A recombinant CYP11B1 dependent Escherichia coli biocatalyst for selective cortisol production and optimization towards a preparative scale

Abstract

BackgroundHuman mitochondrial CYP11B1 catalyzes a one-step regio- and stereoselective 11β-hydroxylation of 11-deoxycortisol yielding cortisol which constitutes not only the major human stress hormone but also represents a commercially relevant therapeutic drug due to its anti-inflammatory and immunosuppressive properties. Moreover, it is an important intermediate in the industrial production of synthetic pharmaceutical glucocorticoids. CYP11B1 thus offers a great potential for biotechnological application in large-scale synthesis of cortisol. Because of its nature as external monooxygenase, CYP11B1-dependent steroid hydroxylation requires reducing equivalents which are provided from NADPH via a redox chain, consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx).ResultsWe established an Escherichia coli based whole-cell system for selective cortisol production from 11-deoxycortisol by recombinant co-expression of the demanded 3 proteins. For the subsequent optimization of the whole-cell activity 3 different approaches were pursued: Firstly, CYP11B1 expression was enhanced 3.3-fold to 257 nmol∗L−1 by site-directed mutagenesis of position 23 from glycine to arginine, which was accompanied by a 2.6-fold increase in cortisol yield. Secondly, the electron transfer chain was engineered in a quantitative manner by introducing additional copies of the Adx cDNA in order to enhance Adx expression on transcriptional level. In the presence of 2 and 3 copies the initial linear conversion rate was greatly accelerated and the final product concentration was improved 1.4-fold. Thirdly, we developed a screening system for directed evolution of CYP11B1 towards higher hydroxylation activity. A culture down-scale to microtiter plates was performed and a robot-assisted, fluorescence-based conversion assay was applied for the selection of more efficient mutants from a random library.ConclusionsUnder optimized conditions a maximum productivity of 0.84 g cortisol∗L−1∗d−1 was achieved, which clearly shows the potential of the developed system for application in the pharmaceutical industry.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0209-5) contains supplementary material, which is available to authorized users.

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

“…This progress can directly be of use for efficient and timesaving production of human drug metabolites. High yields and conversion rates can already be achieved by using corresponding human (Rushmore et al, 2000;Vail et al, 2005;Schroer et al, 2010;Geier et al, 2012;Schiffer et al, 2015) or suitable nonhuman (Taylor et al, 1999;Otey et al, 2006;Sawayama et al, 2009;Reinen et al, 2011;Di Nardo and Gilardi, 2012;Kiss et al, 2015;Ren et al, 2015) P450 enzymes in a whole-cell system to produce respective metabolites. The majority of published bacterial P450 enzymes used for the conversion of drugs are mutants of CYP102A1 (BM3) from Bacillus megaterium.…”

“…Optimizations such as changing expression and reaction conditions or engineering P450 enzymes could be potential topics of interest. Several approaches for increasing the performance of the whole-cell system have been published, describing that an increased number of ferredoxin gene copies (Schiffer et al, 2015) or coexpressing a NADPH regenerating system (Zehentgruber et al, 2010) could enhance product formation. In a recent review, numerous approaches and examples were presented to enhance the catalytic activity of P450 enzymes toward potential practical purposes (Gillam, 2008).…”

Identification and expression of the 11β‐steroid hydroxylase from Cochliobolus lunatus in Corynebacterium glutamicum

“…Hydroxylation performed by fungi has acquired special relevance for the pharmaceutical industries. Among them, the hydroxyl group in position 11β of the cortisol molecule and its synthetic derivatives are the key functionalization that provides their glucocorticoid effects (Mahato and Garai, ; Schiffer et al ., ). Currently, the 11β‐hydroxylation in the industrial production of corticosteroid precursors is catalysed by fungal cultures of Cochliobolus lunatus (Paraszkiewicz and Długon, ; Lu et al ., ).…”

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

“…Preparative scale production of the OT metabolites formed by the CYP11B isoforms for NMR characterization and further investigation of the major metabolites was performed with an E. coli-based wholecell conversion system as previously reported for CYP11B1-dependent transformation of 11-deoxycortisol to cortisol (Schiffer et al, 2015b). Because CYP11B1 products are also formed by CYP11B2, application of CYP11B2 was chosen.…”

“…Large-scale substrate conversion by human CYP11B2 was conducted with a recombinant E. coli whole-cell system as previously described for CYP11B1 (Schiffer et al, 2015b). Briefly, E. coli C43(DE3) [F-ompT gal hsdSB (rB2 mB2) dcm ion l] cells were transformed by electroporation with the pET-17b-based plasmid Twin_11B2 encoding human CYP11B2, bovine AdR, and bovine Adx 1-108 and the plasmid pGro12 for the cosynthesis of the molecular chaperones GroEL/ES (Nishihara et al, 1998).…”