Wei sheng wu xue bao = Acta microbiologica Sinica · 2014
[Reconstruction of N-glycosylation pathway for producing human glycoproteins in Saccharomyces cerevisiae].
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He Tiefan, Xu Sha, Zhang Geyuan, Nakanishi Hideki, Gao Xiaodong
Аннотация
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 N-glycosylation 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 delta och1 delta alg3 delta mnn1 strain that produces Man5 GlcNAc2 intermediate of human N-glycosylation. Our approach for adaptive evolution resulted a remarkable improvement on the growth phenotype of delta och1 delta alg3 delta mnn1 strain. In addition, we also confirmed a small amount of unexpected Man6GlcNAc2 intermediate from delta och1delta alg3 delta mnnl strain. Treatment with alpha-1,2-mannosidase converted both Man,GlcNAc2 and Man,GlcNAc2 products to a single Man3GlcNAc, form, indicating that the additional mannose on Man,GlcNAc, product comes from an alpha-1, 2 modification. Our results demonstrate that delta och1 delta alg3 deltamnn1 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.
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 delta och1 delta alg3 delta mnn1 strain that produces Man5 GlcNAc2 intermediate of human N-glycosylation. Our approach for adaptive evolution resulted a remarkable improvement on the growth phenotype of delta och1 delta alg3 delta mnn1 strain. In addition, we also confirmed a small amount of unexpected Man6GlcNAc2 intermediate from delta och1delta alg3 delta mnnl strain. Treatment with alpha-1,2-mannosidase converted both Man,GlcNAc2 and Man,GlcNAc2 products to a single Man3GlcNAc, form, indicating that the additional mannose on Man,GlcNAc, product comes from an alpha-1, 2 modification. Our results demonstrate that delta och1 delta alg3 deltamnn1 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.