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)₁₈ and 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 HypGP module secreted up to 56‐fold greater levels of EGFP, compared with an EGFP control lacking any HypGP module, supporting the function of HypGP modules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered (SP4)₁₈ and (SP)₃₂ modules underwent Hyp‐O‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fused EGFP protein. Distinct non‐Hyp‐O‐glycosylated (SP4)₁₈‐EGFP and (SP)₃₂‐EGFP intermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered HypGP modules. An updated model depicting the intracellular trafficking, Hyp‐O‐glycosylation and extracellular secretion of extensin‐styled (SP4)₁₈ module and AGP‐styled (SP)₃₂ module is proposed.