Disruption of phacA, an Aspergillus nidulans Gene Encoding a Novel Cytochrome P450 Monooxygenase Catalyzing Phenylacetate 2-Hydroxylation, Results in Penicillin Overproduction

Abstract

Aspergillus nidulans utilizes phenylacetate as a carbon source via homogentisate, 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 aromatic ring. Using cDNA substraction techniques, we have characterized a gene, denoted phacA, whose transcription is strongly induced by phenylacetate and which putatively encodes a cytochrome P450 protein. A disrupted phacA strain does not grow on phenylacetate but grows on 2-hydroxy-or 2,5-dihydroxyphenylacetate. Microsomal extracts of the disrupted strain are deficient in the NADPH-dependent conversion of phenylacetate to 2-hydroxyphenylacetate. We conclude that PhacA catalyzes the ortho-hydroxylation of phenylacetate, the first step of A. nidulans phenylacetate catabolism. The involvement of a P450 enzyme in the ortho-hydroxylation of a monoaromatic compound has no precedent. In addition, PhacA shows substantial sequence divergence with known cytochromes P450 and defines a new family of these enzymes, suggesting that saprophytic fungi may represent a source of novel cytochromes P450.Phenylacetate is a precursor for benzylpenicillin production. phacA disruption increases penicillin production 3-5-fold, indicating that catabolism competes with antibiotic biosynthesis for phenylacetate and strongly suggesting strategies for Penicillium chrysogenum strain improvement by reverse genetics.Aerobic degradation of aromatic hydrocarbons by microbes involves the action of oxygenases (enzymes that incorporate one or two atoms from dioxygen into substrates) acting at two different levels in specific catabolic pathways (1, 2). First, oxygenase enzymes acting at the upstream segment of these pathways incorporate one (monooxygenases, aromatic ring hydroxylases) or two (aromatic ring dioxygenases) oxygen atoms into the aromatic substrate as hydroxyl groups, preparing the ring for a subsequent ring-opening step. In this second step, the dihydroxylated aromatic ring is opened by ring-cleavage dioxygenases.Monooxygenases are a mechanistically diverse group of enzymes (1) including, for example, flavoproteins such as p-hydroxybenzoate hydroxylase (3), multicomponent enzymes such as Pseudomonas mendocina toluene 4-monoxygenase, in which one of the terminal hydroxylase polypeptides contains a binuclear iron cluster (4), or heme-containing cytochrome P450 systems. Monooxygenases of the cytochrome P450 superfamily (5, 6) are widely distributed among living organisms and catalyze a multiplicity of biosynthetic and catabolic reactions, usually with narrow substrate specificity, including the hydroxylation of a variety of lipophylic drugs.In common with other saprophytic microbes, the genetically amenable, obligate aerobic fungus Aspergillus nidulans shows notable metabolic versatility. For example, it can use the aromatic hydrocarbon compound phenylacetate (PhAc) 1 as sole carbon source. Despite the abundant information available on the cataboli...

Reduced Function of a Phenylacetate-Oxidizing Cytochrome P450 Caused Strong Genetic Improvement in Early Phylogeny of Penicillin-Producing Strains

“…The pahA gene from Wisconsin 54-1255 was isolated by hybridization of a genomic library with a 1.2-kb NotI probe corresponding to the A. nidulans homologue phacA, which encodes an enzyme with PA 2-hydroxylase activity (19). Sequence analysis of a 2.5-kb XhoI fragment hybridizing to the phacA probe with the codon preference algorithm (Geneplot, DNASTAR) showed a coding region of 1,723 nucleotides interrupted by three introns of 57, 64, and 55 bp.…”

“…Competition between PA catabolism and penicillin G biosynthesis in P. chrysogenum. The PA catabolic pathway is the same proposed for A. nidulans (19). The enzyme phenylacetate 2-hydroxylase controls the incorporation of PA into a catabolic detoxification pathway, reducing the pool available for penicillin G biosynthesis.…”

“…A genomic library of P. chrysogenum Wisconsin 54-1255 constructed in EMBL4 was screened using as a probe a 1.2-kb NotI cDNA fragment from the phacA gene of A. nidulans (19) according to standard procedures (23). Three recombinant phages were isolated and mapped, locating the hybridization signal in a 2.5-kb XhoI fragment that was subcloned in both orientations in pBluescript KS(ϩ), giving pALP519 and pALP520.…”

“…They catalyze diverse enzymatic reactions involved in the metabolism of endogenous compounds as sterols and vitamins together with the catabolism of toxic and xenobiotic compounds (21). Targeted disruption of the phacA gene in A. nidulans resulted in a partial inability to catabolyze PA by the transformants, together with a fivefold-increased penicillin G productivity (19). These results prompted us to characterize the homologous pahA (PA 2-hydroxylase) gene from P. chry-sogenum and to study its influence on PA oxidation and penicillin G production.…”

“…1). The first step of this pathway involves a 2-hydroxylation of the PA ring by a microsomal cytochrome P450 [PA 2-hydroxylase; phenylacetate, NAD(P):oxygen oxidoreductase (2-hydroxylating); EC number 1.14.13], encoded by the phacA gene (phenylacetate catabolism) (19). The cytochrome P450 superfamily groups more than 500 hemoproteins that are widely distributed in nature.…”