Paclitaxel, an FDA-approved anticancer drug derived from the yew tree (*Taxus brevifolia*), has a complex biosynthetic pathway involving multiple enzymes. Over the past two decades, significant challenges have been faced in reconstituting this pathway due to the promiscuity of heterologously expressed cytochromes P450, such as taxadiene 5α-hydroxylase (T5αH), which forms multiple products. This study addresses these challenges by structurally characterizing four new products of T5αH and demonstrating that overexpression of T5αH can lead to over-oxidation of the primary mono-oxidized products. By tuning the promoter strength for *T5αH* expression in *Nicotiana* plants, the authors observe a threefold increase in the accumulation of taxadien-5α-ol, the precursor to paclitaxel, while reducing the formation of confounding side-products. This optimized system allows for the successful reconstitution of an early paclitaxel biosynthetic network involving six characterized *Taxus* enzymes, with the final products purified and structurally characterized. The findings provide a crucial platform for the discovery of the remaining biosynthetic genes involved in paclitaxel biosynthesis.Paclitaxel, an FDA-approved anticancer drug derived from the yew tree (*Taxus brevifolia*), has a complex biosynthetic pathway involving multiple enzymes. Over the past two decades, significant challenges have been faced in reconstituting this pathway due to the promiscuity of heterologously expressed cytochromes P450, such as taxadiene 5α-hydroxylase (T5αH), which forms multiple products. This study addresses these challenges by structurally characterizing four new products of T5αH and demonstrating that overexpression of T5αH can lead to over-oxidation of the primary mono-oxidized products. By tuning the promoter strength for *T5αH* expression in *Nicotiana* plants, the authors observe a threefold increase in the accumulation of taxadien-5α-ol, the precursor to paclitaxel, while reducing the formation of confounding side-products. This optimized system allows for the successful reconstitution of an early paclitaxel biosynthetic network involving six characterized *Taxus* enzymes, with the final products purified and structurally characterized. The findings provide a crucial platform for the discovery of the remaining biosynthetic genes involved in paclitaxel biosynthesis.