Abstract
Flavanone 3β-hydroxylase plays very important role in the biosynthesis of flavonoids. A putative flavanone 3β-hydroxylase gene (Pef3h) from Populus euphratica was cloned and over-expressed in Escherichia coli. Induction performed with 0.1 mM IPTG at 20°C led to localization of PeF3H in the soluble fraction. Recombinant enzyme was purified by Ni-NTA affinity. The optimal activity of PeF3H was revealed at pH 7.6 and 35°C. The purified enzyme was stable over pH range of 7.6–8.8 and had a half-life of 1 h at 50°C. The activity of PeF3H was significantly enhanced in the presence of Fe2+ and Fe3+. The K M and V max for the enzyme using naringenin as substrate were 0.23 mM and 0.069 μmoles mg–1min-1, respectively. The K m and V max for eriodictyol were 0.18 mM and 0.013 μmoles mg–1min–1, respectively. The optimal conditions for naringenin bioconversion in dihydrokaempferol were obtained: OD600 of 3.5 for cell concentration, 0.1 mM IPTG, 5 mM α-ketoglutaric acid and 20°C. Under the optimal conditions, naringenin (0.2 g/L) was transformed into 0.18 g/L dihydrokaempferol within 24 h by the recombinant E. coli with a corresponding molar conversion of 88%. Thus, this study provides a promising flavanone 3β-hydroxylase that may be used in biosynthetic applications.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Tahara, S., Biosci. Biotech. Bioch., 2007, vol. 71, no. 6, pp. 1387–1404.
Dixon. R.A. and Steele, C.L., Trends Plant Sci., 1999, vol. 4, no. 10, pp. 394–400.
Harborne, J.B. and Williams, C.A., Phytochemistry, 2000, vol. 55, no. 6, pp. 481–504.
Martens, S. and Mithofer, A., Phytochemistry, 2005, vol. 66, no. 20, pp. 2399–2407.
Winkel-Shirley, B.W., Plant Physiol., 2001, vol. 126, no. 2, pp. 485–493.
Prescott, A.G., and John, P., Annu. Rev. Plant Physiol. Plant Mol. Biol., 1996, vol. 47, no. 4, pp. 245–271.
Lee, J.W., Kim, N.H., Kim, J.Y., Park, J.H., Shin, S.Y., Kwon, Y.S., et al., Biomol. Ther., 2013, vol. 21, no. 3, pp. 216–221.
Ma, C., Yang, L., Wang W., Zhao, C., and Zu., Y., Int. J. Mol. Sci., 2012, vol. 13, pp. 8789–8804.
Kiehlmann, E. and Li, E.P.M., J. Nat. Prod., 1995, vol. 58, no. 3, pp. 450–455.
Britsch, L., Ruhnau-Brich, B., and Forkmann, G., J. Biol. Chem., 1992, vol. 267, no. 8, pp. 5380–5387.
Davies, K.M., Plant Physiol., 1993, vol. 103, no. 1, pp. 291–291.
Kim, J.H., Lee, Y.J., Kim, B.G., Lim, Y., and Ahn, J.H., Mol. Cells, 2008, vol. 25, no. 2, pp. 312–316.
Pelletier, M.K. and Shirley B.W., Plant Physiol., 1996, vol. 111, no. 1, pp. 339–345.
Jin, Z., Grotewold, E., Qu, W., Fu, G., and Zhao, D., DNA Seq., 2005, vol. 16, no. 2, pp. 121–129.
Shen, G., Pang, Y., Wu, W., Deng, Z., Zhao, L., Cao, Y., et al., Biosci. Rep., 2006, vol. 26, no. 1, pp. 19–29.
Charrier, B., Coronado, C., Kondorosi, A., and Ratet, P., Plant Mol. Biol., 1995, vol. 29, no. 4, pp. 773–786.
Baek, M.H., Chung, B.Y., Kim, J.H., Wi, S.G., An, B.C., Kim, J.S., et al., J. Hort. Sci. Biotech., 2008, vol. 83, pp. 595–602.
Martens, S., Forkmann, G., Britsch, L., Wellmann, F., Matern, U., and Lukacin, R., FEBS Lett., 2003, vol. 544, no. 1–3, pp. 93–98.
Owens, D.K., Crosby, K.C., Runac, J., Howard, B.A., and Winkel, B.S.J., Plant Physiol. Biochem., 2008, vol. 46, no. 10. pp. 833–843.
Britsch, L. and Grisebach, H., Eur. J. Biochem., 1986, vol. 156, no. 3, pp. 569–577.
Lukacin, R., Groning, I., Schiltz, E., Britsch, L., and Matern, U., Arch. Biochem. Biophys., 2003, vol. 375, no. 2, pp. 364–370.
Punyasiri, P.A., Abeysinghe, I.S., Kumar, V., Treutter, D., Duy, D., Gosch, C., et al., Arch. Biochem. Biophys., 2004, vol. 431, no. 1, pp. 22–30.
Halbwirth, H., Fischer, T.C., Schlangen, K., Rademacher, W., Schleifer, K.J., Forkmann, G., and Stich, K., Plant Sci., 2006, vol. 171, no. 2, pp. 194–205.
Song, X., Diao, J., Ji, J., Wang, G., Guan C., Jin, C., and Wang, Y., Plant Physiol. Biochem., 2016, vol. 98, pp. 89–100.
Mahajan, M. and Yadav, S.K., Plant Mol. Biol., 2014, vol. 85, pp. 551–573.
Stahlhut, S.G., Siedler, S., Malla, S., Harrison, S.J., Maury, J., Neves, A.R., and Forster, J., Metab. Eng., 2015, vol. 31, pp. 84–93.
Chen, Y.N., Wang, Q., Ruan, X., Li, W.H., and Chen, Y.P., Acta Bot. Sin., 2004, vol. 46, no. 12, pp. 1393–1401.
Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., et al., Bioinformatics, 2007, vol. 23, no. 21, pp. 2947–2948.
Pan, K.L., Hsiao, H.C., Weng, C.L., Wu, M.S., and Chou, C.P., J. Bacteriol., 2003, vol. 185, no. 10, pp. 3020–3030.
Sevastsyanovich, Y.R., Alfasi, S.N., and Cole, J.A., Biotechnol. Appl. Biochem., 2010, vol. 55, no. 1, pp. 9–28.
Xu, P., Ranganathan, S., Fowler, Z.L., Maranas, C.D., and Koffas, M.A., Metab. Eng., 2011, vol. 13, no. 5, pp. 578–587.
Fowler, Z.L., Gikandi, W.W., and Koffas, M.A., Appl. Environ. Microbiol., 2009, vol. 75, no. 18, pp. 5831–5839.
Miyahisa, I., Funa, N., Ohnishi, Y., Martens, S., Moriguchi, T., and Horinouchi, S., Appl. Microbiol. Biotechnol., 2006, vol. 71, no. 1, pp. 53–58.
Author information
Authors and Affiliations
Corresponding author
Additional information
The article is published in the original.
Rights and permissions
About this article
Cite this article
Pei, J., Dong, P., Wu, T. et al. Characterization flavanone 3β-hydroxylase expressed from Populus euphratica in Escherichia coli and its application in dihydroflavonol production. Appl Biochem Microbiol 53, 318–324 (2017). https://doi.org/10.1134/S0003683817030127
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0003683817030127