Abstract
Main conclusion
An alkenal double-bond reductase enzyme (CaDBR1) was cloned from Colchicum autumnale L. The encoded enzyme catalysed 4-coumaraldehyde to 4-hydroxydihydrocinnamaldehyde (4-HDCA). Its functional characterization increased the understanding of colchicine biosynthesis.
Abstract
As a traditional medical plant, Colchicum autumnale L. is famous for producing colchicine, a widely used drug for alleviating gout attacks. The biosynthetic pathway of colchicine was revealed most recently, and 4-hydroxydihydrocinnamaldehyde (4-HDCA) has been verified as a crucial intermediate derived from L-phenylalanine. However, the functional gene that catalyses the formation of 4-HDCA remains controversial. In this study, the alkenal double-bond reductase (DBR) gene member CaDBR1 was cloned and characterized from C. autumnale. Bioinformatics analysis predicted and characterized the basic physicochemical properties of CaDBR1. Recombinant CaDBR1 protein was heterologously expressed in Escherichia coli and purified by a Ni-NTA column. In vitro enzyme assays indicated that CaDBR1 could catalyse 4-coumaraldehyde to form 4-HDCA but could not generate 4-HDCA by taking cinnamaldehyde as a substrate. Stable transformation into tobacco BY-2 cells revealed that CaDBR1 localized in the cytoplasm, and tissue-specific expression results showed that CaDBR1 had the highest expression in bulbs. All these results verify and confirm the participation and contribution of CaDBR1 in the biosynthesis pathway of 4-HDCA and colchicine alkaloids in C. autumnale.
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Data availability
The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- DBR:
-
Alkenal double-bond reductase
- C. autumnale :
-
Colchicum autumnale L.
- 4-HDCA:
-
4-Hydroxydihydrocinnamaldehyde
- qRT-PCR:
-
The quantitative real-time PCR
- MDR family:
-
Medium-chain reductase/dehydrogenase family
- LB:
-
Luria–Bertani
- IPTG:
-
Isopropyl-β-d-thiogalactoside
- UPLC:
-
Ultrahigh-pressure liquid chromatography
- UPLC–MS:
-
Ultra-performance liquid chromatograph–mass spectrometry
- Ox:
-
Overexpression
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Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (32070364), the Hainan Provincial Natural Science Foundation of China (319MS084 and 2019RC309), and the Central Public-interest Scientific Institution Basal Research Fund for the Chinese Academy of Tropical Agricultural Sciences (1630032019038).
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Xiong, Z., Wang, L., Sun, J. et al. Functional characterization of a Colchicum autumnale L. double-bond reductase (CaDBR1) in colchicine biosynthesis. Planta 256, 95 (2022). https://doi.org/10.1007/s00425-022-04003-0
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DOI: https://doi.org/10.1007/s00425-022-04003-0