title: Nonlinear biosynthetic gene cluster dose effect on penicillin production by Penicillium chrysogenum.
abstract: Industrial penicillin production levels by the filamentous fungus Penicillium chrysogenum increased dramatically by classical strain improvement. High-yielding strains contain multiple copies of the penicillin biosynthetic gene cluster that encodes three key enzymes of the β-lactam biosynthetic pathway. We have analyzed the gene cluster dose effect on penicillin production using the high-yielding P. chrysogenum strain DS17690 that was cured from its native clusters. The amount of penicillin V produced increased with the penicillin biosynthetic gene cluster number but was saturated at high copy numbers. Likewise, transcript levels of the biosynthetic genes pcbAB [δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase], pcbC (isopenicillin N synthase), and penDE (acyltransferase) correlated with the cluster copy number. Remarkably, the protein level of acyltransferase, which localizes to peroxisomes, was saturated already at low cluster copy numbers. At higher copy numbers, intracellular levels of isopenicillin N increased, suggesting that the acyltransferase reaction presents a limiting step at a high gene dose. Since the number and appearance of the peroxisomes did not change significantly with the gene cluster copy number, we conclude that the acyltransferase activity is limiting for penicillin biosynthesis at high biosynthetic gene cluster copy numbers. These results suggest that at a high penicillin production level, productivity is limited by the peroxisomal acyltransferase import activity and/or the availability of coenzyme A (CoA)-activated side chains.
authors.affiliation: Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, Zernike Institute for Advanced Materials, Centre for Synthetic Biology, and Kluyver Centre for Genomics of Industrial Fermentation, University of Groningen, Kerklaan 30, 9751 NN Haren, Netherlands.
authors.forename: Jeroen G
authors.initials: JG
authors.lastname: Nijland
authors.forename: Bjorg
authors.initials: B
authors.lastname: Ebbendorf
authors.forename: Marta
authors.initials: M
authors.lastname: Woszczynska
authors.forename: Rémon
authors.initials: R
authors.lastname: Boer
authors.forename: Roel A L
authors.initials: RA
authors.lastname: Bovenberg
authors.forename: Arnold J M
authors.initials: AJ
authors.lastname: Driessen
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D018628:Gene Dosage / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D005810:Multigene Family / Q000235:genetics* / Q000502:physiology; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010404:Penicillin V / Q000378:metabolism; D046915:Penicillin-Binding Proteins / Q000235:genetics / Q000378:metabolism; D010406:Penicillins / Q000096:biosynthesis* / Q000378:metabolism; D010408:Penicillium chrysogenum / Q000235:genetics* / Q000378:metabolism; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction
chemical_list: D046915:Penicillin-Binding Proteins; D010406:Penicillins; D010088:Oxidoreductases; C038370:isopenicillin N synthetase; D000217:Acyltransferases; C018756:acyl-CoA-6-aminopenicillanic acid acyltransferase; D010453:Peptide Synthases; C095368:alpha-aminoadipyl-cysteinyl-valine synthetase; C006414:penicillin N; D010404:Penicillin V