van den Brink J M, van den Hondel C A, van Gorcom R F
TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene Technology, Rijswijk, The Netherlands.
Appl Microbiol Biotechnol. 1996 Nov;46(4):360-4. doi: 10.1007/BF00166230.
Introduction in the fungus Aspergillus niger of multiple copies of the A. niger bphA gene, encoding the cytochrome P450 enzyme benzoate p-hydroxylase, did not result in increased activities of this enzyme [Gorcom RFM van, et al. Mol Gen Genet (1990) 223: 192-197] probably because of low expression levels of the gene encoding the second component of the microsomal cytochrome P450 enzyme system, cytochrome P450 reductase. For improvement of this and other cytochrome-P450-dependent reactions, A. niger strains were constructed in which the copy number of the A. niger cprA gene (encoding cytochrome-P450 reductase) or the copy numbers of both cprA and the cytochrome-P450-encoding gene were increased. Expression of both genes was controlled by their own transcription control regions. Benzoate p-hydroxylase activity of different transformants was determined in microsomal fractions using a newly developed indirect in vitro assay. In transformants containing multiple copies of both genes, benzoate p-hydroxylase activity was significantly higher than in the wild-type strain or in transformants in which the copy number of only one of the genes was increased. These results clearly indicate the importance of co-expression of cytochrome-P450 reductase for achieving maximal cytochrome P450 activities in cytochrome-P450-overproducing filamentous fungi.
将编码细胞色素P450酶苯甲酸对羟基化酶的黑曲霉bphA基因的多个拷贝导入黑曲霉中，并未导致该酶活性增加[Gorcom RFM van等人，《分子遗传学与普通遗传学》（1990年）223: 192 - 197]，这可能是因为编码微粒体细胞色素P450酶系统第二个组分即细胞色素P450还原酶的基因表达水平较低。为了改善这种情况以及其他细胞色素P450依赖的反应，构建了黑曲霉菌株，其中黑曲霉cprA基因（编码细胞色素P450还原酶）的拷贝数或cprA和细胞色素P450编码基因的拷贝数均增加。两个基因的表达均由其自身的转录控制区调控。使用新开发的间接体外测定法在微粒体组分中测定了不同转化体的苯甲酸对羟基化酶活性。在含有两个基因多个拷贝的转化体中，苯甲酸对羟基化酶活性显著高于野生型菌株或仅增加其中一个基因拷贝数的转化体。这些结果清楚地表明，在细胞色素P450过量产生的丝状真菌中，细胞色素P450还原酶的共表达对于实现最大细胞色素P450活性至关重要。
