Disruption of vacuolar protein sorting components of the HOPS complex leads to enhanced secretion of recombinant proteins in Pichia pastoris

Abstract

Background The yeast Pichia pastoris is a widely used host for the secretion of heterologous proteins. Despite being an efficient producer, we observed previously that certain recombinant proteins were mistargeted to the vacuole on their route to secretion. Simultaneous disruption of one vacuolar sorting pathway together with vacuolar proteases prevented this mis-sorting and resulted in higher levels of secreted heterologous protein. Inspired by the positive results, we now set out to investigate the influence of further parts of the vacuolar pathway, namely the Cvt-pathway and the homotypic fusion and protein sorting (HOPS) complex. Results Strains impaired in the Cvt pathway (∆ atg11 , ∆ atg8 ) had no effect on secretion of the model protein carboxylesterase (CES), but resulted in lower secretion levels of the antibody fragment HyHEL-Fab. Disruption of genes involved in the HOPS complex led to vacuole-like compartments of the B category of vps mutants, which are characteristic for the deleted genes YPT7, VPS41 and VAM6 . In particular ∆ ypt7 and ∆ vam6 strains showed an improvement in secreting the model proteins HyHEL-Fab and CES. Additional disruption of the vacuolar protease Pep4 and the potential protease Vps70 led to even further enhanced secretion in ∆ ypt7 and ∆ vam6 strains. Nevertheless, intracellular product accumulation was still observed. Therefore, the secretory route was strengthened by overexpression of early or late secretory genes in the vacuolar sorting mutants. Thereby, overexpression of Sbh1, a subunit of the ER translocation pore, significantly increased HyHEL-Fab secretion, leading up to fourfold higher extracellular Fab levels in the ∆ ypt7 strain. The beneficial impact on protein secretion and the suitability of these strains for industrial applicability was confirmed in fed-batch cultivations. Conclusions Disruption of genes involved in the HOPS complex, especially YPT7 , has a great influence on the secretion of the two different model proteins HyHEL-Fab and CES. Therefore, disruption of HOPS genes shows a high potential to increase secretion of other recombinant proteins as well. Secretion of HyHEL-Fab was further enhanced when overexpressing secretion enhancing factors. As the positive effect was also present in fed-batch cultivations, these modifications likely have promising industrial relevance. Electronic supplementary material The online version of this article (10.1186/s12934-019-1155-4) contains supplementary material, which is available to authorized users.

Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system

“…reported that disruption of the autophagy-related Cvt pathway genes ATG8 and ATG11 did not affect the secretion of heterologous proteins in P . pastoris [ 47 ].…”

“…A recent report found that deletion of YPT7 increased secretion of the model proteins HyHEL-Fab and CES in the yeast P . pastoris [ 47 ], and of model proteins cellulase (Ctcel8A) and exocellulase (PCX) in S . cerevisiae [ 49 , 50 ].…”

Comprehensive comparison of Yarrowia lipolytica and Pichia pastoris for production of Candida antarctica lipase B

“…3, the specific fluorescence was on average 2.5-fold increased in the presence of the proteasome inhibitor (3 vs 8 FU.g DCW −1 after 32 h, and 55 vs 23 FU.g DCW −1 after 48 h). This highlighted that some CalB was lost due to intracellular degradation either in the cytosol 32 or in vacuoles 33,34 prior to secretion, thus explaining the low lipase titer observed in the culture supernatant of P. pastoris strain RIY311, despite higher expression levels of CalB than in Y. lipolytica strain RIY368.…”

Production and secretion dynamics of prokaryotic Penicillin G acylase in Pichia pastoris

“…Elevated upregulation of the endoplasmic reticulum associated protein degradation (ERAD) pathway may have resulted in rapid degradation of the accumulated product. However, this is rather improbable since the upregulation of this pathway is mostly expected in the first phase of production (Aw and Polizzi 2013;Marsalek et al 2019;Vogl et al 2014). The more probable explanation is partial translational arrest, as previously described by Edwards-Jones et al (2015).…”