Rodríguez-Limas William A, Tannenbaum Victoria, Tyo Keith E J
Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, 60208, Evanston, Illinois.
Biotechnol Bioeng. 2015 Feb;112(2):376-85. doi: 10.1002/bit.25360. Epub 2014 Oct 10.
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.
酿酒酵母是生产生物制药和工业酶的有用平台。迄今为止，大量工作集中在缓解表达和分泌途径中的几个瓶颈。最近，研究表明，高度活跃的内吞作用会降低上清液中的总蛋白滴度。在本研究中，我们使用改良的烟草蚀纹病毒蛋白酶介导的蛋白质不稳定性诱导（mTIPI）系统来阻断内吞作用和向液泡的运输，以破坏内吞和液泡复合体。我们报告说，与对照相比，内吞作用基因Rvs161的条件性敲低使酿酒酵母培养物上清液中α-淀粉酶的浓度提高了63.7%。通过适应性进化，我们获得了Rvs161和End3基因的敲低突变体，与未进化的对照相比，α-淀粉酶浓度分别提高了2倍和3倍。我们的研究表明，内吞机制的基因阻断可以提高酿酒酵母中异源蛋白的产量。这一结果可能适用于其他真核分泌宿主。
