Cell-free protein synthesis for next-generation biomanufacturing.

Abstract

Chemicals with diverse molecular structures are essential to the pharmaceutical, food, and specialty chemical industries; however, their sustainable production remains challenging due to complex cellular regulation, limited metabolic flexibility, and the scalability constraints of purified enzyme-based systems. Extract-based cell-free protein synthesis (CFPS) has emerged as a versatile biomanufacturing platform that enables the production of functional enzymes in vitro and provides an open, programmable environment for constructing synthetic biosynthetic systems beyond the constraints of living cells. By decoupling chemical synthesis from cellular growth, CFPS allows precise control over enzyme composition, pathway stoichiometry, and cofactor utilization, thereby improving catalytic efficiency and resource utilization. In this review, we summarize recent advances in extract-based CFPS for chemical biosynthesis and discuss key engineering strategies, including cofactor regeneration, pathway balancing, and semicontinuous operation, to enhance system performance. Furthermore, we highlight emerging opportunities to integrate CFPS with artificial intelligence-guided design, renewable feedstocks, and modular reactor technologies toward more sustainable and scalable biomanufacturing processes.

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