Abstract：
Ursolic acid has gradually attracted much attention due to its unique pharmacological activities and valuable market value in recent years. Currently, ursolic acid is mostly extracted from loquat leaves, but the plant extraction method has low yield and high cost, and chemical synthesis is not readily available, so the biosynthesis method provides a new source for ursolic acid. α-amyrin acts as the main precursor for the synthesis of ursolic acid, and its yield is positively correlated with ursolic acid yield. Oxidosqualene cyclase(OSC) belongs to a multigene family which can catalyze the common precursor 2,3-oxidosqualene to generate different types of triterpene backbones, and plays a decisive role in the synthesis of triterpenoids. However, there are fewer reported key genes catalyzing the synthesis of α-amyrin in medicinal plants, and the yield and proportion of α-amyrin in the catalyzed products have always been a focus of research. In this study, ItOSC2, MdOSC1, AaOSC2 and CrAS, four enzymes capable of catalyzing the production of α-amyrin from 2,3-oxidosqualene, were cloned from Iris tectorum, Malus domestica, Artemisia annua and Catharanthus roseus, subject to sequence alignment and phylogenetic tree analyses, and transformed into Saccharomyces cerevisiae as plasmids. After 7 days of fermentation, the yield and proportions of α-amyrin, β-amyrin and ergosterol were measured. Finally, AaOSC2 with the best ability to catalyze the generation of α-amyrin was filtered out, providing a key gene element for the later construction of engineered yeast strains with high production of α-amyrin and ursolic acid.
摘要：
熊果酸由于其独特的药理活性和宝贵的市场价值,近年来逐渐受到人们的关注。目前,熊果酸主要从枇杷叶中提取，但植物提取法收率低、成本高,化学合成并不容易获得，因此，生物合成方法为熊果酸提供了新的来源。α-Amyrin是合成熊果酸的主要前体，其产量与熊果酸产量呈正相关。氧化角鲨烯环化酶（OSC）属于多基因家族，可以催化共同的前体2,3-氧化角鲨烯生成不同类型的三萜主链，在三萜类化合物的合成中起着决定性的作用。然而,在药用植物中催化α-amyrin合成的关键基因报道较少，催化产物中α-Amyrin的产率和比例一直是研究的重点。在这项研究中,ItOSC2，MdOSC1，AaOSC2和CrAS，四种能够催化从2,3-氧化角鲨烯生产α-amyrin的酶，是从Iristectorum克隆的，家蝇,黄花蒿和长春花,进行序列比对和系统发育树分析，并作为质粒转化为酿酒酵母。发酵7天后，α-amyrin的产量和比例，测定β-淀粉肽和麦角甾醇。最后,筛选出催化α-amyrin生成能力最好的AaOSC2,为后期构建高产α-amyrin和熊果酸的工程酵母菌株提供了关键基因元件。
