Modification And Optimization Of Saccharomyces Cerevisiae To Produce Humanized Glycoproteins

The N-glycosylation pathway of yeast cells are partially similarity to mammalian cells. The glycosylation process in Sacchromyces cerevisiae exhibited an ultra-glycosylation process, which is significantly different from that of human beings or other mammals. To humanize the glycoproteins in Sacchromyces cerevisiae, the N-glycosylation pathway should be genetically engineered into human-type. In this study, Sacchromyces cerevisiae W303 a used as an original strain was constructed by disrupting several special glycosyltransferases and expressing exogenous genes. We also tried to resolve the problems which we have met in these progresses, such as the cell wall defect, growth inhibition, and underglycosylation site occupy. The main results are as follows:(1) The growth performance, N-glycosylation site occupancy and cell wall defect of Δalg3Δoch1 were improved by RHO1 overexpression. The results showed that with the overexpression of RHO1 in Δalg3Δoch1, the β-1,3-glucan and mannoproteins layer of yeast cell wall were thicker and cell growth was partially recovered. The N-glycosylation site occupancy and secretion of foreign proteins of Δalg3Δoch1 were also enhanced by overexpression of RHO1.(2) The cell growth and N-glycosylation sites occupancy of Δalg3Δoch1Δmnn1 were improved by growth adaptive evolution. ALG6 and WBP1 gene were detected to upregulate in Δalg3Δoch1Δmnn1 adaptive evolution strain. Overexpression of ALG6 and WBP1 in Δalg3Δoch1Δmnn1 not only enhanced the N-glycosylation site occupancy but also improved the growth performance. The overexpression of ALG6 in Δalg3Δoch1Δmnn1 also increased the secretion of human lysozyme.