Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli.
Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli.
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DOI：
10.1186/1475-2859-10-18
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发表时间：
2011-03-25
影响因子：
6.4
通讯作者：
Lin XN
中科院分区：
文献类型：
--
作者：
Minty JJ;Lesnefsky AA;Lin F;Chen Y;Zaroff TA;Veloso AB;Xie B;McConnell CA;Ward RJ;Schwartz DR;Rouillard JM;Gao Y;Gulari E;Lin XN
异丁醇是一种很有前途的下一代生物燃料，具有高产微生物产量，但这种分子的毒性降低了发酵的体积生产力和最终滴度。有机溶剂耐受性是一种复杂的多基因表型，难以采用合理的工程方法。我们采用实验进化、基因组重测序和基因表达研究来阐明适应外源异丁醇胁迫的遗传基础。在我们进化的谱系中获得的适应性表现出在最小和丰富培养基之间的拮抗多效性，并且似乎是针对长链醇的影响。通过研究多个独立谱系的基因型适应，我们发现了marC、hfq、mdh、acrAB、gatYZABCD和rph基因平行进化的证据。许多异丁醇耐受性谱系显示RpoS活性降低，可能与hfq或acrAB突变有关。与溶剂耐受性的复杂，多基因性质一致，我们观察到适应的多样性细胞过程。许多适应似乎涉及不同突变之间的上位性，这意味着异丁醇耐受性的崎岖适应性景观。我们观察到一种针对转录后调控和生化网络高中心节点的进化趋势。总的来说，我们观察到的基因型适应提示了基于重塑细胞包膜和令人惊讶的应激反应衰减的适应机制。我们已经发现了一组基因型适应，赋予外源异丁醇应激增加耐受性。我们的结果对进一步设计更多的异丁醇耐受大肠杆菌宿主菌株用于异丁醇生产具有直接意义。我们认为rpoS和转录后调控因子，如hfq、RNA解旋酶和sRNAs可能是未来全球表型工程中有趣的突变靶点。
Isobutanol is a promising next-generation biofuel with demonstrated high yield microbial production, but the toxicity of this molecule reduces fermentation volumetric productivity and final titer. Organic solvent tolerance is a complex, multigenic phenotype that has been recalcitrant to rational engineering approaches. We apply experimental evolution followed by genome resequencing and a gene expression study to elucidate genetic bases of adaptation to exogenous isobutanol stress. The adaptations acquired in our evolved lineages exhibit antagonistic pleiotropy between minimal and rich medium, and appear to be specific to the effects of longer chain alcohols. By examining genotypic adaptation in multiple independent lineages, we find evidence of parallel evolution in marC, hfq, mdh, acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced RpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the complex, multigenic nature of solvent tolerance, we observe adaptations in a diversity of cellular processes. Many adaptations appear to involve epistasis between different mutations, implying a rugged fitness landscape for isobutanol tolerance. We observe a trend of evolution targeting post-transcriptional regulation and high centrality nodes of biochemical networks. Collectively, the genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol stress based on remodeling the cell envelope and surprisingly, stress response attenuation. We have discovered a set of genotypic adaptations that confer increased tolerance to exogenous isobutanol stress. Our results are immediately useful to further efforts to engineer more isobutanol tolerant host strains of E. coli for isobutanol production. We suggest that rpoS and post-transcriptional regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis targets for future global phenotype engineering.
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