Development of valuable yeast strains using a novel mutagenesis technique for the effective production of therapeutic glycoproteins

• Published in: Glycobiology (Oxford) 2009-04, Vol.19 (4), p.428-436
• Main Authors: Abe, Hiroko, Takaoka, Yuki, Chiba, Yasunori, Sato, Natsuko, Ohgiya, Satoru, Itadani, Akiko, Hirashima, Mitsuomi, Shimoda, Chikashi, Jigami, Yoshifumi, Nakayama, Ken-ichi
• Format: Article
• Language: English
• Subjects: DNA Polymerase beta - genetics; DNA Polymerase beta - metabolism; error-prone DNA polymerase; Gene Deletion; Genetic Engineering - methods; Glycoproteins - biosynthesis; Glycoproteins - genetics; Glycoproteins - therapeutic use; Humans; Mannosyltransferases - genetics; Mannosyltransferases - metabolism; Mutagenesis; novel mutagenesis technique; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - therapeutic use; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Schizosaccharomyces - enzymology; Schizosaccharomyces - genetics; Schizosaccharomyces pombe; Schizosaccharomyces pombe Proteins - genetics; Schizosaccharomyces pombe Proteins - metabolism; therapeutic glycoproteins; yeast
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwn157

Yeast cells producing mammalian-type N-linked oligosaccharide show severe growth defects and the decreased protein productivity because of the 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, the development of appropriate strains is highly desirable. We applied a novel mutagenesis technique that utilized the proofreading-deficient DNA polymerase δ 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 (och1Δ, mnn1Δ, mnn4Δ), 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. The 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.