Ideas Lab: CFIRE: PRESENT: PRotein Evolution in Spore-ENabled TXTL Systems

This proposal introduces a new way to grow the bioeconomy by making useful products using biology without relying on living cells. These “cell-free systems” have the potential to lower costs and speed up the production of essential chemicals if the right biological tools, or proteins, exist in the first place. The research combines three advanced technologies to that end: computational design of thousands of proteins at once; a shelf-stable system to test, transport, and use the designed proteins; and new-to-nature building blocks that expand what these proteins can do beyond the limits of basic biology. The team will test this approach in three key areas: reducing the cost of DNA synthesis, creating affordable vaccines for livestock and pets, and designing a powerful enzyme for chemical manufacturing. By creating robust techno-economic analyses (TEA), lowering the cost of protein production, and improving protein stability, the project can make cell-free systems more adaptable and resilient. Ultimately, the project aims to spark a wave of innovation that supports industry and reinforces U.S. leadership in biotechnology. This project proposes a hybrid in-vivo, in-vitro, and in-silico platform to overcome the high cost of product engineering and poor scalability of cell-free biosynthesis (CFB). By integrating four advanced technologies—(1) chip-based gene synthesis for large-scale sequence exploration, (2) bacterial spore display for both high-throughput functional screening and final product deployment, (3) incorporation of non-standard amino acids to expand protein functionalities, and (4) machine learning for design optimization—this work will establish a universal workflow for protein engineering and application from design to functional validation while significantly increasing throughput. Optimized proteins will be evolved using genetic code expansion and extensive sequence exploration, then deployed on spore particles that are physically stable, manufacturable, and functionally preserved. The generalizable design-to-display framework will be tested through three independent target applications: improving DNA synthesis via error removal, developing cost-effective viral antigen vaccines for animal health, and engineering specific enzymes for scalable production of industrially relevant chemicals. TEA is embedded throughout to inform R&D priorities, guide design choices, and ensure industrial relevance. Altogether, this approach has the potential to accelerate a new generation of cell-free manufacturing systems with transformative potential in biomaterials, healthcare, and agriculture—while also training the next generation of biomanufacturing talent. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.