Discovery of Novel p-Hydroxybenzoate-m-hydroxylase, Protocatechuate 3,4 Ring-Cleavage Dioxygenase, and Hydroxyquinol 1,2 Ring-Cleavage Dioxygenase from the Filamentous Fungus Aspergillus niger

摘要

The aromatic compound benzoic acid and its derivatives are frequently used preservatives in food and beverages due to their strong antimicrobial properties. Benzoic acid naturally occurs in plants and serves as a building block for primary and secondary metabolites. Several Aspergilli are able to degrade benzoic acid to p-hydroxybenzoic acid by p-hydroxylation followed by m-hydroxylation to protocatechuic acid. The aromatic ring of protocatechuic acid is then cleaved and further converted through the oxoadipate pathway where it is used as a carbon source. The benzoic acid pathway is well studied in the filamentous fungus Aspergillus niger; however, only one enzyme, benzoate-4-monooxygenase, has been characterized. In this study, three novel enzymes of the benzoic acid pathway were identified and characterized. Transcriptome analysis of A. niger on p-hydroxybenzoic acid, protocatechuic acid, and the related compounds p-coumaric acid and caffeic acid revealed two genes of unknown function, which were highly expressed on all four aromatic compounds. Analysis of A. niger deletion mutants of these genes revealed their essential role in the utilization of benzoic acid, p-hydroxybenzoic acid, and protocatechuic acid. Biochemical analysis confirmed that the corresponding enzymes function as the p-hydroxybenzoate-m-hydroxylase and protocatechuate 3,4 ring-cleavage dioxygenase in the benzoic acid pathway. In addition, a hydroxyquinol 1,2 ring-cleavage dioxygenase, involved in an alternative protocatechuic acid metabolic pathway, was identified.