Adrenodoxin—A versatile ferredoxin

Ewen, Kerstin Maria; Ringle, Michael; Bernhardt, Rita

doi:10.1002/iub.1029

Cited by 52 publications

(47 citation statements)

References 52 publications

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“…Therefore, a focused steroid library, consisting of 11 non-3-oxo-Δ 4 steroids was screened in an in vitro enzyme assay. The mitochondrial electron transfer system from bovine adrenals was used to reconstitute the CYP106A2 enzyme system, because the bovine electron transfer proteins are well established as efficient electron suppliers for CYP106A2 and other bacterial P450s [24,25]. …”

Section: Resultsmentioning

confidence: 99%

Steroid conversion with CYP106A2 – production of pharmaceutically interesting DHEA metabolites

2014

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BackgroundSteroids are lipophilic compounds with a gonane skeleton and play an important role in higher organisms. Due to different functionalizations - mainly hydroxylations - at the steroid molecule, they vary highly in their mode of action. The pharmaceutical industry is, therefore, interested in hydroxysteroids as therapeutic agents. The insertion of hydroxyl groups into a steroid core, however, is hardly accomplishable by classical chemical means; that is because microbial steroid hydroxylations are investigated and applied since decades. CYP106A2 is a cytochrome P450 monooxygenase from Bacillus megaterium ATCC 13368, which was first described in the late 1970s and which is capable to hydroxylate a variety of 3-oxo-delta4 steroids at position 15beta. CYP106A2 is a soluble protein, easy to express and to purify in high amounts, which makes this enzyme an interesting target for biotechnological purposes.ResultsIn this work a focused steroid library was screened in vitro for new CYP106A2 substrates using a reconstituted enzyme assay. Five new substrates were identified, including dehydroepiandrosterone and pregnenolone. NMR-spectroscopy revealed that both steroids are mainly hydroxylated at position 7beta. In order to establish a biotechnological system for the preparative scale production of 7beta-hydroxylated dehydroepiandrosterone, whole-cell conversions with growing and resting cells of B. megaterium ATCC1336 the native host of CYP1062 and also with resting cells of a recombinant B. megaterium MS941 strain overexpressing CYP106A2 have been conducted and conversion rates of 400 muM/h (115 mg/l/h) were obtained. Using the B. megaterium MS941 overexpression strain, the selectivity of the reaction was improved from 0.7 to 0.9 for 7beta-OH-DHEA.ConclusionsIn this work we describe CYP106A2 for the first time as a regio-selective hydroxylase for 3-hydroxy-delta5 steroids. DHEA was shown to be converted to 7beta-OH-DHEA which is a highly interesting human metabolite, supposed to act as neuroprotective, anti-inflammatory and immune-modulatory agent. Optimization of the whole-cell system using different B. megaterium strains lead to a conversion of DHEA with B. megaterium showing high selectivity and conversion rates and displaying a volumetric yield of 103 mg/l/h 7beta-OH-DHEA.

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Section: Resultsmentioning

confidence: 99%

Steroid conversion with CYP106A2 – production of pharmaceutically interesting DHEA metabolites

2014

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“…The catalytic activity of CYP106A2 towards CPA was first tested in vitro . The activity was reconstituted using bovine adrenal redox partners (Adx 4–108 and AdR) proven to be highly efficient electron suppliers for CYP106A2 [14,22]. The CYP106A2-dependent conversion of CPA was analyzed by high-performance liquid chromatography (HPLC) and resulted in one main product.…”

Section: Resultsmentioning

confidence: 99%

Process development for the production of 15β-hydroxycyproterone acetate using Bacillus megaterium expressing CYP106A2 as whole-cell biocatalyst

et al. 2015

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BackgroundCYP106A2 from Bacillus megaterium ATCC 13368 was first identified as a regio- and stereoselective 15β-hydroxylase of 3-oxo-∆4-steroids. Recently, it was shown that besides 3-oxo-∆4-steroids, 3-hydroxy-∆5-steroids as well as di- and triterpenes can also serve as substrates for this biocatalyst. It is highly selective towards the 15β position, but the 6β, 7α/β, 9α, 11α and 15α positions have also been described as targets for hydroxylation. Based on the broad substrate spectrum and hydroxylating capacity, it is an excellent candidate for the production of human drug metabolites and drug precursors.ResultsIn this work, we demonstrate the conversion of a synthetic testosterone derivative, cyproterone acetate, by CYP106A2 under in vitro and in vivo conditions. Using a Bacillus megaterium whole-cell system overexpressing CYP106A2, sufficient amounts of product for structure elucidation by nuclear magnetic resonance spectroscopy were obtained. The product was characterized as 15β-hydroxycyproterone acetate, the main human metabolite. Since the product is of pharmaceutical interest, our aim was to intensify the process by increasing the substrate concentration and to scale-up the reaction from shake flasks to bioreactors to demonstrate an efficient, yet green and cost-effective production. Using a bench-top bioreactor and the recombinant Bacillus megaterium system, both a fermentation and a transformation process were successfully implemented. To improve the yield and product titers for future industrial application, the main bottlenecks of the reaction were addressed. Using 2-hydroxypropyl-β-cyclodextrin, an effective bioconversion of 98% was achieved using 1 mM substrate concentration, corresponding to a product formation of 0.43 g/L, at a 400 mL scale.ConclusionsHere we describe the successful scale-up of cyproterone acetate conversion from shake flasks to bioreactors, using the CYP106A2 enzyme in a whole-cell system. The substrate was converted to its main human metabolite, 15β-hydroxycyproterone acetate, a highly interesting drug candidate, due to its retained antiandrogen activity but significantly lower progestogen properties than the mother compound. Optimization of the process led to an improvement from 55% to 98% overall conversion, with a product formation of 0.43 g/L, approaching industrial process requirements and a future large-scale application.

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“…1), the redox partners of bacterial P450s might differ significantly [8]. However, it was shown that heterologous reconstitution of enzymatic activity using redox partners from other organisms, especially using bovine adrenodoxin can be very efficient [47]. In addition, strategies using fusion proteins with known redox domains may be a promising tool to overcome this obstacle [9].…”

Section: Discussionmentioning

confidence: 98%

“…Its usefulness for the conversion of terpenoids by bacterial CYPs needs to be investigated. However, the truncated Adx variant proved to be a very efficient heterologous redox partner in many bacterial P450 bioconversion systems, also in those converting terpenoids [47].…”

Section: Engineering Improved Redox Partner Interactionmentioning

confidence: 99%

Terpene Hydroxylation with Microbial Cytochrome P450 Monooxygenases

Janocha

Schmitz

Bernhardt

2015

Biotechnology of Isoprenoids

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Terpenoids comprise a highly diverse group of natural products. In addition to their basic carbon skeleton, they differ from one another in their functional groups. Functional groups attached to the carbon skeleton are the basis of the terpenoids' diverse properties. Further modifications of terpene olefins include the introduction of acyl-, aryl-, or sugar moieties and usually start with oxidations catalyzed by cytochrome P450 monooxygenases (P450s, CYPs). P450s are ubiquitously distributed throughout nature, involved in essential biological pathways such as terpenoid biosynthesis as well as the tailoring of terpenoids and other natural products. Their ability to introduce oxygen into nonactivated C-H bonds is unique and makes P450s very attractive for applications in biotechnology. Especially in the field of terpene oxidation, biotransformation methods emerge as an attractive alternative to classical chemical synthesis. For this reason, microbial P450s depict a highly interesting target for protein engineering approaches in order to increase selectivity and activity, respectively. Microbial P450s have been described to convert industrial and pharmaceutically interesting terpenoids such as ionones, limone, valencene, resin acids, and triterpenes (including steroids) as well as vitamin D3. Highly selective and active mutants have been evolved by applying classical site-directed mutagenesis as well as directed evolution of proteins. As P450s usually depend on electron transfer proteins, mutagenesis has also been applied to improve the interactions between P450s and their respective redox partners. This chapter provides an overview of terpenoid hydroxylation reactions catalyzed by bacterial P450s and highlights the achievements made by protein engineering to establish productive hydroxylation processes.

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Adrenodoxin—A versatile ferredoxin

Cited by 52 publications

References 52 publications

Steroid conversion with CYP106A2 – production of pharmaceutically interesting DHEA metabolites

Steroid conversion with CYP106A2 – production of pharmaceutically interesting DHEA metabolites

Process development for the production of 15β-hydroxycyproterone acetate using Bacillus megaterium expressing CYP106A2 as whole-cell biocatalyst

Terpene Hydroxylation with Microbial Cytochrome P450 Monooxygenases

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