title: Disruption of genes involved in CORVET complex leads to enhanced secretion of heterologous carboxylesterase only in protease deficient Pichia pastoris.
abstract: 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 Δvps8 and Δvps21 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 Δvps8 and Δvps21 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 Δvps21Δprb1 strains, thus confirming that modifying early steps in the vacuolar pathway has a positive impact on heterologous protein secretion.
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria.|Department of Biotechnology, University of Natural Resources and Life Sciences Vienna (BOKU), Vienna, Austria.
authors.forename: Lukas
authors.initials: L
authors.lastname: Marsalek
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria.|Department of Chemistry, University of Natural Resources and Life Sciences Vienna (BOKU), Vienna, Austria.
authors.forename: Clemens
authors.initials: C
authors.lastname: Gruber
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria.|Department of Chemistry, University of Natural Resources and Life Sciences Vienna (BOKU), Vienna, Austria.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.affiliation: BIOMIN Research Center, Technologiezentrum Tulln, Tulln, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Aleschko
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria.|Department of Biotechnology, University of Natural Resources and Life Sciences Vienna (BOKU), Vienna, Austria.
authors.forename: Diethard
authors.initials: D
authors.lastname: Mattanovich
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria.|Department of Biotechnology, University of Natural Resources and Life Sciences Vienna (BOKU), Vienna, Austria.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gasser
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria.|Department of Biotechnology, University of Natural Resources and Life Sciences Vienna (BOKU), Vienna, Austria.
authors.forename: Verena
authors.initials: V
authors.lastname: Puxbaum
mesh_terms: D001709:Biotechnology / Q000379:methods; D043182:Carboxylesterase / Q000235:genetics* / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics* / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C103153:VPS8 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D043182:Carboxylesterase; C086866:VPS21 protein, S cerevisiae; D020691:rab GTP-Binding Proteins
keywords: methylotrophic yeast; missorting; recombinant protein production; secretion; vacuolar protein sorting