title: Application of the Saccharomyces cerevisiae FLP/FRT recombination system in filamentous fungi for marker recycling and construction of knockout strains devoid of heterologous genes.
abstract: To overcome the limited availability of antibiotic resistance markers in filamentous fungi, we adapted the FLP/FRT recombination system from the yeast Saccharomyces cerevisiae for marker recycling. We tested this system in the penicillin producer Penicillium chrysogenum using different experimental approaches. In a two-step application, we first integrated ectopically a nourseothricin resistance cassette flanked by the FRT sequences in direct repeat orientation (FRT-nat1 cassette) into a P. chrysogenum recipient. In the second step, the gene for the native yeast FLP recombinase, and in parallel, a codon-optimized P. chrysogenum flp (Pcflp) recombinase gene, were transferred into the P. chrysogenum strain carrying the FRT-nat1 cassette. The corresponding transformants were analyzed by PCR, growth tests, and sequencing to verify successful recombination events. Our analysis of several single- and multicopy transformants showed that only when the codon-optimized recombinase was present could a fully functional recombination system be generated in P. chrysogenum. As a proof of application of this system, we constructed a DeltaPcku70 knockout strain devoid of any heterologous genes. To further improve the FLP/FRT system, we produced a flipper cassette carrying the FRT sites as well as the Pcflp gene together with a resistance marker. This cassette allows the controlled expression of the recombinase gene for one-step marker excision. Moreover, the applicability of the optimized FLP/FRT recombination system in other fungi was further demonstrated by marker recycling in the ascomycete Sordaria macrospora. Here, we discuss the application of the optimized FLP/FRT recombination system as a molecular tool for the genetic manipulation of filamentous fungi.
authors.affiliation: Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Germany.
authors.forename: Katarina
authors.initials: K
authors.lastname: Kopke
authors.forename: Birgit
authors.initials: B
authors.lastname: Hoff
authors.forename: Ulrich
authors.initials: U
authors.lastname: Kück
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D025141:Drug Resistance, Fungal; D055786:Gene Knockout Techniques / Q000379:methods; D005800:Genes, Fungal; D016254:Mutagenesis, Insertional; D010408:Penicillium chrysogenum / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013309:Streptothricins / Q000494:pharmacology
chemical_list: D000935:Antifungal Agents; D004271:DNA, Fungal; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013309:Streptothricins; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase
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