【摘要】 目的在大肠杆菌中建立一个能够生物合成紫杉烯(紫杉醇生物合成的第一个重要中间体)的代谢途径。方法利用PCR方法克隆编码大肠杆菌1-脱氧-5-磷酸木酮糖(D-1-deoxyxylulose 5-phosphate,DXP)合酶和异戊烯二磷酸异构酶(isopentenyl diphosphate isomerase,IDI)的基因,以及编码辣椒(Capsicum annuum)牻牛儿基牻牛儿基二磷酸(geranylgeranyl diphosphate,GGDP) 合酶的基因。分别构建含DXP合酶基因的表达载体pSLB208/DXP和含GGDP合酶与IDI异构酶基因的融合表达载体pAIG。将含紫杉烯合酶的表达载体pETB3和本研究构建的这2个表达栽体共转化大肠杆菌,诱导4个基因在大肠杆菌中共表达;并通过GC-MS分析大肠杆菌工程菌的代谢产物。结果在大肠杆菌中构建了一个合成紫杉烯的途径,通过GC-MS分析检测到紫杉烯的合成。结论利用代谢途径工程合成紫杉醇中间体是可行的,可为其他萜类化合物中间体代谢工程研究提供坚实的物质基础。更多还原

【Abstract】 OBJECTIVE To construct a metabolic pathway for producing taxadiene in Escherichia coli. METHODS The genes encoding isopentenyl diphosphate (IDP) isomerase, 1-deoxyxylulose 5-phosphate (DXP) synthase and the gene encoding geranylgeranyl diphosphate (GGDP) synthase were cloned using PCR from E. coli and Capsicum annuum, respectively. The fusion expression vector pAIG harboring the genes of IDP isomerase and GGDP synthase and the expression vector pSLB208/DXP bearing the gene of DXP synthase were constructed. Two expression plasmids and expression plasmid pETB3 harboring the gene of taxadiene synthase from Taxus chinensis were co-transformed into E. coli. The four genes were simultaneously expressed by inducing, and the metabolic products were analyzed by gas chromatography-mass spec-trometry (GC-MS) .RESULTS A metabolic pathway for producing taxadiene was established in the engineered E. cocli. The synthesis of taxadiene was detected by GC-MS analysis.CONCLUSION The intermediate of paclitaxel (Taxol) can be obtained by metabolic engineering. The strains developed in this study can serve as a platform for the production of some terpenoid compound while a terpene synthase gene is available.更多还原