Journal
BIOTECHNOLOGY JOURNAL
Volume 12, Issue 5, Pages -Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.201600584
Keywords
methylotrophic yeast; missorting; recombinant protein production; secretion; vacuolar protein sorting
Funding
- Federal Ministry of Science, Research and Economy (BMWFW)
- Federal Ministry of Traffic, Innovation and Technology (bmvit)
- Styrian Business Promotion Agency SFG
- Standortagentur Tirol
- Government of Lower Austria
- ZIT - Technology Agency of the City of Vienna
- BIOMIN Research Center
- Boehringer Ingelheim RCV GmbH Co KG
- Lonza AG
- Biocrates Life Sciences AG
- VTU Technology GmbH
- Sandoz GmbH
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The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Previous studies have shown that mis-sorting to the vacuole can be a bottleneck during production of recombinant secretory proteins in yeast, however, no information was available for P. pastoris. In this work the authors have therefore generated vps (vacuolar protein sorting) mutant strains disrupted in genes involved in the CORVET (class C core vacuole/endosome tethering) complex at the early stages of endosomal sorting. Both vps8 and vps21 strains contained lower extracellular amounts of heterologous carboxylesterase (CES) compared to the control strain, which could be attributed to a high proteolytic activity present in the supernatants of CORVET engineered strains due to rerouting of vacuolar proteases. Serine proteases were identified to be responsible for this proteolytic degradation by liquid chromatography-mass spectrometry and protease inhibitor assays. Deletion of the major cellular serine protease Prb1 in vps8 and vps21 strains did not only rescue the extracellular CES levels, but even outperformed the parental CES strain (56 and 80% higher yields, respectively). Further deletion of Ybr139W, another serine protease, did not show a further increase in secretion levels. Higher extracellular CES activity and low proteolytic activity were detected also in fed batch cultivation of vps21prb1 strains, thus confirming that modifying early steps in the vacuolar pathway has a positive impact on heterologous protein secretion.
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