Xu Sha, Zhang Ge-Yuan, Zhang Huijie, Kitajima Toshihiko, Nakanishi Hideki, Gao Xiao-Dong
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.
Microb Cell Fact. 2016 Oct 21;15(1):179. doi: 10.1186/s12934-016-0575-7.
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.
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.
Overexpression of RHO1 in 'humanized' glycoprotein producing yeasts could significantly facilitate its future industrial applications for the production of therapeutic glycoproteins.
为了使酵母N-糖基化途径人源化，必须破坏参与酵母特异性高甘露糖基化的基因，然后引入催化合成、转运和添加人源糖类的基因。然而，删除这些基因，例如启动高甘露糖基化的OCH1基因，可能会导致细胞生长、形态发生以及对环境挑战的反应出现严重缺陷。
在本研究中，编码Rho1p小GTP酶的RHO1基因的过表达被证实可部分恢复酿酒酵母Δalg3Δoch1双突变菌株的生长缺陷。此外，透射电子显微镜照片显示，RHO1基因表达细胞的细胞壁结构具有增强的葡聚糖层以及恢复的甘露糖蛋白层，揭示了Rho1p GTP酶对细胞壁生物合成的影响。编码1,3-β-葡聚糖合酶催化亚基Fks2蛋白的基因的过表达也证实了类似的互补表型。除了细胞壁结构的恢复，RHO1基因过表达的Δalg3Δoch1菌株在耐温性、渗透势和药物敏感性方面也表现出改善，而在Δalg3Δoch1-FKS2细胞中未观察到这些现象。此外，RHO1基因的过表达还可以增加N-聚糖位点占有率和分泌糖蛋白的量程。
在“人源化”糖蛋白生产酵母中过表达RHO1基因可显著促进其未来在治疗性糖蛋白生产中的工业应用。
