Process development for high-level secretory production of carboxypeptidase Y by Saccharomyces cerevisiae.
In order to develop a production process for carboxypeptidase Y (CPY, yeast vacuolar protease) secreted by Saccharomyces cerevisiae KS58-2D, medium composition, culture conditions, and expression systems were investigated. We found that the addition of histidine to thiamine-free medium, in which CPY production was almost negligible, raised the intracellular thiamine level, resulting in the increase of CPY production. On the basis of the choice of an expression system that uses an inducible GAL10 promoter, reassessment of histidine concentration in the medium, and optimization of the pH level during cultivation (pH 6.5), active CPY was secreted in a quantity of over 400 mg/l, which was more than tenfold that higher than that previously reported. The process developed could be easily scaled-up to industrial-scale fermentation.
Shiba
Y
Y
Biomedical Research Laboratories, Sankyo Co. Ltd., Fukushima, Japan. shibay@ona.sankyo.co.jp
Fukui
F
F
Ichikawa
K
K
Serizawa
N
N
Yoshikawa
H
H
D018533:Biomass; D002268:Carboxypeptidases / Q000096:biosynthesis* / Q000378:metabolism; D043402:Cathepsin A; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D006639:Histidine / Q000032:analysis / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010865:Pilot Projects; D010957:Plasmids / Q000737:chemistry; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013831:Thiamine / Q000378:metabolism
D003470:Culture Media; D012710:Serum Albumin, Bovine; D006639:Histidine; D002268:Carboxypeptidases; D043402:Cathepsin A; D013831:Thiamine