Secretion and Overproduction of Carboxypeptidase Y by aSaccharomyces cerevisiae ssl1Mutant Strain

Abstract

Carboxypeptidase Y (CPY; EC 3.4.16.1) is the yeast vacuolar protease. To have CPY secreted and to increase its secretion level, we tried to express the prepro-CPY gene under the control of the inducible GAL10 promoter or constitutive ENO1 promoter on a multicopy plasmid. In the strains KK4, PEP4, and A2-1-1A, carrying the CPY expression plasmid, active CPY was not detected in the culture broth although the CPY activity was greatly increased inside the cells. In contrast, when we used a strain that contained the ssl1 (super-secretion of lysozyme) mutation, a large amount of active CPY (about 10-50 mg/liter) was detected in the culture broth. The ssl1 mutants secreted active CPY when the CPY level was increased by expressing it under the control of a strong promoter on a multicopy plasmid, while the endogenous expression of chromosomal CPY gene in the same ssl1 mutant caused a deficiency in the processing of pro-CPY to mature CPY.