title: Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae.
abstract: The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.
authors.affiliation: Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Yarimizu
authors.forename: Sanom
authors.initials: S
authors.lastname: Nonklang
authors.forename: Junpei
authors.initials: J
authors.lastname: Nakamura
authors.forename: Shuya
authors.initials: S
authors.lastname: Tokuda
authors.forename: Takaaki
authors.initials: T
authors.lastname: Nakagawa
authors.forename: Sasithorn
authors.initials: S
authors.lastname: Lorreungsil
authors.forename: Surasit
authors.initials: S
authors.lastname: Sutthikhumpha
authors.forename: Charida
authors.initials: C
authors.lastname: Pukahuta
authors.forename: Takao
authors.initials: T
authors.lastname: Kitagawa
authors.forename: Mikiko
authors.initials: M
authors.lastname: Nakamura
authors.forename: Kamonchai
authors.initials: K
authors.lastname: Cha-Aim
authors.forename: Savitree
authors.initials: S
authors.lastname: Limtong
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hoshida
authors.forename: Rinji
authors.initials: R
authors.lastname: Akada
mesh_terms: D004271:DNA, Fungal / Q000235:genetics*; D005816:Genetic Complementation Test; D007716:Kluyveromyces / Q000166:cytology / Q000235:genetics*; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: D004271:DNA, Fungal
keywords: Kluyveromyces marxianus; Saccharomyces cerevisiae; auxotrophic mutants; mating; non-homologous end-joining; tetrad