Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase

摘要

A gene, phl, encoding a phenylacetyl-CoA ligase was cloned from a phage library of Penicillium chrysogenum AS-P-78. The presence of five introns in the phi gene was confirmed by reverse transcriptase-PCR. The phi gene encoded an aryl-CoA ligase closely related to Arabidopsis thaliana 4-coumaroyl-CoA ligase. The Phi protein contained most of the amino acids defining the aryl-CoA (4-coumaroyl-CoA) ligase substrate-specificity code and differed from acetyl-CoA ligase and other acyl-CoA ligases. The phl gene was not linked to the penicillin gene cluster. Amplification of phi in an autonomous replicating plasmid led to an 8-fold increase in phenylacetyl-CoA ligase activity and a 35 % increase in penicillin production. Transformants containing the amplified phl gene were resistant to high concentrations of phenylacetic acid (more than 2.5 g/l). Disruption of the phi gene resulted in a 40 %, decrease in penicillin production and a similar reduction of phenylacetyl-CoA ligase activity. The disrupted mutants were highly susceptible to phenylacetic acid. Complementation of the disrupted mutants with the phi gene restored normal levels of penicillin production and resistance to phenylacetic acid. The plenylacetyl-CoA ligase encoded by the phi gene is therefore involved in penicillin production, although a second aryl-CoA ligase appears to contribute partially to phenylacetic acid activation. The Phi protein lacks a peptide-carrier-protein domain and behaves as an aryl-capping enzyme that activates phenylacetic acid and transfers it to the isopenicillin N acyltransferase. The Phi protein contains the peroxisome-targeting sequence that is also present in the isopenicillin N acyltransferase. The peroxisomal colocalization of these two proteins indicates that the last two enzymes of the penicillin pathway form a peroxisomal functional complex.