Journal
YEAST
Volume 34, Issue 12, Pages 483-494Publisher
WILEY
DOI: 10.1002/yea.3251
Keywords
Pseudozyma antarctica; auxotrophic mutant; transformation marker; gene disruption; electroporation; lithium acetate treatment
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Funding
- Ministry of Agriculture, Forestry and Fisheries (Japan) [25017AB]
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The basidiomycetous yeast Pseudozyma antarctica is a remarkable producer of industrially valuable enzymes and extracellular glycolipids. In this study, we developed a method for targeted gene replacement in P. antarctica. In addition, transformation conditions were optimized using lithium acetate, single-stranded carrier DNA and polyethylene glycol (lithium acetate treatment), generally used for ascomycetous yeast transformation. In the rice-derived P. antarctica strain GB-4(0), PaURA3, a homologue of the Saccharomyces cerevisiae orotidine-5-phosphate decarboxylase gene (URA3), was selected as the target locus. A disruption cassette was constructed by linking the nouseothricine resistance gene (natMX4) to homologous DNA fragments of PaURA3, then electroporated into the strain GB-4(0). We obtained strain PGB015 as one of the PaURA3 disruptants (Paura3::natMX4). Then the PCR-amplified PaURA3 fragment was introduced into PGB015, and growth of transformant colonies but not background colonies was observed on selective media lacking uracil. The complementation of uracil-auxotrophy in PGB015 by introduction of PaURA3 was also performed using lithium acetate treatment, which resulted in a transformation efficiency of 985CFU/6.8g DNA and a gene-targeting ratio of two among 30 transformants. Copyright (c) 2017 John Wiley & Sons, Ltd.
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