 Yeast 15, 1609–1618 (1999) Transformation of Candida albicans by Electroporation MARIANNE D. DE BACKER 1 *, DIRK MAES 1 , SANDY VANDONINCK 1 , MARC LOGGHE 2 , ROLAND CONTRERAS 2 AND WALTER H. M. L. LUYTEN 1 1 Department of Advanced Biotechnologies, Janssen Research Foundation, Turnhoutseweg 30, B2340 Beerse, Belgium 2 Department of Fundamental and Applied Molecular Biology, University of Gent, K.L. Ledeganckstraat 35, B9000 Gent, Belgium In contrast to a variety of other yeasts, Candida albicans has proved difficult to transform with high efficiency. Lithium acetate transformation is fast and simple but provides a very low efficiency of DNA transfer (50–100 transformants/g DNA), while spheroplast transformation, although more efficient (300 transformants/g integrative DNA and 10 3 –10 4 transformants/g replicative DNA), is complicated and time-consuming. In this study we applied various yeast transformation techniques to C. albicans and selected an electroporation procedure for further optimization. Transformation efficiencies of up to 300 transformants/g were obtained for an integrative plasmid and up to 4500 transformants/g for a CARS-carrying plasmid. This reasonably high transformation efficiency, combined with the ease and speed of electroporation in comparison to alternative techniques, make it the preferred method for transformation of C. albicans. Copyright  1999 John Wiley & Sons, Ltd.   — Candida albicans; transformation; electroporation; homologous recombination; integrative vector; episomal vector INTRODUCTION Candida (C.) albicans is an important opportunis- tic pathogen of humans and belongs to the genus Candida, a group of yeasts united only by the fact that none has a known natural sexual cycle. Many Candida species are diploid or aneuploid and their genetics and molecular biology have there- fore proved difficult to study. A prerequisite for molecular biological manipulation is, of course, a reliable and efficient means for introducing exogenous DNA into the cell. Lithium acetate transformation (Ito et al., 1983) and spheroplast transformation (Hinnen et al., 1978), both widely used methods, suffer from significant limitations, especially when applied to C. albicans. Lithium acetate transformation is fast and simple but gives low transformation efficiencies (50–100 transformants/g DNA) for C. albicans (Sanglard et al., 1996). Spheroplast transformation, although more efficient (300 transformants/g integrative DNA and 10 3 –10 4 transformants/g replicative DNA; Herreros et al., 1992), is laborious and time-consuming. Development of alternative transformation methods which do not rely on spheroplasts may simplify genetic engineering in Candida. When using the spheroplast PEG procedure, the risk remains of generating protoplast fusions, with resulting polypoidy. By far the fastest and simplest method of transformation is electroporation. Unlike the lithium acetate procedure, however, electroporation saturates at low DNA levels (Ausubel et al., 1992). Successful high-efficiency electroporation has been described for a variety of yeasts, such as S. cerevisiae (Hill, 1989; Delorme, 1989; Becker and Guarente, 1991; Manivasakam and Schiestl, 1993; Simon, 1993), Hansenula poly- morpha (Faber et al., 1993), C. maltosa (Kasuske et al., 1992; Becher and Oliver, 1995) and C. utilis (Kondo et al., 1995). However, C. albicans, unlike many other yeasts, has proved difficult to transform by electropora- tion with high efficiency (Brown et al., 1996; *Correspondence to: M. D. De Backer, Department of Ad- vanced Biotechnologies, Janssen Research Foundation, Turn- houtseweg 30, B2340 Beerse, Belgium. Tel: +32-14-60.38.81; fax: +32-14-60.61.11; e-mail: mdbacker@janbe.jnj.com Contract/grant sponsor: Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de In- dustrie (IWT), Belgium; Contract/grant number: 960192. CCC 0749–503X/99/151609–10$17.50 Copyright  1999 John Wiley & Sons, Ltd. Received 4 May 1999 Accepted 12 July 1999