Abe Hiroko, Takaoka Yuki, Chiba Yasunori, Sato Natsuko, Ohgiya Satoru, Itadani Akiko, Hirashima Mitsuomi, Shimoda Chikashi, Jigami Yoshifumi, Nakayama Ken-ichi
Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14, Hayashi, Takamatsu, Kagawa 761-0395, Japan.
Glycobiology. 2009 Apr;19(4):428-36. doi: 10.1093/glycob/cwn157. Epub 2009 Jan 7.
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.
由于酵母特异性糖基转移酶的破坏，产生哺乳动物型N-连接寡糖的酵母细胞表现出严重的生长缺陷和蛋白质生产率下降。工程酵母菌株中这种蛋白质生产率的下降是酵母中高效糖蛋白生产发展的障碍。为了在酵母中经济有效地合成此类治疗性糖蛋白，非常需要开发合适的菌株。我们分别将一种利用酿酒酵母pol3 - 01基因或粟酒裂殖酵母cdc6 - 1基因编码的校对缺陷型DNA聚合酶δ变体的新型诱变技术应用于工程化的酿酒酵母TIY20菌株和粟酒裂殖酵母KT97菌株。TIY20由于三个基因（och1Δ、mnn1Δ、mnn4Δ）的破坏而缺乏甘露聚糖外链，KT97是och1破坏株，由于它们表现出温度敏感（ts）表型、生长缺陷表型和蛋白质生产率下降，因此作为生产治疗性糖蛋白的宿主并不实用。我们成功分离出了缓解TIY20菌株生长缺陷的YAB突变体。令人惊讶的是，这些突变体通常比野生型菌株能更好地分泌外源蛋白。此外，我们也成功分离出了缓解KT97菌株生长缺陷的YPAB突变体。通过酵母基因工程与这种诱变技术相结合开发这些新突变体是工程酵母细胞中生产治疗性糖蛋白的重大突破。
