Abstract
Glycosylation is an effective way to improve the water solubility of natural products. In this work, a novel glycosyltransferase gene (BbGT) was discovered from Beauveria bassiana ATCC 7159 and heterologously expressed in Escherichia coli. The purified enzyme was functionally characterized through in vitro enzymatic reactions as a UDP-glucosyltransferase, converting quercetin to five monoglucosylated and one diglucosylated products. The optimal pH and temperature for BbGT are 35 ℃ and 8.0, respectively. The activity of BbGT was stimulated by Ca2+, Mg2+, and Mn2+, but inhibited by Zn2+. BbGT enzyme is flexible and can glycosylate a variety of substrates such as curcumin, resveratrol, and zearalenone. The enzyme was also expressed in other microbial hosts including Saccharomyces cerevisiae, Pseudomonas putida, and Pichia pastoris. Interestingly, the major glycosylation product of quercetin in E. coli, P. putida, and P. pastoris was quercetin-7-O-β-d-glucoside, while the enzyme dominantly produced quercetin-3-O-β-d-glucoside in S. cerevisiae. The BbGT-harboring E. coli and S. cerevisiae strains were used as whole-cell biocatalysts to specifically produce the two valuable quercetin glucosides, respectively. The titer of quercetin-7-O-β-d-glucosides was 0.34 ± 0.02 mM from 0.83 mM quercetin in 24 h by BbGT-harboring E. coli. The yield of quercetin-3-O-β-d-glucoside was 0.22 ± 0.02 mM from 0.41 mM quercetin in 12 h by BbGT-harboring S. cerevisiae. This work thus provides an efficient way to produce two valuable quercetin glucosides through the expression of a versatile glucosyltransferase in different hosts.
Key points
• A highly versatile glucosyltransferase was identified from B. bassiana ATCC 7159.
• BbGT converts quercetin to five mono- and one di-glucosylated derivatives in vitro.
• Different quercetin glucosides were produced by BbGT in E. coli and S. cerevisiae.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
All data generated or analyzed during this study are included in this published article (and its supplementary information files).
References
Anuradha R, Sukumar D (2013) In vitro anti-inflammatory compound quercimeritrin isolated from tithonia diversifolia flowers by hrbc membrane stabilization. World J Pharm Res 3(1):426–431
Cai X, Fang Z, Dou J, Yu A, Zhai G (2013) Bioavailability of quercetin: problems and promises. Curr Med Chem 20(20):2572–2582. https://doi.org/10.2174/09298673113209990120
Cao H, Chen X, Jassbi AR, Xiao J (2015) Microbial biotransformation of bioactive flavonoids. Biotechnol Adv 33(1):214–223. https://doi.org/10.1016/j.biotechadv.2014.10.012
Caputi L, Lim EK, Bowles DJ (2008) Discovery of new biocatalysts for the glycosylation of terpenoid scaffolds. Chemistry 14(22):6656–6662. https://doi.org/10.1002/chem.200800548
Cartwright AM, Lim E-K, Kleanthous C, Bowles DJ (2008) A kinetic analysis of regiospecific glucosylation by two glycosyltransferases of Arabidopsis thaliana: domain swapping to introduce new activities. J Biol Chem 283(23):15724–15731. https://doi.org/10.1074/jbc.M801983200
Chang A, Singh S, Phillips GN Jr, Thorson JS (2011) Glycosyltransferase structural biology and its role in the design of catalysts for glycosylation. Curr Opin Biotechnol 22(6):800–808. https://doi.org/10.1016/j.copbio.2011.04.013
Chang T-S, Wu J-Y, Wang T-Y, Wu K-Y, Chiang C-M (2018) Uridine diphosphate-dependent glycosyltransferases from Bacillus subtilis ATCC 6633 catalyze the 15-O-glycosylation of ganoderic acid A. Int J Mol Sci 19(11):3469. https://doi.org/10.3390/ijms19113469
Chen Q, Li P, Xu Y, Li Y, Tang B (2015) Isoquercitrin inhibits the progression of pancreatic cancer in vivo and in vitro by regulating opioid receptors and the mitogen-activated protein kinase signalling pathway. Oncol Rep 33(2):840–848. https://doi.org/10.3892/or.2014.3626
Cheng Y, Zhang J, Shao Y, Xu Y, Ge H, Yu B, Wang W (2019) Enzyme-catalyzed glycosylation of curcumin and its analogues by glycosyltransferases from Bacillus subtilis ATCC 6633. Catalysts 9(9):734. https://doi.org/10.3390/catal9090734
Chiang C-M, Wang T-Y, Yang S-Y, Wu J-Y, Chang T-S (2018) Production of new isoflavone glucosides from glycosylation of 8-hydroxydaidzein by glycosyltransferase from Bacillus subtilis ATCC 6633. Catalysts 8(9):387. https://doi.org/10.3390/catal8090387
Cregg JM, Russell KA (1998) Transformation Pichia protocols. Springer 27−39.
Dai L, Li J, Yang J, Zhu Y, Men Y, Zeng Y, Cai Y, Dong C, Dai Z, Zhang X (2018) Use of a promiscuous glycosyltransferase from Bacillus subtilis 168 for the enzymatic synthesis of novel protopanaxatriol-type ginsenosides. J Agric Food Chem 66(4):943–949. https://doi.org/10.1021/acs.jafc.7b03907
Dai L, Qin L, Hu Y, Huang J-w, Hu Z, Min J, Sun Y, Guo R-T (2021) Structural dissection of unnatural ginsenoside-biosynthetic UDP-glycosyltransferase Bs-YjiC from Bacillus subtilis for substrate promiscuity. Biochem Biophys Res Commun 534:73–78. https://doi.org/10.1016/j.bbrc.2020.11.104
Day AJ, Mellon F, Barron D, Sarrazin G, Morgan MR, Williamson G (2001) Human metabolism of dietary flavonoids: identification of plasma metabolites of quercetin. Free Radic Res 35(6):941–952. https://doi.org/10.1080/10715760100301441
Devulapalle KS, Mooser G (1994) Subsite specificity of the active site of glucosyltransferases from Streptococcus sobrinus. J Biol Chem 269(16):11967–11971
Dou F, Wang Z, Li G, Dun B (2019) Microbial transformation of flavonoids by Isaria fumosorosea ACCC 37814. Molecules 24(6):1028. https://doi.org/10.3390/molecules24061028
Erb A, Weiss H, Härle J, Bechthold A (2009) A bacterial glycosyltransferase gene toolbox: generation and applications. Phytochemistry 70(15–16):1812–1821. https://doi.org/10.1016/j.phytochem.2009.05.019
Fan J, Chen C, Yu Q, Li Z-G, Gmitter FG (2010) Characterization of three terpenoid glycosyltransferase genes in ‘Valencia’ sweet orange (Citrus sinensis L. Osbeck). Genome 53(10):816–823. https://doi.org/10.1139/G10-068
Fidan O, Zhan J (2015) Recent advances in engineering yeast for pharmaceutical protein production. RSC Adv 5(105):86665–86674. https://doi.org/10.1039/C5RA13003D
Fidan O, Zhan J (2019) Discovery and engineering of an endophytic Pseudomonas strain from Taxus chinensis for efficient production of zeaxanthin diglucoside. J Biol Eng 13:66. https://doi.org/10.1186/s13036-019-0196-x
Gatti-Lafranconi P, Hollfelder F (2013) Flexibility and reactivity in promiscuous enzymes. ChemBioChem 14(3):285–292. https://doi.org/10.1002/cbic.201200628
Griesser M, Vitzthum F, Fink B, Bellido ML, Raasch C, Munoz-Blanco J, Schwab W (2008) Multi-substrate flavonol O-glucosyltransferases from strawberry (Fragaria×ananassa) achene and receptacle. J Exp Bot 59(10):2611–2625. https://doi.org/10.1093/jxb/ern117
Grogan G, Holland H (2000) The biocatalytic reactions of Beauveria spp. J Mol Catal B Enzym 9(1–3):1–32. https://doi.org/10.1016/S1381-1177(99)00080-6
Hirotani M, Kuroda R, Suzuki H, Yoshikawa T (2000) Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis. Planta 210(6):1006–1013. https://doi.org/10.1007/PL00008158
Hu Y, Chen L, Ha S, Gross B, Falcone B, Walker D, Mokhtarzadeh M, Walker S (2003) Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases. Proc Natl Acad Sci USA 100(3):845–849. https://doi.org/10.1073/pnas.0235749100
Ikeda K, Taguchi R (2010) Highly sensitive localization analysis of gangliosides and sulfatides including structural isomers in mouse cerebellum sections by combination of laser microdissection and hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry with theoretically expanded multiple reaction monitoring. Rapid Commun Mass Spectrom 24(20):2957–2965. https://doi.org/10.1002/rcm.4716
Jiang J-r, Yuan S, Ding J-f, Zhu S-c, Xu H-d, Chen T, Cong X-d, Xu W-p, Ye H, Dai Y-j (2008) Conversion of puerarin into its 7-O-glycoside derivatives by Microbacterium oxydans (CGMCC 1788) to improve its water solubility and pharmacokinetic properties. Appl Microbiol Biotechnol 81(4):647–657. https://doi.org/10.1007/s00253-008-1683-z
Ko JH, Kim BG, Joong-Hoon A (2006) Glycosylation of flavonoids with a glycosyltransferase from Bacillus cereus. FEMS Microbiol Lett 258(2):263–268. https://doi.org/10.1111/j.1574-6968.2006.00226.x
Lairson L, Henrissat B, Davies G, Withers S (2008) Glycosyltransferases: structures, functions, and mechanisms. Annu Rev Biochem 77:521–555. https://doi.org/10.1146/annurev.biochem.76.061005.092322
Lee S, Park HS, Notsu Y, Ban HS, Kim YP, Ishihara K, Hirasawa N, Jung SH, Lee YS, Lim SS (2008) Effects of hyperin, isoquercitrin and quercetin on lipopolysaccharide-induced nitrite production in rat peritoneal macrophages. Phytother Res 22(11):1552–1556. https://doi.org/10.1002/ptr.2529
Legault J, Perron T, Mshvildadze V, Girard-Lalancette K, Perron S, Laprise C, Sirois P, Pichette A (2011) Antioxidant and anti-inflammatory activities of quercetin 7-O-β-D-glucopyranoside from the leaves of Brasenia schreberi. J Med Food 14(10):1127–1134. https://doi.org/10.1089/jmf.2010.0198
Li C, Ban X, Gu Z, Li Z (2013) Calcium ion contribution to thermostability of cyclodextrin glycosyltransferase is closely related to calcium-binding site CaIII. J Agric Food Chem 61(37):8836–8841. https://doi.org/10.1021/jf4024273
Li D, Park J-H, Park J-T, Park CS, Park K-H (2004) Biotechnological production of highly soluble daidzein glycosides using Thermotoga maritima maltosyltransferase. J Agric Food Chem 52(9):2561–2567. https://doi.org/10.1021/jf035109f
Li J, Li Z, Li C, Gou J, Zhang Y (2014) Molecular cloning and characterization of an isoflavone 7-O-glucosyltransferase from Pueraria lobata. Plant Cell Rep 33(7):1173–1185. https://doi.org/10.1007/s00299-014-1606-7
Li Y, Li X-L, Lai C-J-S, Wang R-S, Kang L-P, Ma T, Zhao Z-H, Gao W, Huang L-Q (2019) Functional characterization of three flavonoid glycosyltransferases from Andrographis paniculata. R Soc Open Sci 6(6):190150. https://doi.org/10.1098/rsos.190150
Liang H, Hu Z, Zhang T, Gong T, Chen J, Zhu P, Li Y, Yang J (2017) Production of a bioactive unnatural ginsenoside by metabolically engineered yeasts based on a new UDP-glycosyltransferase from Bacillus subtilis. Metab Eng 44:60–69. https://doi.org/10.1016/j.ymben.2017.07.008
Lim E-K, Ashford DA, Hou B, Jackson RG, Bowles DJ (2004) Arabidopsis glycosyltransferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides. Biotech Bioeng 87(5):623–631. https://doi.org/10.1002/bit.20154
Lin-Cereghino J, Wong WW, Xiong S, Giang W, Luong LT, Vu J, Johnson SD, Lin-Cereghino GP (2005) Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. Biotechniques 38(1):44–48. https://doi.org/10.2144/05381BM04
Lu Y, Ma B-W, Gao J, Tu L-C, Hu T-Y, Zhou J-W, Liu Y, Tu Y-H, Lin Z-S, Huang L-Q (2020) Isolation and characterization of a glycosyltransferase with specific catalytic activity towards flavonoids from Tripterygium wilfordii. J Asian Nat Prod Res 22(6):537–546. https://doi.org/10.1080/10286020.2019.1642330
Ma B, Zeng J, Shao L, Zhan J (2013) Efficient bioconversion of quercetin into a novel glycoside by Streptomyces rimosus subsp. rimosus ATCC 10970. J Biosci Bioeng 115(1):24–26. https://doi.org/10.1016/j.jbiosc.2012.07.020
Makris DP, Rossiter JT (2000) Heat-induced, metal-catalyzed oxidative degradation of quercetin and rutin (quercetin 3-O-rhamnosylglucoside) in aqueous model systems. J Agric Food Chem 48(9):3830–3838. https://doi.org/10.1021/jf0001280
Matsubara K, Ishihara K, Mizushina Y, Mori M, Nakajima N (2004) Anti-angiogenic activity of quercetin and its derivatives. Lett Drug Des Discov 1(4):329–333. https://doi.org/10.2174/1570180043398533
Meesapyodsuk D, Balsevich J, Reed DW, Covello PS (2007) Saponin biosynthesis in Saponaria vaccaria. cDNAs encoding β-amyrin synthase and a triterpene carboxylic acid glucosyltransferase. Plant Physiol 143(2):959–969. https://doi.org/10.1104/pp.106.088484
Méndez C, Salas JA (2001) Altering the glycosylation pattern of bioactive compounds. Trends Biotechnol 19(11):449–456. https://doi.org/10.1016/S0167-7799(01)01765-6
Mfonku NA, Mbah JA, Kodjio N, Gatsing D, Zhan J (2020) Isolation and selective glycosylation of antisalmonellal anthraquinones from the stem bark of Morinda lucida Benth. (Rubiaceae). Phytochem Lett 37:80–84. https://doi.org/10.1016/j.phytol.2020.04.011
Mu S, Li J, Liu C, Zeng Y, Men Y, Cai Y, Chen N, Ma H, Sun Y (2020) Effective glycosylation of cucurbitacin mediated by UDP-glycosyltransferase UGT74AC1 and molecular dynamics exploration of its substrate binding conformations. Catalysts 10(12):1466. https://doi.org/10.3390/catal10121466
Offen W, Martinez-Fleites C, Yang M, Kiat-Lim E, Davis BG, Tarling CA, Ford CM, Bowles DJ, Davies GJ (2006) Structure of a flavonoid glucosyltransferase reveals the basis for plant natural product modification. EMBO J 25(6):1396–1405. https://doi.org/10.1038/sj.emboj.7600970
Ono E, Fukuchi-Mizutani M, Nakamura N, Fukui Y, Yonekura-Sakakibara K, Yamaguchi M, Nakayama T, Tanaka T, Kusumi T, Tanaka Y (2006) Yellow flowers generated by expression of the aurone biosynthetic pathway. Proc Natl Acad Sci USA 103(29):11075–11080. https://doi.org/10.1073/pnas.0604246103
Overwin H, Wray V, Hofer B (2015) Flavonoid glucosylation by non-Leloir glycosyltransferases: formation of multiple derivatives of 3,5,7,3′,4′-pentahydroxyflavane stereoisomers. Appl Microbiol Biotechnol 99(22):9565–9576. https://doi.org/10.1007/s00253-015-6760-5
Paulke A, Eckert GP, Schubert-Zsilavecz M, Wurglics M (2012) Isoquercitrin provides better bioavailability than quercetin: comparison of quercetin metabolites in body tissue and brain sections after six days administration of isoquercitrin and quercetin. Pharmazie 67(12):991–996
Riordan JF (1977) The role of metals in enzyme activity. Ann Clin Lab Sci 7(2):119–129
Schmid J, Heider D, Wendel NJ, Sperl N, Sieber V (2016) Bacterial Glycosyltransferases: challenges and opportunities of a highly diverse enzyme class toward tailoring natural products. Front Microbiol 7:182. https://doi.org/10.3389/fmicb.2016.00182
Sharma LK, Madina BR, Chaturvedi P, Sangwan RS, Tuli R (2007) Molecular cloning and characterization of one member of 3β-hydroxy sterol glucosyltransferase gene family in Withania somnifera. Arch Biochem Biophys 460(1):48–55. https://doi.org/10.1016/j.abb.2007.01.024
Sigel RK, Pyle AM (2007) Alternative roles for metal ions in enzyme catalysis and the implications for ribozyme chemistry. Chem Rev 107(1):97–113. https://doi.org/10.1021/cr0502605
Sordon S, Popłoński J, Tronina T, Huszcza E (2019) Regioselective O-glycosylation of flavonoids by fungi Beauveria bassiana, Absidia coerulea and Absidia glauca. Bioorg Chem 93:102750. https://doi.org/10.1016/j.bioorg.2019.01.046
Strugała P, Tronina T, Huszcza E, Gabrielska J (2017) Bioactivity in vitro of quercetin glycoside obtained in Beauveria bassiana culture and its interaction with liposome membranes. Molecules 22(9):1520. https://doi.org/10.3390/molecules22091520
Valentová K, Vrba J, Bancířová M, Ulrichová J, Křen V (2014) Isoquercitrin: pharmacology, toxicology, and metabolism. Food Chem Toxicol 68:267–282. https://doi.org/10.1016/j.fct.2014.03.018
Vogt T, Jones P (2000) Glycosyltransferases in plant natural product synthesis: characterization of a supergene family. Trends Plant Sci 5(9):380–386. https://doi.org/10.1016/S1360-1385(00)01720-9
Wang D-D, Jin Y, Wang C, Kim Y-J, Perez ZEJ, Baek NI, Mathiyalagan R, Markus J, Yang D-C (2018) Rare ginsenoside Ia synthesized from F1 by cloning and overexpression of the UDP-glycosyltransferase gene from Bacillus subtilis: Synthesis, characterization, and in vitro melanogenesis inhibition activity in BL6B16 cells. J Ginseng Res 42(1):42–49. https://doi.org/10.1016/j.jgr.2016.12.009
Warnecke D, Erdmann R, Fahl A, Hube B, Müller F, Zank T, Zähringer U, Heinz E (1999) Cloning and functional expression of UGT genes encoding sterol glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum. J Biol Chem 274(19):13048–13059. https://doi.org/10.1074/jbc.274.19.13048
Warnecke DC, Baltrusch M, Buck F, Wolter FP, Heinz E (1997) UDP-glucose:sterol glucosyltransferase: cloning and functional expression in Escherichia coli. Plant Mol Biol 35(5):597–603. https://doi.org/10.1023/A:1005806119807
Wen C, Huang W, He M-M, Deng W-L, Yu H-H (2020) Cloning and characterization of a glycosyltransferase from Catharanthus roseus for glycosylation of cardiotonic steroids and phenolic compounds. Biotechnol Lett 42(1):135–142. https://doi.org/10.1007/s10529-019-02756-5
Wen L, Zhao Y, Jiang Y, Yu L, Zeng X, Yang J, Tian M, Liu H, Yang B (2017) Identification of a flavonoid C-glycoside as potent antioxidant. Free Radic Biol Med 110:92–101. https://doi.org/10.1016/j.freeradbiomed.2017.05.027
Xia T, Eiteman MA (2017) Quercetin glucoside production by engineered Escherichia coli. Appl Biochem Biotechnol 182(4):1358–1370. https://doi.org/10.1007/s12010-017-2403-x
Xu F, Gage D, Zhan J (2015) Efficient production of indigoidine in Escherichia coli. J Ind Microbiol Biotechnol 42(8):1149–1155. https://doi.org/10.1007/s10295-015-1642-5
Yang B, Liu H, Yang J, Gupta VK, Jiang Y (2018) New insights on bioactivities and biosynthesis of flavonoid glycosides. Trends Food Sci Technol 79:116–124. https://doi.org/10.1016/j.tifs.2018.07.006
Yang Y, Wu T, Yang WX, Aisa HA, Zhang TY, Ito Y (2008) Preparative isolation and purification of four flavonoids from Flos Gossypii by high-speed countercurrent chromatography. J Liq Chromatogr Relat Technol 31(10):1523–1531. https://doi.org/10.1080/10826070802039663
Yonekura-Sakakibara K, Hanada K (2011) An evolutionary view of functional diversity in family 1 glycosyltransferases. Plant J 66(1):182–193. https://doi.org/10.1111/j.1365-313X.2011.04493.x
Yu D, Xu F, Valiente J, Wang S, Zhan J (2013) An indigoidine biosynthetic gene cluster from Streptomyces chromofuscus ATCC 49982 contains an unusual IndB homologue. J Ind Microbiol Biotechnol 40(1):159–168. https://doi.org/10.1007/s10295-012-1207-9
Yu D, Xu F, Zhang S, Zhan J (2017) Decoding and reprogramming fungal iterative nonribosomal peptide synthetases. Nat Commun 8(1):1–11. https://doi.org/10.1038/ncomms15349
Zeng J, Yang N, Li XM, Shami PJ, Zhan J (2010) 4’-O-Methylglycosylation of curcumin by Beauveria bassiana. Nat Prod Commun 5(1):77–80. https://doi.org/10.1177/1934578X1000500119
Zhan J, Gunatilaka AA (2006) Microbial transformation of amino- and hydroxyanthraquinones by Beauveria bassiana ATCC 7159. J Nat Prod 69(10):1525–1527. https://doi.org/10.1021/np060339k
Zhan J, Leslie Gunatilaka AA (2006) Selective 4’-O-methylglycosylation of the pentahydroxy-flavonoid quercetin by Beauveria bassiana ATCC 7159. Biocatal Biotransfor 24(5):396–399. https://doi.org/10.1080/10242420600792169
Zhao X, Wang P, Li M, Wang Y, Jiang X, Cui L, Qian Y, Zhuang J, Gao L, Xia T (2017) Functional characterization of a new tea (Camellia sinensis) flavonoid glycosyltransferase. J Agric Food Chem 65(10):2074–2083. https://doi.org/10.1021/acs.jafc.6b05619
Funding
This work was supported by the National Science Foundation Award CBET-2044558. The Bruker Avance III HD Ascend-500 NMR instrument used in this research was funded by the National Science Foundation Award CHE–1429195.
Author information
Authors and Affiliations
Contributions
JR, WT, and JZ conceived and designed research. JR, WT, CDB, OP, MWM, AP, BW, SEH, and LPS conducted experiments. JR, WT, JH, and JZ analyzed data. JR, WT, and JZ wrote the manuscript. All authors read and approved the manuscript.
Corresponding author
Ethics declarations
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Jie Ren and Wenzhu Tang contributed equally to this work.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Ren, J., Tang, W., Barton, C.D. et al. A highly versatile fungal glucosyltransferase for specific production of quercetin-7-O-β-d-glucoside and quercetin-3-O-β-d-glucoside in different hosts. Appl Microbiol Biotechnol 106, 227–245 (2022). https://doi.org/10.1007/s00253-021-11716-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-021-11716-x