A mutated hygromycin resistance gene is functional in the n-alkane-assimilating yeast Candida tropicalis

Development of a transformation system in the n-alkane-assimilating diploid yeast Candida tropicalis requires an antibiotic resistance gene in order t o establish a selectable marker. The resistance gene for hygromycin B has o ften been used as a selectable marker in yeast transformation. However, C. tropicalis harboring the hygromycin resistance gene (HYG) was as sensitive to hygromycin B as the wild-type strain. Nine CTG codons were found in the ORF of the HYG gene. This codon has been reported to be translated as serin e rather than leucine in Candida species. Analysis of the tRNA gene in C. t ropicalis with the anticodon CAG [tRNA(CAG) gene], which is complementary t o the codon CTG, showed that the sequence was highly similar to that of the C. maltosa tRNA(CAG) gene. Ln C. maltosa, the codon CTG is read as serine and not leucine. These results suggested that the HYG gene was not function al due to the nonuniversal usage of the CTG codon, Each of the nine CTG cod ons in the ORF of the HYG gene was changed to a CTC codon, which is read as leucine: by site-directed mutagenesis. When a plasmid containing the mutat ed HYG gene (HYG#) was constructed and introduced into C. tropicalis, hygro mycin-resistant transformants were successfully obtained. This mutated hygr omycin resistance gene may be useful for direct selection sf C. tropicalis transformants.