Identification of Additive Formulations for Enhanced Shelf-Life and Productivity of E. coli Cell Extracts

Cell-Free Protein Synthesis (CFPS) is a platform biotechnology that enables a breadth of applications. However, field applications remain limited due to the poor shelf-stability of aqueous cell extracts required for CFPS. Lyophilization of E. coli extracts improves shelf-life but remains insufficient for extended storage at room temperature. To address this limitation, we mapped the chemical space of ten low-cost additives with four distinct mechanisms of action in a combinatorial manner to identify formulations capable of stabilizing lyophilized cell extract. We report three key findings: 1) unique additive formulations that maintain full productivity of cell extracts stored at 4ËšC and 23ËšC; 2) additive formulations that enhance extract productivity by nearly 2-fold; 3) a machine learning algorithm that provides predictive capacity for the stabilizing effects of additive formulations. These findings provide a simple and low-cost advance toward making CFPS field-ready and cost-competitive for biomanufacturing.