雷斯青霉NADPH-细胞色素P450还原酶基因的克隆及生物信息学分析

摘要

Filamentous fungusPenicillium raistrickii is employed for commercial production of 15α-hydroxyl-13-ethyl-estr-4-ene-3,17-dione,a key intermediate for the production of Gestodene,a potent oral contraceptive.It is documented that steroid 15α-hydroxylase involved in the transformation is a P450,enzyme which is believed to be composed of the cyto-chrome P450 hydroxylase and the NADPH-cytochrome P450 reductase(CPR),although the molecular mechanism is yet to be elucidated.Based on the RNA sequencing data set,aP.raistrickii NADPH-cytochrome P450 reductase was cloned through RT-PCR amplification.The full open reading frame of the CPR is 2,082 bp,predicted to encode a polypeptide of 694 amino acids with a molecular weight of 7.63×104,which contains the structural domains typical of CPR,such as FMN binding pocket,FAD and NADPH binding pocket.NCBI BLAST search reveals thatP.raistrickii CPR has the highest homology and 93%, identity withP.italicum.%工业上利用丝状真菌雷斯青霉(Penicillium raistrickii)转化生产高效避孕药孕二烯酮的关键中间体15α–羟基左旋乙基甾烯双酮．已知参与雷斯青霉甾体转化反应的关键酶为P450羟化酶，该羟化酶体系由细胞色素P450羟化酶和 NADPH–细胞色素 P450还原酶(CPR)组成，但有关其15α–羟基化反应的分子基础尚不清楚．根据转录组测序数据库，通过RT-PCR扩增克隆了一个雷斯青霉NADPH-细胞色素P450还原酶基因．该基因的开放阅读框为2,082,bp，编码694个氨基酸的多肽链，预测的蛋白相对分子质量为7.63×104，具有CPR蛋白的典型结构域(FMN结合域、FAD和NADPH结合域)．NCBI BLAST结果显示雷斯青霉CPR与意大利青霉(P.,italicum)NADPH–细胞色素P450还原酶具有较高的同源性，一致性为93%.