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首页> 外文期刊>Applied Microbiology >Combinatorial Strategies for Improving Multiple-Stress Resistance in Industrially Relevant Escherichia coli Strains
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Combinatorial Strategies for Improving Multiple-Stress Resistance in Industrially Relevant Escherichia coli Strains

机译：改善工业相关大肠杆菌菌株耐多重压力的组合策略

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High-cell-density fermentation for industrial production of chemicals can impose numerous stresses on cells due to high substrate, product, and by-product concentrations; high osmolarity; reactive oxygen species; and elevated temperatures. There is a need to develop platform strains of industrial microorganisms that are more tolerant toward these typical processing conditions. In this study, the growth of six industrially relevant strains of Escherichia coli was characterized under eight stress conditions representative of fed-batch fermentation, and strains W and BL21(DE3) were selected as platforms for transposon (Tn) mutagenesis due to favorable resistance characteristics. Selection experiments, followed by either targeted or genome-wide next-generation-sequencing-based Tn insertion site determination, were performed to identify mutants with improved growth properties under a subset of three stress conditions and two combinations of individual stresses. A subset of the identified loss-of-function mutants were selected for a combinatorial approach, where strains with combinations of two and three gene deletions were systematically constructed and tested for single and multistress resistance. These approaches allowed identification of (i) strain-background-specific stress resistance phenotypes, (ii) novel gene deletion mutants in E. coli that confer single and multistress resistance in a strain-background-dependent manner, and (iii) synergistic effects of multiple gene deletions that confer improved resistance over single deletions. The results of this study underscore the suboptimality and strain-specific variability of the genetic network regulating growth under stressful conditions and suggest that further exploration of the combinatorial gene deletion space in multiple strain backgrounds is needed for optimizing strains for microbial bioprocessing applications.
机译：由于高底物，产物和副产物的浓度高，用于工业化生产化学品的高细胞密度发酵会给细胞带来许多压力。高渗透压活性氧和高温。需要开发对这些典型加工条件更具耐受性的工业微生物的平台菌株。在这项研究中，在代表补料分批发酵的八个胁迫条件下表征了6株与工业相关的大肠杆菌的生长，并且由于其良好的抗性特性，选择了W和BL21（DE3）菌株作为转座子（Tn）诱变的平台。进行选择实验，然后进行针对性的或基于基因组的下一代测序的Tn插入位点确定，以鉴定在三种胁迫条件和两种个体胁迫组合下具有改善的生长特性的突变体。选择已鉴定的功能丧失突变体的一个子集用于组合方法，其中系统构建具有两个和三个基因缺失组合的菌株并测试其单抗和多胁迫抗性。这些方法允许鉴定（i）菌株背景特异性抗逆性表型，（ii）大肠杆菌中以菌株背景依赖性方式赋予单和多抗性的新基因缺失突变体，以及（iii）协同作用多个基因缺失赋予了比单个缺失更高的抗性。这项研究的结果强调了在压力条件下调节生长的遗传网络的亚最优性和菌株特异性变异性，并建议在多种菌株背景下进一步探索组合基因缺失空间，以优化用于微生物生物加工应用的菌株。

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