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)‐
Reconstructing the chemical and structural characteristics of the plant cell wall represents a promising solution to overcoming lignocellulosic biomass recalcitrance to biochemical deconstruction. This study aims to leverage hydroxyproline (Hyp)‐
- NSF-PAR ID:
- 10131804
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology and Bioengineering
- Volume:
- 117
- Issue:
- 4
- ISSN:
- 0006-3592
- Page Range / eLocation ID:
- p. 945-958
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary O ‐glycosylated peptides (HypGP s) 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. HypGP s 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 (SP 4)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 HypGP module secreted up to 56‐fold greater levels ofEGFP , compared with anEGFP 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 (SP 4)18and (SP )32modules underwent Hyp‐O ‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fusedEGFP protein. Distinct non‐Hyp‐O ‐glycosylated (SP 4)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 (SP 4)18module andAGP ‐styled (SP )32module is proposed. -
null (Ed.)A wide range of proteins with diverse functions in development, defense, and stress responses are O -arabinosylated at hydroxyprolines (Hyps) within distinct amino acid motifs of continuous stretches of Hyps, as found in the structural cell wall extensins, or at non-continuous Hyps as, for example, found in small peptide hormones and a variety of plasma membrane proteins involved in signaling. Plant O -glycosylation relies on hydroxylation of Prolines to Hyps in the protein backbone, mediated by prolyl-4-hydroxylase (P4H) which is followed by O -glycosylation of the Hyp C 4 -OH group by either galactosyltransferases (GalTs) or arabinofuranosyltranferases (Ara f Ts) yielding either Hyp-galactosylation or Hyp-arabinosylation. A subset of the P4H enzymes with putative preference to hydroxylation of continuous prolines and presumably all Ara f T enzymes needed for synthesis of the substituted arabinose chains of one to four arabinose units, have been identified and functionally characterized. Truncated root-hair phenotype is one common denominator of mutants of Hyp formation and Hyp-arabinosylation glycogenes, which act on diverse groups of O -glycosylated proteins, e.g., the small peptide hormones and cell wall extensins. Dissection of different substrate derived effects may not be regularly feasible and thus complicate translation from genotype to phenotype. Recently, lack of proper arabinosylation on arabinosylated proteins has been shown to influence their transport/fate in the secretory pathway, hinting to an additional layer of functionality of O -arabinosylation. Here, we provide an update on the prevalence and types of O -arabinosylated proteins and the enzymatic machinery responsible for their modifications.more » « less
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Summary HYDROXYPROLINE
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