Glycobiology Advance Access published online on January 7, 2009
Glycobiology, doi:10.1093/glycob/cwn157
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Development of valuable yeast strains using a novel mutagenesis technique for the effective production of therapeutic glycoproteins
2 Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 2217–14, Hayashi, Takamatsu, Kagawa 761–0395, Japan
3 Research Center for Medical Glycoscience, AIST, Central 2, 1–1-1, Umezono, Tsukuba, Ibaraki 305–8568, Japan
4 Research Institute of Genome-based Biofactory, AIST, 2–17-2–1, Tsukisamu-higashi, Toyohira-ku, Sapporo, Hokkaido 062–8517, Japan
5 Department of Biology, Graduate School of Science, Osaka City University, 3–3-138 Sugimoto, Sumiyoshi-ku, Osaka 558–8585, Japan
6 Neo-Morgan Laboratory Inc., The Imperial Hotel Tower 6th Floor, 1–1-1, Uchisaiwai-cho, Chiyoda-ku, Tokyo 100–0011, Japan
7 Department of Immunology and Immunopathology, Faculty of Medicine, Kagawa University, 1750–1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761–0793, Japan
1 Corresponding author: Ken-ichi Nakayama, Tel and Fax; +81–87-869–3593, e-mail; k-nakayama{at}aist.go.jp
Received on September 10, 2008; accepted on December 27, 2008
Yeast cells producing mammalian-type N-linked oligosaccharide show severe growth defects and the decreased protein productivity because of disruption of yeast-specific glycosyltransferases. This decreased protein productivity in engineered yeast strains is an obstacle to the development of efficient glycoprotein production in yeast. For economic and effective synthesis of such therapeutic glycoproteins in yeast, development of appropriate strains is highly desirable. We applied a novel mutagenesis technique that utilized the proofreading-deficient DNA polymerase 
variant encoded by the pol3–01 gene of Saccharomyces cerevisiae or the cdc6–1 gene of Schizosaccharomyces pombe, to the engineered S. cerevisiae TIY20 strain and S. pombe KT97 strain, respectively. TIY20, which is deficient in the outer chain of mannan due to the disruption of three genes (och1
mnn1 mnn4), and KT97, which is an och1 disruptant, are impractical as hosts for the production of therapeutic glycoproteins since they show a temperature-sensitive (ts) phenotype, a growth defect phenotype and decreased protein productivity. We successfully isolated YAB mutants that alleviated the growth defect of the TIY20 strain. Surprisingly, these mutants generally secreted foreign proteins better than the wild-type strain. Furthermore, we successfully isolated YPAB mutants that alleviated the growth defect of the KT97 strain, too. Development of these new mutants by the combination of genetic engineering of yeast and this mutagenesis technique are major breakthroughs for the production of therapeutic glycoproteins in engineered yeast cells.
Key words: therapeutic glycoproteins / yeast / error-prone DNA polymerase / novel mutagenesis technique