Mutagenesis of the glucoamylase signal peptide of Saccharomyces diastaticus and functional analysis in Saccharomyces cerevisiae

To improve the efficiency of the glucoamylase signal peptide (GSP) of Sacch aromyces diastaticus for the secretion of foreign proteins, hybrid plasmids containing one of four types of GSP mutant (ml, pro(-18)-->Leu(-18); m(2), Tyr(-13)-->Leu(-13); m(3), Ser(-9) -->Leu(-9); m(4), Asn-5-->pro(-5)) were constructed and evaluated in Saccharomyces cerevisiae using Bacillus endo- 1,4-beta -D-glucanase (CMCase) as a reporter gene. CMCase secretion by ml, m2 and m3 GSP mutants was increased, likely resulting from a higher probabi lity of the modified GSP to assume an alpha -helical structure. Especially in the case of m3, the substitution of Leu for a polar residue, Ser(-9), in the hydrophobic region resulted in approximately a twofold increase in ext racellular CMCase activity. In mutant 4, which disrupts the alpha -helix of GSP, CMCase was less efficiently secreted. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights r eserved.