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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2022, Vol. 42 Issue (10): 1-8    DOI: 10.13523/j.cb.2205009
研究报告     
细胞色素P450还原酶与CYP17的共表达及其功能分析*
吴琼,赵昕,杜玉瑶,毛淑红**()
工业发酵微生物教育部重点实验室 天津科技大学生物工程学院 天津 300457
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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摘要：

17α-羟基黄体酮(17α-OH-PROG)是甾体激素类药物的关键中间体,其生物合成主要由细胞色素单加氧酶(CYP17)催化生成。在此过程中,细胞色素 P450还原酶(cytochrome P450 reductase,CPR)作为细胞色素P450 酶电子传递链的重要组成部分,直接影响CYP17的催化效率。为研究不同来源CPR与17α-羟化酶的适配性,首先以人源17α-羟化酶作为研究对象,构建了表达质粒pPIC3.5k-hCYP17,获得了重组毕赤酵母菌株。其次筛选获得3种不同来源CPR,构建了表达质粒 pPICZX-CPR,获得17α-羟化酶与CPR共表达菌株,并在毕赤酵母中进行转化实验,对转化产物进行薄层色谱(TLC)和高效液相色谱(HPLC)分析。结果显示,重组菌株具有17α-羟化酶活性,能够催化黄体酮生成目标产物17α-OH-PROG 以及副产物16α-羟基黄体酮(16α-OH-PROG)。不同来源的CPR与17α-羟化酶共表达与仅表达17α-羟化酶的产率相比均有所提高,其中hCPR-CYP17共表达菌株表现出最高的转化水平,17α-OH-PROG产率提高42%。上述结果表明:17α-羟化酶基因与CPR共表达能够提高其黄体酮17α-羟基化水平。为甾体黄体酮17α-羟基化的生物催化研究提供思路,对甾体药物的工业生产具有重要意义。
关键词： 毕赤酵母;  黄体酮;  17α-羟化酶;  异源表达;  细胞色素P450还原酶    
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
收稿日期: 2022-05-04 出版日期: 2022-11-04
ZTFLH:  Q814  
基金资助: * 国家重点研发计划(2019YFA0905300);天津市合成生物技术创新能力提升行动(TSBICIP-KJGG-001-09)
通讯作者: 毛淑红     E-mail: shuhongmao@tust.edu.cn
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引用本文:

吴琼,赵昕,杜玉瑶,毛淑红. 细胞色素P450还原酶与CYP17的共表达及其功能分析*[J]. 中国生物工程杂志, 2022, 42(10): 1-8.

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.

链接本文:

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

图1  pPIC3.5k-hCYP17质粒示意图及其PCR验证结果
图2  毕赤酵母重组菌基因组PCR验证结果
图3  毕赤酵母重组菌发酵TLC及HPLC分析
Strains Product EV/% hCYP17/% A105L/%
P.pastoris 16α-OH-P4 0 11.0 10.3
17α-OH-P4 0 28.5 34.45
表1  毕赤酵母重组菌表达水平
图4  毕赤酵母重组质粒CPR基因PCR验证结果
图5  毕赤酵母重组菌CPR基因与CYP基因 PCR验证
菌株 17α-OH-P4 /% 16α-OH-P4 /%
EV 0 0
WT 28.50 11.20
hCYP17-hCPR 40.44 10.39
hCYP17-ScCPR 34.67 11.00
hCYP17-ratCPR 34.60 11.60
A105L 34.45 10.30
A105L-hCPR 35.60 10.30
A105L-ScCPR 36.24 2.50
A105L-ratCPR 30.00 7.90
表2  CPR毕赤酵母重组菌对黄体酮的转化率
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