Recombinant Production of Human Microsomal Cytochrome P450 2D6 in the Methylotrophic Yeast <i>Pichia pastoris</i>

Dietrich, Matthias; Grundmann, Lisa; Kurr, Katja; Valinotto, Laura; Saussele, Tanja; Schmid, Rolf D.; Lange, Stefan

doi:10.1002/cbic.200500200

Cited by 36 publications

(27 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…cerevisiae is incompatible with human CYP3A4, CYP2B6, and CYP2D6. A similar incompatibility had been observed in Pichia pastoris [21]. Regarding the enzymatic efficiency of coexpressing clones measured by the fluorogenic substrate conversion, the 2D6+ clone exhibited a higher catalytic activity in biotransformation of the substrate into the fluorescence product than the 3A4+ and 2B6+ clones (Fig 1B).…”

Section: Discussionsupporting

confidence: 80%

“…; in phase II (conjugation), transferases use those groups to couple charged molecules making the modified compounds more water soluble so that they can be excreted in the urine [20]. Moreover, for catalytic activity, CYP reactions require the cofactor NADPH as the source of electrons and CPR as the electron transfer partner [21, 22]. In addition to their detoxification function, many CYPs (CYP2B6, CYP2C9, CYP2D6, CYP2E1, CYP3A4, etc.)…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Procarcinogens – Determination and Evaluation by Yeast-Based Biosensor Transformed with Plasmids Incorporating RAD54 Reporter Construct and Cytochrome P450 Genes

Bui¹,

Nguyễn²,

Thanh³

et al. 2016

PLoS ONE

View full text Add to dashboard Cite

In Vietnam, a great number of toxic substances, including carcinogens and procarcinogens, from industrial and agricultural activities, food production, and healthcare services are daily released into the environment. In the present study, we report the development of novel yeast-based biosensor systems to determine both genotoxic carcinogens and procarcinogens by cotransformation with two plasmids. One plasmid is carrying human CPR and CYP (CYP3A4, CYP2B6, or CYP2D6) genes, while the other contains the RAD54-GFP reporter construct. The three resulting coexpression systems bearing both CPR-CYP and RAD54-GFP expression cassettes were designated as CYP3A4/CYP2B6/CYP2D6 + RAD54 systems, respectively and used to detect and evaluate the genotoxic potential of carcinogens and procarcinogens by selective activation and induction of both CPR-CYP and RAD54-GFP expression cassettes in response to DNA damage. Procarcinogens were shown to be predominantly, moderately or not bioactivated by one of the CYP enzymes and thus selectively detected by the specific coexpression system. Aflatoxin B1 and benzo(a)pyrene were predominantly detected by the CYP3A4 + RAD54 system, while N-nitrosodimethylamine only moderately activated the CYP2B6 + RAD54 reporter system and none of them was identified by the CYP2D6 + RAD54 system. In contrast, the genotoxic carcinogen, methyl methanesulfonate, was detected by all systems.Our yeast-reporter system can be performed in 384-well microplates to provide efficient genotoxicity testing to identify various carcinogenic compounds and reduce chemical consumption to about 53% as compared with existing 96-well genotoxicity bioassays. In association with a liquid handling robot, this platform enables rapid, cost-effective, and high-throughput screening of numerous analytes in a fully automated and continuous manner without the need for user interaction.

show abstract

Section: Discussionsupporting

confidence: 80%

Section: Introductionmentioning

confidence: 99%

Procarcinogens – Determination and Evaluation by Yeast-Based Biosensor Transformed with Plasmids Incorporating RAD54 Reporter Construct and Cytochrome P450 Genes

Bui¹,

Nguyễn²,

Thanh³

et al. 2016

PLoS ONE

View full text Add to dashboard Cite

show abstract

“…Therefore, S. pombe strain CAD65 heterologously coexpressing human CYP2B6 and hCPR was chosen for the semipreparative fermentation of PCEEA. Co-expression of the accessory protein hCPR was found to be crucial for sufficient activity of CYP2B6 expressed in fission yeast (Dragan et al, in preparation), which is in line with the findings of other authors that coexpression of hCPR considerably enhances the activity of heterologously expressed human CYPs [16][17][18][19].…”

Section: Resultssupporting

confidence: 76%

Use of fission yeast heterologously expressing human cytochrome P450 2B6 in biotechnological synthesis of the designer drug metabolite N-(1-phenylcyclohexyl)-2-hydroxyethanamine

Peters

Dragan²,

Schwaninger

et al. 2009

Forensic Science International

View full text Add to dashboard Cite

“…They are also used to synthesize drug metabolites in order to assess the toxicity of potential pharmaceuticals [120][121][122]. Recent achievements in the expression of recombinant human P450s in Saccharomyces cerevisiae [76,123,124], Yarrowia lipolytica [125] and Pichia pastoris [126,127] facilitate their use for the synthesis of drug metabolites.…”

Section: Application Of Mammalian P450s For Drug Developmentmentioning

confidence: 99%

Biooxidation with Cytochrome P450 Monooxygenases

Girhard

Urlacher

2010

Modern Oxidation Methods

View full text Add to dashboard Cite

Recombinant Production of Human Microsomal Cytochrome P450 2D6 in the Methylotrophic Yeast Pichia pastoris

Abstract: Abstract:Microsomal cytochrome P450 monooxygenases of the groups 1 -3 are mainly

Cited by 36 publications

References 43 publications

Procarcinogens – Determination and Evaluation by Yeast-Based Biosensor Transformed with Plasmids Incorporating RAD54 Reporter Construct and Cytochrome P450 Genes

Procarcinogens – Determination and Evaluation by Yeast-Based Biosensor Transformed with Plasmids Incorporating RAD54 Reporter Construct and Cytochrome P450 Genes

Use of fission yeast heterologously expressing human cytochrome P450 2B6 in biotechnological synthesis of the designer drug metabolite N-(1-phenylcyclohexyl)-2-hydroxyethanamine

Biooxidation with Cytochrome P450 Monooxygenases

Contact Info

Product

Resources

About