EXPRESSION AND SECRETION OF THE CANDIDA-WICKERHAMII EXTRACELLULAR BETA-GLUCOSIDASE GENE, BGLB, IN SACCHAROMYCES-CEREVISIAE

The yeast Candida wickerhamii exports a cell-associated beta-glucosida se that is active against cellobiose and all soluble cellodextrins. Be cause of its unique ability to tolerate end-product inhibition by gluc ose, the bglB gene that encodes this enzyme was previously cloned and sequenced in this laboratory Using several different promoters and con structs, bglB was expressed in the hosts Escherichia coli, Pichia past oris, and Saccharomyces cerevisiae. Expression was initially performed in E. coli using either the lacZ or tac promoter. This resulted in in tracellular expression of the BglB protein with the protein being rapi dly fragmented. Secretion and glycosylation of active beta-glucosidase was achieved with several different S. cerevisiae constructs utilizin g either the adh1 or the gal1 promoter on 2-mu replicating plasmids. W hen either the invertase (Suc2) or the BglB secretion signal was used, BglB protein remained associated with the cell wall and appeared to b e hyperglycosylated. Expression in P. pastoris was also examined to de termine if higher activity and expression could be achieved in a yeast host that usually does not hyperglycosylate. Using the alcohol oxidas e promoter in conjunction with either the pho1 or the alpha-factor sec retion signal, the recombinant enzyme was successfully secreted and gl ycosylated in P. pastoris. However, levels of protein expression from the chromosomally integrated vector were insufficient to detect activi ty.