Schotte Carsten, Florean Matilde, Czechowski Tomasz, Gilday Alison, Alam Ryan M, Ploss Kerstin, Wurlitzer Jens, Li Yi, Sonawane Prashant, Graham Ian A, O'Connor Sarah E
Department of Natural Product Biosynthesis, Max Planck Institute for Chemical Ecology, 07745, Jena, Germany.
Department of Biology, Centre for Novel Agricultural Products, University of York, Heslington, York, YO10 5DD, UK.
New Phytol. 2025 Sep;247(6):2591-2600. doi: 10.1111/nph.70388. Epub 2025 Jul 17.
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.
大戟科植物是结构复杂的二萜类化合物的丰富来源，其中许多化合物具有抗癌、抗HIV和抗炎活性。其中，从泽漆中分离出的 ingenol-3-当归酸酯（1a；商品名：Picato®）具有强大的抗肿瘤活性。我们报告了在泽漆中与ingenol-3-当归酸酯（1a）生物合成关键步骤相关的首批基因的发现和表征。通过在本氏烟草中进行途径重建以及使用重组酶进行体外试验，我们鉴定出两个基因，其产物催化将当归酰辅酶A（9a）添加到ingenol（5）支架上，生成ingenol-3-当归酸酯（1a）。我们还鉴定出三个基因，其产物催化ingenol-3-当归酸酯（1a）乙酰化为ingenol-3-当归酸酯-20-乙酸酯（2）。病毒诱导基因沉默（VIGS）表明，在泽漆基因组中，这一酶促步骤存在相当程度的功能冗余。我们还鉴定出三个基因，其产物可催化ingenol-3-当归酸酯（1a）乙酰化为ingenol-3-当归酸酯-20-乙酸酯（2）。在这种情况下，病毒诱导基因沉默（VIGS）表明，在泽漆基因组中，编码这一酶促步骤的基因存在相当程度的功能冗余。我们使用VIGS证明，在泽漆中，这些基因中只有一个，即EpBAHD-08，对于这一当归酰化反应至关重要。第二个基因EpBAHD-06的VIGS对泽漆中的麻风树烷类化合物而非贝壳杉烷类化合物有显著影响。这项工作为提高植物中ingenol-3-当归酸酯（1a）的水平铺平了道路，并为发现这些重要分子生物合成途径中的其余基因奠定了基础。
