J Integr Plant Biol. ›› 2002, Vol. 44 ›› Issue (2): 181-187.

• Research Articles • Previous Articles     Next Articles

cDNA Cloning, Expression and Characterization of Taxadiene Synthase, a Diterpene Cyclase from Taxus chinensis

WANG Wei, SHI Qing, ZHU Ping, OUYANG Tao, LI Nong and CHENG Ke-Di   

Abstract:

Taxadiene synthase, a diterpene cyclase, catalyzes the conversion of geranylgeranyl diphosphate (GGPP) to taxadiene, a key intermediate in Taxol biosynthesis in yew. A 2 151 bp cDNA fragment encoding taxadiene synthase of Taxus chinensis (Pilg.) Rehd. was cloned by homology-based PCR and cDNA library screening. The 5′-terminal 611 bp cDNA fragment of taxadiene synthase was isolated by PCR. The two fragments were ligated together and gave a 2712 bp cDNA fragment with a 2586 bp open reading frame (ORF), encoding 862 amino acid residues including a presumptive plastidial transit peptide. The taxadiene synthase of T. chinensis most closely resembles the one from T. brevifolia(97% identity). Heterologous overexpression of 2.5 kb cDNA fragment from T. chinensis was obtained using a fusion expression vector pET-32a and the Escherichia coli strain BL21trxB. The expressed proteins from E. coli BL21trxB were present as inclusion bodies. After the inclusion bodies were denatured, renatured and refolded, the recombinant enzyme was purified by a single step with a His binding metal affinity column. The catalytic product of taxadiene synthase of T. chinensis was detected by capillary gas chromatography mass spectrometry (GC-MS) and identified as taxa 4(5),11(12)-diene.

中国红豆杉紫杉烯合酶cDNA的分离、表达和鉴定

王伟  石青  朱平  欧阳涛  李秾  程克棣*

(中国医学科学院、中国协和医科大学药物研究所,卫生部天然药物生物合成重点实验室,北京 100050)

摘要:紫杉烯合酶是一种二萜环化酶,催化牻牛儿基牛儿基焦磷酸形成紫杉醇生物合成过程中的中间体紫杉烯.利用PCR扩增同源探针筛选cDNA文库,克隆了一个编码中国红豆杉(Taxus chinensis (Pilg.) Rehd.)紫杉烯合酶3′端的2 151 bpcDNA片段,也通过PCR扩增得到了该基因5′端的611 bpcDNA片段,将这两个cDNA片段拼接在一起,得到长2 712 bpcDNA片段,具有一个2 586个碱基的开放阅读框架(ORF),编码包括质体转移肽在内的共862个氨基酸残基;该酶与太平洋红豆杉紫杉烯合酶有97%的同源性(identity),与其他植物萜类环化酶也有较高的同源性.利用融合表达载体pET-32a在大肠杆菌BL21trxB中表达,所表达的融合蛋白以包含体形式存在.包含体经过变性、复性和再折叠,利用His残基亲和凝胶柱层析得到融合的紫杉烯合酶.用毛细管气相色谱-质谱联用对酶促反应产物进行分析,结果表明,融合的紫杉烯合酶能催化产生4(5),11(12)-紫杉烯.

 

关键词 中国红豆杉 紫杉醇生物合成 紫杉烯合酶

*通讯作者 Email: chengkd@imm.ac.cn

 

Key words: Taxus chinensis, Taxol biosynthesis, taxadiene synthase

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