Pichia pastoris is the most frequently used yeast system for heterologous protein production today. The last few years have seen several products based on this platform reach approval as biopharmaceutical drugs. Successful glycoengineering to humanize N-glycans is further fuelling this development. However, detailed understanding of the yeast's physiology, genetics and regulation has only developed rapidly in the last few years since published genome sequences have become available. An expanding toolbox of genetic elements and strains for the improvement of protein production is being generated, including promoters, gene copy-number enhancement, gene knockout and high-throughput methods. Protein folding and secretion have been identified as significant bottlenecks in yeast expression systems, pinpointing a major target for strain optimization. At the same time, it has become obvious that P. pastoris, as an evolutionarily more 'ancient' yeast, may in some cases be a better model for human cell biology and disease than Saccharomyces cerevisiae.

The development of Pichia pastoris as a production platform for recombinant proteins has been a remarkable success story over the last three decades. Stable cheap production processes and the good protein secretion abilities were pacemakers of this development. However, limitations of protein folding, glycosylation or secretion have been identified quite early on. With the availability of genome sequences and the development of systems biology characterization in the last 5 years, remarkable success in strain improvement was achieved. Here, we focus on recent developments of characterization and improvement of P. pastoris production strains regarding protein folding, intracellular trafficking, glycosylation and proteolytic degradation.

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The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) mediates biosynthetic sorting and endocytosis of various factors that impinge on the proliferation, migration and invasiveness of tumour cells. The gene encoding M6P/IGF2R is frequently lost or mutated in a wide range of malignant tumours including squamous cell carcinomas. We have previously shown that M6P/IGF2R-deficient SCC-VII murine squamous cell carcinoma cells secrete large amounts of pro-invasive lysosomal proteinases. Furthermore, the formation of mature lysosomes is impaired in SCC-VII cells. To assess the link between M6P/ IGF2R status and tumour invasion, we have now generated SCC-VII lines stably transfected with human M6P/IGF2R cDNA. Reconstitution of functional M6P/IGF2R expression in SCC-VII cells strongly improves the intracellular retention of lysosomal proteinases and restores the formation of mature lysosomes. In addition, the presence of heterologous M6P/IGF2R compromises the growth of SCC-VII cells both in vitro and in vivo. Remarkably, M6P/IGF2R expression also reduces the invasive capacity of SCC-VII cells in response to various chemoattractants. These results indicate that the M6P/IGF2R status influences the metastatic propensity of squamous cell carcinomas. ' 2008 Wiley-Liss, Inc.Key words: cathepsin; lysosome; proteolysis; squamous cell carcinoma; tumour invasion A key requirement for metastatic cancer cell invasion is the penetration of extracellular matrix (ECM) barriers. This process involves the degradation of different ECM proteins and proteoglycans. 1,2 Various proteinases have been implicated in ECM degradation associated with tumour invasion and metastasis, including urokinase-type plasminogen activator, matrix metalloproteinases and cathepsins. [3][4][5] The involvement of the latter enzymes in ECM proteolysis is perplexing, since cathepsins are normally localized in lysosomes. However, tumour cells often secrete significant amounts of these proteinases into the pericellular fluid, as first observed for cathepsin B. 6 As typical for lysosomal enzymes, the N-glycan moieties of cathepsins are modified during their biosynthesis with mannose 6-phosphate (M6P) residues which permit interaction with the main lysosomal sorting receptors, the 300-kDa mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) and the 46-kDa mannose 6-phosphate receptor (MPR46). 7 Evidence has been provided that excessive secretion of cathepsins by tumour cells may be due to transformation-induced changes to the M6P receptor pathway. [8][9][10] Lysosomal sorting via M6P/IGF2R is generally far more efficient than by MPR46, demonstrating that the former is the main lysosomal targeting receptor in mammalian cells. 11,12 However, M6P/IGF2R also binds a variety of other factors that impinge on the proliferation, migration and invasiveness of tumour cells, including insulin-like growth factor II (IGF-II), 13 transforming growth factor b, 14 urokinase-type plasminogen activator receptor 15 and plasminogen. 16 Hence, it is o...

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.

BackgroundBacterial inclusion bodies (IBs) are non-toxic protein aggregates commonly produced in recombinant bacteria. They are formed by a mixture of highly stable amyloid-like fibrils and releasable protein species with a significant extent of secondary structure, and are often functional. As nano structured materials, they are gaining biomedical interest because of the combination of submicron size, mechanical stability and biological activity, together with their ability to interact with mammalian cell membranes for subsequent cell penetration in absence of toxicity. Since essentially any protein species can be obtained as IBs, these entities, as well as related protein clusters (e.g., aggresomes), are being explored in biocatalysis and in biomedicine as mechanically stable sources of functional protein. One of the major bottlenecks for uses of IBs in biological interfaces is their potential contamination with endotoxins from producing bacteria.ResultsTo overcome this hurdle, we have explored here the controlled production of functional IBs in the yeast Pichia pastoris (Komagataella spp.), an endotoxin-free host system for recombinant protein production, and determined the main physicochemical and biological traits of these materials. Quantitative and qualitative approaches clearly indicate the formation of IBs inside yeast, similar in morphology, size and biological activity to those produced in E. coli, that once purified, interact with mammalian cell membranes and penetrate cultured mammalian cells in absence of toxicity.ConclusionsStructurally and functionally similar from those produced in E. coli, the controlled production of IBs in P. pastoris demonstrates that yeasts can be used as convenient platforms for the biological fabrication of self-organizing protein materials in absence of potential endotoxin contamination and with additional advantages regarding, among others, post-translational modifications often required for protein functionality.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0565-9) contains supplementary material, which is available to authorized users.

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.

Background & AimsThe mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R), a multifunctional protein, plays a central role in intracellular targeting of lysosomal enzymes and control of insulin-like growth factor II (IGF-II) bioactivity. Importantly, the gene encoding this receptor is frequently inactivated in a wide range of malignant tumors including hepatocellular carcinomas. Thus, M6P/IGF2R is considered a putative liver tumor suppressor. The aim of this study was to establish the impact of the receptor on the invasive properties of liver cells.MethodsReconstitution experiments were performed by expression of wild type and mutant M6P/IGF2R in receptor-deficient FRL14 fetal rat liver cells. RNA interference was used to induce M6P/IGF2R downregulation in receptor-positive MIM-1–4 mouse hepatocytes.ResultsWe show that the M6P/IGF2R status exerts a strong impact on the invasiveness of tumorigenic rodent liver cells. M6P/IGF2R-deficient fetal rat liver cells hypersecrete lysosomal cathepsins and penetrate extracellular matrix barriers in a cathepsin-dependent manner. Forced expression of M6P/IGF2R restores intracellular transport of cathepsins to lysosomes and concomitantly reduces the tumorigenicity and invasive potential of these cells. Conversely, M6P/IGF2R knock-down in receptor-positive mouse hepatocytes causes increased cathepsin secretion as well as enhanced cell motility and invasiveness. We also demonstrate that functional M6P-binding sites are important for the anti-invasive properties of M6P/IGF2R, whereas the capacity to bind IGF-II is dispensable for the anti-invasive activity of the receptor in liver cells.ConclusionsM6P/IGF2R restricts liver cell invasion by preventing the pericellular action of M6P-modified proteins.