Identification of BAHD-acyltransferase enzymes involved in ingenane diterpenoid biosynthesis

Abstract

The plant family Euphorbiaceae is an abundant source of structurally complex diterpenoids, many of which have reported anticancer, anti-HIV, and anti-inflammatory activities. Among these, ingenol-3-angelate (1a; tradename: Picato®), isolated from Euphorbia peplus, has potent antitumor activity. We report the discovery and characterization of the first genes linked to committed steps of ingenol-3-angelate (1a) biosynthesis in E. peplus. Using pathway reconstitution in Nicotiana benthamiana and in vitro assays with recombinant enzymes, we identified two genes whose products catalyze the addition of angelyl-CoA (9a) to the ingenol (5) scaffold, producing ingenol-3-angelate (1a). We also identified three genes whose products catalyze acetylation of ingenol-3-angelate (1a) to ingenol-3-angelate-20-acetate (2). Virus induced gene silencing (VIGS) suggests considerable functional redundancy in the E. peplus genome for this enzymatic step. We also identified three genes whose products can catalyze acetylation of ingenol-3-angelate (1a) to ingenol-3-angelate-20-acetate (2). In this case, virus-induced gene silencing (VIGS) indicates considerable functional redundancy in the E. peplus genome of genes encoding this enzymatic step. We demonstrate using VIGS that just one of these genes, EpBAHD-08, is essential for this angeloylation in E. peplus. VIGS of the second gene, EpBAHD-06, has a significant effect on jatrophanes rather than ingenanes in E. peplus. This work paves the way for increasing ingenol-3-angelate (1a) levels in planta and provides a foundation for the discovery of the remaining genes in the biosynthetic pathway of these important molecules.

Keywords: BAHD‐acyltransferase; Euphorbia peplus; diterpene; ingenol‐3‐angelate; medicinal plant; natural product biosynthesis.

Fig. 1

Diterpene biosynthesis in the Euphorbia genus. (a) Clinically important diterpenoids isolated from various Euphorbia species. (b) Known, proposed, and newly discovered steps in Euphorbia peplus diterpene biosynthesis. Genes identified in this study catalyze formation of ingenol‐3‐angelate (1a) from ingenol (5) (EpBAHD‐06 and EpBAHD‐08); and ingenol‐3‐angelate‐20‐acetate (2) from ingenol‐3‐angelate (1a) (EpBAHD‐07 and EpBAHD‐11). (c) Expression profiles of genes involved in jolkinol C (7) biosynthesis and BAHD‐acyltransferases identified in the course of this study. Red corresponds to maximum expression, and blue corresponds to minimum expression levels per gene across different tissues (expression measured in fragments per kilobase of exon per million of mapped fragments). The heatmap was created with Morpheus (https://software.broadinstitute.org/morpheus).

Fig. 2

Enzyme activity assays of acyltransferases from Euphorbia peplus. (a) Enzymatic reactions observed in this study. (b) Tobacco infiltration of a dedicated angelyl‐CoA ligase (EpCCL2) (Callari et al., 2018), angelic acid (8), ingenol (5), and EpBAHD‐06 and EpBAHD‐08. Both enzymes catalyze formation of ingenol‐3‐angelate (1a) in planta (peak B). (c) Tobacco infiltration of a dedicated angelyl‐CoA ligase (EpCCL2), angelic acid (8), ingenol (5), EpBAHD‐08 (together affording ingenol‐3‐angelate [1a]), and EpBAHD‐07 and EpBAHD‐11. Both EpBAHD‐07 and EpBAHD‐11 catalyze formation of ingenol‐3‐angelate‐20‐acetate (2). Note that chromatography methods in panel (b) and (c) are different and that 1a and 2 can be differentiated. (d) In vitro assays with EpBAHD‐06 and EpBAHD‐08 using angelyl‐CoA (9a) and ingenol (5) as substrates lead to formation of ingenol‐3‐angelate (1a) as a minor product (peak B), with the isomer ingenol‐3‐tiglate (1b) (peak A) as the major product. (e) In vitro assays with EpBAHD‐06 and EpBAHD‐08 using tiglyl‐CoA (9b) and ingenol (5) as substrates lead to formation of ingenol‐3‐tiglate (1b) (peak A). (f) In vitro assays with EpBAHD‐07 and EpBAHD‐11 using ingenol‐3‐angelate (1a) and acetyl‐CoA (10) as substrates lead to formation of ingenol‐3‐angelate‐20‐acetate (2). Plant art and depiction of microcentrifuge tube in Scheme 2 (b–f) are from biorender.com (https://BioRender.com/1qhnqih).

Fig. 3

Phylogenetic analysis of acyltransferases identified from Euphorbia peplus in this study. Sequence alignment of amino acid sequences was performed using Muscle v.3.8.425 (Edgar, 2022). The displayed tree was then constructed with Bayesian analyses using MrBayes v.3.2.7a (Ronquist et al., 2012). Posterior probabilities were reported as supporting values for nodes in the trees, and the scale bar represents substitutions per site (bar indicates a branch length of 0.2.). Lines indicate the edges of the clades.

Fig. 4

Virus‐induced gene silencing (VIGS) analysis of EpBAHD‐06 and EpBAHD‐08 genes in Euphorbia peplus. Metabolite levels in VIGS material were measured for stem and leaves in VIGS marker‐only (EpCH42_A, black bars) and marker plus selected BAHD genes: EpCH42:EpBAHD‐06 (cyan bars) and EpCH42:EpBAHD‐08 (red bars). Triterpenes represent the sum of four major triterpenes annotated in Datasets S1 and S2. Error bars – SEM (n = 6). Letters represent Tukey's range test results after one‐way ANOVA was performed separately within the ‘leaf’ and ‘stem’ datasets. Groups not sharing letters within ‘leaf’ or ‘stem’ datasets indicate statistically significant differences (P < 0.05).