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.

Upregulation of Genes Involved in Gluconeogenesis and the Glyoxylate Cycle Suppressed the Drug Sensitivity of anN-Glycan-DeficientSaccharomyces cerevisiaeMutant

“…A number of glycosylation mutants have been reported to be hygromycin B sensitive, 21) and YAB103 exhibited less sensitivity to hygromycin B than TIY20. 8) As expected, TIY20 cells harboring the CAT8 expression plasmid grew on YPAD medium containing hygromycin B, whereas TIY20 cells harboring the original vector failed to grow on the same medium (Fig. 2B).…”

“…We have isolated YAB103, which was able to suppress the drug sensitivity of TIY20. 8) In the present study, we found that some genes involved in gluconeogenesis and in the glyoxylate cycle were up-regulated in YAB103. Up-regulation of these genes led to an increase in glucan content, resulting in an altered cell wall able to suppress the drug sensitivity of the parent strain.…”

“…8) To determine why YAB103 lacked the growth defect and drug sensitivity of the parental strain, gene expression profiles of YAB103 and TIY20 were analyzed using DNA microarrays. For this purpose, the two strains were first pre-cultured in YPAD liquid medium for 15 h at 30 C, after which a portion of the cultures was used to inoculate fresh YPAD liquid medium, and then they were grown at 34 C. The cell densities of the two strains were adjusted by the inoculum size such that both strains subsequently grew until OD600 = 5 for almost the same time.…”

“…8) Collectively, the drug sensitivity analysis, the cell-wall sugar composition data, and cell lysis and tranformation efficiency analyses suggest that the cell-wall structure of YAB103 is significantly different from that of TIY20. Furthermore, we showed that the improved hygromycin B sensitivity was probably due to induction of gluconeogenesis, which increased the cellwall glucan content.…”

The role of disparity-mutagenesis model on tumor development with special reference to increased mutation rate

“…There are several other experiments in which yeast disparity-mutators displayed high adaptabilities to different conditions [29][30][31] .…”