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.
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Research Article|
March 15 2006
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
Mónica Lamas-Maceiras;
Mónica Lamas-Maceiras
*Área de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, Campus de Vegazana, s/n, 24071 León, Spain
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Inmaculada Vaca;
Inmaculada Vaca
†Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, 1 Av. Real, 24006 León, Spain
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Esther Rodríguez;
Esther Rodríguez
*Área de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, Campus de Vegazana, s/n, 24071 León, Spain
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Javier Casqueiro;
Javier Casqueiro
*Área de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, Campus de Vegazana, s/n, 24071 León, Spain
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Juan F. Martín
Juan F. Martín
1
*Área de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, Campus de Vegazana, s/n, 24071 León, Spain
†Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, 1 Av. Real, 24006 León, Spain
1To whom correspondence should be addressed (email degjmm@unileon.es).
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Publisher: Portland Press Ltd
Received:
September 28 2005
Revision Received:
December 01 2005
Accepted:
December 02 2005
Accepted Manuscript online:
December 02 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 395 (1): 147–155.
Article history
Received:
September 28 2005
Revision Received:
December 01 2005
Accepted:
December 02 2005
Accepted Manuscript online:
December 02 2005
Citation
Mónica Lamas-Maceiras, Inmaculada Vaca, Esther Rodríguez, Javier Casqueiro, Juan F. Martín; 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. Biochem J 1 April 2006; 395 (1): 147–155. doi: https://doi.org/10.1042/BJ20051599
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