Production of a recombinant peroxidase in different glyco-engineered Pichia pastoris strains: a morphological and physiological comparison

BackgroundThe methylotrophic yeast Pichia pastoris is a common host for the production of recombinant proteins. However, hypermannosylation hinders the use of recombinant proteins from yeast in most biopharmaceutical applications. Glyco-engineered yeast strains produce more homogeneously glycosylated proteins, but can be physiologically impaired and show tendencies for cellular agglomeration, hence are hard to cultivate. Further, comprehensive data regarding growth, physiology and recombinant protein production in the controlled environment of a bioreactor are scarce.ResultsA Man5GlcNAc2 glycosylating and a Man8–10GlcNAc2 glycosylating strain showed similar morphological traits during methanol induced shake-flask cultivations to produce the recombinant model protein HRP C1A. Both glyco-engineered strains displayed larger single and budding cells than a wild type strain as well as strong cellular agglomeration. The cores of these agglomerates appeared to be less viable. Despite agglomeration, the Man5GlcNAc2 glycosylating strain showed superior growth, physiology and HRP C1A productivity compared to the Man8–10GlcNAc2 glycosylating strain in shake-flasks and in the bioreactor. Conducting dynamic methanol pulsing revealed that HRP C1A productivity of the Man5GlcNAc2 glycosylating strain is best at a temperature of 30 °C.ConclusionThis study provides the first comprehensive evaluation of growth, physiology and recombinant protein production of a Man5GlcNAc2 glycosylating strain in the controlled environment of a bioreactor. Furthermore, it is evident that cellular agglomeration is likely triggered by a reduced glycan length of cell surface glycans, but does not necessarily lead to lower metabolic activity and recombinant protein production. Man5GlcNAc2 glycosylated HRP C1A production is feasible, yields active protein similar to the wild type strain, but thermal stability of HRP C1A is negatively affected by reduced glycosylation.

[1]  Y Jigami,et al.  OCH1 encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine‐linked oligosaccharides. , 1992, The EMBO journal.

[2]  J. Cregg,et al.  Recombinant protein expression in Pichia pastoris , 2000, Molecular biotechnology.

[3]  Lin Liu,et al.  Effects of N-glycosylation on the biochemical properties of recombinant bEKL expressed in Pichia pastoris. , 2018, Enzyme and microbial technology.

[4]  Oliver Spadiut,et al.  A fast approach to determine a fed batch feeding profile for recombinant Pichia pastoris strains , 2011, Microbial cell factories.

[5]  S. Strahl,et al.  Protein O-mannosylation: conserved from bacteria to humans. , 2009, Glycobiology.

[6]  J. Cregg,et al.  Expression in the yeast Pichia pastoris. , 2009, Methods in enzymology.

[7]  Yu Shen,et al.  N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae , 2016, Scientific Reports.

[8]  E. Redwan,et al.  Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System , 2016, BioMed research international.

[9]  B. Söderström.  Vital staining of fungi in pure cultures and in soil with fluorescein diacetate , 1977 .

[10]  Oliver Spadiut,et al.  A dynamic method based on the specific substrate uptake rate to set up a feeding strategy for Pichia pastoris , 2011, Microbial cell factories.

[11]  Roland Contreras,et al.  Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology , 2008, Nature Protocols.

[12]  Christoph Herwig,et al.  At-line determination of spore inoculum quality in Penicillium chrysogenum bioprocesses , 2016, Applied Microbiology and Biotechnology.

[13]  W. Cook,et al.  Production of sialylated O-linked glycans in Pichia pastoris. , 2013, Glycobiology.

[14]  David A. Johnson,et al.  Expression of recombinant human mast cell chymase with Asn-linked glycans in glycoengineered Pichia pastoris. , 2014, Protein expression and purification.

[15]  T. Vogl,et al.  Optimizing cofactor availability for the production of recombinant heme peroxidase in Pichia pastoris , 2015, Microbial Cell Factories.

[16]  D. Maresch,et al.  Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase , 2015, Microbial Cell Factories.

[17]  F. Képès,et al.  Three‐dimensional structure of tubular networks, presumably Golgi in nature, in various yeast strains: A comparative study , 1995, The Anatomical record.

[18]  Shuping Zou,et al.  N-linked glycosylation influences on the catalytic and biochemical properties of Penicillium purpurogenum β-d-glucuronidase. , 2012, Journal of biotechnology.

[19]  Jack Hoopes,et al.  Humanization of Yeast to Produce Complex Terminally Sialylated Glycoproteins , 2006, Science.

[20]  J. Garin,et al.  Yap1 and Skn7 Control Two Specialized Oxidative Stress Response Regulons in Yeast* , 1999, The Journal of Biological Chemistry.

[21]  A. Depicker,et al.  Comparison of VHH-Fc antibody production in Arabidopsis thaliana, Nicotiana benthamiana and Pichia pastoris. , 2015, Plant biotechnology journal.

[22]  L. Skov,et al.  Interrelationship of steric stabilization and self-crowding of a glycosylated protein. , 2009, Biophysical journal.

[23]  G. Dubelaar,et al.  CytoBuoy: a step forward towards using flow cytometry in operational oceanography* , 2000 .

[24]  F. Altmann,et al.  Site-Specific Glycosylation Profiling Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS). , 2015, Methods in molecular biology.

[25]  R. Contreras,et al.  Fed-batch fermentation of GM-CSF-producing glycoengineered Pichia pastoris under controlled specific growth rate , 2010, Microbial cell factories.

[26]  Y. Hannun,et al.  Cellular Morphogenesis Under Stress Is Influenced by the Sphingolipid Pathway Gene ISC1 and DNA Integrity Checkpoint Genes in Saccharomyces cerevisiae , 2011, Genetics.

[27]  A. Glieder,et al.  Purification and basic biochemical characterization of 19 recombinant plant peroxidase isoenzymes produced in Pichia pastoris , 2014, Protein expression and purification.

[28]  Roland Contreras,et al.  In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans in Pichia pastoris , 2004, Applied and Environmental Microbiology.

[29]  O. Spadiut,et al.  Purification of a recombinant plant peroxidase produced in Pichia pastoris by a simple 2-step strategy. , 2012, Protein expression and purification.

[30]  K. Welinder,et al.  Glycosylation and thermodynamic versus kinetic stability of horseradish peroxidase , 1998, FEBS letters.

[31]  Chi‐Huey Wong,et al.  Glycoengineering of antibody (Herceptin) through yeast expression and in vitro enzymatic glycosylation , 2018, Proceedings of the National Academy of Sciences.

[32]  Oliver Spadiut,et al.  Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway , 2012, Microbial Cell Factories.

[33]  K. Śmietanka,et al.  The Length of N-Glycans of Recombinant H5N1 Hemagglutinin Influences the Oligomerization and Immunogenicity of Vaccine Antigen , 2017, Front. Immunol..

[34]  T. Nagasu,et al.  Isolation of new temperature‐sensitive mutants of Saccharomyces cerevisiae deficient in mannose outer chain elongation , 1992, Yeast.

[35]  A. Glieder,et al.  Deletion of the Pichia pastoris KU70 Homologue Facilitates Platform Strain Generation for Gene Expression and Synthetic Biology , 2012, PloS one.

[36]  Byung-Kwon Choi,et al.  Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[37]  O. Spadiut,et al.  Development of a mixed feed strategy for a recombinant Pichia pastoris strain producing with a de-repression promoter , 2015, Microbial Cell Factories.

[38]  A. Glieder,et al.  Knockout of an endogenous mannosyltransferase increases the homogeneity of glycoproteins produced in Pichia pastoris , 2013, Scientific Reports.

[39]  Susan Dean,et al.  The eukaryotic two-component histidine kinase Sln1p regulates OCH1 via the transcription factor, Skn7p. , 2002, Molecular biology of the cell.

[40]  O. Spadiut,et al.  Effects of different media supplements on the production of an active recombinant plant peroxidase in a Pichia pastoris Δoch1 strain , 2015, Bioengineered.

[41]  A. Schaller,et al.  Expression in Pichia pastoris and characterization of two novel dirigent proteins for atropselective formation of gossypol , 2017, Applied Microbiology and Biotechnology.

[42]  T. Gerngross,et al.  A combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris , 2011, Yeast.

[43]  T. Gerngross,et al.  Glycosylation engineering in yeast: the advent of fully humanized yeast. , 2007, Current opinion in biotechnology.

[44]  Dietmar Haltrich,et al.  Improving thermostability and catalytic activity of pyranose 2‐oxidase from Trametes multicolor by rational and semi‐rational design , 2009, The FEBS journal.

[45]  Brigitte Gasser,et al.  Quo vadis? The challenges of recombinant protein folding and secretion in Pichia pastoris , 2015, Applied Microbiology and Biotechnology.