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Off-target glycans encountered along the synthetic biology route toward humanized N-glycans in Pichia pastoris

(2020) BIOTECHNOLOGY AND BIOENGINEERING. 117(8). p.2479-2488
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Abstract
The 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. When expressing mouse interleukin-22 in a Pichia pastoris (syn. Komagataella phaffii) GlycoSwitchM5 strain, which had been optimized to produce Man(5)GlcNAc(2)N-glycans, glycan profiling revealed two major species: Man(5)GlcNAc(2) and an unexpected, partially alpha-mannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis, and nuclear magnetic resonance revealed that this novel glycan was Man(5)GlcNAc(2) modified with a Glc alpha-1,2-Man beta-1,2-Man beta-1,3-Glc alpha-1,3-R tetrasaccharide. Expression of a Golgi-targeted GlcNAc transferase-I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan(5)GlcNAc(2) structure. Our findings reinforce accumulating evidence that robustly customizing the N-glycosylation pathway in P. 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.
Keywords
INDUCIBLE FACTOR, GLYCOSYLATION, EXPRESSION, CYTOKINE, INTERLEUKIN-22, IDENTIFICATION, MANNOSYLATION, RECEPTOR, CLONING, IL-22, GlycoSwitch, N-glycan engineering, Pichia pastoris, synthetic biology

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MLA
Laukens, Bram, et al. “Off-Target Glycans Encountered along the Synthetic Biology Route toward Humanized N-Glycans in Pichia Pastoris.” BIOTECHNOLOGY AND BIOENGINEERING, vol. 117, no. 8, 2020, pp. 2479–88, doi:10.1002/bit.27375.
APA
Laukens, B., Jacobs, P., Gheysen, K., Martins, J., De Wachter, C., Ameloot, P., … Callewaert, N. (2020). Off-target glycans encountered along the synthetic biology route toward humanized N-glycans in Pichia pastoris. BIOTECHNOLOGY AND BIOENGINEERING, 117(8), 2479–2488. https://doi.org/10.1002/bit.27375
Chicago author-date
Laukens, Bram, Pieter Jacobs, Katelijne Gheysen, José Martins, Charlot De Wachter, Paul Ameloot, Willy Morelle, et al. 2020. “Off-Target Glycans Encountered along the Synthetic Biology Route toward Humanized N-Glycans in Pichia Pastoris.” BIOTECHNOLOGY AND BIOENGINEERING 117 (8): 2479–88. https://doi.org/10.1002/bit.27375.
Chicago author-date (all authors)
Laukens, Bram, Pieter Jacobs, Katelijne Gheysen, José Martins, Charlot De Wachter, Paul Ameloot, Willy Morelle, Jurgen Haustraete, Jean-Christophe Renauld, Bart Samyn, Roland Contreras, Simon Devos, and Nico Callewaert. 2020. “Off-Target Glycans Encountered along the Synthetic Biology Route toward Humanized N-Glycans in Pichia Pastoris.” BIOTECHNOLOGY AND BIOENGINEERING 117 (8): 2479–2488. doi:10.1002/bit.27375.
Vancouver
1.
Laukens B, Jacobs P, Gheysen K, Martins J, De Wachter C, Ameloot P, et al. Off-target glycans encountered along the synthetic biology route toward humanized N-glycans in Pichia pastoris. BIOTECHNOLOGY AND BIOENGINEERING. 2020;117(8):2479–88.
IEEE
[1]
B. Laukens et al., “Off-target glycans encountered along the synthetic biology route toward humanized N-glycans in Pichia pastoris,” BIOTECHNOLOGY AND BIOENGINEERING, vol. 117, no. 8, pp. 2479–2488, 2020.
@article{8666927,
  abstract     = {{The 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. When expressing mouse interleukin-22 in a Pichia pastoris (syn. Komagataella phaffii) GlycoSwitchM5 strain, which had been optimized to produce Man(5)GlcNAc(2)N-glycans, glycan profiling revealed two major species: Man(5)GlcNAc(2) and an unexpected, partially alpha-mannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis, and nuclear magnetic resonance revealed that this novel glycan was Man(5)GlcNAc(2) modified with a Glc alpha-1,2-Man beta-1,2-Man beta-1,3-Glc alpha-1,3-R tetrasaccharide. Expression of a Golgi-targeted GlcNAc transferase-I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan(5)GlcNAc(2) structure. Our findings reinforce accumulating evidence that robustly customizing the N-glycosylation pathway in P. 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.}},
  author       = {{Laukens, Bram and Jacobs, Pieter and Gheysen, Katelijne and Martins, José and De Wachter, Charlot and Ameloot, Paul and Morelle, Willy and Haustraete, Jurgen and Renauld, Jean-Christophe and Samyn, Bart and Contreras, Roland and Devos, Simon and Callewaert, Nico}},
  issn         = {{0006-3592}},
  journal      = {{BIOTECHNOLOGY AND BIOENGINEERING}},
  keywords     = {{INDUCIBLE FACTOR,GLYCOSYLATION,EXPRESSION,CYTOKINE,INTERLEUKIN-22,IDENTIFICATION,MANNOSYLATION,RECEPTOR,CLONING,IL-22,GlycoSwitch,N-glycan engineering,Pichia pastoris,synthetic biology}},
  language     = {{eng}},
  number       = {{8}},
  pages        = {{2479--2488}},
  title        = {{Off-target glycans encountered along the synthetic biology route toward humanized N-glycans in Pichia pastoris}},
  url          = {{http://dx.doi.org/10.1002/bit.27375}},
  volume       = {{117}},
  year         = {{2020}},
}

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