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Functional identification of hyoscyamine 6β-hydroxylase from Anisodus acutangulus and overproduction of scopolamine in genetically-engineered Escherichia coli

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Abstract

Hyoscyamine 6β-hydroxylase (H6H; EC 1.14.11.11) converts hyoscyamine to scopolamine in the last step of scopolamine biosynthetic pathway. The gene encoding H6H in Anisodus acutangulus was cloned and expressed in Escherichia coli and the recombinant proteins fused with His-tag or GST-tag at its N-terminal were purified and then confirmed by Western bolt analysis. The biofunctional assay revealed that the His-AaH6H and GST-AaH6H converted hyoscyamine (40 mg/l) to scopolamine at 32 and 31 mg/l, respectively. This is the first report on AaH6H expression, purification and functional characterization facilitates further genetic improvement of scopolamine yield in A. acutangulus.

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Acknowledgments

This work was supported by National Natural Science Fund (30900110), National Transgenic Organism New Variety Culture Key Project (2009ZX08012-002B), Project from Ministry of Science and Technology of China (NC2010AE0075, NC2010AE0372), Shanghai Science and Technology Committee Project (10JC1412000, 09QH1401900, 06QA14038, 08391911800, 073158202, 075405117, 065458022, 05ZR14093), Kunshan Municipal Science & Technology Project (KS0914), Zhejiang Provincial Natural Science Fund (Y2080621), Program from Shanghai Municipal Education Commission (06DZ015, 09ZZ138), Fujian Science and Technology Committee Key Special Project (2008NZ0001-4), Leading Academic Discipline Project of Shanghai Municipal Education Commission (J50401) and Project from Shanghai Normal University.

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Authors and Affiliations

  1. Laboratory of Plant Biotechnology, College of Life and Environment Sciences, Shanghai Normal University, Shanghai, 200234, People’s Republic of China

    Guoyin Kai, Yuanyuan Liu, Sheng Yang, Xueqing Fu, Xiuqin Luo & Pan Liao

  2. Department of Gynaecology and Obstetrics, Kunshan Hospital of Traditional Chinese Medicine, Kunshan, 215300, Jiangsu Province, People’s Republic of China

    Xiaoyun Wang

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  1. Guoyin Kai
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  2. Yuanyuan Liu
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  3. Xiaoyun Wang
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  4. Sheng Yang
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  5. Xueqing Fu
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  6. Xiuqin Luo
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  7. Pan Liao
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Correspondence to Guoyin Kai.

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Kai, G., Liu, Y., Wang, X. et al. Functional identification of hyoscyamine 6β-hydroxylase from Anisodus acutangulus and overproduction of scopolamine in genetically-engineered Escherichia coli . Biotechnol Lett 33, 1361–1365 (2011). https://doi.org/10.1007/s10529-011-0575-y

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  • DOI: https://doi.org/10.1007/s10529-011-0575-y

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