title: Development of valuable yeast strains using a novel mutagenesis technique for the effective production of therapeutic glycoproteins.
abstract: 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 delta 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 (och1Delta, mnn1 Delta, mnn4 Delta), 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.
authors.affiliation: Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14, Hayashi, Takamatsu, Kagawa 761-0395, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Abe
authors.forename: Yuki
authors.initials: Y
authors.lastname: Takaoka
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Chiba
authors.forename: Natsuko
authors.initials: N
authors.lastname: Sato
authors.forename: Satoru
authors.initials: S
authors.lastname: Ohgiya
authors.forename: Akiko
authors.initials: A
authors.lastname: Itadani
authors.forename: Mitsuomi
authors.initials: M
authors.lastname: Hirashima
authors.forename: Chikashi
authors.initials: C
authors.lastname: Shimoda
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Jigami
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Nakayama
mesh_terms: D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods*; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000627:therapeutic use; D006801:Humans; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000627:therapeutic use; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C491233:cdc6 protein, S pombe; D008364:Mannosyltransferases; C539723:Och1 protein, S pombe; D019951:DNA Polymerase beta