Production and secretion patterns of cloned glucoamylase in plasmid-harboring and chromosome-integrated recombinant yeasts employing an SUC2 promoter.
To understand the differences in production and secretion patterns between plasmid-harboring and chromosome-integrated recombinant yeasts, the two recombinant Saccharomyces cerevisiae yeasts, containing the structural glucoamylase STA gene and the SUC2 promoter, were investigated. Both systems were regulated by glucose concentration in the culture broth. First, the glucoamylase activity per gene copy number of the chromosome-integrated recombinant yeast was 2.8- to 5.6-fold higher than that of the plasmid-harboring recombinant yeast. Overburdened owing to high copy number, the plasmid-harboring recombinant yeast gave lower glucoamylase activity per gene copy number. Second, the efficiency of signal sequence was compared; the secretion efficiency of glucoamylase in the plasmid-harboring recombinant yeast was higher than that in the chromosome-integrated recombinant yeast at 96 h of cultivation (74 vs 65%). We postulated that the higher level of secretion efficiency of the plasmid-harboring recombinant yeast resulted because the production level did not reach the capacity of the secretory apparatus of the host yeast. However, the specific secretion rate was much higher in the chromosome-integrated recombinant yeast even though the final secretion efficiency was lower. The lower secretion rate in the plasmid-harboring recombinant yeast could be explained by an adverse effect caused by higher production rate. Finally, the optimal glucose concentration for glucoamylase production in the chromosome-integrated recombinant yeast culture was lower than that in the plasmid-harboring recombinant yeast culture owing to gene dosage effect.
Cha
H J
HJ
Department of Chemical Engineering, Pohang University of Science and Technology, Korea. hjcha@postech.ac.kr
Chae
H J
HJ
Choi
S S
SS
Yoo
Y J
YJ
D002455:Cell Division; D002458:Cell Fractionation; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D005285:Fermentation; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis* / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D013053:Spectrophotometry; D013997:Time Factors; D043324:beta-Fructofuranosidase
D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D005087:Glucan 1,4-alpha-Glucosidase