Transformation of Candida maltosa by Electroporation

The genus Candida comprises a group of yeasts united by nothing more than the fact that none has a natural sexual cycle. If these yeasts do not form a natural grouping, they nonetheless contain a number of species of considerable scientific and practical interest. Candida albicans is an important opportunistic pathogen of humans, it is a dimorphic organism, and the transition from the yeast to the mycelial form is an essential prerequisite for the establishment of an infection. Commercially important species include Candida utilis and Candida maltosa , which have applications as food and feed organisms and can be used in the processing of a wide range of substrates, including, in the latter case, crude oil. Candida species are diploid or aneuploid in their genetic constitution and have proven to be difficult organisms to study in terms of their genetics and molecular biology. Traditional approaches to mutation and genetic mapping have provided very limited information, and only a few of the 166 species of Candida (1 ) have yielded to molecular genetic methods. Nevertheless, recombinant DNA technology represents the only efficient route to analyzing their genomes, and this requires an efficient DNA transformation method. Electroporation has become the preferred method for gene transfer owing to its ease and efficiency in comparison to alternative techniques. Moreover, electroporation permits the introduction of DNA into organisms that are refractory to other transformation techniques (2 ). However, experience with Saccharomyces cerevisiae has indicated that the electroporation conditions may need to be adapted for each strain used (3 –6 ), and our findings with Candida maltosa support this view (7 ). We have found that optimal electroporation conditions show significant variation even between different mutants derived from a single strain, and thus, it is impossible to give a general transformation protocol for the extremely heterogeneous genus Candida . Therefore, in this chapter, we will focus on the hydrocarbon-utilizing yeast C. maltosa , a member of the Candida genus whose molecular genetics has advanced significantly of late (8 –18 ).