ArticleApplied and environmental microbiology2010
Application of the Saccharomyces cerevisiae FLP/FRT recombination system in filamentous fungi for marker recycling and construction of knockout strains devoid of heterologous genes.
Article in Applied and environmental microbiology, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 35 papers.
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Who cites it
35 citing papers in PubMed, 84 citations in OpenAlex.
- STRIPAK-associated greenbeard proteins DOC1 and DOC2 regulate MAK2 signaling and sexual development in Sordaria macrospora.PLoS biology · 2026Article
- FLP/FRT-Mediated Recombination for Resistance Marker Recycling.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Copper acquisition is essential for plant colonization and virulence in a root-infecting vascular wilt fungus.PLoS pathogens · 2024Article
- NHEJ and HDR can occur simultaneously during gene integration into the genome of Aspergillus niger.Fungal biology and biotechnology · 2024Article
- Recent Advances in Antibiotic-Free Markers; Novel Technologies to Enhance Safe Human Food Production in the World.Molecular biotechnology · 2023Review
- Establishment, optimization, and application of genetic technology inFrontiers in microbiology · 2023Review
- Characterization and engineering of the xylose-inducibleMetabolic engineering communications · 2022Article
- A Straightforward Approach to Synthesize 7-Aminocephalosporanic Acid In Vivo in the Cephalosporin C ProducerJournal of fungi (Basel, Switzerland) · 2022Article
- Review
- TheFrontiers in fungal biology · 2022Article
- Codon Optimization, Soluble Expression and Purification of PE_PGRS45 Gene fromJournal of microbiology and biotechnology · 2021Article
- Golden Gate vectors for efficient gene fusion and gene deletion in diverse filamentous fungi.Current genetics · 2021Article
- Conditional gene expression reveals stage-specific functions of the unfolded protein response in the Ustilago maydis-maize pathosystem.Molecular plant pathology · 2020Article
- Crosstalk Between Pheromone Signaling and NADPH Oxidase Complexes Coordinates Fungal Developmental Processes.Frontiers in microbiology · 2020Article
- High production of valencene in Saccharomyces cerevisiae through metabolic engineering.Microbial cell factories · 2019Article
- Advances in targeting and heterologous expression of genes involved in the synthesis of fungal secondary metabolites.RSC advances · 2019Review
- Construction of a Codon-Adapted Nourseotricin-Resistance Marker Gene for Efficient Targeted Gene Deletion in the Mycophenolic Acid ProducerJournal of fungi (Basel, Switzerland) · 2019Article
- A new strategy for seamless gene editing and marker recycling in Saccharomyces cerevisiae using lethal effect of Cwp1.MicrobiologyOpen · 2019Article
- Development of a novel selection/counter-selection system for chromosomal gene integrations and deletions in lactic acid bacteria.BMC molecular biology · 2019Article
- Establishment of a selection marker recycling system for sequential transformation of the plant-pathogenic fungus Colletotrichum orbiculare.Molecular plant pathology · 2019Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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