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Production of isoorientin and isovitexin from luteolin and apigenin using coupled catalysis of glycosyltransferase and sucrose synthase

  • Jianjun Pei
  • Qing Sun
  • Na Gu
  • Linguo ZhaoEmail author
  • Xianying Fang
  • Feng Tang
  • Fuliang Cao
Article
  • 25 Downloads

Abstract

Isoorientin and isovitexin, kinds of flavone C-glycosides, exhibit a number of biological properties. In this work, The C-glucosyltransferase (Gt6CGT) gene from Gentiana triflora was cloned and expressed in Escherichia coli BL21(DE3). The optimal activity of Gt6CGT was at pH 7.5 and 50° C. The enzyme was stable over pH range of 6.5–9.0, and had a 1-h half-life at 50° C. The Vmax for luteolin and apigenin was 21.1 nmol/min/mg and 31.7 nmol/min/mg, while the Km was 0.21 mM and 0.22 mM, respectively. Then, we developed an environmentally safe and efficient method for isoorientin and isovitexin production using the coupled catalysis of Gt6CGT and Glycine max sucrose synthase (GmSUS). By optimizing coupled reaction conditions, the titer of isoorientin and isovitexin reached 3820 mg/L with a corresponding molar conversion of 94.7% and 3772 mg/L with a corresponding molar conversion of 97.1%, respectively. The maximum number of UDP-glucose regeneration cycles (RCmax) reached 28.4 for isoorientin and 29.1 for isovitexin. The coupled catalysis reported herein represents a promising method to meet industrial requirements for large-scale isoorientin and isovitexin production in the future.

Graphical Abstract

Keywords

Isoorientin Isovitexin Glycosyltransferase Sucrose synthase Coupled catalysis 

Notes

Funding information

This work was supported by the National Key R&D Program of China (2017YFD0600805), the National Natural Science Foundation of China (31570565), the Natural Science Foundation of the Jiangsu Province (BK20160929), the Open Foundation of Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration (JPELBCPI2017002), and the Qing Lan Project and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

The article does not contain any studies with human participants performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study.

References

  1. 1.
    Dixon, R. A., & Paiva, N. L. (1995). Stress-induced phenylpropanoid metabolism. Plant Cell, 7(7), 1085–1097.CrossRefGoogle Scholar
  2. 2.
    Yonekura-Sakakibara, K., Tohge, T., Niida, R., & Saito, K. (2007). Identification of a flavonol 7-O-rhamnosyltransferase gene determining flavonoid pattern in Arabidopsis by transcriptome coexpression analysis and reverse genetics. The Journal of Biological Chemistry, 282(20), 14932–14941.CrossRefGoogle Scholar
  3. 3.
    Nijveldt, R. J., van Nood, E., van Hoorn, D. E., Boelens, P. G., van Norren, K., & van Leeuwen, P. A. (2001). Flavonoids: a review of probable mechanisms of action and potential applications. The American Journal of Clinical Nutrition, 74(4), 418–425.CrossRefGoogle Scholar
  4. 4.
    Wu, X., Chu, J., Wu, B., Zhang, S., & He, B. (2013). An efficient novel glycosylation of flavonoid by beta-fructosidase resistant to hydrophilic organic solvents. Bioresource Technology, 129, 659–662.CrossRefGoogle Scholar
  5. 5.
    An, D. G., Yang, S. M., Kim, B. G., & Ahn, J. H. (2016). Biosynthesis of two quercetin O-diglycosides in Escherichia coli. Journal of Industrial Microbiology & Biotechnology, 43(6), 841–849.CrossRefGoogle Scholar
  6. 6.
    Durr, C., Hoffmeister, D., Wohlert, S. E., Ichinose, K., Weber, M., Von Mulert, U., Thorson, J. S., & Bechthold, A. (2004). The glycosyltransferase UrdGT2 catalyzes both C- and O-glycosidic sugar transfers. Angewandte Chemie International Edition, 43(22), 2962–2965.CrossRefGoogle Scholar
  7. 7.
    Pacifico, S., Scognamiglio, M., D’Abrosca, B., Piccolella, S., Tsafantakis, N., Gallicchio, M., Ricci, A., & Fiorentino, A. (2010). Spectroscopic characterization and antiproliferative activity on HepG2 human hepatoblastoma cells of flavonoid C-glycosides from Petrorhagia velutina. Journal of Natural Products, 73(12), 1973–1978.CrossRefGoogle Scholar
  8. 8.
    Yuan, L., Wang, J., Xiao, H., Wu, W., Wang, Y., & Liu, X. (2013). MAPK signaling pathways regulate mitochondrial-mediated apoptosis induced by isoorientin in human hepatoblastoma cancer cells. Food and Chemical Toxicology, 53(3), 62–68.CrossRefGoogle Scholar
  9. 9.
    Yuan, L., Wei, S., Wang, J., & Liu, X. (2014). Isoorientin induces apoptosis and autophagy simultaneously by reactive oxygen species (ROS)-related p53, PI3K/Akt, JNK, and p38 signaling pathways in HepG2 cancer cells. Journal of Agricultural and Food Chemistry, 62(23), 5390–5400.CrossRefGoogle Scholar
  10. 10.
    Zhang, Y., Jiao, J., Liu, C., Wu, X., & Zhang, Y. (2007). Isolation and purification of four flavone C -glycosides from antioxidant of bamboo leaves by macroporous resin column chromatography and preparative high-performance liquid chromatography. Food Chemistry, 107(3), 1326–1336.Google Scholar
  11. 11.
    Hu, C., Zhang, Y., & Kitts, D. D. (2000). Evaluation of antioxidant and prooxidant activities of bamboo Phyllostachys nigra var. Henonis leaf extract in vitro. Journal of Agricultural and Food Chemistry, 48(8), 3170–3176.CrossRefGoogle Scholar
  12. 12.
    Yuan, L., Han, X., Li, W., Ren, D., & Yang, X. (2016). Isoorientin prevents hyperlipidemia and liver injury by regulating lipid metabolism, antioxidant capability, and inflammatory cytokine release in high-fructose-fed mice. Journal of Agricultural and Food Chemistry, 64(13), 2682–2689.CrossRefGoogle Scholar
  13. 13.
    Luan, G., Wang, Y., Wang, Z., Zhou, W., Hu, N., Li, G., & Wang, H. (2018). Flavonoid glycosides from fenugreek seeds regulate glycolipid metabolism by improving mitochondrial function in 3T3-L1 adipocytes in vitro. Journal of Agricultural and Food Chemistry, 66(12), 3169–3178.CrossRefGoogle Scholar
  14. 14.
    Yuan, L., Li, X., He, S., Gao, C., Wang, C., & Shao, Y. (2018). Effects of natural flavonoid isoorientin on growth performance and gut microbiota of mice. Journal of Agricultural and Food Chemistry, 66(37), 9777–9784.CrossRefGoogle Scholar
  15. 15.
    Zhang, Y., Bao, B., Lu, B., Ren, Y., & Tie, X. (2005). Determination of flavone C-glucosides in antioxidant of bamboo leaves (AOB) fortified foods by reversed-phase high-performance liquid chromatography with ultraviolet diode array detection. Journal of Chromatography A, 1065(2), 177–185.CrossRefGoogle Scholar
  16. 16.
    Kim, Y. C., Jun, M., Jeong, W. S., & Chung, S. K. (2010). Antioxidant properties of flavone C-glycosides from Atractylodes japonica leaves in human low-density lipoprotein oxidation. Journal of Food Science, 70(9), S575–S580.CrossRefGoogle Scholar
  17. 17.
    De Bruyn, F., Van Brempt, M., Maertens, J., Van Bellegem, W., Duchi, D., & De Mey, M. (2015). Metabolic engineering of Escherichia coli into a versatile glycosylation platform: production of bio-active quercetin glycosides. Microbial Cell Factories, 14(1), 138.CrossRefGoogle Scholar
  18. 18.
    Kim, H. J., Kim, B. G., & Ahn, J. H. (2013). Regioselective synthesis of flavonoid bisglycosides using Escherichia coli harboring two glycosyltransferases. Applied Microbiology and Biotechnology, 97(12), 5275–5282.CrossRefGoogle Scholar
  19. 19.
    Pei, J., Dong, P., Wu, T., Zhao, L., Fang, X., Cao, F., Tang, F., & Yue, Y. (2016). Metabolic engineering of Escherichia coli for astragalin biosynthesis. Journal of Agricultural and Food Chemistry, 64(42), 7966–7972.CrossRefGoogle Scholar
  20. 20.
    Brazier-Hicks, M., & Edwards, R. (2013). Metabolic engineering of the flavone-C-glycoside pathway using polyprotein technology. Metabolic Engineering, 16, 11–20.CrossRefGoogle Scholar
  21. 21.
    Sasaki, N., Nishizaki, Y., Yamada, E., Tatsuzawa, F., Nakatsuka, T., Takahashi, H., & Nishihara, M. (2015). Identification of the glucosyltransferase that mediates direct flavone C-glucosylation in Gentiana triflora. FEBS Letters, 589(1), 182–187.CrossRefGoogle Scholar
  22. 22.
    Shrestha, A., Pandey, R. P., Dhakal, D., Parajuli, P., & Sohng, J. K. (2018). Biosynthesis of flavone C-glucosides in engineered Escherichia coli. Applied Microbiology and Biotechnology, 102(3), 1251–1267.CrossRefGoogle Scholar
  23. 23.
    Pei, J., Sun, Q., Zhao, L., Shi, H., Tang, F., & Cao, F. (2019). Efficient biotransformation of luteolin to isoorientin through adjusting induction strategy, controlling acetic acid and increasing UDP-glucose supply in Escherichia coli. Journal of Agricultural and Food Chemistry, 67(1), 331–340.CrossRefGoogle Scholar
  24. 24.
    Pei, J., Chen, A., Zhao, L., Cao, F., Ding, G., & Xiao, W. (2017). One-pot synthesis of hyperoside by a three-enzyme cascade using a UDP-galactose regeneration system. Journal of Agricultural and Food Chemistry, 65(29), 6042–6048.CrossRefGoogle Scholar
  25. 25.
    Bungaruang, L., Gutmann, A., & Nidetzky, B. (2013). Leloir glycosyltransferases and natural product glycosylation: biocatalytic synthesis of the C-glucoside nothofagin, a major antioxidant of Redbush Herbal Tea. Advanced Synthesis & Catalysis, 355(14-15), 2757–2763.CrossRefGoogle Scholar
  26. 26.
    Gutmann, A., Bungaruang, L., Weber, H., Leypold, M., Breinbauer, R., & Nidetzky, B. (2014). Towards the synthesis of glycosylated dihydrochalcone natural products using glycosyltransferase-catalysed cascade reactions. Green Chemistry, 16(9), 4417–4425.CrossRefGoogle Scholar
  27. 27.
    Pei, J., Chen, A., Sun, Q., Zhao, L., Cao, F., & Tang, F. (2018). Construction of a novel UDP-rhamnose regeneration system by a two-enzyme reaction system and application in glycosylation of flavonoid. Biochemical Engineering Journal, 139, 33–42.CrossRefGoogle Scholar
  28. 28.
    Qian, Y., Liu, J., Song, W., Chen, X., Luo, Q., & Liu, L. (2018). Production of β-alanine from fumaric acid using a dual-enzyme cascade. ChemCatChem, 10(21), 4984–4991.CrossRefGoogle Scholar
  29. 29.
    Zhang, Y., Gu, H., Shi, H., Wang, F., & Li, X. (2017). Green synthesis of conjugated linoleic acids from plant oils using a novel synergistic catalytic system. Journal of Agricultural and Food Chemistry, 65(26), 5322–5329.CrossRefGoogle Scholar
  30. 30.
    Lepak, A., Gutmann, A., Kulmer, S. T., & Nidetzky, B. (2015). Creating a water-soluble resveratrol-based antioxidant by site-selective enzymatic glucosylation. ChemBioChem, 16(13), 1870–1874.CrossRefGoogle Scholar
  31. 31.
    Schmolzer, K., Lemmerer, M., Gutmann, A., & Nidetzky, B. (2017). Integrated process design for biocatalytic synthesis by a Leloir glycosyltransferase: UDP-glucose production with sucrose synthase. Biotechnology and Bioengineering, 114(4), 924–928.CrossRefGoogle Scholar
  32. 32.
    Pei, J., Chen, A., Dong, P., Shi, X., Zhao, L., Cao, F., & Tang, F. (2019). Modulating heterologous pathways and optimizing fermentation conditions for biosynthesis of kaempferol and astragalin from naringenin in Escherichia coli. Journal of Industrial Microbiology & Biotechnology, 46(2), 171–186.CrossRefGoogle Scholar
  33. 33.
    Baroja-Fernandez, E., Munoz, F. J., Li, J., Bahaji, A., Almagro, G., Montero, M., Etxeberria, E., Hidalgo, M., Sesma, M. T., & Pozueta-Romero, J. (2012). Sucrose synthase activity in the sus1/sus2/sus3/sus4 Arabidopsis mutant is sufficient to support normal cellulose and starch production. Proceedings of the National Academy of Sciences of the United States of America, 109(1), 321–326.CrossRefGoogle Scholar
  34. 34.
    Schmolzer, K., Gutmann, A., Diricks, M., Desmet, T., & Nidetzky, B. (2016). Sucrose synthase: a unique glycosyltransferase for biocatalytic glycosylation process development. Biotechnology Advances, 34(2), 88–111.CrossRefGoogle Scholar
  35. 35.
    Oka, T., Nemoto, T., & Jigami, Y. (2007). Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDP-D-glucose to UDP-L-rhamnose conversion. The Journal of Biological Chemistry, 282(8), 5389–5403.CrossRefGoogle Scholar
  36. 36.
    Brazier-Hicks, M., Evans, K. M., Gershater, M. C., Puschmann, H., Steel, P. G., & Edwards, R. (2009). The C-glycosylation of flavonoids in cereals. The Journal of Biological Chemistry, 284(27), 17926–17934.CrossRefGoogle Scholar
  37. 37.
    Falcone Ferreyra, M. L., Rodriguez, E., Casas, M. I., Labadie, G., Grotewold, E., & Casati, P. (2013). Identification of a bifunctional maize C- and O-glucosyltransferase. The Journal of Biological Chemistry, 288(44), 31678–31688.CrossRefGoogle Scholar
  38. 38.
    Nagatomo, Y., Usui, S., Ito, T., Kato, A., Shimosaka, M., & Taguchi, G. (2014). Purification, molecular cloning and functional characterization of flavonoid C-glucosyltransferases from Fagopyrum esculentum M. (buckwheat) cotyledon. The Plant Journal: for Cell and Molecular Biology, 80(3), 437–448.CrossRefGoogle Scholar
  39. 39.
    Hirade, Y., Kotoku, N., Terasaka, K., Saijo-Hamano, Y., Fukumoto, A., & Mizukami, H. (2015). Identification and functional analysis of 2-hydroxyflavanone C-glucosyltransferase in soybean (Glycine max). FEBS Letters, 589(15), 1778–1786.CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Co-Innovation Center for Sustainable Forestry in Southern ChinaNanjing Forestry UniversityNanjingChina
  2. 2.College of Chemical EngineeringNanjing Forestry UniversityNanjingChina
  3. 3.Jiangsu Key Lab for the Chemistry & Utilization of Agricultural and Forest BiomassNanjingChina
  4. 4.International Centre for Bamboo and RattanBeijingChina

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