CERULENIN-RESISTANT MUTANTS OF SACCHAROMYCES-CEREVISIAE WITH AN ALTERED FATTY-ACID SYNTHASE GENE

Cerulenin, an antifungal antibiotic produced by Cephalosporium caerule ns, is a potent inhibitor of fatty acid synthase in various organisms, including Saccharomyces cerevisiae. The antibiotic inhibits the enzym e by binding covalently to the active center cysteine of the condensin g enzyme domain. We isolated 12 cerulenin-resistant mutants of S. cere visiae following treatment with ethyl methanesulfonate. The mechanism of cerulenin resistance in one of the mutants, KNCR-1, was studied. Gr owth of the mutant was over 20 times more resistant to cerulenin than that of the wild-type strain. Tetrad analysis suggested that all mutan ts mapped at the same locus, F AS2, the gene encoding the a subunit of the fatty acid synthase. The isolated fatty acid synthase, purified f rom the mutant KNCR-1, was highly resistant to cerulenin. The ceruleni n concentration causing 50% inhibition (IC50) of the enzyme activity w as measured to be 400 mu M, whereas the IC50 value was 15 mu M for the enzyme isolated from the wild-type strain, indicating a 30-fold incre ase in resistance to cerulenin. The F AS2 gene was cloned from the mut ant. Sequence replacement experiments suggested that an 0.8 kb EcoRV-H indIII fragment closely correlated with cerulenin resistance. Sequence analysis of this region revealed that the GGT codon encoding Gly-1257 of the F AS2 gene was altered to AGT in the mutant, resulting in the codon for Ser. Furthermore, a recombinant F AS2 gene, in which the 0.8 Kb EcoRV-HindIII fragment of the wild-type F AS2 gene was replaced wi th the same region from the mutant, when introduced into F AS2-defecti ve S. cerevisiae complemented the F AS2 phenotype and showed cerulenin resistance. These data indicate that one amino acid substitution (Gly -->Ser) in the a subunit of fatty acid synthase is responsible for the cerulenin resistance of the mutant KNCR-1.