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

Abstract

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 phl gene was confirmed by reverse transcriptase-PCR. The phl gene encoded an aryl-CoA ligase closely related to Arabidopsis thaliana 4-coumaroyl-CoA ligase. The Phl 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 phl 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 phl 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 phl gene restored normal levels of penicillin production and resistance to phenylacetic acid. The phenylacetyl-CoA ligase encoded by the phl gene is therefore involved in penicillin production, although a second aryl-CoA ligase appears to contribute partially to phenylacetic acid activation. The Phl 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 Phl protein contains the peroxisome-targeting sequence that is also present in the isopenicillin N acyltransferase. The peroxisomal co-localization of these two proteins indicates that the last two enzymes of the penicillin pathway form a peroxisomal functional complex.

Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum

“…This gene was not transcribed under the conditions studied (Supplementary Table 6). As deletion of phl, which encodes phenylacetyl-CoA ligase, resulted only in a partial loss of penicillinG production 23 , other phenylacetyl-CoA ligases must be present 24 . These may include identified orthologs of 4-coumarateCoA ligase.…”

Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells

“…The importance of microbodies in penicillin production is underscored by the identification of a PTS1-related sequence on key enzymes of penicillin biosynthesis, isopenicillin N:acyl CoA acyltransferase (IAT; -ARL-COOH; Barredo et al 1989) and two phenylacetyl-CoA ligases (PclA, -SKI-COOH and PhlB, -AKL-COOH; Lamas-Maceiras et al 2006;Wang et al 2007). Indeed, IAT and PclA have been shown to be located in the microbody matrix (Müller et al 1991;Gidijala et al 2007; W.H.…”

Transport of substrates into peroxisomes: the paradigm of β-lactam biosynthetic intermediates

“…The pH of the cytosol in filamentous fungi has been reported to be between 6.5 and 7.0 (106) , whereas the internal pH of the peroxisomes was shown to be 7.5 (107) . This peroxisomal pH is near the optimal pH for the IAT, which was found to be 7.5 -8.0 (108, 109) , for phenylacetyl-CoA ligase (86) and for the linear fatty acid CoA ligase (88) .…”

“…It is now well established that the last steps of penicillin biosynthesis performed by the IAT and the phenylacetylCoA ligase occur in peroxisomes (16,85,86,110) . These enzymes are synthesized in ribosomes in the cytoplasm and targeted to the peroxisomal matrix in a PTS1-dependent manner.…”