Uracil-auxotrophic marker recycling system for multiple gene disruption in Pseudozyma antarctica
Abstract The basidiomycetous yeast Pseudozyma antarctica, which has multiple auxotrophic markers, was constructed, without inserting a foreign gene, as the host strain for the introduction of multiple useful genes. P. antarctica was more resistant to ultraviolet (UV) irradiation than the model yeast...
| Published in: | Bioscience, Biotechnology, and Biochemistry |
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| Main Authors: | , , , , |
| Format: | Article in Journal/Newspaper |
| Language: | English |
| Published: |
Oxford University Press (OUP)
2022
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| Subjects: | |
| Online Access: | http://dx.doi.org/10.1093/bbb/zbac075 https://academic.oup.com/bbb/advance-article-pdf/doi/10.1093/bbb/zbac075/43815511/zbac075.pdf |
| Summary: | Abstract The basidiomycetous yeast Pseudozyma antarctica, which has multiple auxotrophic markers, was constructed, without inserting a foreign gene, as the host strain for the introduction of multiple useful genes. P. antarctica was more resistant to ultraviolet (UV) irradiation than the model yeast Saccharomyces cerevisiae, and a Paura3 mutant (C867T) was obtained after 3 min of UV exposure. A uracil-auxotrophic marker (URA3) recycling system developed in ascomycetous yeasts and fungi was applied to the P. antarctica Paura3 strain. The PaLYS12 and PaADE2 loci were disrupted via site-directed homologous recombination of PaURA3 (pop-in), followed by the removal of PaURA3 (pop-out). In the obtained double auxotrophic strain (Palys12Δ, Paura3), PaADE2 was further disrupted, and PaURA3 was removed to obtain the triple auxotrophic strain PGB800 (Paura3, Palys12Δ, Paade2Δ). The whole-genome sequence of the PGB800 strain did not contain foreign genes used for genetic manipulation and disrupted PaADE2 and PaLYS12 and removed PaURA3, as planned. |
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