Gene identification and semisynthesis of the anti-inflammatory oleanane-type triterpenoid wilforlide A
Gene identification and semisynthesis of the anti-inflammatory oleanane-type triterpenoid wilforlide A
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DOI：
10.1111/nph.19427
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发表时间：
2023-11-28
期刊：
影响因子：
9.4
通讯作者：
Liu，Changli
中科院分区：
文献类型：
--
作者：
Liu，Yuan;Zhou，Jiawei;Liu，Changli
雷公藤内酯甲是雷公藤中微量产生的主要活性成分之一，具有良好的抗炎和免疫抑制作用。尽管雷公藤内酯甲的结构看似简单，但其生物合成途径仍不清楚，本研究采用基因特异性表达分析和基因组挖掘技术鉴定候选基因，并在体外和体内研究其功能。利用模块化两步（M2 S）技术和CRISPR-Cas9方法构建工程酵母，发现了一个催化雷公藤内酯甲生物合成过程中C-22羟基化的细胞色素P450基因TwCYP 82 AS 1。我们还发现，TwCYP 712 K1至K3可以进一步氧化除无栓烷型三萜之外的齐墩果烷型三萜的C-29羧化。工程酵母中生物合成途径的重建增加了前体供应，并将TwCYP 82 AS 1和TwCYP 712 KS结合产生芽生酸，将其短暂酸化以实现雷公藤内苷A的半合成。我们的工作提出了一种替代的代谢工程方法来获得雷公藤内苷A，而不依赖于从植物中提取。
Wilforlide A is one of the main active constituents produced in trace amounts inTripterygium wilfordiiHook F, which has excellent anti‐inflammatory and immune suppressive effects. Despite the seeming structural simplicity of the compound, the biosynthetic pathway of wilforlide A remains unknown.Gene‐specific expression analysis and genome mining were used to identify the gene candidates, and their functions were studiedin vitroandin vivo. A modularized two‐step (M2S) technique and CRISPR–Cas9 methods were used to construct engineering yeast.Here, we identified a cytochrome P450, TwCYP82AS1, that catalyses C‐22 hydroxylation during wilforlide A biosynthesis. We also found that TwCYP712K1 to K3 can further oxidize the C‐29 carboxylation of oleanane‐type triterpenes in addition to friedelane‐type triterpenes. Reconstitution of the biosynthetic pathway in engineered yeast increased the precursor supply, and combining TwCYP82AS1 and TwCYP712Ks produced abrusgenic acid, which was briefly acidified to achieve the semisynthesis of wilforlide A.Our work presents an alternative metabolic engineering approach for obtaining wilforlide A without relying on extraction from plants.