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Abstract Biosynthesis of paclitaxel (Taxol™) is a hot topic with extensive and durable interests for decades. However, it is severely hindered due to the very low titers of intermediates. In this study, Escherichia coli was employed to de novo synthesize a key intermediate of paclitaxel, taxadien-5α-yl-acetate (T5OAc). Plasmid-based pathway reconstruction and optimization were conducted for T5OAc production. The endogenous methylerythritol phosphate pathway was enhanced to increase the precursor supply. Three taxadien-5α-ol O-acetyltransferases were tested to obtain the best enzyme for the acetylation step. Metabolic burden was relieved to restore cell growth and promote production through optimizing the plasmid production system. In order to achieve metabolic balance, the biosynthesis pathway was regulated precisely by multivariate-modular metabolic engineering. Finally, in a 5-L bioreactor, the T5OAc titer was enhanced to reach 10.9 mg/L. This represents an approximately 272-fold increase in production compared to the original strain, marking the highest yield of T5OAc ever documented in E. coli, which is believed to be helpful for promoting the progress of paclitaxel biosynthesis.