Pichia pastoris is an important host for recombinant protein production. As a protein production platform, further development for therapeutic glycoproteins has been hindered by the high-mannose-type N-glycosylation common to yeast and fungi. Such N-glycans can complicate downstream processing, might be immunogenic or cause the rapid clearance of the glycoprotein from circulation. In recent years, much effort has gone to engineering the N-glycosylation pathway of Pichia pastoris to mimic the human N-glycosylation pathway. This can be of pivotal importance to generate the appropriate glycoforms of therapeutically relevant glycoproteins or to gain a better understanding of structure-function relationships.This chapter describes the methodology to create such glyco-engineered Pichia pastoris strains using the GlycoSwitch(®). This strategy consists of the disruption of an endogenous glycosyltransferase and the heterologous expression of a glycosidase or glycosyltransferase targeted to the Endoplasmic Reticulum or the Golgi of the host. For each step in the process, we describe the transformation procedure, small-scale screening and we also describe how to perform DNA-Sequencer-Aided Fluorophore-Assisted Capillary Electrophoresis (DSA-FACE) to select for clones with the appropriate N-glycosylation profile. The steps described in this chapter can be followed in an iterative fashion in order to generate clones of Pichia pastoris expressing heterologous proteins with humanized N-glycans.
Background:The glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized applicationcustomized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N-glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized.Results: When expressing mouse interleukin-22 (mIL-22) in a Pichia pastoris (syn. Komagataella phaffi) GlycoSwitchM5 strain which had been optimized to produce Man5GlcNAc2 N-glycans, glycan profiling revealed two major species: Man5GlcNAc2 and an unexpected, partially αmannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis and NMR, revealed that this novel glycan was Man5GlcNAc2 modified with a Glcα-1,2-Manβ-1,2-Manβ-1,3-Glcα-1,3-R tetra-saccharide. Also the biosynthetic intermediates of this off-target modification were detected. Expression of a Golgi-targeted GlcNAc Transferase-I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan5GlcNAc2 structure. We have also observed the off-target glycan on other glycoproteins produced in the GlycoSwitchM5 strain. This illustrates the intricacies of Golgi glycosylation pathways and cautions that the use of glycoengineered expression host cells should always be accompanied by detailed glycan analysis of the particular therapeutic proteins being produced.
Conclusions:Our findings reinforce accumulating evidence that robustly customizing the Nglycosylation pathway in Pichia pastoris to produce particular human-type structures is still an incompletely solved synthetic biology challenge, which will require further innovation to enable safe glycoprotein pharmaceutical production.
ABSTRACTBackgroundThe glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized application-customized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N-glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized.ResultsWhen expressing mouse interleukin-22 (mIL-22) in a Pichia pastoris (syn. Komagataella phaffi) GlycoSwitchM5 strain which had been optimized to produce Man5GlcNAc2 N-glycans, glycan profiling revealed two major species: Man5GlcNAc2 and an unexpected, partially α-mannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis and NMR, revealed that this novel glycan was Man5GlcNAc2 modified with a Glcα-1,2-Manβ-1,2-Manβ-1,3-Glcα-1,3-R tetra-saccharide. Also the biosynthetic intermediates of this off-target modification were detected. Expression of a Golgi-targeted GlcNAc Transferase-I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan5GlcNAc2 structure. We have also observed the off-target glycan on other glycoproteins produced in the GlycoSwitchM5 strain. This illustrates the intricacies of Golgi glycosylation pathways and cautions that the use of glyco-engineered expression host cells should always be accompanied by detailed glycan analysis of the particular therapeutic proteins being produced.ConclusionsOur findings reinforce accumulating evidence that robustly customizing the N-glycosylation pathway in Pichia pastoris to produce particular human-type structures is still an incompletely solved synthetic biology challenge, which will require further innovation to enable safe glycoprotein pharmaceutical production.
The structural organization of the Golgi stacks in mammalian cells is intrinsically linked to function, including glycosylation, but the role of morphology is less clear in lower eukaryotes. Here we investigated the link between the structural organization of the Golgi and secretory pathway function using Pichia pastoris as a model system. To unstack the Golgi cisternae, we disrupted 18 genes encoding proteins in the secretory pathway without loss of viability. Using biosensors, confocal microscopy and transmission electron microscopy we identified three strains with irreversible perturbations in the stacking of the Golgi cisternae, all of which had disruption in genes that encode proteins with annotated function as or homology to calcium/calcium permeable ion channels. Despite this, no variation in the secretory pathway for ER size, whole cell glycomics or recombinant protein glycans was observed. Our investigations showed the robust nature of the secretory pathway in P. pastoris and suggest that Ca2+ concentration, homeostasis or signalling may play a significant role for Golgi stacking in this organism and should be investigated in other organisms.
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