Design and multidimensional optimization of cell-free system for efficient (2S)-naringenin production.

Naringenin is a valuable flavonoid with diverse pharmacological activities, but its industrial production is limited by low yields in microbial cell factories. Cell-free biosynthesis offers a promising alternative by decoupling product synthesis from cell growth. In this study, we established and systematically optimized an Escherichia coli-based cell-free system for naringenin production. We first compared three system architectures: cell-free metabolic engineering based on crude cell extraction (CFME), cell-free protein synthesis-driven metabolic engineering (CFPS-ME) and cell-free biosynthesis based on the 4CL plasmid (CFPS-4CL) and identified the CFPS-4CL system as the most efficient, achieving a 4-fold higher yield than the CFME system. We then screened orthologs of the four pathway enzymes (TAL, 4CL, CHS, CHI) from different sources and determined the optimal combination as RgTAL (Rhodotorula glutinis), At4CL4 (Arabidopsis thaliana), PhCHSA (Petunia hybrida), and ZmCHI (Zea mays). Subsequent optimization of enzyme concentrations, buffer components, cofactor availability, and substrate supply led to a final (2S)-naringenin yield of 2153.8 ± 60.3 mg/L with a productivity of 89.7 mg/L/h. This study constructs a stable, high-efficiency cell-free synthesis system for naringenin and provides solid theoretical foundations and practical experimental references for the in vitro bioproduction of various flavonoid compounds.