title: Penicillin production in industrial strain Penicillium chrysogenum P2niaD18 is not dependent on the copy number of biosynthesis genes.
abstract: Multi-copy gene integration into microbial genomes is a conventional tool for obtaining improved gene expression. For Penicillium chrysogenum, the fungal producer of the beta-lactam antibiotic penicillin, many production strains carry multiple copies of the penicillin biosynthesis gene cluster. This discovery led to the generally accepted view that high penicillin titers are the result of multiple copies of penicillin genes. Here we investigated strain P2niaD18, a production line that carries only two copies of the penicillin gene cluster.
  
  
  
  We performed pulsed-field gel electrophoresis (PFGE), quantitative qRT-PCR, and penicillin bioassays to investigate production, deletion and overexpression strains generated in the P. chrysogenum P2niaD18 background, in order to determine the copy number of the penicillin biosynthesis gene cluster, and study the expression of one penicillin biosynthesis gene, and the penicillin titer. Analysis of production and recombinant strain showed that the enhanced penicillin titer did not depend on the copy number of the penicillin gene cluster. Our assumption was strengthened by results with a penicillin null strain lacking pcbC encoding isopenicillin N synthase. Reintroduction of one or two copies of the cluster into the pcbC deletion strain restored transcriptional high expression of the pcbC gene, but recombinant strains showed no significantly different penicillin titer compared to parental strains.
  
  
  
  Here we present a molecular genetic analysis of production and recombinant strains in the P2niaD18 background carrying different copy numbers of the penicillin biosynthesis gene cluster. Our analysis shows that the enhanced penicillin titer does not strictly depend on the copy number of the cluster. Based on these overall findings, we hypothesize that instead, complex regulatory mechanisms are prominently implicated in increased penicillin biosynthesis in production strains.
authors.affiliation: Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, ND7/131, Universitätsstraße 150, 44780, Bochum, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Ziemons
authors.affiliation: Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, ND7/131, Universitätsstraße 150, 44780, Bochum, Germany.
authors.forename: Katerina
authors.initials: K
authors.lastname: Koutsantas
authors.affiliation: Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, ND7/131, Universitätsstraße 150, 44780, Bochum, Germany.
authors.forename: Kordula
authors.initials: K
authors.lastname: Becker
authors.affiliation: Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, ND7/131, Universitätsstraße 150, 44780, Bochum, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Dahlmann
authors.affiliation: Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, ND7/131, Universitätsstraße 150, 44780, Bochum, Germany. ulrich.kueck@rub.de.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Kück
mesh_terms: D053898:Biosynthetic Pathways / Q000235:genetics; D018628:Gene Dosage / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods; D007218:Industrial Microbiology; D005810:Multigene Family / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics; D010406:Penicillins / Q000096:biosynthesis* / Q000302:isolation & purification; D010408:Penicillium chrysogenum / Q000145:classification* / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics*; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D010406:Penicillins; D011994:Recombinant Proteins; D010088:Oxidoreductases; C038370:isopenicillin N synthetase
keywords: Beta-lactam antibiotics; Gene cluster amplification; Penicillium chrysogenum; Production strain