EFFICIENT SECRETION OF BACILLUS-SUBTILIS LEVANASE BY SACCHAROMYCES-CEREVISIAE

The secretion of Bacillus subtilis (Bs) levanase (Lev) was studied in the yeast Saccharomyces cerevisiae. A set of different yeast expressio n plasmids, based on the constitutive PGK promoter and harbouring the Bs Lev-encoding gene (sacC), was constructed. In these plasmids, the o riginal Bs signal sequence was either intact, partially deleted or ent irely missing. With all constructs, Lev was produced from yeast transf ormants, However, only when the intact bacterial signal peptide was pr esent was the synthesized enzyme secreted; around 20% was found in the periplasm and 30% in the culture medium. The secreted protein found i n the periplasmic space was mainly core-glycosylated and unglycosylate d, and had a size of 80-90 and 74 kDa, respectively, In contrast, Lev found in the culture medium was mainly hyper-glycosylated and had a si ze of 180-200 kDa. Yeast transformants harbouring sacC, but lacking pa rts of the bacterial signal sequence, only produced cytoplasmic protei n which was not glycosylated and had a size of about 74 kDa. The delet ion of the entire signal peptide and a further 22 amino acids at the N terminus of mature Lev resulted in a 71-kDa cytoplasmic protein which was not active.