Improved n-butanol tolerance in Escherichia coli by controlling membrane related functions
Improved n-butanol tolerance in Escherichia coli by controlling membrane related functions
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DOI：
10.1016/j.jbiotec.2015.03.025
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发表时间：
2015-06-20
影响因子：
4.1
通讯作者：
Kim, Sun Chang
中科院分区：
文献类型：
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作者：
Bui, Le Minh;Lee, Ju Young;Kim, Sun Chang
随着化学品和燃料市场需求的不断增长，人们对利用生物途径生产正丁醇的兴趣重新燃起，以设计经济的发酵工艺，与目前占主导地位的化学合成竞争。正丁醇传统上是通过丙酮丁醇梭菌的 ABE 发酵生产的。然而，由于效率有限且缺乏进一步改进的基因改造工具，该系统在经济上不可行。或者，正丁醇合成途径成功转移到大肠杆菌中并迅速改进以达到与本地生产者相当的生产水平。然而，正丁醇的毒性已成为任何一种方法都必须解决的共同问题。此前，我们报道了通过工程化人工转录因子（ATF）成功提高了大肠杆菌的正丁醇耐受性，该转录因子可以同时修改多个靶标的表达水平，并将MG1655菌株的正丁醇耐受性提高至1.5%（vol/vol）正丁醇。然而，据观察，一些可能的正丁醇耐受机制在ATF表达时并未发生，特别是与膜相关的功能，如同卵粘性适应、铁摄取系统和外排泵系统。在这项工作中，我们试图通过将 ATF 与大肠杆菌中的膜相关功能相结合来增强与 ATF 相关的正丁醇耐受性，包括脂肪酸合成基因、铁摄取蛋白 FeoA 的过表达，以及将来自恶臭假单胞菌的 SrpABC 外排泵引入大肠杆菌。这种组合方法的协同效应使用于生产正丁醇的 MG1655 敲除菌株在含有 1%、1.5% 和 2%（体积/体积）正丁醇的培养物中的生长分别提高了 4、5 和 9 倍，并将耐受极限扩大到 2%（体积/体积）正丁醇。 (C) 2015 Elsevier B.V. 保留所有权利。
As the increasing demand from both chemical and fuel markets, the interest in producing n-butanol using biological route has been rejuvenated to engineer an economical fermentation process, competing with the currently-dominant chemical synthesis. n-Butanol has been traditionally produced from the ABE fermentation of Clostridium acetobutylicum. This system, however, is not economically feasible due to its limited efficiency and the lack of genetic modification tools for further improvements. Alternatively, n-butanol synthesis pathway was successfully transferred into Escherichia coli and rapidly improved to reach a level of production comparable to the native producer. Nevertheless, the toxicity of n-butanol has become a common issue that either approach has to deal with. Previously, we reported our success in improving n-butanol tolerance in E. coli by engineering an Artificial Transcription Factor (ATF) that can modify the expression level of multiple targets simultaneously and improved the n-butanol tolerance of MG1655 strain to 1.5% (vol/vol) n-butanol. However, it was observed that some possible n-butanol tolerance mechanisms did not occurred upon the ATF expression, especially the membrane-related functions such as the homeoviscous adaptation, iron uptaking system, and efflux pump system. In this work, we attempted to enhance the n-butanol tolerance associated with the ATF by combining it with the membrane-related functions in E. coli, including the overexpression of fatty acid synthesis genes, iron-uptaking protein FeoA, and introducing a SrpABC efflux pump from Pseudomonas putida into E. coli. The synergistic effect of this combinatorial approach led to 4, 5, and 9-fold improved growths in the cultures containing 1, 1.5, and 2% (vol/vol) n-butanol, respectively, of an MG1655 knockout strain engineered for n-butanol production, and expanded the tolerance limit to 2% (vol/vol) n-butanol. (C) 2015 Elsevier B.V. All rights reserved.