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Applied and Environmental Microbiology, November 2002, p. 5693-5697, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5693-5697.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Engineering of Polyploid Saccharomyces cerevisiae for Secretion of Large Amounts of Fungal Glucoamylase

Keisuke Ekino,1,2 Hiroyuki Hayashi,3 Masahiro Moriyama,3 Minoru Matsuda,3 Masatoshi Goto,2 Sadazo Yoshino,2 and Kensuke Furukawa2*

Department of Applied Microbial Technology, Sojo University, Kumamoto, 860-0082,1 Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581,2 Fukutokucho Co., Ltd., Kurume 830-0063, Japan3

Received 31 May 2002/ Accepted 19 August 2002

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.

* Corresponding author. Mailing address: Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan. Phone and fax: 81-92-642-2849. E-mail: kfurukaw{at}agr.kyushu-u.ac.jp.

Applied and Environmental Microbiology, November 2002, p. 5693-5697, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5693-5697.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Nevoigt, E. (2008). Progress in Metabolic Engineering of Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 72: 379-412 [Abstract] [Full Text]  


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