Chitin-Functionalized Cell-Free System Enables Sustainable Biocatalysis and Gene Expression

Cell-free protein synthesis (CFPS) has proven invaluable for expressing a wide array of proteins and enzymes, boasting significant advantages, such as facile manipulation, rapid mass transfer, and high productivity. However, traditional cell-free expression systems typically operate in batch format reactions, posing challenges for enzyme reuse in biocatalysis and rendering supplemented gene templates nonrecyclable. To overcome this limitation, we introduce a chitin-functionalized CFPS system designed to immobilize in vitro-expressed enzymes and gene templates for sustainable biocatalysis and protein synthesis. At its core, this system capitalizes on the strong binding affinity between a chitin-binding domain (ChBD) and crystalline chitin nanofibers (ChNFs). Specifically, we engineer ChBD fusion with target proteins and enzymes, allowing for their cell-free expression and in situ immobilization on ChNFs. This facilitates the effortless recycling of enzymes for multiple biocatalytic reactions, while ChNFs-immobilized enzymes can also be deployed in continuous flow biocatalysis setups. Leveraging the ChBD-ChNF pairing, gene templates can likewise be immobilized and recycled for sustained gene expression. Our results demonstrate that the chitin-functionalized cell-free system significantly enhances CFPS performance through immobilization with ChNF materials. This work underscores the flexibility and robustness of cell-free systems, which can seamlessly integrate with advanced techniques from fields such as chemistry and materials science for impactful applications.