Biosynthetic pathway of prescription bergenin from Bergenia purpurascens and Ardisia japonica

Abstract

Bergenin is a typical carbon glycoside and the primary active ingredient in antitussive drugs widely prescribed for central cough inhibition in China. The bergenin extraction industry relies on the medicinal plant species Bergenia purpurascens and Ardisia japonica as their resources. However, the bergenin biosynthetic pathway in plants remains elusive. In this study, we functionally characterized a shikimate dehydrogenase (SDH), two O-methyltransferases (OMTs), and a C-glycosyltransferase (CGT) involved in bergenin synthesis through bioinformatics analysis, heterologous expression, and enzymatic characterization. We found that BpSDH2 catalyzes the two-step dehydrogenation process of shikimic acid to form gallic acid (GA). BpOMT1 and AjOMT1 facilitate the methylation reaction at the 4-OH position of GA, resulting in the formation of 4-O-methyl gallic acid (4-O-Me-GA). AjCGT1 transfers a glucose moiety to C-2 to generate 2-Glucosyl-4-O-methyl gallic acid (2-Glucosyl-4-O-Me-GA). Bergenin production ultimately occurs in acidic conditions or via dehydration catalyzed by plant dehydratases following a ring-closure reaction. This study for the first time uncovered the biosynthetic pathway of bergenin, paving the way to rational production of bergenin in cell factories via synthetic biology strategies.

Keywords: C-glycosyltransferase; O-methyltransferases; bergenin; biosynthetic pathway; carbon glycosides.

Figure 1

Possible biosynthesis pathway of bergenin in plants. SA, Shikimic acid; 3-DHS, 3-Dehydroshikimic acid; GA, Gallic acid; 4-O-Me-GA, 4-O-Methyl gallic acid. SDH, Shikimate dehydrogenases; OMT, O-methyltransferase; CGT, C-glycosyltransferases. SAM, S-adenosy-L-methionine; UDP-Glc, Uridine diphosphate-Glucose.

Figure 2

Phylogenetic tree of SDHs involved in GA biosynthesis. A phylogenetic tree was constructed based on the amino acid sequences containing the shikimate dehydrogenases domain from B. purpurascens, A. japonica, and other species. The red box represents SDH found in B. purpurascens, A. japonica, while the blue box represents reported SDH with GA-producing function. All other species’ SDH sequences were obtained from the NCBI database ( Supplementary Table S3 ).

Figure 3

HPLC detection of substrate activity for shikimic acid with BpSDH1 protein. (A) The biosynthetic pathway for the conversion of shikimic acid to gallic acid, as well as the possible pathway for the synthesis of protocatechuic acid. (B) In vitro enzyme activity assay products of shikimic acid with BpSDH2 and NADP+. (C) The reaction of inactivated BpSDH2 protein with shikimic acid and NADP+ serves as a negative control. (D) The retention times of SA, 3-DHS, GA, and PCA.

Figure 4

Phylogenetic tree of OMTs involved in 4-O-Me-GA biosynthesis. A phylogenetic tree was constructed based on the amino acid sequences containing the O-methyltransferase domain from B. purpurascens, A. japonica, and other species. The blue boxes represent the candidate OMTs identified from B. purpurascens, A. japonica, which are potential enzymes involved in catalyzing the conversion of GA to 4-O-Me-GA. The OMTs depicted in red boxes serve as the main reference sequences, as they have been previously reported to have the methylating catalytic function on structures similar to GA. All other species’ OMTs were obtained from the NCBI database ( Supplementary Table S3 ).

Figure 5

HPLC detection of the C-4 methylation activity of OMT protein on GA. (A) The biosynthetic pathway of 4-O-Me-GA through the C-4 methylation of GA. (B) The enzymatic activity assay of GA with BpOMT1 and SAM in vitro. (C) Inactivated BpOMT1 protein was used as a negative control in the reaction with GA and SAM. (D) The retention times of GA and 4-O-Me-GA standard. (E) The enzymatic activity assay of GA with AjOMT1 and SAM in vitro. (F) The reaction of inactivated AjOMT1 protein with GA and SAM serves as a negative control. (G) The retention time of authentic standard of GA and 4-O-Me-GA.

Figure 6

Phylogenetic tree of CGTs involved in bergenin biosynthesis. The sector (A) highlights the CGT sequences of glycosyltransferases found in animals, while sector (B) highlights those found in bacteria. Sector (C) represents the reported CGTs with glycosyltransferase function in plants. Sector (D) represents the UGTs identified in B. purpurascens and A. japonica by transcriptomics. The red pentagrams represent the candidate CGTs identified in B. purpurascens and A. japonica. The CGTs of other species in sectors A, B, and C were obtained from the NCBI database ( Supplementary Table S3 ).

Figure 7

HPLC detection of the C-2 glycosylation activity of CGT protein on 4-O-Me-GA. (A) The biosynthetic pathway of bergenin through the C-2 glycosylation of 4-O-Me-GA. (B) The enzymatic activity assay of 4-O-Me-GA with AjCGT1 and UDP-Glc in vitro. (C) Inactivated AjCGT1 protein was used as a negative control in the reaction with 4-O-Me-GA and UDP-Glc. (D) The retention times of the standard compounds 4-O-Me-GA and bergenin.