CLONING AND EXPRESSION OF AN ARXULA-ADENINIVORANS GLUCOAMYLASE GENE IN SACCHAROMYCES-CEREVISIAE

The glucoamylase gene of the yeast Arxula adeninivorans Ls3 has been c loned from a genomic library and sequenced. The gene could be localize d on chromosome 2 from A. adeninivorans and comprises 1875 bp. The fir st 16 N-terminal amino acids represent the signal sequence for enterin g the endomembrane system. Comparing the amino acid sequence from this glucoamylase with those of other fungal glucoamylases shows that the glucoamylase of strain Ls3 has a homology to the glucoamylases from Rh izopus oryzae (32.6%), Saccharomycopsis fibuligera (23.1%), Aspergillu s niger (22.1%), and Saccharomyces diastaticus (15.4%). No homology co uld be detected to the glucoamylase of Schwanniomyces occidentalis. By using the GAL1 promoter from Saccharomyces cerevisiae within an auton omously replicating plasmid it was possible to express the isolated Ar xula, glucoamylase gene in Saccharomyces cerevisiae. The transformants secreted 95% of the enzyme into the culture medium. The N termini of glucoamylases synthesized in A. adeninivorans and S. cerevisiae transf ormants are identical, which means that the signal sequences were clea ved at the same positions during maturation of the proteins. The highe st glucoamylase activities were reached in the culture medium of S. ce revisiae transformants after 36 h of fermentation. Northern hybridizat ion showed that the glucoamylase transcripts were formed continuously for up to 70 h. These results reveal that the glucoamylase is expresse d and secreted more rapidly in the S. cerevisiae transformants than in A. adeninivorans Ls3.