Optimization of 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-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.
van den Brink
J M
JM
TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene Technology, Rijswijk, The Netherlands.
van den Hondel
C A
CA
van Gorcom
R F
RF
D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics*; D001565:Benzoates / Q000494:pharmacology*; D019817:Benzoic Acid; D018628:Gene Dosage; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects*; D005800:Genes, Fungal / Q000235:genetics; D008861:Microsomes / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010105:Oxygenases / Q000235:genetics*; D014170:Transformation, Genetic
D001565:Benzoates; D019817:Benzoic Acid; D010105:Oxygenases; C019766:benzoate 1,2-dioxygenase; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase