OPTIMIZATION EF THE BENZOATE INDUCIBLE BENZOATE P-HYDROXYLASE CYTOCHROME-P450 ENZYME-SYSTEM IN ASPERGILLUS-NIGER

Introduction in the fungus Aspergillus niger of multiple copies of the A. niger bphA gene, encoding the cytochrome P450 enzyme benzoate p-hy droxylase, did not result in increased activities of this enzyme [Gorc om 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 reducta se. For improvement of this and other cytochrome-P450-dependent reacti ons. A. niger strains were constructed in which the copy number of the A. niger cprA gene (encoding cytochrome-P450 reductase) or the copy n umbers of both cprA and thr cytochrome-P450-encoding gene were increas ed. Expression of both genes was controlled by their own transcription control regions. Benzoate p-hydroxylase activity of different transfo rmants was determined in microsomal fractions using a newly developed indirect in vitro assay. In transformants containing multiple copies o f both genes, benzoate p-hydroxylase activity was significantly higher than in the wild-type strain or in transformants in which the copy nu mber of only one of the gents was increased. These results clearly ind icate the importance of co-expression of cytochrome-P450 reductase for achieving maximal cytochrome P450 activities in cytochrome-P450-overp roducing filamentous fungi.