Metabolism of cholesterol with Escherichia coli whole cells coexpressing P450 27A1 with adrenodoxin and adrenodoxin reductase

27‐Hydroxycholesterol (27HC) is a biologically active metabolite. Its levels are elevated in hypercholesterolemia and have been found in atherosclerotic lesions. 27HC is produced by the action of cytochrome P450 (P450) 27A1 through an alternative (extrahepatic) pathway of oxysterol biosysnthesis from cholesterol. In eukaryotic cells P450 27A1 is a membrane bound protein located on the inner mitochondrial membrane that requires two auxiliary reduction partners, adrenodoxin (Adx) and NADPH‐adrenodoxin reductase (Adr), for catalysis in the bile acid biosyntesis pathway. We developed a strategy for the functional coexpression of the P450 27A1 with Adr and Adx in a tricistronic fashion (single RNA, three proteins), mimicking the mitochondrial P450 system in Escherichia coli . Intact bacterial cells coexpressing the P450 system efficiently hydroxylated cholesterol in the 27 position when supplemented with glycerol as a carbon source. Chemical derivatization with dansyl chloride was used for the detection of the hydroxylated product by LC‐MS/MS in selected reaction monitoring (SRM) mode. This system maybe useful in the study of other mitochondrial P450s and demonstrates the advantage of a self‐sufficient P450 catalytic system for the metabolism of cholesterol derivatives. Supported in part by NIH R37 CA090426