在大肠杆菌内引入甲羟戊酸途径高效合成抗疟药青蒿素前体--紫穗槐-4,11-二烯

Biosynthesis of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli through introducing mevalonate pathway

以青蒿素为基础的联合药物疗法(ACTs)被认为是目前治疗恶性疟疾的最有效方法。然而青蒿素供应不足且价格昂贵,限制了ACTs的广泛使用。采用基因工程手段构建异源类异戊二烯生物合成途径,利用大肠杆菌发酵能高效合成抗疟药青蒿素前体——紫穗槐-4,11-二烯。首先在大肠杆菌Escherichia coli DHGT7中引入人工合成的紫穗槐-4,11-二烯合酶基因,利用大肠杆菌内源的法尼基焦磷酸,成功获得了紫穗槐-4,11-二烯。为提高前体供给,引入粪肠球菌的甲羟戊酸途径,紫穗槐-4,11-二烯的产量提高了13.3倍,达到151 mg/L。进一步研究发现了3个限制酶,分别是紫穗槐-4,11-二烯合酶、HMG-COA还原酶和甲羟戊酸激酶;通过调节这些酶的水平,紫穗槐-4,11-二烯产量提高了7.2倍,在摇瓶中达到235 mg/L。研究结果为高效生物合成抗疟药青蒿素前体——紫穗槐-4,11-二烯提供了参考。

Artemisinin-based combination therapies（ACTs） are recommended to be the most effective therapies for the first-line treatment of uncomplicated falciparum malaria.However,artemisinin is often in short supply and unaffordable to most malaria patients,which limits the wide use of ACTs.Production of amorpha-4,11-diene,an artemisinin precursor,was investigated by engineering a heterologous isoprenoid biosynthetic pathway in Escherichia coli.The production of amorpha-4,11-diene was achieved by expression of a synthetic amorpha-4,11-diene synthase gene in Escherichia coli DHGT7 and further improved by about 13.3 fold through introducing the mevalonate pathway from Enterococcus faecalis.After eliminating three pathway bottlenecks including amorpha-4,11-diene synthase,HMG-CoA reducase and mevalonate kinase by optimizing the metabolic flux,the yield of amorpha-4,11-diene was increased by nearly 7.2 fold and reached at 235 mg/L in shaking flask culture.In conclusion,an engineered Escherichia coli was constructed for high-level production of amorpha-4,11-diene.