Random and targeted gene integrations through the control of non-homologous end joining in the yeast Kluyveromyces marxianus.
Kluyveromyces marxianus DMKU3-1042 is a thermotolerant yeast strain suitable for high-temperature ethanol fermentation and genetic engineering with linear DNA. We have developed a highly efficient random gene integration method with a frequency that exceeds 2.5 x 10(6) transformants/microg linear DNA, a figure comparable to what is observed with autonomously replicating plasmid transformation in Saccharomyces cerevisiae. To establish the mechanism of random integration in DMKU3-1042, we identified and deleted the K. marxianus KU70 gene, which is known to be involved in the non-homologous end-joining (NHEJ) pathway. In yeast lacking KU70, high-frequency non-homologous gene integration was abolished and the Kmku70 mutants showed 82-95% homologous gene targeting efficiencies using homologous sequences of 40-1000 bp. These results indicate that the highly efficient NHEJ pathway can be utilized with random gene disruption techniques such as transposon mutagenesis and plasmid-free gene manipulations in K. marxianus.
Abdel-Banat
Babiker M A
BM
Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, Tokiwadai, Ube 755-8611, Japan.
Nonklang
Sanom
S
Hoshida
Hisashi
H
Akada
Rinji
R
D000595:Amino Acid Sequence; D004271:DNA, Fungal / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D005818:Genetic Engineering; D007716:Kluyveromyces / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D004271:DNA, Fungal; D005656:Fungal Proteins