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

Cytochrome P450 enzyme systems are found throughout nature and are involved in many different, often complex, bioconversions. In the endoplasmic retic ulum of the filamentous fungus Aspergillus niger a cytochrome P450 enzyme s ystem is present that is capable of the para-hydroxylation of benzoate. The expression of the two genes encoding the components of this system, the cy tochrome P450 gene encoding benzoate para-hydroxylase (bphA) and the gene e ncoding cytochrome P450 reductase (cprA), is inducible by benzoate. The BPH system was used as a model system to study the mechanisms that result in c o-regulation of both components of an eukaryote cytochrome P450 enzyme syst em. Deletion analysis of the transcription control regions of cprA and bphA resulted in the identification of a region that was involved in benzoate i nduction of gene expression. The functional competence of the cprA Benzoate Responsive Region thus defined was demonstrated directly by cloning this f ragment upstream of a constitutively expressed mini-promoter and analysing expression of the hybrid transcription control region in a lacZ reporter sy stem. Further analysis of cprA gene expression revealed a clear quantitativ e discrepancy between induction at the protein level (approximately 4-fold) and at the transcription level (> 20-fold). The majority of the transcript s observed after benzoate induction (cprA beta) were larger then the consti tutively expressed cprA alpha transcript. The difference in size between th e cpr da and cprA beta transcript is caused by differential promoter use. A s the longer cprA beta 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 resul ts show that regulation of CPR expression is particularly complex, involvin g regulatory promoter elements, differential promoter use and regulation at the post-transcriptional level.