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Stable isotope labeling and 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucopyranoside biosynthetic pathway characterization in Fallopia multiflora

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Abstract

Main conclusion

The THSG biosynthetic pathway in F. multiflora was characterized, and enzymatic activities responsible for the resveratrol synthesis, hydroxylation, and glycosylation reactions involved in THSG biosynthesis were confirmed in vitro.

The biosynthetic origin of 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucopyranoside (THSG) and the enzymes involved in THSG biosynthesis in Fallopia multiflora were studied using stable isotope labeling and biocatalytic methods. UPLC-MS-based analyses were used to unravel the isotopologue composition of the biosynthetic intermediates and products, as well as to detect the products of the enzyme assay experiments. In this study, 13C-labeled l-phenylalanine (l-PHE), sodium pyruvate (SP), and sodium bicarbonate (SB) were used as putative precursors in the feeding experiment. Labeling of polydatin (PD) and THSG using [13C9]L-PHE and [13C1]l-PHE confirmed that the p-coumaric moiety of PD and THSG was derived from PHE. The results of the feeding experiments with [13C] SB and [2, 3-13C2] SP suggested that PD and THSG were derivatives of resveratrol that were synthesized by glycosylation and hydroxylation. We developed methods using total crude protein extracts (soluble and microsomal) for comprehensive and simultaneous analysis of resveratrol synthase, glycosyltransferase, and hydroxylase activities in various tissue types of wild F. multiflora and callus cultures. The activity of each tested enzyme was confirmed in one or more tissue types or cell cultures in vitro. The results of the enzyme activity experiments and the distributions of PD and THSG were used to determine the main site and pathway of THSG biosynthesis in F. multiflora.

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Abbreviations

THSG:

2,3,5,4′-Tetrahydroxystilbene-2-O-β-d-glucopyranoside

l-PHE:

l-Phenylalanine

SP:

Sodium pyruvate

SB:

Sodium bicarbonate

PD:

Polydatin

RS:

Resveratrol synthase

GT:

Glycosyltransferase

STS:

Stilbene synthase

UDPG:

Uridine 5′-diphosphoglucose disodium salt

NADPH:

β-Micotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt

CA:

Cinnamic acid

References

  1. Austin MB, Noel A (2003) The chalcone synthase superfamily of type III polyketide synthases. Nat Prod Rep 20:79–110

  2. Austin MB, Bowman ME, Ferrer JL, Schroder J, Noel JP (2004) An aldol switch discovered in stilbene synthases mediates cyclization specificity of type III polyketide synthases. Chem Biol 11:1179–1194

  3. Cullen JP, Morrow D, Jin Y, von Offenberg Sweeney N, Sitzmann JV, Cahill PA, Redmond EM (2007) Resveratrol inhibits expression and binding activity of the monocyte chemotactic protein-1 receptor, CCR2, on THP-1 monocytes. Atherosclerosis 195:e125–e133

  4. Delaunois B, Cordelier S, Conreux A, Clément C, Jeandet P (2009) Molecular engineering of resveratrol in plants. Plant Biotechnol J 7:2–12

  5. Furuya T, Kino K (2014) Regioselective synthesis of piceatannol from resveratrol: catalysis by two-component flavin-dependent monooxygenase HpaBC in whole cells. Tetrahedron Lett 55:2853–2855

  6. Hall D, De Luca V (2007) Mesocarp localization of a bi-functional resveratrol/hydroxycinnamic acid glucosyltransferase of Concord grape (Vitis labrusca). Plant J 49:579–591

  7. Han L, Wu B, Pan G, Wang Y, Song X, Gao X (2009) UPLC-PDA analysis for simultaneous quantification of four active compounds in crude and processed rhizome of Polygonum multiflorum Thunb. Chromatographia 70:657–659

  8. Jeong Y, An CH, Woo SG, Park JH, Lee K, Lee S, Rim Y, Jeong HJ, Ryu YB, Kim CY (2016) Enhanced production of resveratrol derivatives in tobacco plants by improving the metabolic flux of intermediates in the phenylpropanoid pathway. Plant Mol Biol 92:117–129

  9. Jez JM, Austin MB, Ferrer JL, Bowman ME, Schroder J, Noel JP (2000a) Structural control of polyketide formation in plant-specific polyketide synthases. Chem Biol 7:919–930

  10. Jez JM, Ferrer JL, Bowman ME, Dixon RA, Noel JP (2000b) Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase. Biochemistry 39:890–902

  11. Krasnow MN, Murphy TM (2004) Polyphenol glucosylating activity in cell suspensions of grape (Vitis vinifera). J Agr Food Chem 52:3467–3472

  12. Lanz T, Der GS, Der JS (1990) Differential regulation of genes for resveratrol synthase in cell cultures of Arachis hypogaea L. Planta 181(2):169–175

  13. Le T, Jang H, Nguyen HTH, Doan TTM, Lee G, Park KD, Ahn T, Joung YH, Kang H, Yun C (2017) Highly regioselective hydroxylation of polydatin, a resveratrol glucoside, for one-step synthesis of astringin, a piceatannol glucoside, by P450 BM3. Enzyme Microb Tech 97:34–42

  14. Lim CG, Fowler ZL, Hueller T, Schaffer S, Koffas MAG (2011) High-yield resveratrol production in engineered Escherichia coli. Appl Environ Microb 77:3451–3460

  15. Lin L, Ni B, Lin H, Zhang M, Li X, Yin X, Qu C, Ni J (2015) Traditional usages, botany, phytochemistry, pharmacology and toxicology of Polygonum multiflorum Thunb.: a review. J Ethnopharmacol 159:158–183

  16. Li-Shuang LV, Gu XH, Ho CT, Tang J (2006) Stilbene glycosides from the roots of Polygonum multiflorum Thunb and their in vitro antioxidant activities. J Food Lipids 13:131–144

  17. Lv L, Gu X, Tang J, Ho C (2007) Antioxidant activity of stilbene glycoside from Polygonum multiflorum Thunb in vivo. Food Chem 104:1678–1681

  18. Ma L, Pang X, Shen H, Pu G, Wang H, Lei C, Wang H, Li G, Liu B, Ye H (2009) A novel type III polyketide synthase encoded by a three-intron gene from Polygonum cuspidatum. Planta 229:457–469

  19. Olas B, Wachowicz B, Saluk-Juszczak J, Zieliński T (2002) Effect of resveratrol, a natural polyphenolic compound, on platelet activation induced by endotoxin or thrombin. Thromb Res 107:141–145

  20. Shao L, Zhao S, Cui T, Liu Z, Zhao W (2012) 2,3,5,4′-Tetrahydroxystilbene-2-O-β-d-glycoside biosynthesis by suspension cells cultures of Polygonum multiflorum Thunb and production enhancement by methyl jasmonate and salicylic acid. Molecules 17:2240–2247

  21. Sheng S, Liu Z, Zhao W, Shao L, Zhao S (2010) Molecular analysis of a type III polyketide synthase gene in Fallopia multiflora. BIOLOGIA 65:939–946

  22. Shin S, Han NS, Park Y, Kim M, Seo J (2011) Production of resveratrol from p-coumaric acid in recombinant Saccharomyces cerevisiae expressing 4-coumarate:coenzyme A ligase and stilbene synthase genes. Enzyme Microb Tech 48:48–53

  23. Uesugi D, Hamada H, Shimoda K, Kubota N, Ozaki SI, Nagatani N (2017) Synthesis, oxygen radical absorbance capacity, and tyrosinase inhibitory activity of glycosides of resveratrol, pterostilbene, and pinostilbene. Biosci Biotechnol Biochem 81:226–230

  24. Xia W, Lei L, Zhao W, Feng Y, Zhao S (2016) Quantitative analysis of 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glycoside in wild Polygonum multiflorum and suspension cell cultures fed different precursors and elicitors. Phytochem Lett 15:180–185

  25. Yang Y, Paik JH, Cho D, Cho J, Kim C (2008) Resveratrol induces the suppression of tumor-derived CD4+ CD25+ regulatory T cells. Int Immunopharmacol 8:542–547

  26. Zhao W, Sheng S, Liu Z, Di L, Zhu K, Li X, Zhao S, Yao Y (2014a) Isolation of biosynthesis related transcripts of 2,3,5,4′-tetrahydroxy stilbene-2-O-β-d-glucoside from Fallopia multiflora by suppression subtractive hybridization. Acta Soc Bot Pol 83:147–157

  27. Zhao W, Xia W, Li J, Sheng S, Lei L, Zhao S (2014b) Transcriptome profiling and digital gene expression analysis of Fallopia multiflora to discover putative genes involved in the biosynthesis of 2,3,5,4′-tetrahydroxy stilbene-2-O-β-d-glucoside. Gene 547:126–135

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Author information

Correspondence to Shujin Zhao.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Online Resource 1 Extracted ion chromatogram of PD (A), THSG (B), piceatannol (C) and resveratrol (D) Mass spectra under ESI (–) mode of PD (a), THSG (b), piceatannol (c) and resveratrol (d) (TIFF 2220 kb)

Online Resource 2 Total protein concentration of the crude extracts from wild F. multiflora tissues and callus (TIFF 17684 kb)

Online Resource 3 Effects of the UDPG concentration on the piceatannol glycosyltransferase activity of the crude enzyme extract from the roots. The concentration of piceatannol was 40 μM (TIFF 80567 kb)

Online Resource 4 Effects of the piceatannol concentration on the piceatannol glycosyltransferase activity of the crude enzyme extract from the roots. The concentration of UDPG was 0.5 mM (TIFF 78003 kb)

Online Resource 5 Identification of products of enzyme assay experiments (DOCX 15 kb)

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Xia, W., Rui, W., Zhao, W. et al. Stable isotope labeling and 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucopyranoside biosynthetic pathway characterization in Fallopia multiflora . Planta 247, 613–623 (2018). https://doi.org/10.1007/s00425-017-2797-2

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Keywords

  • UPLC-MS
  • 13C-labeled precursor
  • Glycosylation
  • Hydroxylation
  • Enzyme assay
  • Resveratrol synthase