Eukaryotic cell-free protein synthesis: Chassis diversification, system engineering, and emerging applications

The eukaryotic cell-free protein synthesis (CFPS) system, endowed with intrinsic post-translational modification capabilities and a complex molecular chaperone network, efficiently synthesizes functional proteins with correct conformation and biological activity. This effectively compensates for the structural limitations of prokaryotic systems in expressing complex eukaryotic proteins. This paper aims to comprehensively review and analyze the latest advances in the field of eukaryotic CFPS from a systems engineering perspective. The paper delves into the diversification of host chassis, rational design of core reaction components, and the pivotal role of novel biomaterial integration and high-throughput reaction equipment development in system reconfiguration. At the application level, it summarizes the platform's latest achievements, including elucidation of fundamental mechanisms, complex protein engineering, and metabolic synthesis. It particularly highlights its potential in emerging areas such as the construction of artificial cells, the development of bioelectronic interfaces, and the design of microarray chips. Furthermore, addressing the current standardization and cost bottlenecks hindering industrialization, this paper proposes a solution strategy based on artificial intelligence and synthetic biology tools, aligning with the shift from empirical trial-and-error to rational design paradigms. By integrating the current technological landscape with emerging trends, this review aims to provide theoretical references and practical guidance for constructing an economical, high-throughput eukaryotic cell-free biomanufacturing platform.