The parallel biosynthesis routes of hyperoside from naringenin in Hypericum monogynum

Abstract

Hyperoside is a bioactive flavonoid galactoside in both medicinal and edible plants. It plays an important physiological role in the growth of flower buds. However, the hyperoside biosynthesis pathway has not been systematically elucidated in plants, including its original source, Hypericaceae. Our group found abundant hyperoside in the flower buds of Hypericum monogynum, and we sequenced its transcriptome to study the biosynthetic mechanism of hyperoside. After gene screening and functional verification, four kinds of key enzymes were identified. Specifically, HmF3Hs (flavanone 3-hydroxylases) and HmFLSs (flavonol synthases) could catalyze flavanones into dihydroflavonols, as well as catalyzing dihydroflavonols into flavonols. HmFLSs could also convert flavanones into flavonols and flavones with varying efficiencies. HmF3′H (flavonoid 3′-hydroxylase) was found to act broadly on 4′-hydroxyl flavonoids to produce 3′,4′-diydroxylated flavanones, dihydroflavonols, flavonols, and flavones. HmGAT (flavonoid 3- O-galactosyltransferase) would transform flavonols into the corresponding 3- O-galactosides, including hyperoside. The parallel hyperoside biosynthesis routes were thus depicted, one of which was successfully reconstructed in Escherichia coli BL21(DE3) by feeding naringenin, resulting in a hyperoside yield of 25 mg/l. Overall, this research not only helped us understand the interior catalytic mechanism of hyperoside in H. monogynum concerning flower development and bioactivity, but also provided valuable insights into these enzyme families.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 32070389), and the ‘Double First-Class’ University project of China Pharmaceutical University (CPU2022QZ29). We appreciate Dr Hindra from the Department of Biology at McMaster University for helping us organize the manuscript and improve the language.

Author contributions

J.L., Y.W., and L.K. designed the research; Y.W. performed the experiments, and Z.C., Q.L., S.Z., Y.L., and X.L. assisted with the experiments; Y.W. wrote the manuscript; J.L. and L.K. revised the manuscript. All authors read and approved the final version of the manuscript.

Data availability

All data related to this research are available in this paper and its supplementary materials published online. GenBank accession numbers involved in this article are as follows: Query proteins: OsFLS (XP_015624815.1); GbFLS (ACY00393.1); MtF3H (ACR15123.1); PhGAT (AAD55985.1); PhF3′H (AAD56282.1). Phylogenetic tree of plant GATs: PhGAT (AAD55985.10); VmGAT (BAA36972.1); AgGAT (AXU98426.1); DkGAT (BAI40148.1); DcGAT (AKI23632.1); EgGAT (BAF49284.1); EkGAT (QTU90759.1); AcGAT (BAD06514.1); AcGAT (A0A2R6Q8R5.1); SbGT (QBL54224.1); FaGLT (Q2V6K0.1); GmGLT (NP_001304377.2); VvGLT (AAB81682.1); FhFLT (ADK75021.1); EkGLT (QTU90760.1); PfGLT (BAA19659.1). Phylogenetic tree of plant F3Hs and FLSs: AbFLS1 (QBQ58058.1); AcFLS1 S(AQR58515.1); AtF3H (ABB60207.1); AcF3H (AXF74850.1); AtF3H (AAC49176.1); AtFLS1 (NP_196481.1); CcFLS (QCF41219.1); CmF3H (ARI70437.1); CsF3H (BAA36553.1); CsFLS1 (ASU87436.1); CsFLS3 (ASU87438.1); CsFLS2 (ASU87437.1); CuFLS (BAA36554.1); GbFLS (ACY00393.1); GbF3H (AAU93347.1); IbF3H (ANA53146.1); InF3H (BAA21897.1); LaF3H (ARA73611.1); OsF3H (XP_015634635.1); OsFLS (XP_015624815.1); PaF3H (QBI90546.1); PhFLS (CAA80264.1); PrF3H (AGY80772.1); PtF3H (QBI90549.1); PsF3H (ABK25766.1); PtFLS (XP_002325697.1); ZmF3H (AAA91227.1); ZmFLS (NP_ 001140915.1). Sequence data of HmF3H1–2, HmFLS1–3, HmF3′H, and HmGAT identified in this study have been submitted to the GenBank database under accession numbers OP585373, OP585374, OP585375, OP585376, OP585377, OP585378, and OP585379, respectively. The transcriptome database of H. monogynum has been submitted to NCBI under project ID PRJNA907155 [55].

Conflict of interest

The authors declare no conflicts of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

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