UPLC‐PDA coupled HR‐TOF ESI/MS 2 Based Identification of Derivatives Produced by Whole‐Cell Biotransformation of Epothilone A using Nocardia sp. CS692 and a Cytochrome P450 Overexpressing Strain

2021 ◽  
Author(s):  
Ramesh Prasad Pandey ◽  
Dipesh Dhakal ◽  
Samir Bahadur Thapa ◽  
Puspalata Bashyal ◽  
Tae‐Su Kim ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Whole Cell ◽  
Esi Ms ◽  
Epothilone A ◽  
Nocardia Sp ◽  
Whole Cell Biotransformation ◽  
Overexpressing Strain

scholarly journals Heterologous redox partners supporting the efficient catalysis of epothilone B biosynthesis by EpoK in Schlegelella brevitalea

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Junheng Liang ◽  
Huimin Wang ◽  
Xiaoying Bian ◽  
Youming Zhang ◽  
Guoping Zhao ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Electron Transport ◽  
Cytochrome P450 Enzyme ◽  
Whole Cell ◽  
Epothilone B ◽  
Redox Partners ◽  
Conversion Rates ◽  
Epothilone A ◽  
P450 Enzyme ◽  
Whole Cell Biotransformation

Abstract Background Epothilone B is a natural product that stabilizes microtubules, similar to paclitaxel (Taxol); therefore, epothilone B and several derivatives have shown obvious antitumour activities. Some of these products are in clinical trials, and one (ixabepilone, BMS) is already on the market, having been approved by the FDA in 2007. The terminal step in epothilone B biosynthesis is catalysed by the cytochrome P450 enzyme EpoK (CYP167A1), which catalyses the epoxidation of the C12–C13 double bond (in epothilone C and D) to form epothilone A and B, respectively. Although redox partners from different sources support the catalytic activity of EpoK in vitro, the conversion rates are low, and these redox partners are not applied to produce epothilone B in heterologous hosts. Results Schlegelella brevitalea DSM 7029 contains electron transport partners that efficiently support the catalytic activity of EpoK. We screened and identified one ferredoxin, Fdx_0135, by overexpressing putative ferredoxin genes in vivo and identified two ferredoxin reductases, FdR_0130 and FdR_7100, by whole-cell biotransformation of epothilone C to effectively support the catalytic activity of EpoK. In addition, we obtained strain H7029-3, with a high epothilone B yield and found that the proportion of epothilone A + B produced by this strain was 90.93%. Moreover, the whole-cell bioconversion strain 7029-10 was obtained; this strain exhibited an epothilone C conversion rate of 100% in 12 h. Further RT-qPCR experiments were performed to analyse the overexpression levels of the target genes. Gene knock-out experiments showed that the selected ferredoxin (Fdx_0135) and its reductases (FdR_0130 and FdR_7100) might participate in critical physiological processes in DSM 7029. Conclusion Gene overexpression and whole-cell biotransformation were effective methods for identifying the electron transport partners of the P450 enzyme EpoK. In addition, we obtained an epothilone B high-yield strain and developed a robust whole-cell biotransformation system. This strain and system hold promise for the industrial production of epothilone B and its derivatives.

CYP21-catalyzed production of the long-term urinary metandienone metabolite 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one: a contribution to the fight against doping

2010 ◽  
Vol 391 (1) ◽  
Author(s):  
Andy Zöllner ◽  
Maria Kristina Parr ◽  
Călin-Aurel Drăgan ◽  
Stefan Dräs ◽  
Nils Schlörer ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Reference Material ◽  
Anabolic Androgenic Steroids ◽  
Reference Substance ◽  
Cytochrome P450 Enzymes ◽  
Whole Cell ◽  
In Doping ◽  
Whole Cell Biotransformation ◽  
Androgenic Steroids

AbstractAnabolic-androgenic steroids are some of the most frequently misused drugs in human sports. Recently, a previously unknown urinary metabolite of metandienone, 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one (20OH-NorMD), was discovered via LC-MS/MS and GC-MS. This metabolite was reported to be detected in urine samples up to 19 days after administration of metandienone. However, so far it was not possible to obtain purified reference material of this metabolite and to confirm its structure via NMR. Eleven recombinant strains of the fission yeastSchizosaccharomyces pombethat express different human hepatic or steroidogenic cytochrome P450 enzymes were screened for production of this metabolite in a whole-cell biotransformation reaction. 17,17-Dimethyl-18-norandrosta-1,4,13-trien-3-one, chemically derived from metandienone, was used as substrate for the bioconversion, because it could be converted to the final product in a single hydroxylation step. The obtained results demonstrate that CYP21 and to a lesser extent also CYP3A4 expressing strains can catalyze this steroid hydroxylation. Subsequent 5 l-scale fermentation resulted in the production and purification of 10 mg of metabolite and its unequivocal structure determination via NMR. The synthesis of this urinary metandienone metabolite viaS. pombe-based whole-cell biotransformation now allows its use as a reference substance in doping control assays.

Whole-cell biotransformation with recombinant cytochrome P450 for the selective oxidation of Grundmann’s ketone

2014 ◽  
Vol 22 (20) ◽  
pp. 5586-5592 ◽  
Author(s):  
Alba Hernández-Martín ◽  
Clemens J. von Bühler ◽  
Florian Tieves ◽  
Susana Fernández ◽  
Miguel Ferrero ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Selective Oxidation ◽  
Whole Cell ◽  
Whole Cell Biotransformation

Asymmetric reduction of a key intermediate of eslicarbazepine acetate using whole cell biotransformation in a biphasic medium

2012 ◽  
Vol 2 (8) ◽  
pp. 1602 ◽  
Author(s):  
Manpreet Singh ◽  
Sawraj Singh ◽  
Sateesh Deshaboina ◽  
Hare Krishnen ◽  
Richard Lloyd ◽  
...  
Keyword(s):  
Asymmetric Reduction ◽  
Eslicarbazepine Acetate ◽  
Whole Cell ◽  
Whole Cell Biotransformation ◽  
Biphasic Medium

scholarly journals Characterization of P450 FcpC, the Enzyme Responsible for Bioconversion of Diosgenone to Isonuatigenone in Streptomyces virginiae IBL-14

2009 ◽  
Vol 75 (12) ◽  
pp. 4202-4205 ◽  
Author(s):  
Wei Wang ◽  
Feng-Qing Wang ◽  
Dong-Zhi Wei
Keyword(s):  
Escherichia Coli ◽  
Electron Transfer ◽  
Cytochrome P450 ◽  
Cytochrome P450 Monooxygenase ◽  
P450 Monooxygenase ◽  
Whole Cell ◽  
Electron Transfer Chain ◽  
Streptomyces Virginiae ◽  
Transfer Chain

ABSTRACT A new cytochrome P450 monooxygenase, FcpC, from Streptomyces virginiae IBL-14 has been identified. This enzyme is found to be responsible for the bioconversion of a pyrano-spiro steroid (diosgenone) to a rare nuatigenin-type spiro steroid (isonuatigenone), which is a novel C-25-hydroxylated diosgenone derivative. A whole-cell P450 system was developed for the production of isonuatigenone via the expression of the complete three-component electron transfer chain in an Escherichia coli strain.

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