绿玉树羽扇豆醇合酶基因分离及异源表达

Isolation and heterologous expression of lupeol synthase gene from Euphorbia tirucalli L.

羽扇豆醇是一种重要的五环三萜化合物,具有抗肿瘤、抗感染、抑菌等多种药理活性。从非洲药用植物绿玉树(Euphorbia tirucalli L.)中分离出羽扇豆醇合酶基因EtOSC7,测序结果表明该基因全长2301 bp,编码766个氨基酸,与蓖麻(Ricinus communis)和木览(Bruguiera gymnorhiza)的羽扇豆醇合酶氨基酸序列的相似度分别为78%和76%。利用羊毛甾醇缺陷型酵母GIL77菌株对EtOSC7蛋白进行了功能验证,结果表明该蛋白能够催化羽扇豆醇的合成。以EtOSC7基因为基础,在大肠杆菌和酿酒酵母两种模式菌株中进行了羽扇豆醇生物合成的尝试,结果表明,在重组大肠杆菌中未检测到羽扇豆醇的生成,而在重组酿酒酵母中测得羽扇豆醇的产量为0.55 mg/L。研究结果可以为构建三萜化合物微生物细胞工厂提供新的基因元件,并为羽扇豆醇的生物合成提供理论支持。

Lupeol is an important pentacyclic triterpenoid with various pharmacological activities such as anti-tumor, anti-infection, and bacteriostasis. A lupeol synthase gene EtOSC7 was isolated from the medicinal plant Euphorbia tirucalli L.in Africa. The sequencing results showed that the gene was 2 301 bp in length and encoded 766 amino acids. The amino acid sequence similarities with the lupeol synthase of Ricinus communis and Bruguiera gymnorhiza were 78% and 76%, respectively. The EtOSC7 protein was functionally verified using the lanosterol-deficient yeast GIL77 strain, and the results showed that the protein could catalyze the synthesis of lupinol. Based on the EtOSC7 gene, the biosynthesis of lupeol was attempted in two model strains of Escherichia coli and Saccharomyces cerevisiae. The results showed that the production of lupeol was not detected in recombinant E. coli, while the yield of lupeol was 0.55 mg/L in recombinant S. cerevisiae. The results of this study can provide new genetic elements for the construction of triterpenoid microbial cell factories and provide theoretical support for the future biosynthesis of lupeol.