Taxadiene-5α-ol is a minor product of CYP725A4 when expressed in Escherichia coli.

CYP725A4 is a P450 enzyme from Taxus cuspidata that catalyzes the formation of taxadiene-5-ol (T5-ol) from taxadiene in paclitaxel biosynthesis. Past attempts expressing CYP725A4 in heterologous hosts reported the formation of 5(12)-oxa-3(11)-cyclotaxane (OCT) and/or 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) instead of, or in addition to, T5-ol. Here, we report that T5-ol is produced as a minor product by Escherichia coli expressing both taxadiene synthase and CYP725A4. The major products were OCT and iso-OCT, while trace amounts of unidentified monooxygenated taxanes were also detected by gas chromatography-mass spectrometry. Since OCT and iso-OCT had not been found in nature, we tested the hypothesis that protein-protein interaction of CYP725A4 with redox partners, such as cytochrome P450 reductase (CPR) and cytochrome b5, may affect the products formed by CYP725A4, possibly favoring the formation of T5-ol over OCT and iso-OCT. Our results show that coexpression of CYP725A4 with CPR from different organisms did not change the relative ratios of OCT, iso-OCT, and T5-ol, while cytochrome b5 decreased overall levels of the products formed. Although unsuccessful in finding conditions that promote T5-ol formation over other products, we used our results to clarify conflicting claims in the literature and discuss other possible approaches to produce paclitaxel via metabolic and enzyme engineering.