Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin
Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin
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DOI：
10.1073/pnas.1110740109
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发表时间：
2012-01-17
影响因子：
11.1
通讯作者：
Paddon, Chris J.
中科院分区：
文献类型：
--
作者：
Westfall, Patrick J.;Pitera, Douglas J.;Paddon, Chris J.
疟疾是由疟原虫引起的，每年导致近100万人死亡和2亿多新感染。世界卫生组织建议使用以青蒿素为基础的联合疗法来治疗疟疾。青蒿素是从青蒿属植物中分离得到的一种倍半萜内酯。然而，青蒿素的供应和价格波动很大，另一种生产方法对于增加供应将是有价值的。我们描述了基于从工程酿酒酵母发酵生产青蒿素前体4，11-二烯，并将其化学转化为二氢青蒿酸，进而转化为青蒿素，从而开发半合成青蒿素供应的进展。以前生产青蒿素前体的努力使用酿酒酵母S288C过表达甲氧丙酮酸途径的特定基因[Ro等人。(2006)自然440：940-943]。我们现在已经在酿酒酵母CEN.PK2中过表达了甲氧丙酮酸途径到ERG20的每一种酶，并将其与CEN.PK2进行了比较，该CEN.PK2的工程与先前工程的S288C菌株相同。甲氧丙酮酸途径的每一种酶的过度表达使青蒿酸的产量增加了一倍，而紫穗菊-4，11-二烯的产量是青蒿酸的10倍。因此，我们专注于紫穗菊-4，11-二烯的生产。对重组的CEN.PK2紫穗槐-4，11-二烯菌株的发酵工艺进行了改进，生产了>；40g/L的产品。开发了一种将紫穗菊-4，11-二烯转化为二氢青蒿酸的化学工艺，该二氢青蒿酸随后可以转化为青蒿素。所描述的菌株和程序代表了生产半合成青蒿素的完整过程。
Malaria, caused by Plasmodium sp, results in almost one million deaths and over 200 million new infections annually. The World Health Organization has recommended that artemisinin-based combination therapies be used for treatment of malaria. Artemisinin is a sesquiterpene lactone isolated from the plant Artemisia annua. However, the supply and price of artemisinin fluctuate greatly, and an alternative production method would be valuable to increase availability. We describe progress toward the goal of developing a supply of semisynthetic artemisinin based on production of the artemisinin precursor amorpha-4,11-diene by fermentation from engineered Saccharomyces cerevisiae, and its chemical conversion to dihydroartemisinic acid, which can be subsequently converted to artemisinin. Previous efforts to produce artemisinin precursors used S. cerevisiae S288C overexpressing selected genes of the mevalonate pathway [Ro et al. (2006) Nature 440:940-943]. We have now overexpressed every enzyme of the mevalonate pathway to ERG20 in S. cerevisiae CEN.PK2, and compared production to CEN.PK2 engineered identically to the previously engineered S288C strain. Overexpressing every enzyme of the mevalonate pathway doubled artemisinic acid production, however, amorpha-4,11-diene production was 10-fold higher than artemisinic acid. We therefore focused on amorpha-4,11-diene production. Development of fermentation processes for the reengineered CEN.PK2 amorpha-4,11-diene strain led to production of >40 g/L product. A chemical process was developed to convert amorpha-4,11-diene to dihydroartemisinic acid, which could subsequently be converted to artemisinin. The strains and procedures described represent a complete process for production of semisynthetic artemisinin.