Reconstruction of the Early Stage Paclitaxel Biosynthesis Pathway in <i>Escherichia coli</i> by Integrating Enzyme and Metabolic Engineering Approaches

Heterologous biosynthesis of paclitaxel (Taxol) has recently become a hot research topic due to its potential to alleviate resource scarcity and shorten production cycles. However, the poor expression and low efficiency of catalytic components and challenges in combinatorial regulation remain critical bottlenecks, hindering the efficient synthesis of paclitaxel and its intermediates. In this study, we constructed an early stage biosynthesis pathway of paclitaxel from glycerol in an Escherichia coli (E. coli) cell factory. A 10.3-fold increase in taxadiene titer (up to 131 mg/L) was achieved through the engineering of isopentenyl diphosphate isomerase and geranylgeranyl diphosphate synthase, accompanied by a substrate channeling strategy. Promoter and protein engineering were then employed to optimize the production of taxadien-5α-ol and taxadien-5α-yl-acetate, resulting in titers of 17.9 and 7.6 mg/L, respectively. Finally, cofactor engineering strategies involving intracellular heme supplementation and NADPH regeneration were applied to synthesize taxadiene-5α,10β-diol-5-acetate, yielding titers of 8.7 mg/L in shake-flask cultivation and 18.5 mg/L in a 5 L jar fermenter. To the best of our knowledge, this represents the highest level reported to date in E. coli. The engineered strains and enzymes constructed in this study will serve as a solid foundation for subsequent paclitaxel biosynthesis, and the engineering strategies employed here may be applied to the de novo biosynthesis of terpenoids in microbial cell factories.