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

Cloning and Sequence Analysis of a NADPH-cytochrome P450 Reductase Gene from Penicillium raistrickii

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%.

Filamentous fungus Penicillium 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 15a-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, a P. 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 ×10^4,which contains the structural domains typical of CPR, such as FMN binding pocket, FAD and NADPH binding pocket. NCBI BLAST search reveals that P. raistrickii CPR has the highest homology and 93% identity with P. italicum.