Targeted gene replacement at the URA3 locus of the basidiomycetous yeast Pseudozyma antarctica and its transformation using lithium acetate treatment

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 homolog 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 (Paura3Δ::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 985 CFU/6.8 μg DNA and a gene‐targeting ratio of two among 30 transformants.
Source: Yeast - Category: Molecular Biology Authors: Tags: Research Article Source Type: research