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Cloning and characterization of the NADPH cytochrome P450 oxidoreductase gene from the filamentous fungus Aspergillus niger

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Author: Brink, J.M. van den · Zeijl, C.M.J. van · Brons, J.F. · Hondel, C.A.M.J.J. van den · Gorcom, R.F.M. van
Type:article
Date:1995
Institution: Centraal Instituut voor Voedingsonderzoek TNO
Source:DNA and Cell Biology, 8, 14, p. 719-729. Ook in: Cytochrome P450 reductase of Aspergillus niger : a molecular biological study / J.M. van den Brink. - Leiden : Rijksuniversiteit, 1997. - p. 31-48. - Thesis
Identifier: 68172
Keywords: Nutrition · Benzoic acid derivative · Reduced nicotinamide adenine dinucleotide phosphate ferrihemoprotein reductase · Amino acid sequence · Aspergillus niger · Gene isolation · Gene sequence · Genetic analysis · Hybridization · Molecular cloning · Nonhuman · Nucleotide sequence · Polymerase chain reaction · Priority journal · Sequence analysis · Structure activity relation · Amidohydrolases · Amino Acid Sequence · Aspergillus niger · Base Sequence · Benzoates · Benzoic Acid · Cloning, Molecular · Conserved Sequence · Enzyme Induction · Gene Dosage · Gene Expression Regulation, Fungal · Genes, Structural, Fungal · Molecular Sequence Data · NADPH-Ferrihemoprotein Reductase · Polymerase Chain Reaction · Restriction Mapping · Sequence Alignment · Sequence Analysis, DNA · Transformation, Genetic

Abstract

In this paper, we describe the cloning and molecular characterization of the Aspergillus niger cytochrome P450 reductase (CPR) gene, cprA. Attempts to clone the cprA gene by heterologous hybridization techniques were unsuccessful. Using the polymerase chain reaction (PCR) with degenerate primers based on conserved regions found in cpr genes from other organisms, we were able to isolate a fragment that contained part of the gene. With the aid of this fragment, a genomic fragment containing the entire coding region and 5' and 3' untranslated ends of the cprA gene was isolated and sequenced. The cprA gene was introduced in multiple copies in A. niger strain N402 using the amdS transformation system. One of the resulting transformants, AB2-2, showed a 14-fold increase in CPR activity, indicating that the cloned cprA gene is functional. We analyzed the induction of cprA gene expression by several generally used cytochrome P450 inducers but did not find any induction of cprA gene expression. However, A. niger cprA gene expression could be induced by benzoic acid, which is the substrate of the highly inducible A. niger cytochrome P450 gene, bphA (cyp53). On the basis of a comparison of the deduced protein sequence of the A. niger cprA gene with CPR proteins isolated from other organisms, the structure-function relationship of some conserved regions is discussed.