Developing a Minimal and Cost-Effective Cell-Free Biomanufacturing System Using an In Vitro Fluorescent Assay

Cell-free protein synthesis (CFPS) has the potential to reduce the cost of biologics manufacturing through simplified lysates, high-throughput workflows, and streamlined downstream purification; however, high reagent costs and inconsistent product yields remain significant barriers to industrial adoption. A key limitation is the lack of cost-effective, high-throughput methods to monitor metabolite dynamics during the CFPS optimization. To address this challenge, we developed a fluorescent assay that enables the real-time monitoring of transcription, translation, and key metabolite pools. The fluorescent assay was used to identify and address technical challenges and produce insights that were subsequently used to engineer a minimal CFPS platform that costs nearly 97.5% less than commercial systems but still attains comparable protein yields. More broadly, our work introduces a generalizable framework for interrogating and engineering CFPS systems through the real-time observation of transcription and metabolism.