Accurate analysis of fusion expression of Pichia pastoris glycosylphosphatidylinositol-modified cell wall proteins

• Published in: Journal of Industrial Microbiology and Biotechnology
• Main Authors: Wang, Pan, Zhang, Li, Fisher, Rebecca, Chen, Meiqi, Liang, Shuli, Han, Shuangyan, Zheng, Suiping, Sui, Haixin, Lin, Ying
• Other Authors: China National High Technology Research and Development Program, High Technology Research and Development of Guangdong Province, The Recruitment Program of Leading Talents in Innovation and Entrepreneurship of Guangzhou, National Institute of Health
• Format: Article in Journal/Newspaper
• Language: English
• Published: Oxford University Press (OUP) 2017
• Subjects: Applied Microbiology and Biotechnology; Biotechnology; Bioengineering; Antarc*; Antarctica
• Online Access: http://dx.doi.org/10.1007/s10295-017-1962-8; http://link.springer.com/article/10.1007/s10295-017-1962-8/fulltext.html; http://link.springer.com/content/pdf/10.1007/s10295-017-1962-8.pdf; http://academic.oup.com/jimb/article-pdf/44/9/1355/36745738/jimb1355.pdf

Abstract 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.