Abstract
BackgroundTo humanize yeast N-glycosylation pathways, genes involved in yeast specific hyper-mannosylation must be disrupted followed by the introduction of genes catalyzing the synthesis, transport, and addition of human sugars. However, deletion of these genes, for instance, OCH1, which initiates hyper-mannosylation, could cause severe defects in cell growth, morphogenesis and response to environmental challenges.ResultsIn this study, overexpression of RHO1, which encodes the Rho1p small GTPase, is confirmed to partially recover the growth defect of Saccharomyces cerevisiae Δalg3Δoch1 double mutant strain. In addition, transmission electron micrographs indicated that the cell wall structure of RHO1-expressed cells have an enhanced glucan layer and also a recovered mannoprotein layer, revealing the effect of Rho1p GTPase on cell wall biosynthesis. Similar complementation phenotypes have been confirmed by overexpression of the gene that encodes Fks2 protein, a catalytic subunit of a 1,3-β-glucan synthase. Besides the recovery of cell wall structure, the RHO1-overexpressed Δalg3Δoch1 strain also showed improved abilities in temperature tolerance, osmotic potential and drug sensitivity, which were not observed in the Δalg3Δoch1-FKS2 cells. Moreover, RHO1 overexpression could also increase N-glycan site occupancy and the amount of secreted glycoproteins.ConclusionsOverexpression of RHO1 in ‘humanized’ glycoprotein producing yeasts could significantly facilitate its future industrial applications for the production of therapeutic glycoproteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0575-7) contains supplementary material, which is available to authorized users.
Highlights
To humanize yeast N-glycosylation pathways, genes involved in yeast specific hyper-mannosylation must be disrupted followed by the introduction of genes catalyzing the synthesis, transport, and addition of human sugars
Overexpression of RHO1 partially recovers the growth defect of a glycosylation mutant strain Cell growth is enhanced by overexpression of RHO1 In order to test the effects of overexpression of RHO1 and FSK2 genes, plasmids blank pY26, pY26-RHO1 and pY26-FKS2 were transformed in a wild type strain W303A or a glycosylation mutant Δalg3Δoch1 strain, respectively
The results showed that Δalg3Δoch1-RHO1 were grown to logarithmic phase at 17 h comparing to that of W303A-pY26 at 9 h, while Δalg3Δoch1-pY26 exhibited a prolonged lag phase of 26 h
Summary
To humanize yeast N-glycosylation pathways, genes involved in yeast specific hyper-mannosylation must be disrupted followed by the introduction of genes catalyzing the synthesis, transport, and addition of human sugars. Deletion of these genes, for instance, OCH1, which initiates hyper-mannosylation, could cause severe defects in cell growth, morphogenesis and response to environmental challenges. Glycosylation can influence a variety of physiological processes, Most therapeutic glycoproteins are currently produced by mammalian cells. Due to limited growth rate, expensive serum-based media, and the potential spread of infectious diseases, production of glycoproteins in CHO cells results in low productivity and
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