Identification of a novel cytochrome P450 17A2 enzyme catalyzing the C17α hydroxylation of progesterone and its application in engineered Pichia pastoris

Abstract

The 17α-hydroxyprogesterone (17-HP) is an important steroid drug. Progesterone-17α-hydroxylase (CYP17A) transforms progesterone into 17-HP; however, the major limitation in the bioproduction of 17-HP is the lack of CYP17A2 or low activity of the existing enzymes. Herein, a novel CYP17A2 from Mastacembelus armatus (MA_CYP17A2) was obtained through genome mining and expressed in Pichia pastoris GS115. The biotransformation of 17-HP was performed in whole cells and the product analysis indicated that MA_CYP17A2 possessed the activities of progesterone C17-hydroxylate (17-HP titer, 43.5 ± 4.7 mg/L). Moreover, cytochrome P450 oxidoreductase (CPR) was optimized from five different species by co-expressing them in engineered GS115-MA_CYP17A2 with dual plasmids for boosting the electron-transfer. Furthermore, glucose-6-phosphate dehydrogenase (ZWF) was optimized from three different species and used for enhancing the regeneration of NADPH in the presence of glucose. After optimization, a binary vector of pPICZB-cpr-zwf containing optimal cprrat from Rattus norvegicus and zwfk from Kluyveromyces lactis was constructed and electro-transformed into GS115-MA_CYP17A2 for the co-expression of three foreign proteins in one host. Finally, the synthetic ability of 17-HP on GS115-MA_CYP17A2-CPRRAT-ZWFK was determined, the 17-HP titer improved by 2.8-fold and 17-HP specific productivity improved by 3.0-fold compared to GS115-MA_CYP17A2. The heterologous expression system GS115-MA_CYP17A2-CPRRAT-ZWFK has been reported to cause the highest 17-HP bioconversion. This study provides a promising hydroxylation system candidate for the efficient biosynthesis of 17-HP.