Construction and optimization of a cell-free metabolic engineering system for lycopene synthesis

In this study, a cell-free metabolic engineering system was designed for the synthesis of lycopene in vitro. Initially, comprehensive bioinformatic analysis was performed on three key enzymes from Deinococcus wulumuqiensis R12, geranylgeranyl pyrophosphate synthase, phytoene synthase and phytoene desaturase to assess their phylogenetic relationships and characterize functional domains. Subsequently, heterologous expression systems were constructed to enable soluble overexpression of these enzymes in recombinant Escherichia coli. The cell-free metabolic engineering synthesis system was constructed and optimized, based on the purified enzymes and crude extracts from recombinant Escherichia coli for lycopene synthesis. After comparing the two different platforms and optimizing various key parameters, including enzyme concentration, substrate concentration, enzyme ratio, cofactors, and metal ions, a lycopene titer of 14.06 mg/L was achieved in the cell-free system after 24 h. Finally, the synthetic system was employed to validate a rapid enzyme screen and to validate lycopene synthesis in vivo. This construction strategy demonstrates three major applications of CFME in rapid optimization of cell-free systems based on purified enzymes, the optimization of cell-free systems based on crude cell extracts and the rapid enzyme screening and provides a referable research strategy and synthesis platform for the design and optimization of multienzyme pathways for the synthesis of complex compounds such as carotenoids.