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.
(© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)