Protein Engineering, Vol. 15, No. 3, 251-255,
March 2002
© 2002 Oxford University Press
Alternative CUG codon usage (Ser for Leu) in Pichia farinosa and the effect of a mutated killer gene in Saccharomyces cerevisiae
1 National Food Research Institute, 2–1–12, Kannon-dai, Tsukuba, Ibaraki 305-8642 and 2 Central Research Laboratories, Ajinomoto Co., Inc., 1–1, Suzuki-cho, Kawasaki-ku, Kawasaki, Kanagawa 210-8681, Japan
The halotolerant yeast Pichia farinosa KK1 strain produces a killer toxin termed SMKT (salt-mediated killer toxin). Mass spectrometry and Edman sequencing of peptides from the mature SMKT and secreted protoxin demonstrate that positions specified by the CUG codon contain unmodified serine (Ser) in P.farinosa. In order to express the authentic SMK1 product in Saccharomyces cerevisiae, which uses the universal genetic code, the three CUG codons corresponding to Ser87, Ser137 and Ser206 in the SMK1 gene were changed to universal Ser codons by site-directed mutagenesis. The expression of the modified SMK1 gene with universal Ser codons was lethal in S.cerevisiae, as well as that of the unmodified SMK1 gene with the CUG codons. The secretion of protoxin with the authentic amino acid sequence from the modified SMK1 was significantly increased, whereas the transcription level of SMK1 was not affected in the presence or absence of CUG codon. Our results provide the first in vivo evidence that non-universal decoding of CUG is used in a hemiascomycetous yeast, P.farinosa.