代谢工程酵母菌合成紫杉烯的研究

紫杉烯是紫杉醇生物合成的重要中间体,为在酿酒酵母(Saccharomyces cerevisiae)中建立一个生物合成紫杉烯的代谢途径,克隆了酵母的羟甲基戊二酰CoA(3-hydroxy-3-methylglutarylcoenzyme A,HMG-CoA)还原酶基因和=牛儿基=牛儿基二磷酸(geranylgeranyl diphosphate,GGDP)合酶基因,并构建了其融合表达载体pGBT9/HG;同时构建了包含紫杉烯合酶基因的表达载体pADH/TS;将这两个表达载体共转化酵母细胞,通过GC-MS分析检测工程酵母的代谢产物,结果表明获得的工程酵母能够合成紫杉烯,即在酵母细胞中建立了一个合成紫杉烯的代谢途径。

Preliminary Study on Metabolic Engineering of Yeast for Producing Taxadiene

Paclitaxel (taxol) is a potent antimitotic agen t with excellent activity against a range of cancers. The limited supply of this drug from the natural sou rce, the bark of the yew (Taxus), prompted intensive efforts to develop alternate mea ns of taxol production. Great progress has been made in recent years toward understand ing the biosynthesis of taxol. Many functional genes have been cloned and identified in taxol biosynthetic pathway. Among those genes the taxadiene synthase gene is of importance. The enzyme catalyzes the conversion of geranylgeranyl diphosphate (GGDP) to taxadiene, which is the first intermediate with the taxol skeleton, bu t is not easily obtained. A metabolic pathway for producing taxadiene has been constructed in Saccharomyces cerevisiae. The gene fragment of the catal ytic domain of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was cloned. The fusion expression vector pGBT9/HG harboring the HMG-CoA reductase and geranylgeranyl diphosphate synthase was constructed, and the other expressio n vector pADH/TS bearing the gene of taxadiene synthase from Taxus chinensis was also constructed. Then the two expression vectors were co-transformed into S. cerevisiae. The three genes were expressed combinatorially under the control o f alcohol dehydrogenase I promoter, respectively. The synthesis of taxadiene in ye ast cells was identified by gas chromatography-mass spectrometry (GC-MS) analysis. The results showed that the metabolic pathway for producing taxadiene was established in the engineered S. cerevisiae.