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Blocking endocytotic mechanisms to improve heterologous protein titers in Saccharomyces cerevisiae
Author(s) -
RodríguezLimas William A.,
Tannenbaum Victoria,
Tyo Keith E. J.
Publication year - 2015
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25360
Subject(s) - saccharomyces cerevisiae , heterologous , endocytosis , biology , protease , microbiology and biotechnology , yeast , vacuole , secretion , receptor mediated endocytosis , mutant , biochemistry , gene , enzyme , cytoplasm , receptor
Saccharomyces cerevisiae is a useful platform for protein production of biopharmaceuticals and industrial enzymes. To date, substantial effort has focused on alleviating several bottlenecks in expression and the secretory pathway. Recently, it has been shown that highly active endocytosis could decrease the overall protein titer in the supernatant. In this study, we block endocytosis and trafficking to the vacuole using a modified TEV Protease‐Mediated Induction of Protein Instability (mTIPI) system to disrupt the endocytotic and vacuolar complexes. We report that conditional knock‐down of endocytosis gene Rvs161 improved the concentration of α‐amylase in supernatant of S. cerevisiae cultures by 63.7% compared to controls. By adaptive evolution, we obtained knock‐down mutants in Rvs161 and End3 genes with 2‐fold and 3‐fold α‐amylase concentrations compared to controls that were not evolved. Our study demonstrates that genetic blocking of endocytotic mechanisms can improve heterologous protein production in S. cerevisiae . This result is likely generalizable to other eukaryotic secretion hosts. Biotechnol. Bioeng. 2015;112: 376–385. © 2014 Wiley Periodicals, Inc.