The use of Yarrowia lipolytica for the expression of human cytochrome P450 CYP1A1.
Cytochromes P450 constitute a superfamily of haem-thiolate mono-oxygenases that are involved in the oxidative metabolism of lipophilic subtrates. These enzymes require association with cytochrome P450 reductase (CPR) to achieve optimal activities. We have expressed human cytochrome P450 CYP1A1 under the POX2 promoter (pPOX2-CYP1A1) in Y. lipolytica, with or without overproduction of Y. lipolytica CPR expressed under the ICL promoter (pICL-CPR) or the POX2 promoter (pPOX2-CPR). Activity of cytochrome CYP1A1 was analysed by conversion of hydroxyresorufin to resorufin. Strain JMY330 and JMY330-CPR present no activity, the monocopy cytochrome CYP1A1 integrant JMY331 and JMY331-CPR derivatives present an average activity of 32.0 pM/min/dw and 48.3 and 64.6 pM/min/dw for pICL-CPR and pPOX2-CPR, respectively. Increase of CPR expression resulted in about two-fold higher activity. The multicopy 1A1 integrant JMY339 and JMY339-CPR derivatives present an activity of 129 pM/min/dw and 815-1845 pM/min/dw, respectively. Increase of CPR expression resulted in 6.3-12.8-fold higher activity, depending on the CPR transformant. We observed a 50-fold increase of activity between the monocopy integrant JMY331 as compared to the multicopies integrant JMY339-CPR in which CPR was overexpressed.
Nthangeni
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MB
Laboratoire de Microbiologie et Génétique Moléculaire, INRA CNRS INAP-G, UMR2585, Centre de Biotechnologie Agro-Industrielle, 78850 Thiverval-Grignon, France.
Urban
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Pompon
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Smit
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MS
Nicaud
J-M
JM
D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis* / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010078:Oxazines / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic / Q000502:physiology; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics*
D010078:Oxazines; D011994:Recombinant Proteins; C007649:ethoxyresorufin; D019363:Cytochrome P-450 CYP1A1