Systematic screening of glycosylation- and trafficking-associated gene knockouts in Saccharomyces cerevisiaeidentifies mutants with improved heterologous exocellulase activity and host secretion

BMC Biotechnology

Research Article

I-Li Wang¹  Ya-Wun Cai¹  Sz-Kai Ruan¹  Po-Chun Ho¹  Hsin-Liang Chen¹  Tzi-Yuan Wang¹  Kuo-Yen Hung¹  Wen-Hsiung Li²  Chih-Jen Huang³  Huei-Mien Ke⁴  Huang-Mo Sung⁵  Ming-Che Shih⁶  Hsing-Yi Cho⁷ 

[1] Biodiversity Research Center, 115, Academia Sinica, Taipei, Taiwan;Biodiversity Research Center, 115, Academia Sinica, Taipei, Taiwan;Department of Ecology and Evolution, University of Chicago, 60637, Chicago, IL, USA;Biodiversity Research Center, 115, Academia Sinica, Taipei, Taiwan;Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung-Hsing University, 115, Academia Sinica, Taipei, Taiwan;Graduate Institute of Biotechnology, National Chung-Hsing University, 402, Taichung, Taiwan;Biodiversity Research Center, 115, Academia Sinica, Taipei, Taiwan;Program in Microbial Genomics, National Chung Hsing University, 115, Academia Sinica, Taipei, Taiwan;Department of Life Sciences, National Cheng Kung University, 701, Tainan, Taiwan;Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung-Hsing University, 115, Academia Sinica, Taipei, Taiwan;Agricultural Biotechnology Research Center, 115, Academia Sinica, Taipei, Taiwan;Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung-Hsing University, 115, Academia Sinica, Taipei, Taiwan;Graduate Institute of Biotechnology, National Chung-Hsing University, 402, Taichung, Taiwan;Agricultural Biotechnology Research Center, 115, Academia Sinica, Taipei, Taiwan;

关键词: Cellulase production;    Glycosylation;    Protein secretion;   

DOI  :  10.1186/1472-6750-13-71

 received in 2012-12-27, accepted in 2013-08-29,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundAs a strong fermentator, Saccharomyces cerevisiae has the potential to be an excellent host for ethanol production by consolidated bioprocessing. For this purpose, it is necessary to transform cellulose genes into the yeast genome because it contains no cellulose genes. However, heterologous protein expression in S. cerevisiae often suffers from hyper-glycosylation and/or poor secretion. Thus, there is a need to genetically engineer the yeast to reduce its glycosylation strength and to increase its secretion ability.ResultsSaccharomyces cerevisiae gene-knockout strains were screened for improved extracellular activity of a recombinant exocellulase (PCX) from the cellulose digesting fungus Phanerochaete chrysosporium. Knockout mutants of 47 glycosylation-related genes and 10 protein-trafficking-related genes were transformed with a PCX expression construct and screened for extracellular cellulase activity. Twelve of the screened mutants were found to have a more than 2-fold increase in extracellular PCX activity in comparison with the wild type. The extracellular PCX activities in the glycosylation-related mnn10 and pmt5 null mutants were, respectively, 6 and 4 times higher than that of the wild type; and the extracellular PCX activities in 9 protein-trafficking-related mutants, especially in the chc1, clc1 and vps21 null mutants, were at least 1.5 times higher than the parental strains. Site-directed mutagenesis studies further revealed that the degree of N-glycosylation also plays an important role in heterologous cellulase activity in S. cerevisiae.ConclusionsSystematic screening of knockout mutants of glycosylation- and protein trafficking-associated genes in S. cerevisiae revealed that: (1) blocking Golgi-to-endosome transport may force S. cerevisiae to export cellulases; and (2) both over- and under-glycosylation may alter the enzyme activity of cellulases. This systematic gene-knockout screening approach may serve as a convenient means for increasing the extracellular activities of recombinant proteins expressed in S. cerevisiae.

【 授权许可】

Unknown   
© Wang et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.