High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli
High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli
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DOI：
10.1002/bit.21017
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发表时间：
2006-11-05
影响因子：
3.8
通讯作者：
Keasling, Jay D.
中科院分区：
文献类型：
--
作者：
Newman, Jack D.;Marshall, Jessica;Keasling, Jay D.
重建微生物的合成代谢途径对大规模发酵生产药物具有很大的希望。通过在细菌中重建生物合成途径，传统上从稀缺的自然资源中收获的复杂分子可以在微生物培养中产生。在这里，我们报道了一株大肠杆菌含有一种外源的9基因生物合成途径，用于生产抗疟疾药物青蒿素的前体terpene amorpha4,11 -diene。以前的报告低估了该菌株的生产力，因为它具有挥发性。在这里，我们展示了amorphadiene从发酵罐中蒸发，半衰期约为50分钟。使用冷凝器，我们利用这种挥发性，将amorphadiene困在废气中。采用核磁共振波谱法对其进行鉴定，确定其纯度为89%。我们通过采用带有十二烷有机相的两相分配生物反应器在原位生产时捕获了摩菲迪烯。使用先前表征的石竹烯标准来校准amorphadiene的生产和捕获，确定产生的amorphadiene浓度为0.5 g/L培养基。从废气中收集的一项摩菲二烯标准表明，石竹烯标准高估了摩菲二烯的产量约30%。(c) 2006 Wiley期刊公司
Reconstructing synthetic metabolic pathways in microbes holds great promise for the production of pharmaceuticals in large-scale fermentations. By recreating biosynthetic pathways in bacteria, complex molecules traditionally harvested from scarce natural resources can be produced in microbial cultures. Here we report on a strain of Escherichia coli containing a heterologous, nine-gene biosynthetic pathway for the production of the terpene amorpha-4,11-diene, a precursor to the anti-malarial drug artemisinin. Previous reports have underestimated the productivity of this strain due to the volatility of amorphadiene. Here we show that amorphadiene evaporates from a fermentor with a half-life of about 50 min. Using a condenser, we take advantage of this volatility by trapping the amorphadiene in the off-gas. Amorphadiene was positively identified using nuclear magnetic resonance spectroscopy and determined to be 89% pure as collected. We captured amorphadiene as it was produced in situ by employing a two-phase partitioning bioreactor with a dodecane organic phase. Using a previously characterized caryophyllene standard to calibrate amorphadiene production and capture, the concentration of amorphadiene produced was determined to be 0.5 g/L of culture medium. A standard of amorphadiene collected from the off-gas showed that the caryophyllene standard overestimated amorphadiene production by approximately 30%. (c) 2006 Wiley Periodicals, Inc.