Development of a native Escherichia coli induction system for ionic liquid tolerance.
Development of a native Escherichia coli induction system for ionic liquid tolerance.
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DOI：
10.1371/journal.pone.0101115
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Mukhopadhyay A
中科院分区：
文献类型：
--
作者：
Frederix M;Hütter K;Leu J;Batth TS;Turner WJ;Rüegg TL;Blanch HW;Simmons BA;Adams PD;Keasling JD;Thelen MP;Dunlop MJ;Petzold CJ;Mukhopadhyay A
溶解木质纤维素的能力使得某些离子液体（IL）非常有效地用于在生物燃料发酵的糖化之前预处理生物质。然而，糖水溶液中残留的IL可抑制生物燃料生产微生物的生长和功能。在大肠这种毒性可以通过异源表达由来自解木质肠杆菌的eilA编码的IL外排泵来部分克服。在目前的工作中，我们使用微阵列分析来鉴定天然E。coli IL-诱导型启动子，并开发用于调控eilA基因表达的控制系统。选择三种候选启动子PmarR '、Pydf 0'和Pydf A '，并与IPTG诱导型PlacUV 5系统进行比较以控制eilA的表达。基于PydfA'和PmarR'的系统在拯救E.大肠杆菌从典型的毒性水平的IL，从而消除了使用基于IPTG的系统用于这种耐受性工程的需要。我们提出了一个机制模型，表明诱导型控制系统降低靶基因的表达时，IL水平低。选择反应监测质谱分析显示，在高IL浓度下，与其它启动子相比，在PydfA'和PmarR'的控制下，EilA蛋白水平显著升高。此外，在设计用于确定适应性的混合培养物竞争中，含有pPmarR ′-eilA的菌株胜过具有其它启动子构建体的菌株，在IL浓度高于150 mM时最显著。这些结果表明，天然启动子如PmarR ′可提供用于调节宿主工程中异源基因表达的有效系统，并简化工业上有用菌株的开发。
The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In E. coli this toxicity can be partially overcome by the heterologous expression of an IL efflux pump encoded by eilA from Enterobacter lignolyticus. In the present work, we used microarray analysis to identify native E. coli IL-inducible promoters and develop control systems for regulating eilA gene expression. Three candidate promoters, PmarR’, PydfO’, and PydfA’, were selected and compared to the IPTG-inducible PlacUV5 system for controlling expression of eilA. The PydfA’ and PmarR’ based systems are as effective as PlacUV5 in their ability to rescue E. coli from typically toxic levels of IL, thereby eliminating the need to use an IPTG-based system for such tolerance engineering. We present a mechanistic model indicating that inducible control systems reduce target gene expression when IL levels are low. Selected-reaction monitoring mass spectrometry analysis revealed that at high IL concentrations EilA protein levels were significantly elevated under the control of PydfA’ and PmarR’ in comparison to the other promoters. Further, in a pooled culture competition designed to determine fitness, the strain containing pPmarR’-eilA outcompeted strains with other promoter constructs, most significantly at IL concentrations above 150 mM. These results indicate that native promoters such as PmarR’ can provide effective systems for regulating the expression of heterologous genes in host engineering and simplify the development of industrially useful strains.
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影响因子：
3.5
作者：
Ouellet M;Adams PD;Keasling JD;Mukhopadhyay A
通讯作者：
Mukhopadhyay A
影响因子：
3.2
作者：
Chubiz, Lon M.;Rao, Christopher V.
通讯作者：
Rao, Christopher V.
影响因子：
46.9
作者：
Dahl, Robert H.;Zhang, Fuzhong;Keasling, Jay D.
通讯作者：
Keasling, Jay D.
影响因子：
5.6
作者：
BRISSETTE, JL;WEINER, L;MODEL, P
通讯作者：
MODEL, P
影响因子：
5
作者：
Li, Qiang;Jiang, Xinglin;Yang, Jianming
通讯作者：
Yang, Jianming