A cost-effective polyphosphate-based metabolism fuels an all E. coli cell-free expression system
A cost-effective polyphosphate-based metabolism fuels an all E. coli cell-free expression system
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DOI：
10.1016/j.ymben.2014.10.007
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发表时间：
2015-01-01
影响因子：
8.4
通讯作者：
Noireaux, Vincent
中科院分区：
文献类型：
--
作者：
Caschera, Filippo;Noireaux, Vincent
本文介绍了一种新的具有成本效益的代谢方式，它为无细胞蛋白质合成提供了一种ATP再生系统。六偏磷酸钠（一种多聚磷酸盐分子）作为磷酸盐供体，与麦芽糊精（一种用作碳源以刺激糖酵解的多糖）一起使用。值得注意的是，这种代谢不需要添加酶，它是由大肠杆菌粗提物中存在的内源性催化剂来进行的。这种新的ATP再生系统能够有效地循环利用无机磷酸盐，而无机磷酸盐是蛋白质合成的一种强抑制剂。我们表明，在孵育5小时后，分批模式反应中可合成高达1.34 - 1.65毫克/毫升的活性报告蛋白。与基于噬菌体转录的典型混合体外蛋白质合成系统不同，表达是通过大肠杆菌启动子进行的，仅使用提取物提供的内源性转录 - 翻译分子机制。我们证明，传统昂贵的能量再生系统，如磷酸肌酸、磷酸烯醇式丙酮酸或磷酸甘油酸，可以被一种适用于无细胞蛋白质合成应用的具有成本效益的代谢方案所取代。我们的工作还表明，无细胞系统是代谢工程的有用平台。（C）2014国际代谢工程学会。由爱思唯尔公司出版。保留所有权利。
A new cost-effective metabolism providing an ATP-regeneration system for cell-free protein synthesis is presented. Hexametaphosphate, a polyphosphate molecule, is used as phosphate donor together with maltodextrin, a polysaccharide used as carbon source to stimulate glycolysis. Remarkably, addition of enzymes is not required for this metabolism, which is carried out by endogenous catalysts present in the Escherichia coli crude extract. This new ATP regeneration system allows efficient recycling of inorganic phosphate, a strong inhibitor of protein synthesis. We show that up to 1.34-1.65 mg/mL of active reporter protein is synthesized in batch-mode reaction after 5 h of incubation. Unlike typical hybrid in vitro protein synthesis systems based on bacteriophage transcription, expression is carried out through E. colt promoters using only the endogenous transcription-translation molecular machineries provided by the extract. We demonstrate that traditional expensive energy regeneration systems, such as creatine phosphate, phosphoenolpyruvate or phosphoglycerate, can be replaced by a cost-effective metabolic scheme suitable for cell-free protein synthesis applications. Our work also shows that cell-free systems are useful platforms for metabolic engineering. (C) 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.