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

Abstract

BackgroundSynthetic glucocorticoids like methylprednisolone (medrol) are of high pharmaceutical interest and represent powerful drugs due to their anti-inflammatory and immunosuppressive effects. Since the chemical hydroxylation of carbon atom 21, a crucial step in the synthesis of the medrol precursor premedrol, exhibits a low overall yield because of a poor stereo- and regioselectivity, there is high interest in a more sustainable and efficient biocatalytic process. One promising candidate is the mammalian cytochrome P450 CYP21A2 which is involved in steroid hormone biosynthesis and performs a selective oxyfunctionalization of C21 to provide the precursors of aldosterone, the main mineralocorticoid, and cortisol, the most important glucocorticoid. In this work, we demonstrate the high potential of CYP21A2 for a biotechnological production of premedrol, an important precursor of medrol.ResultsWe successfully developed a CYP21A2-based whole-cell system in Escherichia coli by coexpressing the cDNAs of bovine CYP21A2 and its redox partner, the NADPH-dependent cytochrome P450 reductase (CPR), via a bicistronic vector. The synthetic substrate medrane was selectively 21-hydroxylated to premedrol with a max. yield of 90 mg L−1 d−1. To further improve the biocatalytic activity of the system by a more effective electron supply, we exchanged the CPR with constructs containing five alternative redox systems. A comparison of the constructs revealed that the redox system with the highest endpoint yield converted 70 % of the substrate within the first 2 h showing a doubled initial reaction rate compared with the other constructs. Using the best system we could increase the overall yield of premedrol to a maximum of 320 mg L−1 d−1 in shaking flasks. Optimization of the biotransformation in a bioreactor could further improve the premedrol gain to a maximum of 0.65 g L−1 d−1.ConclusionsWe successfully established a CYP21-based whole-cell system for the biotechnological production of premedrol, a pharmaceutically relevant glucocorticoid, in E. coli and could improve the system by optimizing the redox system concerning reaction velocity and endpoint yield. This is the first step for a sustainable replacement of a complicated chemical low-yield hydroxylation by a biocatalytic cytochrome P450-based whole-cell system.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0333-2) contains supplementary material, which is available to authorized users.

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

“…The presented premedrol production here did not exceed 320 mg L -1 as observed by Brixius-Anderko et al using arh1/ etp fd , since the latter yield was obtained by multiple addition of lower substrate concentrations summed up to 1 mM. However, comparing premedrol production obtained by non-successive substrate addition, using the bCPR −27 increased the premedrol production 2.4-fold compared with premedrol production supported by WT CPR (Brixius-Anderko et al, 2015).…”

“…The visibility of the supportive optimizations was limited by single-step substrate application, which we finally uncovered by successive substrate feeding. Applying the enhanced fed-batch protocol together with the use of bCPR −27 and CYP21A2_M210V as well as the coexpression of cytochrome b 5 enabled us to produce up to 691 mg·L −1 ·d −1 premedrol within a biotechnological E. coli based whole-cell system which is a more than a 100% improvement compared with our previously reported system under similar conditions (Brixius-Anderko et al, 2015). This is a big step forward towards application of this system in an industrial process for the sustainable production of the methylprednisolone-precursor premedrol.…”

“…The truncation of 27 amino acids at the N-terminus enables high-yield expression of a soluble catalytically active CPR. The cloning and generation of the vector p21b_bRED(−27) was performed as described in (Brixius-Anderko et al, 2015;Neunzig et al, 2017) ( Figure 1a).…”

An indole‐deficient Escherichia coli strain improves screening of cytochromes P450 for biotechnological applications

“…Since the inhibitory effect of indole on a bacterial CYP was clearly demonstrated, we next aimed to examine its impact on a mammalian CYP, bovine CYP21A2, to show that an indole‐mediated inhibition is independent of the CYP origin. Recently, a new process for the biotechnological production of premedrol, the precursor of the pharmaceutically highly relevant synthetic glucocorticoid medrol (methylprednisolone), by using whole cells of E. coli expressing bovine CYP21A2 was published . Since mammalian CYPs usually exhibit low turnover numbers, a molecular evolution of CYP21A2 could lead to an increased substrate conversion and facilitate an application of this enzyme in an industrial process, replacing the established chemical synthesis.…”

Determinants of thermostability in the cytochrome P450 fold

“…The most thermostable Fd that has been identified to interact with multiple class I P450s is the C-terminal domain of an electron transfer protein (Etp1) from the fission yeast, Schizosaccharomyces pombe, Etp1 fd [122][123][124][125][126]. Etp1 fd is homologous to vertebrate-type Fds [122] and has been characterized with respect to its thermal stability as the full length (Etp1 fd (505-631)) and a truncated form (Etp1 fd (516-618)) created for crystallization [127].…”