BioChem - DOS

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

BMC biotechnology

Wang, Tzi-Yuan; Huang, Chih-Jen; Chen, Hsin-Liang; Ho, Po-Chun; Ke, Huei-Mien; Cho, Hsing-Yi; Ruan, Sz-Kai; Hung, Kuo-Yen; Wang, I-Li; Cai, Ya-Wun; Sung, Huang-Mo; Li, Wen-Hsiung; Shih, Ming-Che

<P><B>Background</B></P><P>As a strong fermentator, <I>Saccharomyces cerevisiae</I> 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 <I>S. cerevisiae</I> 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.</P><P><B>Results</B></P><P><I>Saccharomyces cerevisiae</I> gene-knockout strains were screened for improved extracellular activity of a recombinant exocellulase (PCX) from the cellulose digesting fungus <I>Phanerochaete chrysosporium</I>. 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 <I>mnn10</I> and <I>pmt5</I> 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 <I>chc1</I>, <I>clc1</I> and <I>vps21</I> null mutants, were at least 1.5 times higher than the parental strains. Site-directed mutagenesis studies further revealed that the degree of <I>N</I>-glycosylation also plays an important role in heterologous cellulase activity in <I>S. cerevisiae</I>.</P><P><B>Conclusions</B></P><P>Systematic screening of knockout mutants of glycosylation- and protein trafficking-associated genes in <I>S. cerevisiae</I> revealed that: (1) blocking Golgi-to-endosome transport may force <I>S. cerevisiae</I> 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 <I>S. cerevisiae</I>.</P>

2013

BioMed Central

1472-6750

13

pp.71-71

10.1186/1472-6750-13-71

http://click.ndsl.kr/servlet/OpenAPIDetailView?keyValue=05787966&target=NART&cn=NART71713710

Cellulase production; Glycosylation; Protein secretion