Ideas Lab: CFIRE: Upgrading Cell-free Cascades with Membraneless Compartmentalization

Award ID: 2452698

Upgrading Cell-free Cascades with Membraneless Compartmentalization This project is developing more efficient ways to make valuable organic chemicals outside of living cellsa process called cell-free biomanufacturing. Cell-free approaches can remove the background processes in cells that complicate chemical reactions. However, this comes at the cost of losing the advantages of biology at the same time, such as the different compartments within cells that make these reactions possible. This approach uses custom-designed enzymes and innovative materials that help organize chemical reactions without needing a cell, making the process easier to scale and more cost-effective. This could open the door to producing high-value products that support and enhance supply chain resilience, while training the next generation of biomanufacturing talent. By advancing this technology, the work supports a stronger U.S. bioeconomy and helps build a skilled workforce for the growing biomanufacturing industry. Cell-free enzymatic cascades present a powerful opportunity to operate biomanufacturing systems beyond the constraints of living organisms. However, removing cells also eliminates the natural spatial organization biology uses to separate competing pathways. In conventional one-pot systems, maintaining distinct local redox environments is not feasible, which restricts the simultaneous execution of multiple, directionally opposed redox reactions required to produce chiral chemicals. To address this limitation, this project introduces membrane-less coacervate droplets that encapsulate enzymes and artificial redox cofactors within spatially organized compartments. These coacervates and artificial cofactors are composed of low-cost, stable materials using simple, scalable chemistries. This design enables efficient cofactor recycling and enables precise, low-cost delivery of reducing and oxidizing powerall within a single reaction vessel. The result is a robust, reusable platform capable of sustaining multi-enzyme cascades for efficient synthesis. The project will focus on proving the technology through synthesis of high-value, enantio-enriched products such as chiral amines and alcoholsessential building blocks in pharmaceuticals and agrochemicals. Cell-free biomanufacturing addresses key inefficiencies of traditional synthesis of chiral chemicals, such as high production costs and difficulties in chiral separation, potentially opening a new avenue to produce these molecules. Complementing the experimental efforts, data-driven spatiotemporal modeling will generate predictive design rules and enable automated system optimization. The platform will deliver translatable, modular, and rapidly reconfigurable technology designed for broad applicability across diverse chemical targets. This academicindustry collaboration will accelerate innovation, reduce technical risk, and support workforce development in biochemical engineering and computational biosystemslaying the foundation for widespread adoption and future advances in cell-free biosynthesis. 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.

NSF Program Director: Waleed Nasser