A transformation system for the nonuniversal CUG(Ser) codon usage species Candida rugosa

Since Candida rugosa utilizes a nonuniversal serine codon CUG rather than leucine, no vectors have been constructed to transform this organism. Moreover, it is difficult to design a new transformation system because no selection markers and promoters are available. In this study, Zeocin (400 microg/ml) was demonstrated to inhibit the growth of C. rugosa. The dominant selectable marker bleomycin-resistant determinant (ble) gene containing five CUG codons in an open-reading frame of 375 bp was synthesized by replacing its CUG codons into leucine codons (zeo-n). This marker conferred resistance to Zeocin. GAL1 promoter, transcription elongation factor 1 (TEF1) promoter from Saccharomyces cerevisiae and LIP3 promoter from C. rugosa were then used to drive zeo-n and to examine the function of promoter in C. rugosa. The resulting vectors enabled selection of Zeocin-resistant clones after transformation by LiCl method and electroporation. These results demonstrate that transformation into C. rugosa is feasible under the operation of GAL1, TEF1, and LIP3 promoters. The development of the transformation system for C. rugosa is essential to the genetic analysis of gene regulation and biochemical features of this fungal species and the expression of recombinant proteins in C. rugosa.