Cascade biocatalysis systems for bioactive naringenin glucosides and quercetin rhamnoside production from sucrose

  • Samir Bahadur Thapa
  • Ramesh Prasad Pandey
  • Puspalata Bashyal
  • Tokutaro Yamaguchi
  • Jae Kyung SohngEmail author
Biotechnological products and process engineering


Two sustainable and cost-effective cascade enzymatic systems were developed to regenerate uridine diphosphate (UDP)-α-d-glucose and UDP-β-l-rhamnose from sucrose. The systems were coupled with the UDP generating glycosylation reactions of UDP sugar–dependent glycosyltransferase (UGT) enzymes mediated reactions. As a result, the UDP generated as a by-product of the GT-mediated reactions was recycled. In the first system, YjiC, a UGT from Bacillus licheniformis DSM 13, was used for transferring glucose from UDP-α-d-glucose to naringenin, in which AtSUS1 from Arabidopsis thaliana was used to synthesize UDP-α-d-glucose and fructose as a by-product from sucrose. In the second system, flavonol 7-O-rhamnosyltransferase (AtUGT89C1) from A. thaliana was used to transfer rhamnose from UDP-β-l-rhamnose to quercetin, in which AtSUS1 along with UDP-β-l-rhamnose synthase (AtRHM1), also from A. thaliana, were used to produce UDP-β-l-rhamnose from the same starter sucrose. The established UDP recycling system for the production of naringenin glucosides was engineered and optimized for several reaction parameters that included temperature, metal ions, NDPs, pH, substrate ratio, and enzymes ratio, to develop a highly feasible system for large-scale production of different derivatives of naringenin and other natural products glucosides, using inexpensive starting materials. The developed system showed the conversion of about 37 mM of naringenin into three different glucosides, namely naringenin, 7-O-β-d-glucoside, naringenin, 4′-O-β-d-glucoside, and naringenin, 4′,7-O-β-d-diglucoside. The UDP recycling (RCmax) was 20.10 for naringenin glucosides. Similarly, the conversion of quercetin to quercetin 7-O-α-l-rhamnoside reached a RCmax value of 10.0.


Glucosylation UDP recycling Sucrose synthase Rhamnosylation Cascade biocatalysis 


Funding information

This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant no. PJ013137), Rural Development Administration, Republic of Korea.

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2019_10060_MOESM1_ESM.pdf (6.2 mb)
ESM 1 (PDF 6374 kb)


  1. Antonopoulou I, Varriale S, Topakas E, Rova U, Christakopoulos P, Faraco V (2016) Enzymatic synthesis of bioactive compounds with high potential for cosmeceutical application. Appl Microbiol Biotechnol 100:6519–6543CrossRefGoogle Scholar
  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  3. Cavia-Saiz M, Busto MD, Pilar-Izquierdo MC, Ortega N, Perez-Mateos M, Muñiz P (2010) Antioxidant properties, radical scavenging activity, and biomolecule protection capacity of flavonoid naringenin and its glycoside naringin: a comparative study. J Sci Food Agric 90:1238–1244CrossRefGoogle Scholar
  4. Chouhan S, Sharma K, Zha J, Guleria S, Koffas MA (2017) Recent advances in the recombinant biosynthesis of polyphenols. Front Microbiol 8:2259CrossRefGoogle Scholar
  5. Danishefsky SJ, Bilodeau MT (1996) Glycals in organic synthesis: the evolution of comprehensive strategies for the assembly of oligosaccharides and glycoconjugates of biological consequence. Angew Chem Int Ed 35:1380–1419CrossRefGoogle Scholar
  6. Darsandhari S, Pandey RP, Shrestha B, Parajuli P, Liou K, Sohng JK (2018) One-pot multienzyme cofactors recycling (OPME-CR) system for lactose and non-natural saccharide conjugated polyphenol production. J Agric Food Chem 66:7965–7974CrossRefGoogle Scholar
  7. Daudé D, André I, Monsan P, Remaud-Siméon M (2014) Successes in engineering glucansucrases to enhance glycodiversification. In: Rauter AP, Lindhorst T, Queneau Y (eds) Carbohydrate Chemistry, Volume 40. The Royal Society of Chemistry, Cambridge, pp 624–645CrossRefGoogle Scholar
  8. De Bruyn F, Maertens J, Beauprez J, Soetaert W, De Mey M (2015) Biotechnological advances in UDP-sugar based glycosylation of small molecules. Biotechnol Adv 33:288–302CrossRefGoogle Scholar
  9. Deng X, Wang Z, Liu J, Xiong S, Xiong R, Cao X, Tang G (2017) Design, synthesis and biological evaluation of flavonoid salicylate derivatives as potential anti-tumor agents. RSC Adv 7:38171–38178CrossRefGoogle Scholar
  10. Diricks M, Gutmann A, Debacker S, Dewitte G, Nidetzky B, Desmet T (2017) Sequence determinants of nucleotide binding in sucrose synthase: improving the affinity of a bacterial sucrose synthase for UDP by introducing plant residues. Protein Eng Des Sel 30:143–150Google Scholar
  11. Du G, Jin L, Han X, Song Z, Zhang H, Liang W (2009) Naringenin: a potential immunomodulator for inhibiting lung fibrosis and metastasis. J Cancer Res Ther 69:3205–3212Google Scholar
  12. Felgines C, Texier O, Morand C, Manach C, Scalbert A, Régerat F, Rémésy C (2000) Bioavailability of the flavanone naringenin and its glycosides in rats. Am J Physiol Gastrointest Liver Physiol 279:G1148–G1154CrossRefGoogle Scholar
  13. Figueroa CM, Asención Diez MD, Kuhn ML, McEwen S, Salerno GL, Iglesias AA, Ballicora MA (2013) The unique nucleotide specificity of the sucrose synthase from Thermosynechococcus elongatus. FEBS Lett 587:165–169CrossRefGoogle Scholar
  14. Gantt RW, Peltier-Pain P, Thorson JS (2011) Enzymatic methods for glyco (diversification/randomization) of drugs and small molecules. Nat Prod Rep 28:1811–1853CrossRefGoogle Scholar
  15. Gurung RB, Kim EH, Oh TJ, Sohng JK (2013) Enzymatic synthesis of apigenin glucosides by glucosyltransferase (YjiC) from Bacillus licheniformis. DSM Mol Cell 36:355–361CrossRefGoogle Scholar
  16. 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 Chem 16:4417–4425CrossRefGoogle Scholar
  17. Gutmann A, Lepak A, Diricks M, Desmet T, Nidetzky B (2017) Glycosyltransferase cascades for natural product glycosylation: use of plant instead of bacterial sucrose synthases improves the UDP-glucose recycling from sucrose and UDP. Biotechnol J 12:1600557CrossRefGoogle Scholar
  18. He X, Ou HY, Yu Q, Zhou X, Wu J, Liang J (2015) Analysis of a genomic island housing genes for DNA S-modification system in Streptomyces lividans 66 and its counterparts in other distantly related bacteria. Mol Microbiol 65:1034–1048CrossRefGoogle Scholar
  19. Jang SW, Cho CH, Jung YS, Rha C, Nam TG, Kim DO, Baek NI, Park CS, Lee BH, Shin HS, Seo DH, Lee SY (2018) Enzymatic synthesis of α-flavone glucoside via regioselective transglucosylation by amylosucrase from Deinococcus geothermalis. PLoS One 13:e0207466CrossRefGoogle Scholar
  20. Khan AW, Kotta S, Ansari SH, Sharma RK, Ali J (2015) Enhanced dissolution and bioavailability of grapefruit flavonoid naringenin by solid dispersion utilizing fourth generation carrier. Drug Dev Ind Pharm 41:772–779CrossRefGoogle Scholar
  21. Kim HJ, Kim BG, Ahn JH (2013) Regioselective synthesis of flavonoid bisglycosides using Escherichia coli harboring two glycosyltransferases. Appl Microbiol Biotechnol 97:5275–5282CrossRefGoogle Scholar
  22. Kim B, Park H, Na D, Lee SY (2014) Metabolic engineering of Escherichia coli for the production of phenol from glucose. Biotechnol J 9:621–629CrossRefGoogle Scholar
  23. Kim BG, Yang SM, Kim SY, Cha MN, Ahn JH (2015) Biosynthesis and production of glycosylated flavonoids in Escherichia coli: current state and perspectives. Appl Microbiol Biotechnol 99:2979–2988CrossRefGoogle Scholar
  24. Koirala N, Pandey RP, Parajuli P, Jung HJ, Sohng JK (2014) Methylation and subsequent glycosylation of 7, 8-dihydroxyflavone. J Biotechnol 184:128–137CrossRefGoogle Scholar
  25. Kumar S, Pandey AK (2013) Chemistry and biological activities of flavonoids: an overview. Sci World J 2013:162750Google Scholar
  26. Le TT, Pandey RP, Gurung RB, Dhakal D, Sohng JK (2014) Efficient enzymatic systems for synthesis of novel α-mangostin glycosides exhibiting antibacterial activity against gram-positive bacteria. Appl Microbiol Biotechnol 98:8527–8538CrossRefGoogle Scholar
  27. Lee BH, Pyo MK, Lee JH, Choi SH, Shin TJ, Lee SM, Yun-Choi HS (2008) Differential regulations of quercetin and its glycosides on ligand-gated ion channels. Biol Pharm Bull 31:611–617CrossRefGoogle Scholar
  28. Lee HS, Kim TS, Parajuli P, Pandey RP, Sohng JK (2018) Sustainable production of dihydroxybenzene glucosides using immobilized amylosucrase from Deinococcus geothermalis. J Microbiol Biotechnol 28:1447–1456Google Scholar
  29. Leonardi T, Vanamala J, Taddeo SS, Davidson LA, Murphy ME, Patil BS, Turner ND (2010) Apigenin and naringenin suppress colon carcinogenesis through the aberrant crypt stage in azoxymethane-treated rats. Exp Biol Med 235:710–717CrossRefGoogle Scholar
  30. Lewandowska U, Szewczyk K, Hrabec E, Janecka A, Gorlach S (2013) Overview of metabolism and bioavailability enhancement of polyphenols. J Agric Food Chem 61:12183–12199CrossRefGoogle Scholar
  31. Manach C, Donovan JL (2004) Pharmacokinetics and metabolism of dietary flavonoids in humans. Free Radic Res 38:771–785CrossRefGoogle Scholar
  32. Masada S, Kawase Y, Nagatoshi M, Oguchi Y, Terasaka K, Mizukami H (2007) An efficient chemoenzymatic production of small molecule glucosides with in situ UDP-glucose recycling. FEBS Lett 581:2562–2566CrossRefGoogle Scholar
  33. Mei YZ, Liu RX, WangDP WX, Dai CC (2015) Biocatalysis and biotransformation of resveratrol in microorganisms. Biotechnol Lett 37:9–18CrossRefGoogle Scholar
  34. Muthana MM, Qu J, Li Y, Zhang L, Yu H, Ding L, Chen X (2012) Efficient one-pot multienzyme synthesis of UDP-sugars using a promiscuous UDP-sugar pyrophosphorylase from Bifidobacterium longum (BLUSP). Chem Commun 48:2728–2730CrossRefGoogle Scholar
  35. Myung SK, Kim Y, Ju W, Choi HJ, Bae WK (2009) Effects of antioxidant supplements on cancer prevention: meta-analysis of randomized controlled trials. Ann Oncol 21:166–179CrossRefGoogle Scholar
  36. Nicolaou KC, Mitchell HJ (2001) Adventures in carbohydrate chemistry: new synthetic technologies, chemical synthesis, molecular design, and chemical biology. Angew Chem Int Ed 40:1576–1624CrossRefGoogle Scholar
  37. Panche AN, Diwan AD, Chandra SR (2016) Flavonoids: an overview. J Nutr Sci 5:e47CrossRefGoogle Scholar
  38. Pandey RP, Li TF, Kim EH, Yamaguchi T, Park YI, Kim JS, Sohng JK (2013) Enzymatic synthesis of novel phloretin glucosides. Appl Environ Microbiol 79:3516–3521CrossRefGoogle Scholar
  39. Pandey RP, Parajuli P, Shin JY, Lee J, Lee S, Hong YS, Sohng JK (2014) Enzymatic biosynthesis of novel resveratrol glucoside and glycoside derivatives. Appl Environ Microbiol 80:7235–7243CrossRefGoogle Scholar
  40. Pandey RP, Parajuli P, Chu LL, Darsandhari S, Sohng JK (2015) Biosynthesis of amino deoxy-sugar-conjugated flavonol glycosides by engineered Escherichia coli. Biochem Eng J 101:191–199CrossRefGoogle Scholar
  41. Pandey RP, Parajuli P, Koffas MAG, Sohng JK (2016) Microbial production of natural and non-natural flavonoids: pathway engineering, directed evolution and systems/synthetic biology. Biotechnol Adv 34:634–662CrossRefGoogle Scholar
  42. Parajuli P, Pandey RP, Trang NTH, Oh TJ, Sohng JK (2015) Expanded acceptor substrates flexibility study of flavonol 7-O-rhamnosyltransferase, AtUGT89C1 from Arabidopsis thaliana. Carbohydr Res 418:13–19CrossRefGoogle Scholar
  43. Park H, Kim J, Park JH, Baek NI, Park CS, Lee HS, Cha J (2012) Bioconversion of piceid to piceid glucoside using amylosucrase from Alteromonas macleodii deep ecotype. J Microbiol Biotechnol 22:1698–1704CrossRefGoogle Scholar
  44. 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. J Agric Food Chem 65:6042–6048CrossRefGoogle Scholar
  45. Qin L, Jin L, Lu L, Lu X, Zhang C, Zhang F, Liang W (2011) Naringenin reduces lung metastasis in a breast cancer resection model. Protein Cell 2:507–516CrossRefGoogle Scholar
  46. Roepke J, Bozzo GG (2013) Biocatalytic synthesis of quercetin 3-O-glucoside-7-O-rhamnoside by metabolic engineering of Escherichia coli. ChemBioChem 14:2418–2422CrossRefGoogle Scholar
  47. Rosencrantz RR, Lange B, Elling L (2014) Chemo-enzymatic cascade reactions for the synthesis of glycoconjugates. In: Riva S, Fessner WD (eds) Cascade biocatalysis. Wiley, Weinheim, pp 133–160Google Scholar
  48. Rouseff RL, Martin SF, Youtsey CO (1987) Quantitative survey of narirutin, naringin, hesperidin, and neohesperidin in citrus. J Agric Food Chem 35:1027–1030CrossRefGoogle Scholar
  49. Schmölzer K, Gutmann A, Diricks M, Desmet T, Nidetzky B (2016) Sucrose synthase: a unique glycosyltransferase for biocatalytic glycosylation process development. Biotechnol Adv 34:88–111CrossRefGoogle Scholar
  50. Schmölzer K, Lemmerer M, Gutmann A, Nidetzky B (2017) Integrated process design for biocatalytic synthesis by a leloir glycosyltransferase: UDP-glucose production with sucrose synthase. Biotechnol Bioeng 114:924–928CrossRefGoogle Scholar
  51. Shiloach J, Fass R (2005) Growing E. coli to high cell density – a historical perspective on method development. Biotechnol Adv 23:345–357CrossRefGoogle Scholar
  52. Shomar H, Gontier S, van den Broek NJ, Mora HT, Noga MJ, Hagedoorn PL, Bokinsky G (2018) Metabolic engineering of a carbapenem antibiotic synthesis pathway in Escherichia coli. Nat Chem Biol 1:794–800CrossRefGoogle Scholar
  53. Simkhada D, Kurumbang NP, Lee HC, Sohng JK (2010) Exploration of glycosylated flavonoids from metabolically engineered E. coli. Biotechnol Bioprocess Eng 15:754–760CrossRefGoogle Scholar
  54. Teles Y, Souza M (2018) Sulphated flavonoids: biosynthesis, structures, and biological activities. Molecules 23:480CrossRefGoogle Scholar
  55. Terasaka K, Mizutani Y, Nagatsu A, Mizukami H (2012) In situ UDP-glucose regeneration unravels diverse functions of plant secondary product glycosyltransferases. FEBS Lett 586:4344–4350CrossRefGoogle Scholar
  56. Tian Y, Xu W, Zhang W, Zhang T, Guang C, Mu W (2018) Amylosucrase as a transglucosylation tool: from molecular features to bioengineering applications. Biotechnol Adv 36:1540–1552CrossRefGoogle Scholar
  57. Trantas EA, Koffas MA, Xu P, Ververidis F (2015) When plants produce not enough or at all: metabolic engineering of flavonoids in microbial hosts. Front Plant Sci 6:7CrossRefGoogle Scholar
  58. Xiao J, Muzashvili TS, Georgiev MI (2014) Advances in the biotechnological glycosylation of valuable flavonoids. Biotechnol Adv 32:1145–1156CrossRefGoogle Scholar
  59. Xie L, Zhang L, Wang C, Wang X, Xu YM, YuH WX (2018) Methylglucosylation of aromatic amino and phenolic moieties of drug-like biosynthons by combinatorial biosynthesis. Proc Natl Acad Sci 115:E4980–E4989CrossRefGoogle Scholar
  60. Xu L, Qi T, Xu L, Lu L, Xiao M (2016) Recent progress in the enzymatic glycosylation of phenolic compounds. J Carbohydr Chem 35:1–23CrossRefGoogle Scholar
  61. Yıldız SZ, Küçükislamoğlu M, Tuna M (2009) Synthesis and characterization of novel flavonoid-substituted phthalocyanines using (±) naringenin. J Org Chem 694:4152–4161CrossRefGoogle Scholar
  62. Yin R, Han K, Heller W, Albert A, Dobrev PI, Zažímalová E, Schäffner AR (2014) Kaempferol 3-O-rhamnoside-7-O-rhamnoside is an endogenous flavonol inhibitor of polar auxin transport in Arabidopsis shoots. New Phytol 201:466–475CrossRefGoogle Scholar
  63. Yuan S, Yin S, Liu M, Kong JQ (2018) Isolation and characterization of a multifunctional flavonoid glycosyltransferase from Ornithogalum caudatum with glycosidase activity. Sci Rep 8:5886CrossRefGoogle Scholar
  64. Zhang YX, Chen SL (2006) Molecular identification, polymorphism, and expression analysis of major histocompatibility complex class IIA and B genes of turbot (Scophthalmus maximus). Mar Biotechnol 8:611–623CrossRefGoogle Scholar
  65. Zhang J, Singh S, Hughes RR, Zhou M, Sunkara M, Morris AJ, Thorson JS (2014) A simple strategy for glycosyltransferase-catalyzed aminosugar nucleotide synthesis. ChemBioChem 15:647–651CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019
corrected publication 2019

Authors and Affiliations

  1. 1.Department of Life Science and Biochemical EngineeringSun Moon UniversityChungnamKorea
  2. 2.Department of Pharmaceutical Engineering and BiotechnologySun Moon UniversityChungnamKorea

Personalised recommendations