Department of Forest Resources, College of Forest and Environmental Sciences, Kangwon National University, Chuncheon 200-701, Korea.
Division of Forest Biotechnology, Korea Forest Research Institute, Suwon 441-847, Korea.
Plant Cell Physiol. 2018 Feb 1;59(2):319-330. doi: 10.1093/pcp/pcx188.
Kalopanax septemlobus, commonly named the castor aralia tree, is a highly valued woody medicinal tree belonging to the family Araliaceae. Kalopanax septemlobus contains approximately 15 triterpenoid saponins primarily constituted of hederagenin aglycones. Hederagenin is a representative precursor for hemolytic saponin in plants. In the present study, transcriptome analysis was performed to discover genes involved in hederagenin saponin biosynthesis in K. septemlobus. De novo assembly generated 82,698 unique sequences, including 17,747 contigs and 64,951 singletons, following 454 pyrosequencing. Oxidosqualene cyclases (OSCs) are enzymes that catalyze the formation of diverse triterpene skeletons from 2,3-oxidosqualene. Heterologous expression of an OSC sequence in yeast revealed that KsBAS is a β-amyrin synthase gene. Cytochrome P450 genes (CYPs) make up a supergene family in the plant genome and play a key role in the biosynthesis of sapogenin aglycones. In total, 95 contigs and 110 singletons annotated as CYPs were obtained by sequencing the K. septemlobus transcriptome. By heterologous expression in yeast, we found that CYP716A94 was β-amyrin 28-oxidase involved in oleanolic acid production from β-amyrin, and CYP72A397 was oleanolic acid 23-hydroxylase involved in hederagenin production from oleanolic acid. Engineered yeast co-expressing KsBAS, CYP716A94 and CYP72A397 produced hederagenin. Kalopanax septemlobus CYP72A397 is a novel CYP enzyme that synthesizes hederagenin aglycone from oleanolic acid as a single product. In conclusion, we characterized three genes participating in sequential steps for hederagenin biosynthesis from β-amyrin, which are likely to play a major role in hederagenin saponin biosynthesis in K. septemlobus.
五加科的辽东楤木，俗称刺嫩芽，是一种极具价值的木本药用植物。辽东楤木含有约 15 种三萜皂苷，主要由齐墩果酸苷元组成。齐墩果酸苷元是植物中溶血皂苷的代表性前体。在本研究中，通过转录组分析发现了辽东楤木中参与齐墩果酸皂苷生物合成的基因。454 焦磷酸测序后，从头组装生成 82698 条独特序列，包括 17747 条 contigs 和 64951 条 singletons。鲨烯环氧化酶 (OSC) 是一种酶，可催化 2,3-氧化鲨烯形成多种三萜骨架。在酵母中异源表达 OSC 序列表明，KsBAS 是 β-齐墩果酸合酶基因。细胞色素 P450 基因 (CYP) 是植物基因组中的一个超基因家族，在皂角苷元苷的生物合成中起着关键作用。通过对辽东楤木转录组测序，共获得 95 条 contigs 和 110 条 singlets 注释为 CYP。通过在酵母中的异源表达，我们发现 CYP716A94 是β-齐墩果酸 28-氧化酶，参与从 β-齐墩果酸生成齐墩果酸，CYP72A397 是齐墩果酸 23-羟化酶，参与从齐墩果酸生成齐墩果酸苷元。工程酵母共表达 KsBAS、CYP716A94 和 CYP72A397 可产生齐墩果酸苷元。辽东楤木 CYP72A397 是一种新型 CYP 酶，可将齐墩果酸作为单一产物合成齐墩果酸苷元。总之，我们鉴定了三个参与从 β-齐墩果酸合成齐墩果酸苷元的基因，它们可能在辽东楤木中齐墩果酸皂苷的生物合成中起主要作用。
