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A TRP5/5-fluoroanthranilic acid counter-selection system for gene disruption in Candida guilliermondii

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Abstract

Candida guilliermondii is an interesting biotechnological model for the industrial production of value-added metabolites and also remains an opportunistic emerging fungal agent of candidiasis often associated with oncology patients. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both an ATCC 6260-derived recipient strain and a recyclable selection marker. We first disrupted the TRP5 gene in the wild-type strain and demonstrated that trp5 mutants were tryptophan auxotroph as well as being resistant to the antimetabolite 5-fluoroanthranilic acid (FAA). Following an FAA selection of spontaneous mutants derived from the ATCC 6260 strain and complementation analysis, we demonstrated that trp5 genotypes could be directly recovered on FAA-containing medium. The TRP5 wild-type allele, flanked by two short repeated sequences of its 3′UTR, was then used to disrupt the FCY1 gene in C. guilliermondii trp5 recipient strains. The resulting fcy1 mutants displayed strong flucytosine resistance and a counter-selection on FAA allowed us to pop-out the TRP5 allele from the FCY1 locus. To illustrate the capacity of this blaster system to achieve a second round of gene disruption, we knocked out both the LEU2 and the HOG1 genes in the trp5, fcy1 background. Although all previously described yeast “TRP blaster” disruption systems used TRP1 as counter-selectable marker, this study demonstrated the potential of the TRP5 gene in such strategies. This newly created “TRP5 blaster” disruption system thus represents a powerful genetic tool to study the function of a large pallet of genes in C. guilliermondii.

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Acknowledgments

The authors acknowledge the Broad Institute Fungal Genome Initiative for making the complete genome sequence of C. guilliermondii available. The authors thank “Le STUDIUM” (Agency for Research and Hosting Foreign associated Researchers in the Centre region, France) for the financial support of A. J. Simkin.

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Authors and Affiliations

  1. Biomolécules et Biotechnologies Végétales, UFR Pharmacie, Université François Rabelais de Tours, EA2106, Tours, France

    Emilien Foureau, Marc Clastre, Lucie Cornet, Emeline Marais, Joël Crèche & Nicolas Papon

  2. Centre de Résonance Magnétique des Systèmes Biologiques, UMR 5536, Université Victor Segalen Bordeaux 2, Bordeaux, France

    Yoann Millerioux

  3. Biomolécules et Biotechnologies Végétales, UFR Sciences et Techniques, Université François Rabelais de Tours, EA2106, Tours, France

    Christelle Dutilleul, Sébastien Besseau, Céline Melin, Nathalie Giglioli-Guivarc’h & Vincent Courdavault

  4. Laboratoire de Chimie Organique IC2MP, Faculté de Médecine-Pharmacie, Université de Poitiers, Poitiers, France

    Jérôme Guillard

  5. School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, UK

    Andrew J. Simkin

Authors
  1. Emilien Foureau
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  2. Marc Clastre
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  3. Yoann Millerioux
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  4. Andrew J. Simkin
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  5. Lucie Cornet
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  6. Christelle Dutilleul
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  7. Sébastien Besseau
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  8. Emeline Marais
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  9. Céline Melin
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  10. Jérôme Guillard
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  11. Joël Crèche
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  12. Nathalie Giglioli-Guivarc’h
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  13. Vincent Courdavault
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  14. Nicolas Papon
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Corresponding author

Correspondence to Nicolas Papon.

Additional information

Communicated by C. Gaillardin.

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Foureau, E., Clastre, M., Millerioux, Y. et al. A TRP5/5-fluoroanthranilic acid counter-selection system for gene disruption in Candida guilliermondii . Curr Genet 58, 245–254 (2012). https://doi.org/10.1007/s00294-012-0377-3

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  • DOI: https://doi.org/10.1007/s00294-012-0377-3

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