Evolution, genomic analysis, and reconstruction of isobutanol tolerance in Escherichia coli.
Evolution, genomic analysis, and reconstruction of isobutanol tolerance in Escherichia coli.
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DOI：
10.1038/msb.2010.98
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发表时间：
2010-12-21
影响因子：
9.9
通讯作者：
中科院分区：
文献类型：
--
作者：
大肠杆菌已经被改造为生产异丁醇，滴度达到大于毒性水平。然而，异丁醇对细胞的具体影响从未被完全理解。在这里，我们的目的是确定异丁醇反应的基因型-表型关系。一个异丁醇耐受突变体分离与系列转移。使用全基因组测序，然后进行基因修复和敲除，我们确定了五个突变（acrA，gatY，tnaA，yhbJ和marCRAB），这些突变主要负责增加异丁醇耐受性。我们成功地重建的耐受性表型相结合的这五个位点的缺失，并确定葡糖胺-6-磷酸作为一个重要的代谢产物异丁醇耐受性，这大概增强膜合成。异丁醇耐受性突变体也显示出对正丁醇和2-甲基-1-丁醇的耐受性增加，但与亲本菌株相比，乙醇耐受性没有改善，对己烷和氯霉素的敏感性更高。这些结果表明，与一般溶剂或抗生素胁迫相比，C4，C5酒精胁迫对细胞的影响不同。有趣的是，改善的异丁醇耐受性没有增加异丁醇生产的最终滴度。
Escherichia coli has been engineered to produce isobutanol, with titers reaching greater than the toxicity level. However, the specific effects of isobutanol on the cell have never been fully understood. Here, we aim to identify genotype–phenotype relationships in isobutanol response. An isobutanol-tolerant mutant was isolated with serial transfers. Using whole-genome sequencing followed by gene repair and knockout, we identified five mutations (acrA, gatY, tnaA, yhbJ, and marCRAB) that were primarily responsible for the increased isobutanol tolerance. We successfully reconstructed the tolerance phenotype by combining deletions of these five loci, and identified glucosamine-6-phosphate as an important metabolite for isobutanol tolerance, which presumably enhanced membrane synthesis. The isobutanol-tolerant mutants also show increased tolerance to n-butanol and 2-methyl-1-butanol, but showed no improvement in ethanol tolerance and higher sensitivity to hexane and chloramphenicol than the parental strain. These results suggest that C4, C5 alcohol stress impacts the cell differently compared with the general solvent or antibiotic stresses. Interestingly, improved isobutanol tolerance did not increase the final titer of isobutanol production.
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影响因子：
2.1
作者：
Hollands, Kerry;Busby, Stephen J. W.;Lloyd, Georgina S.
通讯作者：
Lloyd, Georgina S.
影响因子：
9.9
作者：
Brynildsen, Mark P.;Liao, James C.
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影响因子：
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CHAO, YP;LIAO, JC
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Liao, James C.
影响因子：
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作者：
Hirakawa, H;Inazumi, Y;Yamaguchi, A
通讯作者：
Yamaguchi, A