Engineering the paclitaxel biosynthetic pathway in Physcomitrium patens for de novo and stable biosynthesis

Paclitaxel is a highly valuable diterpenoid anticancer drug originally extracted from the yew tree. Due to the low efficiency of plant extraction and the high cost of semi-synthesis, efforts have been made to produce paclitaxel through biosynthesis. However, there have been no reports to date on engineered strains capable of paclitaxel biosynthesis. Here, we engineer a paclitaxel-producing line using the moss ( Physcomitrium patens ) chassis cells by combining transient expression verification, cytoplasmic localization strategies, self-cleaving 2 A peptide-mediated multi-gene co-expression systems and a modular iterative transformation strategy for stable genetic inheritance. Even after continuous subculturing multiple times, the engineered paclitaxel-producing lines obtained through both the initial reconstructed pathway and the optimized pathway are able to stably produce paclitaxel. This study proposes that Physcomitrium patens can serve as a plant chassis for paclitaxel biosynthesis.