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Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the fungal CTG clade. This yeast remains actively studied as a result of its moderate clinical importance and most of all for its potential uses in biotechnology. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both a methionine auxotroph recipient strain and a functional MET gene as selection marker. We first disrupted the MET2 and MET15 genes encoding homoserine-O-acetyltransferase and O-acetylserine O-acetylhomoserine sulphydrylase, respectively. The met2 mutant was shown to be a methionine auxotroph in contrast to met15 which was not. Interestingly, met2 and met15 mutants formed brown colonies when cultured on lead-containing medium, contrary to the wild-type strain, which develop as white colonies on this medium. The MET2 wild-type allele was successfully used to transfer a yellow fluorescent protein (YFP) gene-expressing vector into the met2 recipient strain. In addition, we showed that the loss of the MET2-containing YFP-expressing plasmid can be easily observed on lead-containing medium. The MET2 wild-type allele, flanked by two short repeated sequences, was then used to disrupt the LYS2 gene (encoding the α-aminoadipate reductase) in the C. guilliermondii met2 recipient strain. The resulting lys2 mutants displayed, as expected, auxotrophy for lysine. Unfortunately, all our attempts to pop-out the MET2 marker (following the recombination of the bordering repeat sequences) from a target lys2 locus were unsuccessful using white/brown colony colour screening. Nevertheless, this MET2 transformation/disruption system represents a new versatile genetic tool for C. guilliermondii., (Copyright © 2014 John Wiley & Sons, Ltd.)

The biotechnological potential of C. guilliermondii is now well established. This yeast species currently benefits from the availability of a convenient molecular toolbox including recipient strains, selectable markers and optimized transformation protocols. However, the number of expression systems for biotechnological applications in C. guilliermondii remains limited. We have therefore developed and characterized a new series of versatile controllable expression vectors for this yeast. While previous studies firmly demonstrated that knock-out systems represent efficient genetic strategies to interrupt yeast biochemical pathways at a specific step in C. guilliermondii, the set of expression plasmids described in this study will provide new powerful opportunities to boost homologous or heterologous biosynthetic routes by fine controlled over-expression approaches., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Fungal histidine kinase receptors (HKR) sense and transduce many intra- and extracellular signals that regulate a wide range of physiological processes. Candida CTG clade species commonly possess three types of HKR namely Sln1p (type VI), Nik1p (type III) and Chk1p (type X). Although some recent work has demonstrated the potential involvement of HKR in osmoregulation, morphogenesis, sexual development, adaptation to osmotic stresses and drug resistance in distinct Candida species, little data is available in relation to their subcellular distribution within yeast cells. We describe in this work the comparative subcellular localization of class III, VI, and X HKRs in Candida guilliermondii, a yeast CTG clade species of clinical and biotechnological interest. Using a fluorescent protein fusion approach, we showed that C. guilliermondii Sln1p fused to the yellow fluorescent protein (Sln1p-YFP) appeared to be anchored in the plasma membrane. By contrast, both Chk1p-YFP and YFP-Chk1p were localized in the nucleocytosol of C. guilliermondii transformed cells. Furthermore, while Nik1p-YFP fusion protein always displayed a nucleocytosolic localization, we noted that most of the cells expressing YFP-Nik1p fusion protein displayed an aggregated pattern of fluorescence in the cytosol but not in the nucleus. Interestingly, Sln1p-YFP and Nik1p-YFP fusion protein localization changed in response to hyperosmotic stress by rapidly clustering into punctuated structures that could be associated to osmotic stress signaling. To date, this work provides the first insight into the subcellular localization of the three classes of HKR encoded by CTG clade yeast genomes and constitutes original new data concerning this family of receptors. This represents also an essential prerequisite to open a window into the understanding of the global architecture of HKR-mediated signaling pathways in CTG clade species., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Candida guilliermondii is an ascomycetous yeast widely studied due to its clinical importance, biotechnological interest, and biological control potential. During a series of preliminary experiments aiming at optimizing the electroporation procedure of C. guilliermondii cells, we observed that the efficiency of transformation of an ura5 recipient strain with the corresponding dominant marker URA5 was more than a thousand fold higher as compared with the transformation of an ura3 strain with the URA3 wild type allele. This result allowed the identification of an autonomously replicating sequence (ARS) within an A/T rich region located upstream of the URA5 open reading frame (ORF). Interestingly, linear double strand DNAs (dsDNAs) containing this putative ARS are circularized and then autonomously replicated in C. guilliermondii transformed cells. We demonstrated that the C. guilliermondii Lig4p ligase, involved in the canonical non-homologous end-joining (NHEJ) pathway, was responsible for this phenomenon since a lig4 mutant was unable to circularize and to autonomously maintain transforming dsDNAs containing the putative ARS. Finally, a functional dissection of the C. guilliermondii A/T rich region located upstream of the URA5 ORF revealed the presence of a 60 bp-length sequence essential and sufficient to confer ARS properties to shuttle plasmid and linear dsDNAs., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The yeast, Candida guilliermondii, has been widely studied due to its biotechnological interest as well as its biological control potential. It integrates foreign DNA predominantly via ectopic events, likely through the well-known non-homologous end-joining (NHEJ) pathway involving the Ku70p/Ku80p heterodimer, Lig4p, Nej1p and Lif1p. This phenomenon remains highly deleterious for targeted gene knock-out strategies that require the homologous recombination process. Here, we have constructed a ku70 mutant strain derived from the ATCC 6260 reference strain of C. guilliermondii. Following a series of disruption attempts of various genes (FCY1, ADE2 and TRP5), using several previously described dominant selectable markers (URA5, SAT-1 and HPH#), we demonstrated that the efficiencies of homologous gene targeting in such a NHEJ-deficient strain was very high compared to the wild type strain. The C. guilliermondii ku70 deficient mutant thus represents a powerful recipient strain to knock-out genes efficiently in this yeast.