Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, RDA, Eumseong 27709, South Korea.
Department of Botany, University of British Columbia, 6270 University Blvd, Vancouver V6T 1Z4, Canada.
Plant Cell Physiol. 2018 Jun 1;59(6):1200-1213. doi: 10.1093/pcp/pcy055.
Centella asiatica is widely used as a medicinal plant due to accumulation of the ursane-type triterpene saponins asiaticoside and madecassoside. The molecular structure of both compounds suggests that they are biosynthesized from α-amyrin via three hydroxylations, and the respective Cyt P450-dependent monooxygenases (P450 enzymes) oxidizing the C-28 and C-2α positions have been reported. However, a third enzyme hydroxylating C-23 remained elusive. We previously identified 40,064 unique sequences in the transcriptome of C. asiatica elicited by methyl jasmonate, and among them we have now found 149 unigenes encoding putative P450 enzymes. In this set, 23 full-length cDNAs were recognized, 13 of which belonged to P450 subfamilies previously implicated in secondary metabolism. Four of these genes were highly expressed in response to jasmonate treatment, especially in leaves, in accordance with the accumulation patterns of asiaticoside. The functions of these candidate genes were tested using heterologous expression in yeast cells. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that yeast expressing only the oxidosqualene synthase CaDDS produced the asiaticoside precursor α-amyrin (along with its isomer β-amyrin), while yeast co-expressing CaDDS and CYP716A83 also contained ursolic acid along with oleanolic acid. This P450 enzyme thus acts as a multifunctional triterpenoid C-28 oxidase converting amyrins into corresponding triterpenoid acids. Finally, yeast strains co-expressing CaDDS, CYP716A83 and CYP714E19 produced hederagenin and 23-hydroxyursolic acid, showing that CYP714E19 is a multifunctional triterpenoid oxidase catalyzing the C-23 hydroxylation of oleanolic acid and ursolic acid. Overall, our results demonstrate that CaDDS, CYP716A83 and CYP714E19 are C. asiatica enzymes catalyzing consecutive steps in asiaticoside biosynthesis.
积雪草因其积累熊果烷型三萜皂苷积雪草苷和羟基积雪草苷而被广泛用作药用植物。这两种化合物的分子结构表明它们是由 α-香树脂醇通过三次羟基化生物合成的，并且分别报道了氧化 C-28 和 C-2α 位置的细胞色素 P450 依赖性单加氧酶（P450 酶）。然而，第三个羟基化 C-23 的酶仍然难以捉摸。我们之前从甲基茉莉酸诱导的积雪草转录组中鉴定了 40064 个独特序列，其中现在已经发现了 149 个编码推定 P450 酶的基因。在这一组中，识别出 23 个全长 cDNA，其中 13 个属于先前涉及次生代谢的 P450 亚家族。其中 4 个基因在茉莉酸处理下高度表达，特别是在叶子中，与积雪草苷的积累模式一致。这些候选基因的功能通过在酵母细胞中的异源表达进行了测试。气相色谱-质谱（GC-MS）分析表明，仅表达氧化鲨烯合酶 CaDDS 的酵母产生积雪草苷前体 α-香树脂醇（以及其异构体 β-香树脂醇），而共表达 CaDDS 和 CYP716A83 的酵母也含有熊果酸以及齐墩果酸。这种 P450 酶因此充当多功能三萜 C-28 氧化酶，将香树脂醇转化为相应的三萜酸。最后，共表达 CaDDS、CYP716A83 和 CYP714E19 的酵母菌株产生了栀子苷和 23-羟基熊果酸，表明 CYP714E19 是一种多功能三萜氧化酶，催化齐墩果酸和熊果酸的 C-23 羟基化。总的来说，我们的结果表明 CaDDS、CYP716A83 和 CYP714E19 是催化积雪草苷生物合成中连续步骤的积雪草酶。
