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Cloning, heterologous expression, and functional characterization of a hydroxyphenylpyruvate reductase (HPPR) gene involved in rosmarinic acid biosynthesis in Prunella vulgaris

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Abstract

Rosmarinic acid (RA) and its derivatives have wide medicinal applications. However, the biosynthetic pathway of RA in Prunella vulgaris is still unclear. Here, we isolated and characterized a hydroxyphenylpyruvate reductase cDNA (PvHPPR, KM053279) from P. vulgaris, commonly known as “all-heal”. Based on the high sequence similarity of the predicted protein, PvHPPR was found to belong to the D-isomer-specific 2-hydroxyacid dehydrogenase family. Heterologous expression of the PvHPPR open reading frame was performed in Escherichia coli, and the resulting protein was proved to catalyze the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvate, phenylpyruvate, and pyruvate to the corresponding lactates. The Km values for the various substrates were 0.31 mM for 4-hydroxyphenylpyruvate, 0.73 mM for phenylpyruvate, and 1.02 mM for pyruvate, demonstrating that 4-hydroxyphenylpyruvate is the preferred substrate. The role of PvHPPR in RA biosynthesis was evaluated by using Agrobacterium rhizogenes-mediated overexpression in P. vulgaris hairy roots. PvHPPR transcript levels and RA contents were increased by 0.7–11.8-fold and 24–63% respectively compared with the wild-type counterparts, suggesting PvHPPR is likely involved in RA biosynthesis in P. vulgaris. The positive correlation between the two provides evidence for this claim.

Key message

A hydroxyphenylpyruvate reductase (HPPR) gene involved in rosmarinic acid biosynthesis in Prunella vulgaris was cloned, heterologously expressed, and functionally characterized.

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Data availability

All data generated or analyzed during this study are included in this published article [and its supplementary information fles].

Abbreviations

HPPR:

Hydroxyphenylpyruvate reductase

IPTG:

Isopropyl-b-D-thiogalactoside

SDS:

Sodium dodecyl sulfate

DTT:

Dithiothreitol

RA:

Rosmarinic acid

GFP:

Green fluorescence protein

DW:

Dry weight

HPP:

4-Hydroxyphenylpyruvate

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Acknowledgements

This work was financially supported by the Science & Technology and Special Talents Project of Guangxi (Guike AD19245087) and the National Natural Science Foundation of China (Grant no: 81373908, 82160754).

Funding

National Natural Science Foundation of China, 81373908, Zongsuo Liang, Science & Technology and Special Talents Project of Guangxi, Guike AD19245087,Mei Ru

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Authors and Affiliations

  1. College of Pharmacy, Guangxi University of Chinese Medicine, Nanning, 530200, People’s Republic of China

    Mei Ru, Liuyan Chen, Jiali Liu, Tingting Lan, Chuchu Yang, Xiaoyao Tan, Chuanmei Wu & Pan Liang

  2. College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, 310000, People’s Republic of China

    Zongsuo Liang

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  1. Mei Ru
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Contributions

RM and LZS: conceived and designed research. RM, WCM and LP: conducted experiments. RM, CLY, and LJL: analyzed data. RM, LTT, and LZS: wrote the manuscript. RM, YCC, TXY, and LZS: revised the manuscript. All authors have read and approved the manuscript.

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Correspondence to Zongsuo Liang.

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Communicated by Ali R. Alan.

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Ru, M., Chen, L., Liu, J. et al. Cloning, heterologous expression, and functional characterization of a hydroxyphenylpyruvate reductase (HPPR) gene involved in rosmarinic acid biosynthesis in Prunella vulgaris. Plant Cell Tiss Organ Cult 153, 273–283 (2023). https://doi.org/10.1007/s11240-023-02452-3

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