Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene
Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene
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DOI：
10.1016/j.ymben.2008.07.007
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发表时间：
2009-01-01
影响因子：
8.4
通讯作者：
Keasling, Jay D.
中科院分区：
文献类型：
--
作者：
Anthony, Jennifer R.;Anthony, Larry C.;Keasling, Jay D.
在微生物宿主中引入或创造代谢途径，使得生产具有治疗和工业重要性的复杂化学物质成为可能。然而，这些途径在首次引入宿主生物时很少发挥最佳作用，并且通常会对宿主生长产生有害影响，导致所需产品的产量达不到最佳水平。用于改善工程生物合成途径生产的常用方法包括优化密码子使用，增强限速酶的生产，消除有毒中间体或副产物的积累，以改善细胞生长。我们利用这些技术提高了由大肠杆菌工程菌株生产抗疟疾化合物青蒿素的前体阿莫菲-4,11-二烯（阿莫菲二烯）的产量。首先，我们建立了一个简单的克隆系统，用于在大肠杆菌中表达amorphadiene生物合成途径，并鉴定了两种限速酶（甲戊酸激酶（MK）和amorphadiene合成酶（ADS））。通过优化启动子强度来平衡编码基因的表达，我们缓解了两个通路瓶颈，并将产量提高了5倍。当通过修改质粒拷贝数进一步增加这些基因的表达时，观察到与原始菌株相比，amorphadiene的产量增加了7倍。这里展示的方法适用于识别和消除其他构建的生物合成途径中的限速步骤。(C) 2008爱思唯尔公司版权所有。
The introduction or creation of metabolic pathways in microbial hosts has allowed for the production of complex chemicals of therapeutic and industrial importance. However, these pathways rarely function optimally when first introduced into the host organism and can often deleteriously affect host growth, resulting in suboptimal yields of the desired product. Common methods used to improve production from engineered biosynthetic pathways include optimizing codon usage, enhancing production of rate-limiting enzymes, and eliminating the accumulation of toxic intermediates or by products to improve cell growth. We have employed these techniques to improve production of amorpha-4,11-diene (amorphadiene), a precursor to the anti-malarial compound artemisinin, by an engineered strain of Escherichia coli. First we developed a simple cloning system for expression of the amorphadiene biosynthetic pathway in E. coli, which enabled the identification of two rate-limiting enzymes (mevalonate kinase (MK) and amorphadiene synthase (ADS)). By optimizing promoter strength to balance expression of the encoding genes we alleviated two pathway bottlenecks and improved production five fold. When expression of these genes was further increased by modifying plasmid copy numbers, a seven-fold increase in amorphadiene production over that from the original strain was observed. The methods demonstrated here are applicable for identifying and eliminating rate-limiting steps in other constructed biosynthetic pathways. (C) 2008 Elsevier Inc. All rights reserved.