Cloning of the Schwanniomyces occidentalis glucoamylase gene (GAM1) and its expression in Saccharomyces cerevisiae

Abstract

The Schwanniomyces occidentalis glucoamylase (GAM)-encoding gene (GAM1) was isolated from a lambda Charon4A genomic library using synthetic oligodeoxynucleotides as probes. GAM1 contains an ORF of 2874 nucleotides (nt) coding for 958 amino acids. S1 mapping revealed that the transcript has only a very short 5'-untranslated leader of 8-12 nt. Disruption and displacement of the GAM1 gene in Sc. occidentalis resulted in loss of the ability to grow on starch efficiently. The gam1 strains still exhibit low GAM activity suggesting that at least a second weakly expressed GAM-encoding gene (GAM2) is present in Sc. occidentalis. Expression of the Sc. occidentalis GAM1 gene in Saccharomyces cerevisiae was achieved after promoter exchange. S. cerevisiae cells transformed with centromere plasmids carrying the GAM1 gene fused to promoters of different S. cerevisiae genes, namely GAL1, PDC1 and ADH1, efficiently secrete GAM and are able to grow with soluble starch as a sole carbon source. The essential enzymatic properties of the GAMs secreted from S. cerevisiae and Sc. occidentalis are identical, although the modifications of the proteins are different.