Secretion and overproduction of carboxypeptidase Y by a Saccharomyces cerevisiae ssl1 mutant strain.
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.
Ichikawa
K
K
Fermentation Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan.
Shiba
Y
Y
Jigami
Y
Y
Serizawa
N
N
D001483:Base Sequence; D015153:Blotting, Western; D002268:Carboxypeptidases / Q000096:biosynthesis* / Q000378:metabolism; D043402:Cathepsin A; D003470:Culture Media; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012710:Serum Albumin, Bovine / Q000494:pharmacology
D003470:Culture Media; D029701:Saccharomyces cerevisiae Proteins; D012710:Serum Albumin, Bovine; D002268:Carboxypeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; C022633:serine carboxypeptidase