Skip to main content
SpringerLink
Log in

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

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

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

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Dixon, R. A., & Paiva, N. L. (1995). Stress-induced phenylpropanoid metabolism. Plant Cell, 7(7), 1085–1097.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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. 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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  20. Brazier-Hicks, M., & Edwards, R. (2013). Metabolic engineering of the flavone-C-glycoside pathway using polyprotein technology. Metabolic Engineering, 16, 11–20.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

Download references

Funding

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).

Author information

Author notes

  1. Jianjun Pei, Qing Sun and Na Gu contributed equally to this work.

Authors and Affiliations

  1. Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China

    Jianjun Pei, Qing Sun, Na Gu, Linguo Zhao, Xianying Fang & Fuliang Cao

  2. College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, China

    Jianjun Pei, Qing Sun, Na Gu, Linguo Zhao & Xianying Fang

  3. Jiangsu Key Lab for the Chemistry & Utilization of Agricultural and Forest Biomass, Nanjing, China

    Linguo Zhao, Xianying Fang & Fuliang Cao

  4. International Centre for Bamboo and Rattan, Beijing, China

    Feng Tang

Authors
  1. Jianjun Pei
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qing Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Na Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Linguo Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xianying Fang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Feng Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Fuliang Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Linguo Zhao.

Ethics declarations

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.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pei, J., Sun, Q., Gu, N. et al. Production of isoorientin and isovitexin from luteolin and apigenin using coupled catalysis of glycosyltransferase and sucrose synthase. Appl Biochem Biotechnol 190, 601–615 (2020). https://doi.org/10.1007/s12010-019-03112-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-019-03112-z

Keywords

Search

Navigation