilization of the enzymes upon immobilization on the protein scaffolds. Together, these results establish the groundwork for future protein-based scaffolding of enzyme cascades for in vitro or in vivo biocatalysis.