Department of Chemical Engineering, Texas A&M University, College Station, TX, USA.
Metab Eng. 2012 Sep;14(5):579-90. doi: 10.1016/j.ymben.2012.05.002. Epub 2012 May 29.
Toxicity of products or feedstock components poses a challenge in the biocatalyst-based production of fuels and chemicals. The genetic determinants that are involved in increased resistance to an inhibitor form the adaptive landscape for the phenotype; so in order to engineer more robust biocatalysts, a better understanding of the adaptive landscape is required. Here, we used an adaptive laboratory evolution method called visualizing evolution in real time (VERT) to help map out part of the adaptive landscape of Escherichia coli tolerance to the biofuel n-butanol. VERT enables identification of adaptive events (population expansions triggered by adaptive mutants) via visualization of the relative proportions of different fluorescently-labeled cells. Knowledge of the occurrence of adaptive events allows for a more systematic isolation of adaptive mutants while simultaneously reducing the number of missed adaptive mutants (and the underlying adaptive mechanisms) that result from clonal interference during the course of in vitro evolution. Based on the evolutionary dynamics observed, clonal interference was found to play a significant role in shaping the population structure of E. coli during exposure to n-butanol, and VERT helped to facilitate the isolation of adaptive mutants from the population. We further combined adaptive laboratory evolution with genome shuffling to significantly enhance the desired n-butanol tolerance phenotype. Subsequent transcriptome analysis of the isolated adaptive mutants revealed different mechanisms of n-butanol resistance in different lineages. In one fluorescently-marked subpopulation, members of the Fur regulon were upregulated; which was not observed in the other subpopulation. In addition, genome sequencing of several adaptive mutants revealed the genetic basis for some of the observed transcriptome profiles. We further elucidated the potential role of the iron-related gene in n-butanol tolerance via overexpression and deletion studies and hypothesized that the upregulation of the iron-related genes indirectly led to modifications in the outer membrane, which contributed to enhanced n-butanol tolerance.
产物或原料成分的毒性给基于生物催化剂的燃料和化学品生产带来了挑战。参与提高对抑制剂抗性的遗传决定因素构成了表型的适应性景观；因此，为了设计更稳健的生物催化剂，需要更好地了解适应性景观。在这里，我们使用了一种称为实时可视化进化（VERT）的适应性实验室进化方法来帮助绘制大肠杆菌对生物燃料正丁醇耐受性的部分适应性景观。VERT 通过可视化不同荧光标记细胞的相对比例来识别适应性事件（适应性突变体引发的种群扩张）。适应性事件的发生知识允许更系统地分离适应性突变体，同时减少由于体外进化过程中的克隆干扰而导致的错过适应性突变体（和潜在的适应性机制）的数量。基于观察到的进化动态，发现克隆干扰在暴露于正丁醇时对大肠杆菌的种群结构起着重要作用，VERT 有助于从种群中分离适应性突变体。我们进一步将适应性实验室进化与基因组改组相结合，显著增强了所需的正丁醇耐受性表型。随后对分离的适应性突变体进行转录组分析，揭示了不同谱系中不同的正丁醇抗性机制。在一个荧光标记的亚群中，Fur 调控子的成员上调；而在另一个亚群中则没有观察到这种情况。此外，对几个适应性突变体的基因组测序揭示了一些观察到的转录组图谱的遗传基础。我们通过过表达和缺失研究进一步阐明了与铁相关的基因在正丁醇耐受性中的潜在作用，并假设铁相关基因的上调间接导致了外膜的修饰，这有助于提高正丁醇耐受性。
