Targeted integrative transformation of Candida tropicalis by electroporation

Abstract

A method for integrative transformation of the diploid yeast Candida tropicalis by electroporation has been developed. by linearizing the transforming plasmid DNA containing the URA3 gene prior to electroporation of recipient cells, its integration was targeted to a specific locus in the genome, resulting in single or multiple tandem integrations. The optimal electroporation conditions for this yeast were established and include an electric pulse of 2.25 kV/cm for a duration of 50 ms. Using these conditions, Ura+ transformants were readily obtained at a high frequency (45 transformants/micrograms DNA) as the result of targeted integration of the URA3 gene containing plasmid DNA at the chromosomal ura3 locus. The number of transformants resulting from this procedure is comparable to that achieved with a recently reported spheroplast transformation procedure for C. tropicalis; in addition, it offers the advantages of being simple, rapid and reproducible.

A transformation system for the yeast Candida utilis: use of a modified endogenous ribosomal protein gene as a drug-resistant marker and ribosomal DNA as an integration target for vector DNA

“…The electroporation method was used to transform C. utilis since the method is relatively simple and has been applied successfully to many yeast species (3,5,10,15,21). The results demonstrate that electroporation can be utilized for the efficient transformation of C. utilis.…”

Highly-efficient electrotransformation of the yeast Hansenula polymorpha

“…Therefore, we have investigated whether H. poIymorpha can be transformed by electroporation. Recently, this technique has been successfully applied to introduce foreign DNA into several yeast species, including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, and several Candida species (Meilhoc et al 1990;Becker et al 1991;Kas~ske et al 1992;Rohrer and Picataggio 1992;Nuttley et a1.1993). The advantage of electrotransformation is that it combines simplicity with high transformation frequencies.…”

Development of an integrative DNA transformation system for the yeastCandida utilis

“…The electroporation method was used to transform C. utilis because of its relative simpleness and due to the fact that it has been applied successfully to many yeast species [15–17]. To compare the transformation efficiency obtained by electroporation with other transformation procedures, we have also transformed the C. utilis strain with the LiAc method.…”