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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.

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