Reconstruction of N-glycosylation pathway for producing human glycoproteins in Saccharomyces cerevisiae

Abstract:

Abstract: ［Objective］ To produce human glycoproteins in Saccharomyces cerevisiae，human N-glycosylation pathway must be genetically engineered into the yeast cell．We tried to construct a strain，which can be used to introduce human Nglycosylation reactions，by disrupting several special glycosyltransferases in yeast N-glycosylation pathway．Furthermore，this mutant cell was applied for adaptive evolution to overcome its growth-defect phenotype．［Methods］Three yeast genes ALG3，OCH1 and MNN1 were disrupted． The N-linked oligosaccharides from the mutant cells were analyzed by the activity staining of invertase，and their structure was further confirmed by high-performance liquid chromatography (HPLC) and the treatment with glycosidase．Mutant cells were cultured under a high temperature for their adaptive evolution of growth．［Results ＆ Conclusion］We obtained a Δoch1Δalg3Δmnn1 strain that produces Man5GlcNAc2 intermediate of human Nglycosylation．Our approach for adaptive evolution resulted a remarkable improvement on the growth phenotype of Δoch1Δalg3Δmnn1 strain． In addition，we also confirmed a small amount of unexpected Man6GlcNAc2 intermediate from Δoch1Δalg3Δmnn1 strain．Treatment with α-1，2-mannosidase converted both Man5GlcNAc2 and Man6GlcNAc2 products to a single Man3GlcNAc2 form，indicating that the additional mannose on Man6GlcNAc2 product comes from an α-1，2 modification．Our results demonstrate that Δoch1Δalg3Δmnn1 triple mutant can be used as an initial strain to construct an yeast therapeutic glycoprotein-expression system by introducing various enzymes that are involved in human N-glycosylation pathway.

Key words:Keywords: humanization，N-glycosylation，adaptive evolution，Saccharomyces cerevisiae