Disruption of phacA, an Aspergillus nidulans gene encoding a novel cytochrome P450 monooxygenase catalyzing phenylacetate 2-hydroxylation, results inpenicillin overproduction

Aspergillus nidulans utilizes phenylacetate as a carbon source via homogent isate, which is degraded to fumarate and acetoacetate. Mutational evidence strongly suggested that phenylacetate is converted to homogentisate through two sequential hydroxylating reactions in positions 2 and 5 of the aromati c ring. Using cDNA substraction techniques, we have characterized a gene, d enoted phacA, whose transcription is strongly induced by phenylacetate and which putatively encodes a cytochrome P450 protein. A disrupted phacA strai n does not grow on phenylacetate but grows on 2-hydroxy- or 2,5-dihydroxyph enylacetate. Microsomal extracts of the disrupted strain are deficient in t he NADPH-dependent conversion of phenylacetate to 2-hydroxyphenylacetate. W e conclude that PhacA catalyzes the ortho-hydroxylation of phenylacetate, t he first step of A. nidulans phenylacetate catabolism, The involvement of a P450 enzyme in the ortho-hydroxylation of a monoaromatic compound has no p recedent, In addition, PhacA shows substantial sequence divergence with kno wn cytochromes P450 and defines a new family of these enzymes, suggesting t hat saprophytic fungi may represent a source of novel cytochromes P450, Phenylacetate is a precursor for benzylpenicillin production, phacA disrupt ion increases penicillin production 3-5-fold, indicating that catabolism co mpetes with antibiotic biosynthesis for phenylacetate and strongly suggesti ng strategies for Penicillium chrysogenum strain improvement by reverse gen etics.