Characterization of GMPP from Dendrobium huoshanense yielding GDP-D-mannose

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.

Dendrobium huoshanense C.Z.Tang et S.J.Cheng: A Review of Its Traditional Uses, Phytochemistry, and Pharmacology

“…Polysaccharide is the main active ingredient of D. huoshanense , which is effective in anti-inflammation, anti-oxidant, anti-tumor, and immunoregulation, and is mainly composed of glucose, galactose, xylose, and arabinose and mannose, with the molecular weight distribution range of 3,200–8,090,000 Da ( Zha et al, 2007b ; Xie et al, 2018 ; Hao et al, 2019 ; Dai et al, 2020 ; Yi et al, 2021b ; Zhang Y. et al, 2021 ; Wu et al, 2022 ). At present, a variety of D. huoshanense polysaccharides have been isolated, such as DHAH ( Meng et al, 2013 ), DHP ( Pan et al, 2014 ), DHP1A ( Tian et al, 2013 ), HPS-1B23 ( Zha et al, 2007c ), DHP-W2 ( Pan et al, 2013 ), DHPD1 ( Zha et al, 2013 ), DHPD2 ( Li et al, 2014 ), TC-DHPA4 ( Si et al, 2018 ), GXG ( Xie et al, 2019a ), cDHPS ( Liu B. et al, 2020 ; Shang et al, 2021 ), and DHP-4A ( Li et al, 2015 ).…”

Selection of Suitable Reference Genes for Gene Expression Normalization Studies in Dendrobium huoshanense

“…In order to further confirm the suitability of the selected reference genes, two pairs of the most stable genes ( RPL5 and MDH , and APT1 and TBP ) and their combinations and one least stable gene ( TIP41 ) were selected for the normalization of CESA [ 46 ] and GMPP [ 20 ] involved in the polysaccharide biosynthesis of D. huoshanense under MeJA treatments and in various tissues. Noticeably, when the selected stable reference gene pairs or their combinations were standardized alone, there were only slight differences in the relative expression levels of CESA and GMPP between different tissues and MeJA treatment.…”

“…Firstly, the website tool RefFinder ( (accessed on 1 August 2021)) was used to comprehensively reassess the results of the candidate genes stability analysis from the geNorm, NormFinder, and BestKeeper softwares. Then, in order to examine the reliability of the selected reference genes, the two most stable and one least stable internal reference genes were used for normalizing the expression levels of two target genes ( GMPP and CESA ) [ 20 , 46 ] related to polysaccharide biosynthesis in different tissues and hormone treatment. Sample collections and experiments were conducted as mentioned above.…”

“…In recent years, plenty of studies have carried out gene and protein research on the biosynthetic pathway of D. huoshanense [ 13 , 14 , 15 , 16 , 17 , 20 , 21 ]. With the development of next-generation sequencing (NGS) technology, it has become simpler and faster to analyze the distribution of mRNA and their expression levels in the biosynthetic pathway of this plant [ 22 ].…”

“…Next, geNorm [ 43 ], NormFinder [ 44 ], and BestKeeper [ 45 ] were preliminarily employed to analyze the gene expression stability, and RefFinder ( (accessed on 1 August 2021)) was then used to comprehensively reassess the candidate genes. Finally, two genes related to polysaccharide biosynthesis, guanosine diphosphate (GDP)-mannose pyrophosphorylase ( GMPP ) [ 20 ] and cellulose synthase ( CESA ) [ 46 ], were selected to test and verify the stability of the selected internal reference genes. Our report of the selection of suitable reference genes for gene expression normalization studies in D. huoshanense continues to support that one or more stable and acceptable reference genes is very necessary to guarantee accurate results and eliminate errors.…”