Engineering the Pichia pastoris N-Glycosylation Pathway Using the GlycoSwitch Technology

Abstract

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.

Heterologous Protein Expression in Pichia pastoris: Latest Research Progress and Applications

“…Later Phillips Petroleum contacted Salk Institute Biotechnology/Industrial Associates, Inc. (SIBIA) in order to develop Pichia as a host strain for recombinant protein production . The advantages of using Pichia over S. cerevisiae are 1) Pichia uses an aerobic mode of respiration that allows it to reach a cell density as high as 130 g L −1 ; 2) Protein expression under methanol‐inducible AOXI promoter only reached 22 g L −1 ; 3) It is possible to express recombinant proteins both intracellularly and extracellularly by secretory expression; 4) Traditionally yeasts have been used in the food industry with a “generally recognized as safe” (GRAS) status, and 5) Metabolic engineering of the yeast glycosylation pathway so that it mimics the mammalian system has broadened the spectrum of recombinant proteins to be expressed in this host …”

The synthesis of recombinant membrane proteins in yeast for structural studies

“…Indeed, the high-resolution structure of a glycosylated form of the Caenorhabditis elegans Pglycoprotein (using recombinant protein produced in P. pastoris) demonstrates that yeast glycosylation does not necessarily hinder crystal formation [56]. Nonetheless, in order to overcome potential bottlenecks in producing, purifying, characterizing and crystallizing human proteins in yeast, engineered strains have been developed including strains with ''humanized'' glycosylation [57,58] and sterol pathways (see section 4.3).…”

Smoothing membrane protein structure determination by initial upstream stage improvements

“…Capable of performing many PTM; low cost of culture media; industry-scale fermentation Glycosylation pattern different from mammalian; intracellular recovery of large amount of cells may require specific equipment (French-press); high oxygen demand Improved glyco-engineered strains obtained using the GlycoSwitch® technology; wide range of genetic tools, plasmids, strains, and promoters available; the preference for the respiratory growth allow to be cultivated at high cell densities. Plasma membrane composed of phospholipids, sterols (ergosterol), and sphingolipids (inositol) (Gonçalves et al 2013;Laukens et al 2015;Marredy et al 2011;Pedro et al 2015) Insect (Rajesh et al 2011;Proverbio et al 2013;Zheng et al 2014) Other interesting features to be considered when selecting a host: (1) native intracellular localization of the target protein; proteins that function in specific eukaryotic organelles such as mitochondria, chloroplasts, and peroxisomes will generally benefit from expression hosts that possess such organelles (Fernández and Vega 2016); (2) types of lipids of host membranes; hydrophobic mismatch may occur due to differences in lipid bilayer composition and thickness between hosts, as highlighted for the overexpression of eukaryotic MP in bacteria, where the absence of sterols, sphingolipids and poly-unsaturated fatty acids in E. coli bilayers poses additional challenges to their proper folding (Snijder and Hakulinen 2016); (3) Construct size; proteins larger than 120 kDa are difficult to be efficiently expressed in E. coli, and are typically obtained in very low yields, as inclusion bodies or proteolytically degraded (Fernández and Vega 2016).…”

“…pastoris for the expression of human proteins consists of the disruption of an endogenous glycosyltransferase gene (OCH1) and the stepwise introduction of heterologous glycosylation enzymes, envisaging to largely eliminate the fungal N-type N-glycosylation while avoiding a considerable heterogeneity in the produced protein and their rapid clearance if therapeutics is the main goal (Jacobs et al 2009;Laukens et al 2015). This strategy is generally referred as GlycoSwitch® and can be applied in wild-type strains (e.g., GS115) or GlycoSwitch® Man 5 strain wherein the first glyco-engineering step was already introduced, and encompasses distinct glyco-engineering steps based on the transformation of P. pastoris with GlycoSwitch® vectors under previously reported protocols (Jacobs et al 2009;Laukens et al 2015). Currently, these vectors are commercially available from BioGrammatics (Carlsbad, USA) under the license from Research Corporation Technology (RCT).…”