Disrupting the methionine biosynthetic pathway in <i>Candida guilliermondii</i>: characterization of the <i>MET2</i> gene as counter‐selectable marker

<jats:title>Abstract</jats:title><jats:p><jats:italic>Candida guilliermondii</jats:italic> (teleomorph <jats:italic>Meyerozyma guilliermondii</jats:italic>) 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 <jats:italic>C. guilliermondii</jats:italic> by developing both a methionine auxotroph recipient strain and a functional <jats:italic>MET</jats:italic> gene as selection marker. We first disrupted the <jats:italic>MET2</jats:italic> and <jats:italic>MET15</jats:italic> genes encoding homoserine‐<jats:italic>O</jats:italic>‐acetyltransferase and <jats:italic>O</jats:italic>‐acetylserine <jats:italic>O</jats:italic>‐acetylhomoserine sulphydrylase, respectively. The <jats:italic>met2</jats:italic> mutant was shown to be a methionine auxotroph in contrast to <jats:italic>met15</jats:italic> which was not. Interestingly, m<jats:italic>et2</jats:italic> and <jats:italic>met15</jats:italic> 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 <jats:italic>MET2</jats:italic> wild‐type allele was successfully used to transfer a yellow fluorescent protein (YFP) gene‐expressing vector into the <jats:italic>met2</jats:italic> recipient strain. In addition, we showed that the loss of the <jats:italic>MET2</jats:italic>‐containing YFP‐expressing plasmid can be easily observed on lead‐containing medium. The <jats:italic>MET2</jats:italic> wild‐type allele, flanked by two short repeated sequences, was then used to disrupt the <jats:italic>LYS2</jats:italic> gene (encoding the <jats:italic>α</jats:italic>‐aminoadipate reductase) in the <jats:italic>C. guilliermondii met2</jats:italic> recipient strain. The resulting <jats:italic>lys2</jats:italic> mutants displayed, as expected, auxotrophy for lysine. Unfortunately, all our attempts to pop‐out the <jats:italic>MET2</jats:italic> marker (following the recombination of the bordering repeat sequences) from a target <jats:italic>lys2</jats:italic> locus were unsuccessful using white/brown colony colour screening. Nevertheless, this <jats:italic>MET2</jats:italic> transformation/disruption system represents a new versatile genetic tool for <jats:italic>C. guilliermondii</jats:italic>. Copyright © 2014 John Wiley &amp; Sons, Ltd.</jats:p>