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

作者：Foureau Emilien; Clastre Marc; Millerioux Yoann; Simkin Andrew J; Cornet Lucie; Dutilleul Christelle; Besseau Sebastien; Marais Emeline; Melin Celine; Guillard Jerome; Creche Joel; Giglioli Guivarc' h Nathalie; Courdavault Vincent; Papon Nicolas^*

来源：Current Genetics, 2012, 58(4): 245-254.

DOI：10.1007/s00294-012-0377-3

• Transformation system
• Selectable marker
• TRP5
• Tryptophan synthase
• 5-fluoroanthranilic acid
• Candida guilliermondii

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%26apos;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 %26quot;TRP blaster%26quot; disruption systems used TRP1 as counter-selectable marker, this study demonstrated the potential of the TRP5 gene in such strategies. This newly created %26quot;TRP5 blaster%26quot; disruption system thus represents a powerful genetic tool to study the function of a large pallet of genes in C. guilliermondii.

• 出版日期2012-8