Functional reconstruction of bovine P450scc steroidogenic system in Escherichia coli

Abstract

Mammalian cytochrome P450scc enzyme system cata-lyzes the initial step in steroid hormone biosynthe-sis—cholesterol hydroxylation followed by cleavage of the side-chain to yield pregnenolone. This system consists of three components—the cytochrome P450scc (CYP11A1), a flavoprotein (NADPH-adrenodoxin re-ductase, AdR) and an iron-sulfur protein (adreno-doxin, Adx). In this work, the three-component elec-tron transport chain (AdR/Adx/CYP11A1) from bo-vine adrenal cortex has been implemented in Es-cherichia coli by co-expression of the corresponding coding sequences from a tricistronic plasmid. The cDNAs of AdR, Adx and CYP11A1 are situated in a single transcription unit and separated by ribosome binding sequences. The recombinant strain created was capable of synthesizing functional proteins iden-tical to the bovine CYP11A1, AdR and Adx on mo-lecular weights and immuno-specificity. The experi-ments in vivo showed pregnenolone production from cholesterol by the transformed bacteria. Maximal productivity of 0.42 ± 0.015 mg/l pregnenolone for 24 h has been reached for the induced cells in the pres-ence of cholesterol solubilizing agent—methyl-β-cy-clodextrin. Thus, a stable transgenic E. coli strain with the functional reconstructed bovine cholesterol side-chain cleavage system has been firstly generated in this work. The findings are of importance for stud-ies of mammalian steroidogenic system features, and may open some perspectives for further generation of novel microbial biocatalysts.