A CYP21A2 based whole-cell system in Escherichia coli for the biotechnological production of premedrol

Additional file 4: Fig. S4. In vitro conversion of medrane with the redox systems AdR/Adx/CYP21A2 or arh1/Adx/CYP21A2 with either NADH or NADPH. 400 ÎźM Medrane was converted in a reconstituted in vitro assay with Adx based redox systems containing AdR or arh1 as reductase. To each system either NADH or NADPH was added to verify the ability of arh1 to receive electrons from NADH. AdR served as a negative control. All values represent the mean of triplicates with the respective standard deviation

Identification and circumvention of bottlenecks in CYP21A2-mediated premedrol production using recombinant Escherichia coli

Synthetic glucocorticoids such as methylprednisolone are compounds of fundamental interest to the pharmaceutical industry as their modifications within the sterane scaffold lead to higher inflammatory potency and reduced side effects compared with their parent compound cortisol. In methylprednisolone production, the complex chemical hydroxylation of its precursor medrane in position C21 exhibits poor stereo‐ and regioselectivity making the process unprofitable and unsustainable. By contrast, the use of a recombinant E. coli system has recently shown high suitability and efficiency. In this study, we aim to overcome limitations in this biotechnological medrane conversion yielding the essential methylprednisolone‐precursor premedrol by optimizing the CYP21A2‐based whole‐cell system on a laboratory scale. We successfully improved the whole‐cell process in terms of premedrol production by (a) improving the electron supply to CYP21A2; here we use the N‐terminally truncated version of the bovine NADPH‐dependent cytochrome P450 reductase (bCPR−27) and coexpression of microsomal cytochrome b5; (b) enhancing substrate access to the heme by modification of the CYP21A2 substrate access channel; and (c) circumventing substrate inhibition which is presumed to be the main limiting factor of the presented system by developing an improved fed‐batch protocol. By overcoming the presented limitations in whole‐cell biotransformation, we were able to achieve a more than 100% improvement over the next best system under equal conditions resulting in 691 mg·L−1·d−1 premedrol

Discovery of Novel Non-Steroidal Cytochrome P450 17A1 Inhibitors as Potential Prostate Cancer Agents

The current study presents the design, synthesis, and evaluation of novel cytochrome P450 17A1 (CYP17A1) ligands. CYP17A1 is a key enzyme in the steroidogenic pathway that produces androgens among other steroids, and it is implicated in prostate cancer. The obtained compounds are potent enzyme inhibitors (sub µM) with antiproliferative activity in prostate cancer cell lines. The binding mode of these compounds is also discussed

Nothing wrong with me

An assistant professor and group leader explains how being diagnosed with autism in her early 40s changed her approach to being a scientist

Biotechnologische Anwendung steroidogener Cytochrome P450

Synthetic glucocorticoids are of high pharmaceutical value due to their anti-inflammatory and immunosuppressive effects. The low-yield chemical synthesis of glucocorticoids consists of multiple steps and includes toxic compounds as well as unwished by-product formation. Thus, one intends to replace this process by a more sustainable biocatalytic alternative. The work presented here demonstrates an enzyme-mediated approach to produce the synthetic glucocorticoid premedrol, a precursor of methylprednisolone (medrol), by a one-step hydroxylation at carbon atom 21catalyzed by bovine CYP21A2, which belongs to the protein superfamily of cytochromes P450. Therefore, a CYP21A2-based whole-cell system was established, by which a maximum yield of 0.65 g/L premedrol could be achieved - a promising perspective for an industrial application. With regard to a molecular evolution of CYP21A2 as well as an elucidation of the biocatalytic ability of orphan CYPs in a high-throughput microtiter scale, the indole-deficient Escherichia coli strain C43(DE3)_∆tnaA was generated, since indole is an inhibitor of some CYPs and massively impedes a screening. Applying C43(DE3)_∆tnaA in biotransformation resulted in a significantly higher product formation in case of CYP21A2 as well as of CYP264A1 from the myxobacterium Sorangium cellulosum So ce56 and thus, serves as convenient host in future screening procedures.Synthetische Glucocorticoide sind aufgrund ihrer antiinflammatorischen und immunsuppressiven Wirkung von hohem pharmazeutischem Wert. Die chemische Synthese von Glucocorticoiden besteht aus mehreren Schritten und beinhaltet die Verwendung toxischer Substanzen als auch die Bildung unerwünschter Nebenprodukte. Daher soll dieser Prozess durch ein nachhaltigeres biokatalytisches Verfahren ersetzt werden. Die vorliegende Arbeit demonstriert eine biokatalytische Herangehensweise zur Produktion des synthetischen Glucocorticoids Premedrol, einem Vorläufer von Methylprednisolon (Medrol), durch eine einstufige Hydroxylierung am C21, welche durch das zur Proteinsuperfamilie der Cytochrome P450 gehörende steroidogene bovine CYP21A2 katalysiert wird. Daher wurde ein CYP21A2-basiertes Ganzzellsystem etabliert, mit Hilfe dessen eine maximale Premedrolausbeute von 0,65 g/l erzielt werden konnte – eine vielversprechende Perspektive im Hinblick auf eine industrielle Anwendung. Hinsichtlich einer molekularen Evolution von CYP21A2 und einer Aufklärung des biokatalytischen Potentials unbekannter CYPs durch ein Hochdurchsatz-Screening wurde der Indol defiziente Escherichia coli Stamm C43(DE3)_∆tnaA generiert, da Indol einige CYPs inhibiert und somit ein Screening erschwert. Ein Umsatz mit C43(DE3)_∆tnaA resultierte in einer signifikant höheren Produktbildung im Falle des CYP21A2 und des CYP264A1 aus Sorangium cellulosum So ce56 und ist somit ein geeigneter Wirtsorganismus für künftige Screeningverfahren

Supporting nonlinear careers to diversify science.

Nonlinear careers through academia are increasingly common, but funding agencies and search committees penalize these paths. Why do scientists stray from the beaten path, how do they contribute to science, and how do we level the playing field

Supporting nonlinear careers to diversify science.

Nonlinear careers through academia are increasingly common, but funding agencies and search committees penalize these paths. Why do scientists stray from the beaten path, how do they contribute to science, and how do we level the playing field

Metabolism of Oral Turinabol by Human Steroid Hormone-Synthesizing Cytochrome P450 Enzymes

The human mitochondrial cytochrome P450 enzymes CYP11A1, CYP11B1, and CYP11B2 are involved in the biosynthesis of steroid hormones. CYP11A1 catalyzes the side-chain cleavage of cholesterol, and CYP11B1 and CYP11B2 catalyze the final steps in the biosynthesis of gluco-and mineralocorticoids, respectively. This study reveals their additional capability to metabolize the xenobiotic steroid oral turinabol (OT; 4-chlor-17 beta-hydroxy-17 alpha-methylandrosta-1,4-dien-3-on), which is a common doping agent. By contrast, microsomal steroid hydroxylases did not convert OT. Spectroscopic binding assays revealed dissociation constants of 17.7 mu M and 5.4 mu M for CYP11B1 and CYP11B2, respectively, whereas no observable binding spectra emerged for CYP11A1. Catalytic efficiencies of OT conversion were determined to be 46 min(-1) mM(-1) for CYP11A1, 741 min(-1) mM(-1) for CYP11B1, and 3338 min(-1) mM(-1) for CYP11B2, which is in the same order of magnitude as for the natural substrates but shows a preference of CYP11B2 for OT conversion. Products of OT metabolism by the CYP11B subfamily members were produced at a milligram scale with a recombinant Escherichia coli-based whole-cell system. They were identified by nuclear magnetic resonance spectroscopy to be 11 beta-OH-OT for both CYP11B isoforms, whereby CYP11B2 additionally formed 11 beta, 18-diOH-OT and 11 beta-OH-OT-18-al, which rearranges to its tautomeric form 11 beta, 18-expoxy-18-OH-OT. CYP11A1 produces six metabolites, which are proposed to include 2-OH-OT, 16-OH-OT, and 2,16-diOH-OT based on liquid chromatography-tandem mass spectrometry analyses. All three enzymes are shown to be inhibited by OT in their natural function. The extent of inhibition thereby depends on the affinity of the enzyme for OT and the strongest effect was demonstrated for CYP11B2. These findings suggest that steroidogenic cytochrome P450 enzymes can contribute to drug metabolism and should be considered in drug design and toxicity studies

Metabolism of Oral Turinabol by Human Steroid Hormone-Synthesizing Cytochrome P450 Enzymes

ABSTRACT The human mitochondrial cytochrome P450 enzymes CYP11A1, CYP11B1, and CYP11B2 are involved in the biosynthesis of steroid hormones. CYP11A1 catalyzes the side-chain cleavage of cholesterol, and CYP11B1 and CYP11B2 catalyze the final steps in the biosynthesis of gluco-and mineralocorticoids, respectively. This study reveals their additional capability to metabolize the xenobiotic steroid oral turinabol (OT; 4-chlor-17b-hydroxy-17a-methylandrosta-1,4-dien-3-on), which is a common doping agent. By contrast, microsomal steroid hydroxylases did not convert OT. Spectroscopic binding assays revealed dissociation constants of 17.7 mM and 5.4 mM for CYP11B1 and CYP11B2, respectively, whereas no observable binding spectra emerged for CYP11A1. Catalytic efficiencies of OT conversion were determined to be 46 min 21 mM 21 for CYP11A1, 741 min 21 mM 21 for CYP11B1, and 3338 min 21 mM 21 for CYP11B2, which is in the same order of magnitude as for the natural substrates but shows a preference of CYP11B2 for OT conversion. Products of OT metabolism by the CYP11B subfamily members were produced at a milligram scale with a recombinant Escherichia coli-based whole-cell system. They were identified by nuclear magnetic resonance spectroscopy to be 11b-OH-OT for both CYP11B isoforms, whereby CYP11B2 additionally formed 11b,18-diOH-OT and 11b-OH-OT-18-al, which rearranges to its tautomeric form 11b,18-expoxy-18-OH-OT. CYP11A1 produces six metabolites, which are proposed to include 2-OH-OT, 16-OH-OT, and 2,16-diOH-OT based on liquid chromatography-tandem mass spectrometry analyses. All three enzymes are shown to be inhibited by OT in their natural function. The extent of inhibition thereby depends on the affinity of the enzyme for OT and the strongest effect was demonstrated for CYP11B2. These findings suggest that steroidogenic cytochrome P450 enzymes can contribute to drug metabolism and should be considered in drug design and toxicity studies