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

Abstract

Development of a transformation system in the n-alkane-assimilating diploid yeast Candida tropicalis requires an antibiotic resistance gene in order to establish a selectable marker. The resistance gene for hygromycin B has often 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 serine rather than leucine in Candida species. Analysis of the tRNA gene in C. tropicalis with the anticodon CAG [tRNA(CAG) gene], which is complementary to the codon CTG, showed that the sequence was highly similar to that of the C. maltosa tRNA(CAG) gene. In C. maltosa, the codon CTG is read as serine and not leucine. These results suggested that the HYG gene was not functional due to the nonuniversal usage of the CTG codon. Each of the nine CTG codons 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 mutated HYG gene (HYG#) was constructed and introduced into C. tropicalis, hygromycin-resistant transformants were successfully obtained. This mutated hygromycin resistance gene may be useful for direct selection of C. tropicalis transformants.

Novel and convenient methods for Candida tropicalis gene disruption using a mutated hygromycin B resistance gene

“…The gene is not expressed in Candida species. However, as reported previously (Hara et al 2000), we succeeded in functional expression of a mutated hygromycin B resistance gene (HYG#) in C. tropicalis. As the CUG codon, a universal leucine codon, is translated as serine in several Candida species including C. tropicalis (Ueda et al 1994), all nine CTG codons on the coding sequence of a hygromycin B resistance gene were changed to another leucine codon (CTC) by site-directed mutagenesis.…”

“…To disrupt the URA3 gene, both EcoRI and StyI sites on URA3 were disrupted by bluntend ligations after cutting with each enzyme. Subsequently, HYG# derived from pUCARS-HYG# (Hara et al 2000) was inserted at the NaeI site by blunt-end ligation. All nine CTG codons in the hygromycin B resistance gene were changed to another leucine codon (CTC; Hara et al 2000).…”

Deus ex Candida genetics: overcoming the hurdles for the development of a molecular toolbox in the CTG clade

“…1a; Hara et al, 2000). The authors exchanged the nine CTG codons with CTC codons in the ORF of the bacterial HPH gene using site-directed mutagenesis.…”

“…This cassette was notably used to demonstrate the possibility of homologous recombination in this species. Interestingly, in spite of its potential in the CTG clade, the codon-modified HPH cassette (Hara et al, 2000) was apparently forgotten, only to reappear 10 years later Basso et al, 2010). Basso et al (2010) produced a synthetic Escherichia coli hygromycin B resistance gene with optimized C. albicans codons.…”

“…They placed this sequence under the control of the C. albicans TEF2 promoter and the ACT1 terminator and demonstrated that this cassette could be used as dominant selectable marker in a set of C. albicans wild-type isolates. Millerioux et al (2011) (Hara et al, 2000) to transform a series of C. guilliermondii wild-type strains. This codon-modified HPH marker allowed the selection of hygromycin B-resistant transformants, but only when the HPH sequence was placed under the control of the C. guilliermondii PGK promoter and terminator.…”