Disrupting the methionine biosynthetic pathway in Candida guilliermondii: characterization of the MET2 gene as counter-selectable marker.

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.