Please use this identifier to cite or link to this item: https://repository.ldufk.edu.ua/handle/34606048/10447
Title: Development of a transformation system for gene knock - out in the flavinogenic yeast Pichia guilliermondii
Authors: Boretsky, Yuriy
Pynyaha, Yuriy
Boretsky, Volodymyr
Kutsyaba, Vasyl
Protchenko, Olga
Philpott, Caroline
Sibirny, Andriy
Борецький, Юрій
Keywords: Riboflavin biosynthesis
Codon usage
Deletion cassette
Transformation
URA3 marker
Yeast Pichia guilliermondii
Issue Date: 2007
Citation: Development of a transformation system for gene knock - out in the flavinogenic yeast Pichia guilliermondii / Boretsky Y. R., Pynyaha Y. V., Boretsky V. Y., Kutsyaba V. I ., Protchenko O. V., Philpott C. C., Sibirny A. A. // J. of Microbiol. Methods. – 2007 . – Vol. 70(1). – P. 13 – 19. (Scopus)
Abstract: Pichia guilliermondii is a representative of a yeast species, all of which over-synthesize riboflavin in response to iron deprivation. Molecular genetic studies in this yeast species have been hampered by a lack of strain-specific tools for gene manipulation. Stable P. guilliermondii ura3 mutants were selected on the basis of 5′-fluoroorotic acid resistance. Plasmid carrying Saccharomyces cerevisiae URA3 gene transformed the mutant strains to prototrophy with a low efficiency. Substitution of a single leucine codon CUG by another leucine codon CUC in the URA3 gene increased the efficiency of transformation 100 fold. Deletion cassettes for the RIB1 and RIB7 genes, coding for GTP cyclohydrolase and riboflavin synthase, respectively, were constructed using the modified URA3 gene and subsequently introduced into a P. guilliermondii ura3 strain. Site-specific integrants were identified by selection for the Rib− Ura+ phenotype and confirmed by PCR analysis. Transformation of the P. guilliermondii ura3 strain was performed using electroporation, spheroplasting or lithium acetate treatment. Only the lithium acetate transformation procedure provided selection of uracil prototrophic, riboflavin deficient recombinant strains. Depending on the type of cassette, efficiency of site-specific integration was 0.1% and 3–12% in the case of the RIB1 and RIB7 genes, respectively. We suggest that the presence of the ARS element adjacent to the 3′ end of the RIB1 gene significantly reduced the frequency of homologous recombination. Efficient gene deletion in P. guilliermondii can be achieved using the modified URA3 gene of S. cerevisiae flanked by 0.8–0.9 kb sequences homologous to the target gene.
URI: http://repository.ldufk.edu.ua/handle/34606048/10447
Appears in Collections:Наукові праці професорсько-викладацького складу ЛДУФК в базах даних Scopus, WoS, Tomson Reuters

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