title: Effects of Rho1, a small GTPase on the production of recombinant glycoproteins in Saccharomyces cerevisiae.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  Overexpression of RHO1 in 'humanized' glycoprotein producing yeasts could significantly facilitate its future industrial applications for the production of therapeutic glycoproteins.
authors: School of Biotechnology, Key Laboratory of Glycobiology and Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.|State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
authors: Sha
authors: http://orcid.org/0000-0001-8987-4940
authors: S
authors: Xu
authors: School of Biotechnology, Key Laboratory of Glycobiology and Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
authors: Ge-Yuan
authors: GY
authors: Zhang
authors: School of Biotechnology, Key Laboratory of Glycobiology and Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
authors: Huijie
authors: H
authors: Zhang
authors: School of Biotechnology, Key Laboratory of Glycobiology and Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
authors: Toshihiko
authors: T
authors: Kitajima
authors: School of Biotechnology, Key Laboratory of Glycobiology and Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
authors: Hideki
authors: H
authors: Nakanishi
authors: School of Biotechnology, Key Laboratory of Glycobiology and Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China. xdgao@jiangnan.edu.cn.|State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China. xdgao@jiangnan.edu.cn.
authors: Xiao-Dong
authors: XD
authors: Gao
mesh_terms: D002473:Cell Wall / Q000201:enzymology / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C051302:RHO1 protein, S cerevisiae; D020741:rho GTP-Binding Proteins
keywords: Cell wall integrity; Humanized N-glycosylation; Recombinant-protein production; Rho1p GTPase; Saccharomyces cerevisiae