Department of Chemical Engineering, Tsinghua University, Beijing, China.
PLoS One. 2011 Jan 18;6(1):e16228. doi: 10.1371/journal.pone.0016228.
The tolerance of cells toward different stresses is very important for industrial strains of microbes, but difficult to improve by the manipulation of single genes. Traditional methods for enhancing cellular tolerances are inefficient and time-consuming. Recently, approaches employing global transcriptional or translational engineering methods have been increasingly explored. We found that an exogenous global regulator, irrE from an extremely radiation-resistant bacterium, Deinococcus radiodurans, has the potential to act as a global regulator in Escherichia coli, and that laboratory-evolution might be applied to alter this regulator to elicit different phenotypes for E. coli.
METHODOLOGY/PRINCIPAL FINDINGS: To extend the methodology for strain improvement and to obtain higher tolerances toward different stresses, we here describe an approach of engineering irrE gene in E. coli. An irrE library was constructed by randomly mutating the gene, and this library was then selected for tolerance to ethanol, butanol and acetate stresses. Several mutants showing significant tolerances were obtained and characterized. The tolerances of E. coli cells containing these mutants were enhanced 2 to 50-fold, based on cell growth tests using different concentrations of alcohols or acetate, and enhanced 10 to 100-fold based on ethanol or butanol shock experiments. Intracellular reactive oxygen species (ROS) assays showed that intracellular ROS levels were sharply reduced for cells containing the irrE mutants. Sequence analysis of the mutants revealed that the mutations distribute cross all three domains of the protein.
To our knowledge, this is the first time that an exogenous global regulator has been artificially evolved to suit its new host. The successes suggest the possibility of improving tolerances of industrial strains by introducing and engineering exogenous global regulators, such as those from extremophiles. This new approach can be applied alone or in combination with other global methods, such as global transcriptional machinery engineering (gTME) for strain improvements.
细胞对不同压力的耐受能力对于工业微生物菌株非常重要，但通过单个基因的操作很难提高。传统的提高细胞耐受性的方法效率低且耗时。最近，越来越多的研究采用全局转录或翻译工程方法。我们发现，一种来自极端耐辐射细菌 Deinococcus radiodurans 的外源性全局调节剂 irrE 有可能在大肠杆菌中充当全局调节剂，并且可以通过实验室进化来改变该调节剂，从而引发大肠杆菌的不同表型。
方法/主要发现：为了扩展菌株改进的方法，并获得对不同压力的更高耐受性，我们在此描述了一种在大肠杆菌中工程化 irrE 基因的方法。通过随机突变该基因构建了 irrE 文库，然后对该文库进行了耐乙醇、正丁醇和乙酸盐压力的选择。获得了几个表现出显著耐受性的突变体，并对其进行了表征。基于不同浓度的醇或乙酸盐的细胞生长试验，含有这些突变体的大肠杆菌细胞的耐受性提高了 2 至 50 倍，基于乙醇或正丁醇冲击试验，耐受性提高了 10 至 100 倍。细胞内活性氧（ROS）测定表明，含有 irrE 突变体的细胞内 ROS 水平急剧降低。突变体的序列分析表明，突变分布在蛋白质的三个结构域。
据我们所知，这是第一次人为地进化外源性全局调节剂使其适应新宿主。这些成功表明，通过引入和工程化外源性全局调节剂（例如来自极端微生物的调节剂）来提高工业菌株的耐受性是可能的。这种新方法可以单独使用或与其他全局方法（例如全局转录机制工程（gTME））结合使用，以进行菌株改进。
