Abstract
Main conclusion
The recombinant caffeic acid 3-O-methyltransferase gene has been cloned and characterized from Neem. The gene is involved in ferulic acid biosynthesis, a key intermediate component of lignin biosynthesis.
Abstract
Azadirachta indica (Neem) is a highly reputed traditional medicinal plant and is phytochemically well-known for its limonoids. Besides limonoids, phenolics are also distinctively present, which add more medicinal attributes to Neem. Caffeic acid is one of such phenolic compound and it can be converted enzymatically into another bioactive phytomolecule, ferulic acid. This conversion requires transfer of a methyl group from a donor to caffeic acid under the catalytic action of an appropriate methyltransferase. In this study, caffeic acid 3-O-methyltransferase gene from Neem (NCOMT) fruits has been isolated and heterologously expressed in E. coli. The recombinant NCOMT enzyme was purified, which exhibited efficient catalytic conversion of caffeic acid into ferulic acid, a highly potential pharmaceutical compound. The purified recombinant enzyme was physico-kinetically characterized for its catalysis. The analysis of tissue-wide expression of NCOMT gene revealed interesting pattern of transcript abundance reflecting its role in the development of fruit tissues. Further, NCOMT was heterologously overexpressed in Withania somnifera and Ocimum species, to analyze its role in ferulic acid biosynthesis in planta. Thus, the study provides insight for the endogenous role of NCOMT in ferulic acid biosynthesis en route to lignin, an important structural component. To the best of our knowledge, NCOMT pertains to be the first enzyme of the secondary metabolism that has been purified and kinetically characterized from Neem. This study may also have important prospects of applications as the observation on heterologous expression of NCOMT showed its involvement in the maintenance of the in vivo pool of ferulic acid in the plants. Thus, the study involving NCOMT opens up new dimensions of metabolic engineering approaches for the biosynthesis of potential therapeutically important phytomolecules in heterologous systems.
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Abbreviations
- COMT:
-
Caffeic acid 3-O-methyltransferase
- SAM:
-
S-adenosyl-l-methionine
- IPTG:
-
Isopropyl β-d-1 thiogalactopyranoside
- MJ:
-
Methyl jasmonate
- SA:
-
Salicylic acid
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Acknowledgements
RSS and NSS are thankful to HCP-0010, MLP-5 and BSC-203 CSIR network project for providing financial assistance. LKN, JSJ and SB are thankful to University Grants Commission and CSIR for senior research fellowship.
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425_2020_3514_MOESM1_ESM.tif
Supplementary file1 Fig. S1 Multiple sequence alignment of the deduced amino acid sequences of Azadirachta indica COMT and other plants OMT. Citrus aurantium (ADK97702), Jatropha curcas (ACT87981), Eucalyptus camaldulensis (ACY66932), Rosa chinensis (BAC78827), Gossypium hirsutum (ACT32029), Malus domestica (ABI54119), Theobroma cacao (EOY23716), Fragaria x ananassa (AAF28353), Populus tomentosa (AFZ78575), Medicago truncatula (AES72647), Arabidopsis thaliana (NP_200227), Camellia sinensis (ADN27527) and Salvia miltiorrhiza (AEO14870). Sequence in black box represents the S-adenosyl-L-methionine (SAM) binding motif and red box represents phenolic substrate binding residues (TIF 1431 KB)
425_2020_3514_MOESM2_ESM.tiff
Supplementary file 2 Fig. S2 Western-blot analysis of Ni-NTA purified NCOMT protein. Lane M Pre-stained protein molecular marker; W1 Ni NTA purified fraction 1; W2 Ni NTA purified fraction 2 (TIFF 348 KB)
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Supplementary file3 Fig. S3 NCOMT enzyme assay validation through TLC. CA caffeic acid; FA ferulic acid; C1 control assay without substrate; C2 control assay without protein, E1 and E2 enzyme assay (TIF 245 KB)
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Narnoliya, L.K., Sangwan, N., Jadaun, J.S. et al. Defining the role of a caffeic acid 3-O-methyltransferase from Azadirachta indica fruits in the biosynthesis of ferulic acid through heterologous over-expression in Ocimum species and Withania somnifera. Planta 253, 20 (2021). https://doi.org/10.1007/s00425-020-03514-y
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DOI: https://doi.org/10.1007/s00425-020-03514-y