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.
Bibliographical noteFunding Information:
We thank Dr. Kelly Aukema and Dr. James Christenson (University of Minnesota) for assistance in developing the GC procedures and Dr. Gail Celio at the University of Minnesota Imaging Center for preparing thin cell sections. This research was supported by grants from the National Science Foundation (MCB-12644429 to C.S.-D.) and Defense Threat Reduction Agency (HDTRA-15-0004 to C.S.-D.), and by funding from the Biotechnology Institute at the University of Minnesota through the Biocatalysis Initiative.
- chiral amine
- enzyme cascade
- protein scaffolds