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Molecular characterization and overexpression analyses of secologanin synthase to understand the regulation of camptothecin biosynthesis in Nothapodytes nimmoniana (Graham.) Mabb.

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Abstract

Camptothecin is a high-value anti-cancerous compound produced in many taxonomically unrelated species. Its biosynthesis involves a complex network of pathways and a diverse array of intermediates. Here, we report the functional characterization and regulation of secologanin synthase (NnCYP72A1), a cytochrome P450 involved in camptothecin biosynthesis from Nothapodytes nimmoniana. It comprises an open reading frame of 1566 bp in length. Heterologous expression in Saccharomyces cerevisiae and in vitro enzymatic assays using loganin as substrate confirmed the formation of secologanin. In planta transient overexpression analysis of NnCYP72A1 resulted in 4.21- and 2.73-fold increase in transcript levels of NnCYP72A1 on days 3 and 6 respectively. Phytochemical analysis of transformed tissues revealed ~ 1.13–1.43- and 2.02–2.86-fold increase in secologanin and CPT accumulation, respectively. Furthermore, promoter analysis of NnCYP72A1 resulted in the identification of several potential cis-regulatory elements corresponding to different stress-related components. Methyl jasmonate, salicylic acid, and wounding treatments resulted in considerable modulation of mRNA transcripts of NnCYP72A1 gene. Chemical analysis of elicitor-treated samples showed a significant increase in CPT content which was concordant with the mRNA transcript levels. Overall, the functional characterization and overexpression of NnCYP72A1 may plausibly enhance the pathway intermediates and serve as prognostic tool for enhancing CPT accumulation.

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Acknowledgments

Authors are thankful to Dr. Utpal Nandi at CSIR-IIIM, Jammu for facilitating LC-MS/MS analyses. We are also thankful to Nicolas Navrot, University de Strasbourg, France, for providing pYeDP60 vector and Wat11 strain. GAR is thankful to UGC for providing Senior Research Fellowship. AS thankfully acknowledges the DST-INSPIRE Senior Research Fellowship. This manuscript represents Institutional Communication No. CSIR/IIIM/IPR/0064. 

Funding

This work was supported by a financial grant from Council of Scientific and Industrial Research (CSIR)-Indian Institute of Integrative Medicine under Major Lab Project MLP-3012 (WP 7).

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Authors and Affiliations

  1. Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi, 180001, India

    Gulzar A. Rather, Arti Sharma, Prashant Misra & Surrinder K. Lattoo

  2. Instrumentation Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi, 180001, India

    Amit Kumar

  3. Department of Botany, University of Jammu, Jammu Tawi, 180006, India

    Veenu Kaul

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  1. Gulzar A. Rather
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Contributions

Conceived and designed the experiments: SKL. Performed the experiments: GAR, AS, AK. Analyzed the data: GAR, SKL, VK, PM. Contributed reagents/materials/analysis tools: SKL. Original draft of the manuscript was prepared by GAR. SKL, PM, and VK improved the content and edited the manuscript.

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Correspondence to Surrinder K. Lattoo.

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Supplementary file1

Primers used for amplification and expression of NnCYP72A1. (DOCX 17 kb)

Supplementary file 2

Conserved residue prediction for NnCYP72A1 and multiple sequence alignment. (DOCX 324 kb)

Supplementary file 3

Enzyme kinetics of NnCYP72A1. (XLSX 537 kb)

Supplementary file 4

Nucleotide sequences of NnCYP72A1 gene promoter. (DOCX 1111 kb)

Supplementary file 5

Chemical profiles of tissue extracts for detection and quantification of camptothecin. (XLSX 336 kb)

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Rather, G.A., Sharma, A., Misra, P. et al. Molecular characterization and overexpression analyses of secologanin synthase to understand the regulation of camptothecin biosynthesis in Nothapodytes nimmoniana (Graham.) Mabb.. Protoplasma 257, 391–405 (2020). https://doi.org/10.1007/s00709-019-01440-9

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