Identification of a novel cytochrome P450 17A1 enzyme and its molecular engineering†

Progesterone-17α-hydroxylase (CYP17A) could transform progesterone to 17α-hydroxyprogesterone (17-HP). The main limitation of 17-HP biological production is the lack of CYP17A or the low activity of existing enzymes. A novel CYP17A1 (BT_CYP17A1) from Bovine taurus was identified by genome mining and heterologously expressed in Pichia pastoris GS115. The whole-cell biocatalytic results showed that BT_CYP17A1 possessed high progesterone conversion efficiency (36.8% for 1 mM progesterone) and dual function (17-HP 25.21 ± 1.56 mg L⁻¹ & androstenedione (AD) 85.19 ± 6.81 mg L⁻¹, 22.83% for 17-HP). Enzyme engineering was performed to enhance the 17-HP proportion, and the best mutant of BT_CYP17A1^R347A showed a reverse product ratio (17-HP 113.06 ± 6.72 mg L⁻¹, 96.39% for 17-HP). This phenomenon might be explained by the distance between the substrate and related site being widened for BT_CYP17A1^R347A compared with the parent enzyme (7.0 Å vs. 3.1 Å). Subsequently, cytochrome P450 reductase (CPR_YP) and glucose-6-phosphate dehydrogenase (ZWF_C) were introduced to GS115-BT_CYP17A1R^347A; the 17-HP titer reached 195.86 ± 9.68 mg L⁻¹. Finally, a novel steroid transporter from Cochliobolus heterostrophus (TP_CH) was identified and expressed in GS115-BT_CYP17A1^R347A–CPR_YP–ZWF_C, and the 17-HP titer of GS115-BT_CYP17A1^R347A–CPR_YP–ZWF_C–TP_CH reached 234.82 ± 10.13 mg L⁻¹ with 78.81% conversion efficiency, which is the champion 17-HP bioconversion efficiency for heterologous expression systems.