Title
Cloning and characterization of the NADPH cytochrome P450 oxidoreductase gene from the filamentous fungus Aspergillus niger
Author
van den Brink, J.M.
van Zeijl, C.M.J.
Brons, J.F.
van den Hondel, C.A.M.J.J.
van Gorcom, R.F.M.
Centraal Instituut voor Voedingsonderzoek TNO
Publication year
1995
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.
Subject
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
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TNO identifier
68172
Source
DNA and Cell Biology, 14 (8)
Document type
article