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

Abstract

CYP725A4 is a P450 enzyme from Taxus cuspidata that catalyzes the formation of taxadiene-5α-ol (T5α-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, T5α-ol. Here, we report that T5α-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 T5α-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 T5α-ol, while cytochrome b5 decreased overall levels of the products formed. Although unsuccessful in finding conditions that promote T5α-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.

Chloroplastic metabolic engineering coupled with isoprenoid pool enhancement for committed taxanes biosynthesis in Nicotiana benthamiana

“…These results demonstrate that proper, targeted localization of enzymes is critical to heterologous reconstruction of Taxol biosynthetic pathway, and co-localization in the plastid can lead to higher yields of the oxygenated product, possibly due to the richer isoprenoid precursor pool supplied by the MEP pathway. However, consistent with previous reports on T5αH engineering in E.coli 15,16 , we also identified OCT (Supplementary Fig. 5) and iso-OCT (Supplementary Fig.…”

“…Low expression and poor functionality of CYPs involved in decoration of the taxadiene olefin ring is the major stumbling block hampering efforts to further construct the pathway past taxadiene-5α-ol in yeast and E. coli 13,15–17 . Compared to classical microbial and yeast chassis, plants seem more compatible with heterologous expression of plant enzymes like cytochrome P450s in terms of availability of suitable endomembranes, cofactor availability and protein folding machinery 37 .…”

Enhanced production of taxadiene in Saccharomyces cerevisiae

“…6 . The mass spectra of the peaks at 7.43 and 7.88 min (Additional file 1 : Figures S2-A, C) showed a high degree of similarity to compounds previously identified as verticillene and the taxadiene isomer, taxa-4(20),11(12)-diene (iso-taxadiene), respectively [ 14 , 16 ]. Verticillene is a bridged bicycle product formed as part of a series of carbocation-mediated cyclisations involved in taxadiene synthesis from acyclic GGPP [ 29 ].…”

“…Despite this, to minimise the production of these side products, increasing the metabolic flux towards GGPP formation seems necessary. Although paclitaxel biosynthesis can also proceed through iso-taxadiene, taxadiene is the main precursor in paclitaxel biosynthesis pathway [ 16 ]. However, to increase the selectivity of TASY towards taxadiene formation, enzyme engineering approaches are essential.…”

“…Although taxadiene has been found to be the major product of TASY, with yields over 77%, around 5–13% of the total taxane product has been found to be the isomer taxa-4(20),11(12)-diene (iso-taxadiene) [ 14 – 16 ]. Small quantities of a product tentatively identified as verticillene and an additional taxadiene isomer (taxa-3(4),11(12)-diene) have also been detected in E. coli and Nicotiana benthamiana [ 16 , 17 ].…”