Genetic analysis of Aspergillus niger mutants defective in benzoate-4-hydroxylase function.

This study was prompted by the observation that an Aspergillus niger transformant with a multicopy bphA (benzoate-4-hydroxylase gene) insert did not grow on benzoate, whereas a transformant with only one extra copy could grow. Therefore, an extensive survey has been made for other genes involved in the conversion of benzoate into 4-hydroxy-benzoate. A transformant with two copies of the bphA gene was used in part of the mutation experiments in order to avoid the isolation of many bphA mutants. Filtration enrichment was used to isolate mutants defective in the conversion of benzoate. The Bph mutants that have been isolated belong to six complementation groups. Mutants with a defected structural gene (bphA) were again predominantly found but, in addition, five other groups of mutants that could not grow on benzoate were isolated. Genetic analysis of the mutants showed that the six genes were localized in different parts of the genome. This was used as an additional proof that some mutants involved different genes. Diploids with seven copies of the bphA gene and heterozygous for one of the other bph genes were constructed. No indication has been obtained that any one of the mutant classes is responsible for the growth-limiting factor in bphA multicopy transformants. This study shows that the p-hydroxylation of benzoate is very complex, although the metabolic pathway is straight forward.

Isolation and molecular characterisation of the benzoate-para-hydroxylase gene (bphA) of Aspergillus niger: a member of a new gene family of the cytochrome P450 superfamily.

The gene coding for benzoate-para-hydroxylase (bphA) of Aspergillus niger was cloned using differential hybridisation techniques and complementation of mutants deficient in this enzyme activity. The nucleotide sequence of the gene was determined, the presence of two introns was shown and the transcription start and termination sites were determined. The structure of the mRNA upstream from the long open reading frame (ORF) is unusual. It contains two small, overlapping ORFs whose function is unknown. Comparison of the deduced amino acid sequence of the protein with the sequences present in the databanks, indicated a significant similarity of BPH to the superfamily of cytochrome P450 enzymes. Further analysis revealed that this protein is a member of a new P450 gene family designated P450LIII. The gene is designated CYP53. To increase the BPH activity of A. niger, multiple copies of the bphA gene were introduced into the genome of a recipient strain by transformation. Although increased intracellular levels of the BPH protein could be detected, the BPH enzyme activity was decreased, suggesting titration of another essential component.