Publication:
Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae

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Issued Date

2013

Resource Type

Article

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application/pdf

ISSN

0749503X

DOI

10.1002/yea.2985

Other identifier(s)

2-s2.0-84890183384

Rights Holder(s)

Scopus

Bibliographic Citation

Yeast. Vol 30, No.12 (2013), p.485-500

Suggested Citation

Yarimizu T., Nonklang S., Nakamura J., Tokuda S., Nakagawa T., Lorreungsil S., Sutthikhumpha S., Pukahuta C., Kitagawa T., Nakamura M., Cha-Aim K., Limtong S., Hoshida H., Akada R. Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae. Yeast. Vol 30, No.12 (2013), p.485-500. doi:10.1002/yea.2985 Retrieved from: https://hdl.handle.net/20.500.14740/6508

Title

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae

Author(s)

Yarimizu T.
 Nonklang S.
 Nakamura J.
 Tokuda S.
 Nakagawa T.
 Lorreungsil S.
 Sutthikhumpha S.
 Pukahuta C.
 Kitagawa T.
 Nakamura M.
 Cha-Aim K.
 Limtong S.
 Hoshida H.
 Akada R.

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. © 2013 John Wiley & Sons, Ltd.

Subject(s)

DNA fragment
 Amino acid metabolism
 Article
 Auxotrophic mutant
 Chromosome
 DNA end joining repair
 Fungal strain
 Gene amplification
 Gene library
 Gene mutation
 Gene synthesis
 Genetic code
 Genetic complementation
 Genetic identification
 Genetic transformation
 Kluyveromyces marxianus
 Mating
 Mutation rate
 Nonhuman
 Nucleic acid metabolism
 Nutritional requirement
 Phenotype
 Polymerase chain reaction
 Priority journal
 Saccharomyces cerevisiae
 Sporogenesis
 Kluyveromyces marxianus
 Saccharomyces cerevisiae
 Auxotrophic mutants
 Kluyveromyces marxianus
 Mating
 Non-homologous end-joining
 Saccharomyces cerevisiae
 Tetrad
 DNA, Fungal
 Genetic Complementation Test
 Kluyveromyces
 Mutation
 Plasmids
 Recombination, Genetic
 Saccharomyces cerevisiae
 Transformation, Genetic
 Transgenes

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URI

https://hdl.handle.net/20.500.14740/6508

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