Construction and Optimization of a Lycopene Synthesis Pathway Based on a Cell-Free Synthesis System

Abstract

In this study, a cell-free metabolic engineering system was designed for the synthesis of lycopene in vitro. Firstly, the key enzymes for lycopene synthesis, geranylgeranyl pyrophosphate synthase, phytoene synthase and phytoene desaturase from Deinococcus wulumuqiensis R12 were bioinformatically analyzed to assess their phylogenetic relationships and functional characteristics. Secondly, the source of gene crtB encoding phytoene synthase was screened, after which recombinant strains that could express the three key enzymes in large amounts solubilistically were constructed. Thirdly, a cell-free metabolic engineering synthesis system based on the purified enzymes and crude extracts from recombinant Escherichia coli for lycopene synthesis was constructed and optimized respectively. After optimizing the 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 used to validate a rapid enzyme screen and to validate lycopene synthesis in vivo. This construction strategy 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.

Keywords: Lycopene, multienzyme cascade, purified enzymes, crude extract, cell-free metabolic engineering