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Taxadiene‐5α‐ol is a minor product of CYP725A4 when expressed in Escherichia coli
Author(s) -
SagwanBarkdoll Laxmi,
Anterola Aldwin M.
Publication year - 2017
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1606
Subject(s) - escherichia coli , enzyme , biochemistry , heterologous , cytochrome p450 reductase , paclitaxel , chemistry , cytochrome b5 , taxus , heterologous expression , cytochrome p450 , reductase , biosynthesis , stereochemistry , cytochrome c , biology , apoptosis , recombinant dna , gene , genetics , coenzyme q – cytochrome c reductase , botany , cancer
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.