Wang Pan, Zhang Li, Fisher Rebecca, Chen Meiqi, Liang Shuli, Han Shuangyan, Zheng Suiping, Sui Haixin, Lin Ying
Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, Guangdong, People's Republic of China.
Wadsworth Center, New York State Department of Health, Albany, NY, 12201, USA.
J Ind Microbiol Biotechnol. 2017 Sep;44(9):1355-1365. doi: 10.1007/s10295-017-1962-8. Epub 2017 Jun 28.
Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have diverse intrinsic functions in yeasts, and they also have different uses in vitro. The GPI-modified cell wall proteins GCW21, GCW51, and GCW61 of Pichia pastoris were chosen as anchoring proteins to construct co-expression strains in P. pastoris GS115. The hydrolytic activity and the amount of Candida antarctica lipase B (CALB) displayed on cell surface increased significantly following optimization of the fusion gene dosage and combination of the homogeneous or heterogeneous cell wall proteins. Maximum CALB hydrolytic activity was achieved at 4920 U/g dry cell weight in strain GS115/CALB-GCW (51 + 51 + 61 + 61) after 120 h of methanol induction. Changes in structural morphology and the properties of the cell surfaces caused by co-expression of fusion proteins were observed by transmission electron microscopy (TEM) and on plates containing cell-wall-destabilizing reagent. Our results suggested that both the outer and inner cell layers were significantly altered by overexpression of GPI-modified cell wall proteins. Interestingly, quantitative analysis of the inner layer components showed an increase in β-1,3-glucan, but no obvious changes in chitin in the strains overexpressing GPI-modified cell wall proteins.
糖基磷脂酰肌醇（GPI）锚定糖蛋白在酵母中具有多种内在功能，并且在体外也有不同用途。选择巴斯德毕赤酵母的GPI修饰细胞壁蛋白GCW21、GCW51和GCW61作为锚定蛋白，在毕赤酵母GS115中构建共表达菌株。优化融合基因剂量以及同质或异质细胞壁蛋白的组合后，细胞表面展示的南极假丝酵母脂肪酶B（CALB）的水解活性和量显著增加。甲醇诱导120小时后，在菌株GS115/CALB-GCW（51 + 51 + 61 + 61）中，CALB的最大水解活性达到4920 U/g干细胞重量。通过透射电子显微镜（TEM）以及在含有细胞壁去稳定试剂的平板上观察了融合蛋白共表达引起的细胞表面结构形态和性质变化。我们的结果表明，GPI修饰细胞壁蛋白的过表达显著改变了细胞的外层和内层。有趣的是，对内层成分的定量分析表明，在过表达GPI修饰细胞壁蛋白的菌株中，β-1,3-葡聚糖增加，但几丁质没有明显变化。
