Engineering 'designer' glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline-O-glycosylation process in plants.

Ningning Zhang,Jianfeng Xu,Tristen Wright,Brett J Savary,Xiaoting Wang,Uddhab Karki

doi:10.1111/pbi.13043

Ningning Zhang, Jianfeng Xu + Show 4 more

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https://doi.org/10.1111/pbi.13043

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Abstract

SummaryThe 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.

Highlights

Molecular farming in plants is recognized as an advantageous platform for producing therapeutic proteins through the significant advantages in cost and safety over other eukaryotic systems
Transgenic hairy roots were induced from leaves of the high-expression plantlets expressing (SP4)18-enhanced green fluorescence protein (EGFP), (SP)32-EGFP or EGFP control
Two types of designer HypGP modules, including (SP4)18 comprised of the signature peptide sequence of extensins and (SP)32 comprised of the signature peptide sequence of arabinogalactan proteins (AGPs), were engineered into tobacco hairy roots to evaluate their function in boosting the secreted yields of the fused EGFP protein

Summary

Introduction

Molecular farming in plants is recognized as an advantageous platform for producing therapeutic proteins through the significant advantages in cost and safety over other eukaryotic systems. Increasing concerns over regulatory compliance and product safety have prompted a resurgence of interest in molecular farming with contained in vitro systems such as plant cells and tissues cultured in bioreactors (Santos et al, 2016; Xu et al, 2011). As a fully differentiated organ, hairy roots provide additional benefits that include genotype and phenotype stability and autotrophy for plant hormones (Georgiev et al, 2012; Guillon et al, 2006). Despite these benefits, a bottleneck exits in exploiting this in vitro technology for commercial purposes due to low protein productivity, low secreted protein yields. We seek to exploit an alternative method, engineering novel hydroxyproline (Hyp)-O-glycosylated peptide (HypGP) modules as molecular carriers, for generally boosting secreted protein yields in hairy root culture

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