催化合成α-amyrin的氧化鲨烯环化酶基因克隆及功能表征  

Cloning and functional characterization of oxidosqualene cyclase gene forα-amyrin synthesis

作　　者：梁志鹏 于宛彤 苏新堯 侯红平[2] 薛建平[1,3] 刘佳 王彩霞 LIANG Zhi-peng;YU Wan-tong;SU Xin-yao;HOU Hong-ping;XUE Jian-ping;LIU Jia;WANG Cai-xia(School of Life Sciences,Huaibei Normal University,Huaibei 235000,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Anhui Provincial Engineering Laboratory for Efficient Utilization of Featured Resource Plants,Huaibei 235000,China)

机构地区：[1]淮北师范大学生命科学学院,安徽淮北235000 [2]中国中医科学院中药研究所,北京100700 [3]安徽省特色资源植物利用工程实验室,安徽淮北235000

出　　处：《中国中药杂志》2024年第12期3204-3211,共8页China Journal of Chinese Materia Medica

基　　金：国家重点研发计划项目(2019YFC19066);国家自然科学基金项目(82274048);安徽省高校优秀创新团队项目(2022AH010029)。

摘　　要：熊果酸近年来由于其独特的药理活性以及宝贵的市场价值,逐渐引起人们的广泛关注。目前熊果酸大多从枇杷叶中提取,但植物提取法产率低,成本高,化学合成法又不易获得,因此生物合成法为熊果酸提供了一个新的来源。α-amyrin作为合成熊果酸的主要前体物质,其产量与熊果酸产量呈正相关性。氧化鲨烯环化酶(OSC)属于多基因家族,可以催化常见的前体2,3-氧化鲨烯生成多种不同类型的三萜骨架,为三萜类物质的合成起决定性作用。但药用植物中催化合成α-amyrin的关键基因报道较少,且其催化产物中α-amyrin产量及占比一直是研究者们关注的重点。该文从鸢尾、苹果、青蒿和长春花中克隆出能够催化2,3-氧化鲨烯生成α-amyrin的4种环化酶ItOSC2、MdOSC1、AaOSC2和CrAS基因,系统地对4种环化酶进行序列比对以及进化树分析,并以质粒形式将其转化至酿酒酵母中进行异源表达。发酵7 d后测定其催化产物中α-amyrin、β-amyrin以及麦角固醇的产量及占比,筛选出一个催化生成α-amyrin能力最好的AaOSC2,为后期构建高产α-amyrin及熊果酸的工程酵母菌株提供关键基因元件。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 ofα-amyrin in medicinal plants,and the yield and proportion ofα-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 ofα-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 ofα-amyrin,β-amyrin and ergosterol were measured.Finally,AaOSC2 with the best ability to catalyze the generation ofα-amyrin was filtered out,providing a key gene element for the later construction of engineered yeast strains with high production ofα-amyrin and ursolic acid.

关 键 词：氧化鲨烯环化酶 α-amyrin β-amyrin 酿酒酵母 鲨烯 

分 类 号：R284.1[医药卫生—中药学]