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Paclitaxel is a widely used anti-tumor drug. Currently, paclitaxel can only be extracted from plants or synthesized by chemical semi-synthesis, which cause environmental damage and cannot meet the growing demand. However, the complete biosynthetic pathway of paclitaxel is still not clear, which greatly limits the production of paclitaxel using methods such as synthetic biology. Here, we deduced the paclitaxel biosynthetic pathway by searching all possible intermediates in the paclitaxel synthesis pathway from the natural product databases. In addition, we performed the transcriptome sequencing of Taxus brevifolia and performed co-expression analysis of the identified genes in the paclitaxel synthesis pathway. All these results laid a solid foundation for the elucidation of paclitaxel biosynthetic pathway.