Molecular cloning and nucleotide sequence of the isoamyl acetate-hydrolyzing esterase gene (EST2) from Saccharomyces cerevisiae

A mutant deficient carboxylesterase which hydrolyzes isoamyl acetate (est2) was isolated from a yeast, Saccharomyces cerevisiae. The EST2 gene was cloned and its nucleotide sequence determined. The EST2 gene has an open reading frame of 714 bp (238 amino acids), and the molecular weight of the gene product calculated from the predicted amino acid sequence was 27,304.66. The EST2 gene product lacks the consensus sequence (Gly-Xaa-Ser-Xaa-Gly) that is observed in the serine type esterase or lipase, although a quite similar sequence (Ala-Cys-Ser-Ala-Gly) was found. Genomic Southern analysis revealed that the EST2 gene was present as a single copy on the chromosomal DNA of laboratory strains of S. cerevisiae as well as the sake brewing yeasts. The est2 mutant accumulated approximately 19 times higher amounts of isoamyl acetate compared with the parent strain in laboratory scale sake brewing. Therefore, the EST2 gene product is likely to play a crucial role in the hydrolysis of isoamyl acetate in sake mash.