title: A TRP5/5-fluoroanthranilic acid counter-selection system for gene disruption in Candida guilliermondii.
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.
authors.affiliation: Biomolécules et Biotechnologies Végétales, UFR Pharmacie, Université François Rabelais de Tours, EA2106, Tours, France.
authors.forename: Emilien
authors.initials: E
authors.lastname: Foureau
authors.forename: Marc
authors.initials: M
authors.lastname: Clastre
authors.forename: Yoann
authors.initials: Y
authors.lastname: Millerioux
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Simkin
authors.forename: Lucie
authors.initials: L
authors.lastname: Cornet
authors.forename: Christelle
authors.initials: C
authors.lastname: Dutilleul
authors.forename: Sébastien
authors.initials: S
authors.lastname: Besseau
authors.forename: Emeline
authors.initials: E
authors.lastname: Marais
authors.forename: Céline
authors.initials: C
authors.lastname: Melin
authors.forename: Jérôme
authors.initials: J
authors.lastname: Guillard
authors.forename: Joël
authors.initials: J
authors.lastname: Crèche
authors.forename: Nathalie
authors.initials: N
authors.lastname: Giglioli-Guivarc'h
authors.forename: Vincent
authors.initials: V
authors.lastname: Courdavault
authors.forename: Nicolas
authors.initials: N
authors.lastname: Papon
mesh_terms: D002175:Candida / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005819:Genetic Markers; D005810:Multigene Family; D009154:Mutation; D050052:TRPC Cation Channels / Q000235:genetics* / Q000378:metabolism; D062367:ortho-Aminobenzoates / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D005819:Genetic Markers; D050052:TRPC Cation Channels; D062367:ortho-Aminobenzoates; C031385:anthranilic acid