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Title: Engineering ‘designer’ glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline‐ O ‐glycosylation process in plants

The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐O‐glycosylated peptides (HypGPs) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐O‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. HypGPs representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of ‘Ser‐Hyp‐Hyp‐Hyp‐Hyp’ motif or (SP4)18and an arabinogalactan protein module consisting of 32 tandem repeats of ‘Ser‐Hyp’ motif or (SP)32. Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein (EGFP). Hairy root cultures engineered with a HypGPmodule secreted up to 56‐fold greater levels ofEGFP, compared with anEGFPcontrol lacking any HypGPmodule, supporting the function of HypGPmodules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered (SP4)18and (SP)32modules underwent Hyp‐O‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fusedEGFPprotein. Distinct non‐Hyp‐O‐glycosylated (SP4)18EGFPand (SP)32EGFPintermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered HypGPmodules. An updated model depicting the intracellular trafficking, Hyp‐O‐glycosylation and extracellular secretion of extensin‐styled (SP4)18module andAGP‐styled (SP)32module is proposed.

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Author(s) / Creator(s):
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Publisher / Repository:
Date Published:
Journal Name:
Plant Biotechnology Journal
Page Range / eLocation ID:
p. 1130-1141
Medium: X
Sponsoring Org:
National Science Foundation
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