Resurrection and characterization of ancestral CYP11A1 enzymes.

Mitochondrial cytochromes P450 presumably originated from a common microsomal P450 ancestor. However, it is still unknown how ancient mitochondrial P450s were able to retain their oxygenase function following relocation to the mitochondrial matrix and later emerged as enzymes specialized for steroid hormone biosynthesis in vertebrates. Here, we used the approach of ancestral sequence reconstruction (ASR) to resurrect ancient CYP11A1 enzymes and characterize their unique biochemical properties. Two ancestral CYP11A1 variants, CYP11A_Mammal_N101 and CYP11A_N1, as well as an extant bovine form were recombinantly expressed and purified to homogeneity. All enzymes showed characteristic P450 spectral properties and were able to convert cholesterol as well as other sterol substrates to pregnenolone, yet with different specificities. The vertebrate CYP11A_N1 ancestor preferred the cholesterol precursor, desmosterol, as substrate suggesting a convergent evolution of early cholesterol metabolism and CYP11A1 enzymes. Both ancestors were able to withstand increased levels of hydrogen peroxide but only the ancestor CYP11A_N1 showed increased thermostability (˜ 25 °C increase in T50 ) compared with the extant CYP11A1. The extraordinary robustness of ancient mitochondrial P450s, as demonstrated for CYP11A_N1, may have allowed them to stay active when presented with poorly compatible electron transfer proteins and resulting harmful ROS in the new environment of the mitochondrial matrix. To the best of our knowledge, this work represents the first study that describes the resurrection of ancient mitochondrial P450 enzymes. The results will help to understand and gain fundamental functional insights into the evolutionary origins of steroid hormone biosynthesis in animals.

Development and application of a highly efficient CRISPR-Cas9 system for genome engineering in Bacillus megaterium.

Bacillus megaterium has become increasingly important for the biotechnological production of valuable compounds of industrial and pharmaceutical importance. Despite recent advances in rational strain design of B. megaterium, these studies have been largely impaired by the lack of molecular tools that are not state-of-the-art for comprehensive genome engineering approaches. In the current work, we describe the adaptation of the CRISPR-Cas9 vector pJOE8999 to enable efficient genome editing in B. megaterium. Crucial modifications comprise the exchange of promoter elements and associated ribosomal binding sites as well as the implementation of a 5-fluorouracil based counterselection system to facilitate proper plasmid curing. In addition, the functionality and performance of the new CRISPR-Cas9 vector pMOE was successfully evaluated by chromosomal disruption studies of the endogenous ß-galactosidase gene (BMD_2126) and demonstrated an outstanding efficiency of 100 % based on combinatorial pheno- and genotype analyses. Furthermore, pMOE was applied for the genomic deletion of a steroid esterase gene (BMD_2256) that was identified among several other candidates as the gene encoding the esterase, which prevented accumulation of pharmaceutically important glucocorticoid esters. Recombinant expression of the bacterial chloramphenicol acetyltransferase 1 gene (cat1) in the resulting esterase deficient B. megaterium strain ultimately yielded C21-acetylated as well as novel C21-esterified derivates of cortisone.

Improvement of the 25-hydroxyvitamin D3 production in a CYP109A2-expressing Bacillus megaterium system.

Calcifediol (25(OH)VD3) is a physiologically very important vitamin D3 metabolite and of high pharmaceutical importance, due to its potential for treating not only vitamin D3 deficiencies but also coronary diseases and cancer. Previously, we established a whole-cell Bacillus megaterium-based system using the cytochrome P450 CYP109A2 for the biotransformation of vitamin D3 into its metabolite 25-hydroxyvitamin D3. In this study, we demonstrate the importance of the region between amino acids T103 and A106 for the catalytic activity of CYP109A2 towards vitamin D3 as a substrate. In order to increase the productivity of the system, reaction conditions (xylose, vitamin D3, saponin, 2-hydroxypropyl-ß-cyclodextrin) were optimized for the in vivo production of 25-hydroxyvitamin D3. With cells producing the T103A mutant, a productivity of 282.7 mg/L/48 h was achieved under the optimized conditions. This value is two times higher than that obtained in the control reaction with the wild-type enzyme in this study and five times higher than that obtained in a previous study.

Mixed-culture fermentation for enhanced C21-hydroxylation of glucocorticoids.

Synthetic glucocorticoids are generally preferred over their natural counterparts as these compounds exhibit improved anti-inflammatory potency and glucocorticoid receptor selectivity. However, the biotechnological production of these molecules is often subject to limitations inferred by restricted enzyme stability, selectivity or inhibition thereof. The latter is particularly important during 6α-methylprednisolone production, as the essential C21-hydroxylation of its precursor medrane appears to be hampered by product inhibition of the steroid-21-hydroxylase (CYP21A2). To circumvent this bottleneck, we established a two-step reaction for controlled mixed-culture fermentation, using recombinant E. coli. This process comprises the previously reported C21-hydroxylation of medrane by CYP21A2, followed by an instant derivatization of the hydroxylated product premedrol by chloramphenicol acetyl transferase 1 (CAT1). The CAT1-mediated C21-acetylation prevents the product from regaining access to the enzyme's active site which effectively shifts the chemical equilibrium toward premedrol formation. The successful circumvention of product inhibition at optimized conditions resulted in the formation of more than 1.5 g of product per liter which corresponds to an increase by more than 100 %. Taken together, we demonstrate an efficient system to enhance cytochrome P450-mediated biotransformations, holding great ecologic and economic potential to be applied in industrial processes.

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.

High-yield C11-oxidation of hydrocortisone by establishment of an efficient whole-cell system in Bacillus megaterium.

Steroidal compounds are one of the most widely marketed pharmaceutical products. Chemical synthesis of steroidal compounds faces many challenges, including the requirement for multiple chemical steps, low yield and selectivity in several synthesis steps, low profitability and the production of environmental pollutants. Consequently, in recent decades there has been growing interest in the use of microbial systems to produce pharmaceutical compounds. Several microbial systems have recently been developed for the microbial synthesis of the glucocorticoid hydrocortisone, which serves as a key intermediate in the production of several other pharmaceutically important steroidal compounds. In this study, we sought to establish an efficient, microbial-based system, for the conversion of hydrocortisone into cortisone. To this end, we developed a strategy for high-yield cortisone production based on ectopic expression of the guinea-pig 11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) in Bacillus megaterium. We screened different constructs, containing a variety of promoters tailored for B. megaterium, and created modified versions of the enzyme by protein engineering to optimize cortisone yield. Furthermore, we utilized co-expression of an alcohol dehydrogenase to promote NADP+ regeneration, which significantly improved 11ß-HSD1 activity. The process thereby developed was found to show a remarkably high regioselectivity of >95% and to generate cortisone yields of up to 13.65 g L-1 d-1, which represents a ∼1000-fold improvement over the next-best reported system. In summary, we demonstrate the utility of B. megaterium MS941 as a suitable host for recombinant protein production and its high potential for industrial steroid production.

Expanding the promoter toolbox of Bacillus megaterium.

Over the past decades, Bacillus megaterium has gained significant interest in the biotechnological industry due to its high capacity for protein production. Although many proteins have been expressed efficiently using the optimized xylose inducible system so far, there is a considerable demand for novel promoters with varying activities, particularly for the adjustment of protein levels in multi-enzyme cascades. Genome-wide microarray analyses of the industrially important B. megaterium strain MS941 were applied to identify constitutive and growth phase dependent promoters for the expression of heterologous proteins from the early exponential to the early stationary phase of bacterial growth. Fifteen putative promoter elements were selected based on differential gene expression profiles and signal intensities of the generated microarray data. The corresponding promoter activities were evaluated in B. megaterium via ß-galactosidase screening. ß-Galactosidase expression levels ranged from 15% to 130% compared to the optimized xylose inducible promoter. Apart from these constitutive promoters we also identified and characterized novel inducible promoters, which were regulated by the addition of arabinose, galactose and the commonly used allolactose analog IPTG. The potential application of the identified promoters for biotechnologically relevant processes was demonstrated by overexpression of the cholesterol oxidase II from Brevibacterium sterolicum, thus obtaining product yields of up to 1.13 g/l/d. The provided toolbox of novel promoters offers versatile promoter strengths and will significantly contribute to harmonize protein expression in synthetic metabolic pathways, thereby pushing forward the engineering of B. megaterium as microbial cell factory for the biosynthesis and conversion of valuable compounds.

Characterization and engineering of a carotenoid biosynthesis operon from Bacillus megaterium.

Bacillus megaterium belongs to the group of pigmented bacilli producing carotenoids that ensure self-protection from UV radiation-induced and collateral oxidative damage. Metabolite profiling of strain MS941 revealed the presence of the C30 carotenoids 4,4'-diapophytofluene and 4,4'-diaponeurosporenic acid. A gene function analysis demonstrated the presence of a corresponding C30 carotenoid biosynthetic pathway with pharmaceutical importance. We identified a gene cluster comprising putative genes for a farnesyl diphosphate synthase (IspA), a diapophytoene synthase (CrtM) and three distinct diapophytoene desaturases (CrtN1-3). Intriguingly, crtM was organized in an operon together with two of the identified crtN genes. The individual activities of the encoded enzymes were determined by heterologous expression and product analysis in the non-carotenogenic model organism Escherichia coli. Our experimental data show that the first catalytic steps of C30 carotenoid biosynthesis in B. megaterium share significant similarity to the corresponding biosynthetic pathway of Staphylococcus aureus. The biosynthesis of farnesyl diphosphates and their subsequent condensation to form 4,4'-diapophytoene are catalyzed by the identified IspA and CrtM, respectively. The following desaturation reactions to form 4,4'-diaponeurosporene, however, require the activities of multiple diapophytoene desaturases. A biosynthetic operon was engineered and successfully expressed in an E. coli whole-cell system creating a cell factory for a high-yield production of the C30 carotenoid 4,4'-diaponeurosporene which has promising potential in the treatment of various inflammatory diseases.

Functionalized poly(3-hydroxybutyric acid) bodies as new in vitro biocatalysts.

Cytochromes P450 play a key role in the drug and steroid metabolism in the human body. This leads to a high interest in this class of proteins. Mammalian cytochromes P450 are rather delicate. Due to their localization in the mitochondrial or microsomal membrane, they tend to aggregate during expression and purification and to convert to an inactive form so that they have to be purified and stored in complex buffers. The complex buffers and low storage temperatures, however, limit the feasibility of fast, automated screening of the corresponding cytochrome P450-effector interactions, which are necessary to study substrate-protein and inhibitor-protein interactions. Here, we present the production and isolation of functionalized poly(3-hydroxybutyrate) granules (PHB bodies) from Bacillus megaterium MS941 strain. In contrast to the expression in Escherichia coli, where mammalian cytochromes P450 are associated to the cell membrane, when CYP11A1 is heterologously expressed in Bacillus megaterium, it is located on the PHB bodies. The surface of these particles provides a matrix for immobilization and stabilization of the CYP11A1 during the storage of the protein and substrate conversion. It was demonstrated that the PHB polymer basis is inert concerning the performed conversion. Immobilization of the CYP11A1 onto the PHB bodies allows freeze-drying of the complex without significant decrease of the CYP11A1 activity. This is the first lyophilization of a mammalian cytochrome P450, which allows storage over more than 18days at 4°C instead of storage at -80°C. In addition, we were able to immobilize the cytochrome P450 on the PHB bodies in vitro. In this case the expression of the protein is separated from the production of the immobilization matrix, which widens the application of this method. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

CYP109E1 is a novel versatile statin and terpene oxidase from Bacillus megaterium.

CYP109E1 is a cytochrome P450 monooxygenase from Bacillus megaterium with a hydroxylation activity for testosterone and vitamin D3. This study reports the screening of a focused library of statins, terpene-derived and steroidal compounds to explore the substrate spectrum of this enzyme. Catalytic activity of CYP109E1 towards the statin drug-precursor compactin and the prodrugs lovastatin and simvastatin as well as biotechnologically relevant terpene compounds including ionones, nootkatone, isolongifolen-9-one, damascones, and ß-damascenone was found in vitro. The novel substrates induced a type I spin-shift upon binding to P450 and thus permitted to determine dissociation constants. For the identification of conversion products by NMR spectroscopy, a B. megaterium whole-cell system was applied. NMR analysis revealed for the first time the ability of CYP109E1 to catalyze an industrially highly important reaction, the production of pravastatin from compactin, as well as regioselective oxidations generating drug metabolites (6'ß-hydroxy-lovastatin, 3'α-hydroxy-simvastatin, and 4″-hydroxy-simvastatin) and valuable terpene derivatives (3-hydroxy-α-ionone, 4-hydroxy-ß-ionone, 11,12-epoxy-nootkatone, 4(R)-hydroxy-isolongifolen-9-one, 3-hydroxy-α-damascone, 4-hydroxy-ß-damascone, and 3,4-epoxy-ß-damascone). Besides that, a novel compound, 2-hydroxy-ß-damascenone, produced by CYP109E1 was identified. Docking calculations using the crystal structure of CYP109E1 rationalized the experimentally observed regioselective hydroxylation and identified important amino acid residues for statin and terpene binding.

Biochemical and structural characterization of CYP109A2, a vitamin D3 25-hydroxylase from Bacillus megaterium.

Cytochrome P450 enzymes are increasingly investigated due to their potential application as biocatalysts with high regio- and/or stereo-selectivity and under mild conditions. Vitamin D3 (VD3 ) metabolites are of pharmaceutical importance and are applied for the treatment of VD3 deficiency and other disorders. However, the chemical synthesis of VD3 derivatives shows low specificity and low yields. In this study, cytochrome P450 CYP109A2 from Bacillus megaterium DSM319 was expressed, purified, and shown to oxidize VD3 with high regio-selectivity. The in vitro conversion, using cytochrome P450 reductase (BmCPR) and ferredoxin (Fdx2) from the same strain, showed typical Michaelis-Menten reaction kinetics. A whole-cell system in B. megaterium overexpressing CYP109A2 reached 76 ± 5% conversion after 24 h and allowed to identify the main product by NMR analysis as 25-hydroxylated VD3 . Product yield amounted to 54.9 mg·L-1 ·day-1 , rendering the established whole-cell system as a highly promising biocatalytic route for the production of this valuable metabolite. The crystal structure of substrate-free CYP109A2 was determined at 2.7 Å resolution, displaying an open conformation. Structural analysis predicts that CYP109A2 uses a highly similar set of residues for VD3 binding as the related VD3 hydroxylases CYP109E1 from B. megaterium and CYP107BR1 (Vdh) from Pseudonocardia autotrophica. However, the folds and sequences of the BC loops in these three P450s are highly divergent, leading to differences in the shape and apolar/polar surface distribution of their active site pockets, which may account for the observed differences in substrate specificity and the regio-selectivity of VD3 hydroxylation. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession code 5OFQ (substrate-free CYP109A2). ENZYMES: Cytochrome P450 monooxygenase CYP109A2, EC 1.14.14.1, UniProt ID: D5DF88, Ferredoxin, UniProt ID: D5DFQ0, cytochrome P450 reductase, EC 1.8.1.2, UniProt ID: D5DGX1.

Characterization of cytochrome P450 CYP109E1 from Bacillus megaterium as a novel vitamin D3 hydroxylase.

In this study the ability of CYP109E1 from Bacillus megaterium to metabolize vitamin D3 (VD3) was investigated. In an in vitro system using bovine adrenodoxin reductase (AdR) and adrenodoxin (Adx4-108), VD3 was converted by CYP109E1 into several products. Furthermore, a whole-cell system in B. megaterium MS941 was established. The new system showed a conversion of 95% after 24h. By NMR analysis it was found that CYP109E1 catalyzes hydroxylation of VD3 at carbons C-24 and C-25, resulting in the formation of 24(S)-hydroxyvitamin D3 (24S(OH)VD3), 25-hydroxyvitamin D3 (25(OH)VD3) and 24S,25-dihydroxyvitamin D3 (24S,25(OH)2VD3). Through time dependent whole-cell conversion of VD3, we identified that the formation of 24S,25(OH)2VD3 by CYP109E1 is derived from VD3 via the intermediate 24S(OH)VD3. Moreover, using docking analysis and site-directed mutagenesis, we identified important active site residues capable of determining substrate specificity and regio-selectivity. HPLC analysis of the whole-cell conversion with the I85A-mutant revealed an increased selectivity towards 25-hydroxylation of VD3 compared with the wild type activity, resulting in an approximately 2-fold increase of 25(OH)VD3 production (45mgl-1day-1) compared to wild type (24.5mgl-1day-1).

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

Escherichia coli has developed into an attractive organism for heterologous cytochrome P450 production, but, in some cases, was restricted as a host in view of a screening of orphan cytochromes P450 or mutant libraries in the context of molecular evolution due to the formation of the cytochrome P450 inhibitor indole by the enzyme tryptophanase (TnaA). To overcome this effect, we disrupted the tnaA gene locus of E. coli C43(DE3) and evaluated the new strain for whole-cell substrate conversions with three indole-sensitive cytochromes P450, myxobacterial CYP264A1, and CYP109D1 as well as bovine steroidogenic CYP21A2. For purified CYP264A1 and CYP21A2, the half maximal inhibitory indole concentration was determined to be 140 and 500 µM, which is within the physiological concentration range occurring during cultivation of E. coli in complex medium. Biotransformations with C43(DE3)_∆tnaA achieved a 30% higher product formation in the case of CYP21A2 and an even fourfold increase with CYP264A1 compared with C43(DE3) cells. In whole-cell conversion based on CYP109D1, which converts indole to indigo, we could successfully avoid this reaction. Results in microplate format indicate that our newly designed strain is a suitable host for a fast and efficient screening of indole-influenced cytochromes P450 in complex medium.

A Novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase.

Cytochromes P450 (P450s) require electron transfer partners to catalyze substrate conversions. With regard to biotechnological approaches, the elucidation of novel electron transfer proteins is of special interest, as they can influence the enzymatic activity and specificity of the P450s. In the current work we present the identification and characterization of a novel soluble NADPH-dependent diflavin reductase from Bacillus megaterium with activity towards a bacterial (CYP106A1) and a microsomal (CYP21A2) P450 and, therefore, we referred to it as B. megaterium cytochrome P450 reductase (BmCPR). Sequence analysis of the protein revealed besides the conserved FMN-, FAD- and NADPH-binding motifs, the presence of negatively charged cluster, which is thought to represent the interaction domain with P450s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in Escherichia coli. The purified BmCPR exhibited a characteristic diflavin reductase spectrum, and showed a cytochrome c reducing activity. Furthermore, in an in vitro reconstituted system, the BmCPR was able to support the hydroxylation of testosterone and progesterone with CYP106A1 and CYP21A2, respectively. Moreover, in view of the biotechnological application, the BmCPR is very promising, as it could be successfully utilized to establish CYP106A1- and CYP21A2-based whole-cell biotransformation systems, which yielded 0.3g/L hydroxy-testosterone products within 8h and 0.16g/L 21-hydroxyprogesterone within 6h, respectively. In conclusion, the BmCPR reported herein owns a great potential for further applications and studies and should be taken into consideration for bacterial and/or microsomal CYP-dependent bioconversions.

Biotransformation of the mineralocorticoid receptor antagonists spironolactone and canrenone by human CYP11B1 and CYP11B2: Characterization of the products and their influence on mineralocorticoid receptor transactivation.

Spironolactone and its major metabolite canrenone are potent mineralocorticoid receptor antagonists and are, therefore, applied as drugs for the treatment of primary aldosteronism and essential hypertension. We report that both compounds can be converted by the purified adrenocortical cytochromes P450 CYP11B1 and CYP11B2, while no conversion of the selective mineralocorticoid receptor antagonist eplerenone was observed. As their natural function, CYP11B1 and CYP11B2 carry out the final steps in the biosynthesis of gluco- and mineralocorticoids. Dissociation constants for the new exogenous substrates were determined by a spectroscopic binding assay and demonstrated to be comparable to those of the natural substrates, 11-deoxycortisol and 11-deoxycorticosterone. Metabolites were produced at preparative scale with a CYP11B2-dependent Escherichia coli whole-cell system and purified by HPLC. Using NMR spectroscopy, the metabolites of spironolactone were identified as 11ß-OH-spironolactone, 18-OH-spironolactone and 19-OH-spironolactone. Canrenone was converted to 11ß-OH-canrenone, 18-OH-canrenone as well as to the CYP11B2-specific product 11ß,18-diOH-canrenone. Therefore, a contribution of CYP11B1 and CYP11B2 to the biotransformation of drugs should be taken into account and the metabolites should be tested for their potential toxic and pharmacological effects. A mineralocorticoid receptor transactivation assay in antagonist mode revealed 11ß-OH-spironolactone as pharmaceutically active metabolite, whereas all other hydroxylation products negate the antagonist properties of spironolactone and canrenone. Thus, human CYP11B1 and CYP11B2 turned out to metabolize steroid-based drugs additionally to the liver-dependent biotransformation of drugs. Compared with the action of the parental drug, changed properties of the metabolites at the target site have been observed.

Human CYP27A1 catalyzes hydroxylation of ß-sitosterol and ergosterol.

ß-Sitosterol and ergosterol are the equivalents of cholesterol in plants and fungi, respectively, and common sterols in the human diet. In the current work, both were identified as novel CYP27A1 substrates by in vitro experiments applying purified human CYP27A1 and its redox partners adrenodoxin (Adx) and adrenodoxin reductase (AdR). A Bacillus megaterium based biocatalyst recombinantly expressing the same proteins was utilized for the conversion of the substrates to obtain sufficient amounts of the novel products for a structural NMR analysis. ß-Sitosterol was found to be converted into 26-hydroxy-ß-sitosterol and 29-hydroxy-ß-sitosterol, whereas ergosterol was converted into 24-hydroxyergosterol, 26-hydroxyergosterol and 28-hydroxyergosterol.

Expression of human CYP27A1 in B. megaterium for the efficient hydroxylation of cholesterol, vitamin D3 and 7-dehydrocholesterol.

In the current work the ability of Bacillus megaterium to take up hydrophobic substrates and efficiently express eukaryotic membrane proteins was utilized for establishing a CYP27A1-based biocatalyst. The human mitochondrial cytochrome P450CYP27A1 was co-expressed with its redox partners adrenodoxin reductase (Adr) and adrenodoxin (Adx). CYP27A1 could be localized at the cell's polyhydroxybutyrate (PHB) granules, carbon storage serving organelle-like vesicles that can take up cholesterol, resulting in bioreactor-like structures in B. megaterium . The resulting whole cell system allowed the efficient biotechnological conversion of the CYP27A1 substrates cholesterol, 7-dehydrocholesterol (7-DHC) and vitamin D3. After 48 h, nearly 100% of cholesterol was metabolized producing a final concentration of 113.14 mg/l 27-hydroxycholesterol (27-HC). Moreover, 70% of vitamin D3 was converted into 25-hydroxyvitamin D3 (25-OH-D3) with a final concentration of 80.81 mg/l. Also more than 97% of 7-DHC were found to be metabolized into two products, corresponding to 26/27-hydroxy-7-dehydrocholesterol (P1) and 25-hydroxy-7-dehydrocholesterol (P2). To our knowledge this is the first CYP27A1-based whole-cell system, allowing the efficient and low-cost production of pharmaceutically interesting metabolites of this enzyme from relatively cheap substrates.

Phenotypic, metabolic, and molecular genetic characterization of six patients with congenital adrenal hyperplasia caused by novel mutations in the CYP11B1 gene.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive inherited disorder of steroidogenesis. Steroid 11ß-hydroxylase deficiency (11ß-OHD) due to mutations in the CYP11B1 gene is the second most common form of CAH. In this study, 6 patients suffering from CAH were diagnosed with 11ß-OHD using urinary GC-MS steroid metabolomics analysis. The molecular basis of the disorder was investigated by molecular genetic analysis of the CYP11B1 gene, functional characterization of splicing and missense mutations, and analysis of the missense mutations in a computer model of CYP11B1. All patients presented with abnormal clinical signs of hyperandrogenism. Their urinary steroid metabolomes were characterized by excessive excretion rates of metabolites of 11-deoxycortisol as well as metabolites of 11-deoxycorticosterone, and allowed definite diagnosis. Patient 1 carries compound heterozygous mutations consisting of a novel nonsense mutation p.Q102X (c.304C>T) in exon 2 and the known missense mutation p.T318R (c.953C>G) in exon 5. Two siblings (patient 2 and 3) were compound heterozygous carriers of a known splicing mutation c.1200+1G>A in intron 7 and a known missense mutation p.R448H (c.1343G>A) in exon 8. Minigene experiments demonstrated that the c.1200+1G>A mutation caused abnormal pre-mRNA splicing (intron retention). Two further siblings (patient 4 and 5) were compound heterozygous carriers of a novel missense mutation p.R332G (c.994C>G) in exon 6 and the known missense mutation p.R448H (c.1343G>A) in exon 8. A CYP11B1 activity study in COS-1 cells showed that only 11% of the enzyme activity remained in the variant p.R332G. Patient 6 carried a so far not described homozygous deletion g.2470_5320del of 2850 bp corresponding to a loss of the CYP11B1 exons 3-8. The breakpoints of the deletion are embedded into two typical 6 base pair repeats (GCTTCT) upstream and downstream of the gene. Experiments analyzing the influence of mutations on splicing and on enzyme function were applied as complementary procedures to genotyping and provided a rational basis for understanding the clinical phenotype of CAH.

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-17ß-hydroxy-17α-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 µM and 5.4 µM 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 11ß-OH-OT for both CYP11B isoforms, whereby CYP11B2 additionally formed 11ß,18-diOH-OT and 11ß-OH-OT-18-al, which rearranges to its tautomeric form 11ß,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.

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

BACKGROUND: Synthetic 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. RESULTS: We 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). CONCLUSIONS: We 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.