Abstract
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.
Highlights
Synthetic glucocorticoids are often used as immunosuppressants or for hormone replacement therapy compensating enzymatic disorders or deficiencies to treat various diseases like rheumatoid arthritis (Kadmiel & Cidlowski, 2013; McMaster & Ray, 2008)
High coaffinity of these glucocorticoidal ligands to the mineralocorticoid receptor (MR) causing an aldosterone‐like response (Farman & Rafestin‐Oblin, 2017). Avoiding such side effects while increasing the therapeutic effect of steroidal drugs is a realistic goal and is often achieved by steroid modification. This can be illustrated well using the example of the synthetic glucocorticoid methylprednisolone, which exhibits reduced MR affinity and multiplied glucocorticoid potency compared with its unmethylated derivative, prednisolone
The C21 hydroxylation of medrane has been established in a recombinant Escherichia coli (E. coli) whole‐cell system that enables the CYP21A2‐mediated production of premedrol, the precursor of methylprednisolone (Brixius‐Anderko, Schiffer, Hannemann, Janocha, & Bernhardt, 2015)
Summary
Synthetic glucocorticoids are often used as immunosuppressants or for hormone replacement therapy compensating enzymatic disorders or deficiencies to treat various diseases like rheumatoid arthritis (Kadmiel & Cidlowski, 2013; McMaster & Ray, 2008). These changes in the steroid framework modify the physiological properties such as solubility, skin or intestinal absorption, or steroid receptor affinity (Diederich et al, 2015) The latter influences the suitability of the active substance for targeted medication, as natural glucocorticoids often cause unwanted side effects that typically manifest themselves in hypertension, hypokalemia, osteoporosis, and other symptoms (Henzen, 2003; Schäcke, 2002; Schäcke et al, 2004). There is evidence that the rate‐ limiting transfer of the second electron can be supported by an alternative bypass‐pathway via cytochrome b5, affecting product formation velocity and efficiency (Vergères & Waskell, 1995) Another possible limitation that can occur in recombinant whole‐ cell biocatalysis is the molecular obstruction of substrate access to the active site (Wade, Winn, Schlichting, & Sudarko, 2004), which can be modulated by substitution of the relevant residues that cause steric or polar constraints. We addressed all of these possible obstacles and, (a) improved the efficiency of electron supply by using the modified natural redox partner bCPR−27 and the coexpression of cytochrome b5, (b) improved substrate conversion by molecular modification of the substrate access channel, and (c) bypassed substrate or product inhibition by performing fed‐batch cultivation of E. coli
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
