Abstract

Dendrobium huoshanense has been used for centuries in China and its polysaccharides are the main active components in treating loss of body fluids resulting from fever and asthenic symptoms. However, the biosynthetic pathway of polysaccharides in D. huoshanense remains to be elucidated. In this study, we obtained a guanosine diphosphate (GDP)-mannose pyrophosphorylase (DhGMPP) from D. huoshanense and characterized its function to catalyze the conversion of α-D-mannose-phosphate to GDP-D-mannose involved in the production of polysaccharides. DhGMPP, with the open reading frame of 1,245 bp, was isolated from RNA-Seq data of D. huoshanense. Phylogenetic analysis as well as sequence characterization suggested its involvement in the biosynthesis of GDP-D-mannose. In vitro enzyme assay demonstrated that GMPP encoded a pyrophosphorylase that converted α-D-mannose-phosphate and GTP into GDP-D-mannose. Identification of DhGMPP could provide more insights into the mechanism concerning polysaccharide biosynthesis in D. huoshanense and be utilized for enhancing polysaccharide accumulation through metabolic engineering.

Highlights

  • Embodied in Chinese Pharmacopoeia with the name of Dendrobii Caulis, the stem of Dendrobium huoshanense C

  • D. huoshanense seedlings were collected in April 2019 from Huoshan county, Anhui, China. α-D-Mannose-phosphate and guanosine diphosphate (GDP)-D-mannose were purchased from Sigma-Aldrich, guanosine-5-triphosphoric acid (GTP) from Sangon-Biotech, Total RNA from stem of D. huoshanense was isolated according to the method described previously [9]

  • The size of diphosphate (GDP)-mannose pyrophosphorylase (DhGMPP) protein is 45.77 kDa (415 amino acids) and this is in line with the known GDP-mannose pyrophosphorylases

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Summary

Introduction

Embodied in Chinese Pharmacopoeia with the name of Dendrobii Caulis, the stem of Dendrobium huoshanense C. While the plant produces versatile constituents including flavonoids, bibenzyls, phenanthrenes, polysaccharides, and alkaloids [3–5], water-soluble polysaccharides act as its main active ingredients which by far demonstrated immunostimulating, anti-inflammatory, antipyretic, astringent, and tonic effects [6–8]. One prominent character shared by plants from genus Dendrobium is their high polysaccharide content. Regarding D. huoshanense stems, up to 36% of the dry weight is made of total polysaccharides, 90% of which are water-soluble [9,10]. Despite its high polysaccharide content, the yield of D. huoshanense could hardly meet robust demands of the pharmaceutical market because a dry stem, after 3-year cultivation, weighs only 3–8 g. From the perspective of plant biotechnology, to elaborate its polysaccharide biosynthetic pathway and pinpoint the key catalytic enzymes may help pave the way for producing D. huoshanense polysaccharides (DhPs) adopting bioengineering approaches

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