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.
Université François-Rabelais de Tours, EA2106, Biomolécules et Biotechnologies Végétales, Tours, France; Universidad de Antioquia, Laboratorio de Biotecnología, Sede de Investigación Universitaria, Colombia.
Erika J
EJ
Obando Montoya
Céline
C
Mélin
Nathalie
N
Blanc
Arnaud
A
Lanoue
Emilien
E
Foureau
Leslie
L
Boudesocque
Gildas
G
Prie
Andrew J
AJ
Simkin
Joël
J
Crèche
Lucia
L
Atehortùa
Nathalie
N
Giglioli-Guivarc'h
Marc
M
Clastre
Vincent
V
Courdavault
Nicolas
N
Papon
D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics; D002175:Candida / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003547:Cysteine Synthase / Q000235:genetics / Q000378:metabolism; D005819:Genetic Markers / Q000235:genetics / Q000502:physiology; D008164:Luminescent Proteins / Q000235:genetics; D008715:Methionine / Q000096:biosynthesis* / Q000235:genetics; D008856:Microscopy, Fluorescence; D016254:Mutagenesis, Insertional; D014170:Transformation, Genetic
D005819:Genetic Markers; D008164:Luminescent Proteins; D008715:Methionine; D000123:Acetyltransferases; C021369:homoserine O-acetyltransferase; D003547:Cysteine Synthase
Candida guilliermondii; methionine auxotrophy; selectable marker; transformation system