Co-expression and Functional Analysis of Cytochrome P450 Reductase and CYP17

doi:10.13523/j.cb.2205009

China Biotechnology

2022, Vol. 42

Issue (10): 1-8 DOI: 10.13523/j.cb.2205009

Co-expression and Functional Analysis of Cytochrome P450 Reductase and CYP17

Qiong WU,Xin ZHAO,Yu-yao DU,Shu-hong MAO^**(

)

Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education, School of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China

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Abstract

17α-hydroxyprogesterone (17α-OH-PROG) is a key intermediate in steroidal hormone drugs, and its biosynthesis is mainly catalyzed by cytochrome monooxygenase (CYP17). In this process, cytochrome P450 reductases (CPRs) are important electron transport chain partners of cytochrome P450 enzymes, which directly affect the catalytic efficiency of CYP17. In order to study the effect of different CPRs on the activity of 17α-hydroxylase, the expression vector pPIC3.5k-hCYP17 was constructed with human 17α-hydroxylase gene, and the recombinant Pichia pastoris strain was obtained. Then, three CPRs from different sources were screened, the expression vector pPICZX-CPR was constructed, and then 17α-hydroxylase and CPR co-expression strains were obtained, and the transformation of progesterone was carried out using the recombinant P.pastoris. Finally, the transformation products were analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The results showed that the successfully recombinant strain showed 17α-hydroxylase activity, which was able to catalyze progesterone to produce the target product 17α-OH-PROG and the by-product 16α-hydroxyprogesterone (16α-OH-PROG). Furthermore, compared with strain containing only 17α-hydroxylase, the yield of 17α-OH-PROG all improved when CPR and 17α-hydroxylase were co-expressed in P.pastoris. Of the three CPRs tested, co-expression of hCPR and CYP17 showed the highest progesterone transformation ability, and the yield of 17α-OH-PROG increased by 42%. The above results indicated that the suitable CPR co-expressed with 17α-hydroxylase resulted in the increase of 17α-OH-PROG production. This study provides a promising strategy for the production of 17α-OH-PROG catalyzed by CYP17, which is of great significance for the industrial production of steroid.

Key words： P.pastoris Progesterone CYP17 Heterologous expression Cytochrome P450 reductase

Received: 04 May 2022 Published: 04 November 2022

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Corresponding Authors: Shu-hong MAO E-mail: shuhongmao@tust.edu.cn

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Cite this article:

Qiong WU,Xin ZHAO,Yu-yao DU,Shu-hong MAO. Co-expression and Functional Analysis of Cytochrome P450 Reductase and CYP17. China Biotechnology, 2022, 42(10): 1-8.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2205009 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I10/1

Fig.1 Schematic diagram of pPIC3.5k-hCYP17 plasmid and its PCR verification results M:Marker,1:pPIC3.5k-hCYP17 PCR verification,2:pPIC3.5k-hCYP17-A105L PCR verification

Fig.2 PCR verification results of P. pastoris reorganization genomic M: 10 kb Marker; 1-2: Genome verification of pPIC3.5k-hCYP17 recombinant strain; 3-4: Genome verification of pPIC3.5k-hCYP17-A105L recombinant strain

Fig.3 TLC and HPLC analysis of recombinant P. pastoris fermentation

Table 1 Expression levels of P. pastoris strains

Fig.4 PCR verification results of P.pastoris recombinant plasmid CPR gene M: Marker; 1-3: pPICZX-hCPR, pPICZX-ratCPR, pPICZX-ScCPR, plasmid PCR verification results

Fig.5 PCR verification of CPR gene and CYP gene of P. pastoris recombinant strains M: Marker; 1-2: hCYP17-hCPR was amplified with hCPR primer and hCYP primer; 3-4: hCYP17-ratCPR was amplified with ratCPR primer and hCYP primer; 5-6: hCYP17-ScCPR was amplified with ScCPR Primers and hCYP

Table 2 Conversion rates of progesterone in CPR P.pastoris recombinant strains


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