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Regulation of expression of the Aspergillus niger benzoate para-hydroxylase cytochrome P450 system

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Author: Brink, J.M. van den · Punt, P.J. · Gorcom, R.F.M. van · Hondel, C.A.M.J.J. van den
Institution: Centraal Instituut voor Voedingsonderzoek TNO
Source:Molecular and General Genetics, 4, 263, 601-609
Identifier: 87182
Keywords: Nutrition · Aspergillus niger · Benzoate · Cytochrome P450 · Post-transcriptional regulation · Benzoic acid · Cytochrome P450 · Cytochrome P450 reductase · Oxygenase · Aspergillus niger · DNA responsive element · Enzyme analysis · Enzyme regulation · Gene expression regulation · Gene function · Gene induction · Genetic transcription · Hydroxylation · Nonhuman · Nucleotide sequence · Priority journal · Promoter region · Protein induction · Amino Acid Sequence · Aspergillus niger · Base Sequence · Benzoates · Enzyme Induction · Gene Expression Regulation, Bacterial · Gene Expression Regulation, Enzymologic · Mixed Function Oxygenases · Molecular Sequence Data · NADH, NADPH Oxidoreductases · NADPH-Ferrihemoprotein Reductase · Oxygenases · Promoter Regions (Genetics) · Recombinant Fusion Proteins · Restriction Mapping · Sequence Alignment · Sequence Homology, Amino Acid · Transcription, Genetic · Aspergillus niger · Eukaryota · Fungi


Cytochrome P450 enzyme systems are found throughout nature and are involved in many different, often complex, bioconversions. In the endoplasmic reticulum of the filamentous fungus Aspergillus niger a cytochrome P450 enzyme system is present that is capable of the para-hydroxylation of benzoate. The expression of the two genes encoding the components of this system, the cytochrome P450 gene encoding benzoate para-hydroxylase (bphA) and the gene encoding cytochrome P450 reductase (cprA), is inducible by benzoate. The BPH system was used as a model system to study the mechanisms that result in co-regulation of both components of an eukaryote cytochrome P450 enzyme system. Deletion analysis of the transcription control regions of cprA and bphA resulted in the identification of a region that was involved in benzoate induction of gene expression. The functional competence of the cprA Benzoate Responsive Region thus defined was demonstrated directly by cloning this fragment upstream of a constitutively expressed mini-promoter and analysing expression of the hybrid transcription control region in a lacZ reporter system. Further analysis of cprA gene expression revealed a clear quantitative discrepancy between induction at the protein level. (approximately 4-fold) and at the transcription level (> 20-fold). The majority of the transcripts observed after benzoate induction (cprAβ) were larger then the constitutively expressed cprAα transcript. The difference in size between the cprAα and cprAβ transcript is caused by differential promoter use. As the longer cprAβ transcript carries a small uORF we propose that post-transcriptional regulation of CPR expression underlies the discrepancy in the degree of induction at the protein and transcriptional level. Our results show that regulation of CPR expression is particularly complex, involving regulatory promoter elements, differential promoter use and regulation at the post-transcriptional level. Chemicals/CAS: benzoate 4-monooxygenase, EC; Benzoates; biphenyl-2,3-dioxygenase, EC 1.14.99.-; Mixed Function Oxygenases, EC 1.-; NADH, NADPH Oxidoreductases, EC 1.6.; NADPH-Ferrihemoprotein Reductase, EC; Oxygenases, EC 1.13.; Recombinant Fusion Proteins