title: Engineering of polyploid Saccharomyces cerevisiae for secretion of large amounts of fungal glucoamylase.
abstract: We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var. kawachi. To do this, we used the delta-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. delta-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.
authors.affiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto, 860-0082, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ekino
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Hayashi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Moriyama
authors.forename: Minoru
authors.initials: M
authors.lastname: Matsuda
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Goto
authors.forename: Sadazo
authors.initials: S
authors.lastname: Yoshino
authors.forename: Kensuke
authors.initials: K
authors.lastname: Furukawa
mesh_terms: D015825:Chromosomes, Fungal; D018076:DNA, Complementary / Q000235:genetics; D005818:Genetic Engineering; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000378:metabolism*; D006238:Haploidy; D010957:Plasmids / Q000235:genetics; D011123:Polyploidy; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D005087:Glucan 1,4-alpha-Glucosidase