Expression and Secretion of a Cellulomonas fimi Exoglucanase in Saccharomyces cerevisiae

Abstract

We used the yeast MEL1 gene for secreted α-galactosidase to construct cartridges for the regulated expression of foreign proteins from Saccharomyces cerevisiae. The gene for a Cellulomonas fimi β-1,4-exoglucanase was inserted into one cartridge to create a fusion of the α-galactosidase signal peptide to the exoglucanase. Yeast transformed with plasmids containing this construction produced active extracellular exoglucanase when grown under conditions appropriate to MEL1 promoter function. The cells also produced active intracellular enzyme. The secreted exoglucanase was N-glycosylated and was produced continuously during culture growth. It hydrolyzed xylan, carboxymethyl cellulose, 4-methylumbelliferyl-β- d -cellobiose, and p -nitrophenyl-β- d -cellobiose. A comparison of the recombinant S. cerevisiae enzyme with the native C. fimi enzyme showed the yeast version to have an identical K m and pH optimum but to be more thermostable.