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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 drug therapies (ACTs) are considered to be the most effective method currently available for the treatment of falciparum malaria. However, artemisinin is under-supplied and expensive, limiting the widespread use of ACTs. The genetic engineering method was used to construct the heterologous isoprenoid biosynthesis pathway, and the E.coli fermentation could efficiently synthesize the antimalarial artemisinin precursor amorpha-4,11-diene. Firstly, the synthetic amorpha-4,11-diene synthase gene was introduced into Escherichia coli DHGT7, and the endogenous farnesyl pyrophosphate of Escherichia coli was used to obtain the amorpha-4,11-bis Alkene. In order to increase the supply of precursors and the mevalonate pathway of Enterococcus faecalis, the yield of amorpha-4,11-diene increased by 13.3-fold to 151 mg / L. Further study found three restriction enzymes, namely Amorpha-4,11-diene synthase, HMG-COA reductase and mevalonate kinase; by adjusting the level of these enzymes, amorpha-4,11 - The diene production increased 7.2-fold and reached 235 mg / L in shake flasks. The results provide a reference for the efficient biosynthesis of artemisinin precursor amorpha-4,11-diene.