Whole-cell microtiter plate screening assay for terminal hydroxylation of fatty acids by P450s

Weissenborn, Martin J.; Notonier, Sandra; Lang, Sarah-Luise; Otte, Konrad B.; Herter, Susanne; Turner, Nicholas J.; Flitsch, Sabine L.; Hauer, Bernhard

doi:10.1039/c6cc01749e

Cited by 14 publications

(15 citation statements)

References 31 publications

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“…The measured biocatalytic performance expressed in terms of the total turnover numbers (TTN), that is, the ratio between total product concentration and catalyst concentration, calculated over 24 h is shown in Figure . Here, the variability of P450 expression in E. coli may lead to misjudging the observed yields, thus, a fast, whole‐cell assay was implemented to determine P450 concentration, which was then used to normalize enzyme activity . Finally, the enantiomeric excess values ( ee ) were also determined by comparison with synthesised standards.…”

Section: Resultsmentioning

confidence: 84%

One‐Pot Biocatalytic Double Oxidation of α‐Isophorone for the Synthesis of Ketoisophorone

et al. 2017

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The chemical synthesis of ketoisophorone, a valuable building block of vitamins and pharmaceuticals, suffers from several drawbacks in terms of reaction conditions and selectivity. Herein, the first biocatalytic one‐pot double oxidation of the readily available α‐isophorone to ketoisophorone is described. Variants of the self‐sufficient P450cam‐RhFRed with improved activity have been identified to perform the first step of the designed cascade (regio‐ and enantioselective allylic oxidation of α‐isophorone to 4‐hydroxy‐α‐isophorone). For the second step, the screening of a broad panel of alcohol dehydrogenases (ADHs) led to the identification of Cm‐ADH10 from Candida magnoliae. The crystal structure of Cm‐ADH10 was solved and docking experiments confirmed the preferred position and geometry of the substrate for catalysis. The synthesis of ketoisophorone was demonstrated both as a one‐pot two‐step process and as a cascade process employing designer cells co‐expressing the two biocatalysts, with a productivity of up to 1.4 g L−1 d−1.

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Section: Resultsmentioning

confidence: 84%

One‐Pot Biocatalytic Double Oxidation of α‐Isophorone for the Synthesis of Ketoisophorone

et al. 2017

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“…We started this investigation of a cofactor‐free P450 BM3 system by focusing on an electrochemically active molecule for direct on‐electrode enzyme activity detection. Previous work with P450 assay systems showed that para ‐aminophenol is both electrochemically active and a product of P450‐catalysed aniline hydroxylation .…”

Section: Resultsmentioning

confidence: 99%

Expanding an Efficient, Electrically Driven and CNT‐Tagged P450 System into the Third Dimension: A Nanowired CNT‐Containing and Enzyme‐Stabilising 3 D Sol–Gel Electrode

et al. 2016

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Although electrochemically catalysed P450 reactions have been described, their efficiency and applicability remained limited. This is mostly due to low enzyme activity, laborious protein immobilisation and the small electrode surface. We established a novel protein immobilisation method for a determined orientation and electrical wiring of the enzyme without post-expression modification. By genetic introduction of an anchor-peptide our method is applicable for screening medium to large mutant libraries and detection by an electrode system. The system was expanded by using wired carbon nanotubes within a sol-gel matrix to create a three dimensional electrode.

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“…Some wild-type organisms have found applications in drug synthesis. 9 The solubility and ease of high level heterologous production of bacterial CYPs in Escherichia coli make them attractive for developing new biotechnological applications in, for example, the synthesis of fine chemicals, [10][11][12][13][14][15][16][17][18][19] intermediates, 14, [20][21][22] drugs, [23][24][25][26][27][28] and antibiotics. [29][30][31] However, many of the Class I CYP enzymes are not genetically associated with their Fdx.…”

Section: Introductionmentioning

confidence: 99%

A Structural Model of a P450-Ferredoxin Complex from Orientation-Selective Double Electron–Electron Resonance Spectroscopy

et al. 2018

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Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of chemically inert carbon-hydrogen bonds in diverse endogenous and exogenous organic compounds by atmospheric oxygen. This C-H bond oxy-functionalization activity has huge potential in biotechnological applications. Class I CYPs receive the two electrons required for oxygen activation from NAD(P)H via a ferredoxin reductase and ferredoxin. The interaction of Class I CYPs with their cognate ferredoxin is specific. In order to reconstitute the activity of diverse CYPs, structural characterization of CYP-ferredoxin complexes is necessary, but little structural information is available. Here we report a structural model of such a complex (CYP199A2-HaPux) in frozen solution derived from distance and orientation restraints gathered by the EPR technique of orientation-selective double electron-electron resonance (os-DEER). The long-lived oscillations in the os-DEER spectra were well modeled by a single orientation of the CYP199A2-HaPux complex. The structure is different from the two known Class I CYP-Fdx structures: CYP11A1-Adx and CYP101A1-Pdx. At the protein interface, HaPux residues in the [FeS] cluster-binding loop and the α3 helix and the C-terminus residue interact with CYP199A2 residues in the proximal loop and the C helix. These residue contacts are consistent with biochemical data on CYP199A2-ferredoxin binding and electron transfer. Electron-tunneling calculations indicate an efficient electron-transfer pathway from the [FeS] cluster to the heme. This new structural model of a CYP-Fdx complex provides the basis for tailoring CYP enzymes for which the cognate ferredoxin is not known, to accept electrons from HaPux and display monooxygenase activity.

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Whole-cell microtiter plate screening assay for terminal hydroxylation of fatty acids by P450s

Cited by 14 publications

References 31 publications

One‐Pot Biocatalytic Double Oxidation of α‐Isophorone for the Synthesis of Ketoisophorone

One‐Pot Biocatalytic Double Oxidation of α‐Isophorone for the Synthesis of Ketoisophorone

Expanding an Efficient, Electrically Driven and CNT‐Tagged P450 System into the Third Dimension: A Nanowired CNT‐Containing and Enzyme‐Stabilising 3 D Sol–Gel Electrode

A Structural Model of a P450-Ferredoxin Complex from Orientation-Selective Double Electron–Electron Resonance Spectroscopy

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