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

Obando Montoya, Erika J and Mélin, Céline and Blanc, Nathalie and Lanoue, Arnaud and Foureau, Emilien and Boudesocque, Leslie and Prie, Gildas and Simkin, Andrew J and Crèche, Joël and Atehortùa, Lucia and Giglioli-Guivarc'h, Nathalie and Clastre, Marc and Courdavault, Vincent and Papon, Nicolas (2014) 'Disrupting the methionine biosynthetic pathway in<i>Candida guilliermondii</i>: characterization of the<i>MET2</i>gene as counter-selectable marker.' Yeast, 31 (7). pp. 243-251. ISSN 0749-503X

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Abstract

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. © 2014 John Wiley & Sons, Ltd.

Item Type: Article
Uncontrolled Keywords: transformation system; selectable marker; methionine auxotrophy; Candida guilliermondii
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Faculty of Science and Health
Faculty of Science and Health > Life Sciences, School of
SWORD Depositor: Elements
Depositing User: Elements
Date Deposited: 06 Jul 2015 13:47
Last Modified: 18 Aug 2022 11:15
URI: http://repository.essex.ac.uk/id/eprint/14236

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