High-level production of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli.
High-level production of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli.
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DOI：
10.1371/journal.pone.0004489
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发表时间：
2009
期刊：
影响因子：
3.7
通讯作者：
Newman JD
中科院分区：
文献类型：
--
作者：
Tsuruta H;Paddon CJ;Eng D;Lenihan JR;Horning T;Anthony LC;Regentin R;Keasling JD;Renninger NS;Newman JD
青蒿素衍生物是青蒿素综合疗法的关键活性成分，青蒿素综合疗法是治疗疟疾的最有效疗法。由于原料是从生长季节长的植物中提取的，青蒿素往往供应短缺，发酵将是补充植物来源的一种有吸引力的替代生产方法。以前的工作表明，高水平的紫穗槐-4，11-二烯，青蒿素前体，可以在大肠杆菌中使用来自酵母（酿酒酵母）的异源甲羟戊酸途径，尽管重建的甲羟戊酸途径在特定的酶促步骤中受到限制。通过将异源甲羟戊酸途径中的改进与上级发酵工艺相结合，在补料分批发酵中实现了商业上相关的滴度。HMG-CoA合成酶和HMG-CoA还原酶（途径中的第二和第三种酶）的酵母基因被来自金黄色葡萄球菌的等同基因取代，产量增加一倍以上。通过优化发酵过程中的氮输送，进一步增加了无定形-4，11-二烯滴度。在碳氮限制条件下成功培养的改良菌株的细胞干重稳定在90 g/L，平均效价为27.4 g/L紫穗槐-4，11-二烯。通过发酵生产>25克/升的紫穗槐-4，11-二烯，然后化学转化为青蒿素，可以开发一种工艺，提供青蒿素的替代来源，用于青蒿素综合疗法。
Artemisinin derivatives are the key active ingredients in Artemisinin combination therapies (ACTs), the most effective therapies available for treatment of malaria. Because the raw material is extracted from plants with long growing seasons, artemisinin is often in short supply, and fermentation would be an attractive alternative production method to supplement the plant source. Previous work showed that high levels of amorpha-4,11-diene, an artemisinin precursor, can be made in Escherichia coli using a heterologous mevalonate pathway derived from yeast (Saccharomyces cerevisiae), though the reconstructed mevalonate pathway was limited at a particular enzymatic step. By combining improvements in the heterologous mevalonate pathway with a superior fermentation process, commercially relevant titers were achieved in fed-batch fermentations. Yeast genes for HMG-CoA synthase and HMG-CoA reductase (the second and third enzymes in the pathway) were replaced with equivalent genes from Staphylococcus aureus, more than doubling production. Amorpha-4,11-diene titers were further increased by optimizing nitrogen delivery in the fermentation process. Successful cultivation of the improved strain under carbon and nitrogen restriction consistently yielded 90 g/L dry cell weight and an average titer of 27.4 g/L amorpha-4,11-diene. Production of >25 g/L amorpha-4,11-diene by fermentation followed by chemical conversion to artemisinin may allow for development of a process to provide an alternative source of artemisinin to be incorporated into ACTs.
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影响因子：
2.7
作者：
Geiser, M;Cèbe, R;Schmitz, R
通讯作者：
Schmitz, R
影响因子：
7.7
作者：
RIESENBERG, D
通讯作者：
RIESENBERG, D
影响因子：
5
作者：
Regentin, R;Frykman, S;Licari, P
通讯作者：
Licari, P
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL
影响因子：
64.8
作者：
Ro, DK;Paradise, EM;Keasling, JD
通讯作者：
Keasling, JD