Mapping temperature-sensitive mutations at a genome scale to engineer growth switches in Escherichia coli.

IF 8.5 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Molecular Systems Biology Pub Date : 2023-10-12 Epub Date: 2023-08-29 DOI:10.15252/msb.202311596
Thorben Schramm, Paul Lubrano, Vanessa Pahl, Amelie Stadelmann, Andreas Verhülsdonk, Hannes Link
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引用次数: 1

Abstract

Temperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. 1,269 of these mutants showed temperature-sensitive growth in a time-resolved competition assay. We reconstructed 94 TS mutants and measured their metabolism under growth arrest at 42°C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of nongrowing E. coli is perturbation-dependent. For example, 24 TS mutants of metabolic enzymes overproduced the direct substrate metabolite due to a bottleneck in their associated pathway. A strain with TS homoserine kinase (ThrBF267D ) produced homoserine for 24 h, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaXL289Q ) to decouple growth from arginine overproduction in engineered E. coli. These results provide a strategy to identify TS mutants en masse and demonstrate their large potential to produce bacterial metabolites with nongrowing cells.

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Abstract Image

在基因组范围内绘制温度敏感突变图,以设计大肠杆菌的生长开关。
温度敏感(TS)突变体是干扰和设计细胞系统的独特工具。在这里,我们用15120个大肠杆菌突变体构建了一个CRISPR文库,每个突变体在346种必需蛋白中的一种中都有一个氨基酸变化。这些突变体中的1269个在时间分辨竞争测定中显示出温度敏感性生长。我们重建了94个TS突变体,并使用代谢组学测量了它们在42°C生长停滞下的代谢。代谢组变化强烈且具有突变特异性,表明非生长型大肠杆菌的代谢具有扰动依赖性。例如,代谢酶的24个TS突变体由于其相关途径的瓶颈而过量产生直接底物代谢产物。一株含有TS高丝氨酸激酶(ThrBF267D)的菌株产生高丝氨酸24 h、 并且生产可通过温度进行调节。最后,我们使用DNA聚合酶III的TS亚基(DnaXL289Q)在工程大肠杆菌中使生长与精氨酸过量产生脱钩。这些结果提供了一种大规模鉴定TS突变体的策略,并证明了它们在非赛艇细胞中产生细菌代谢产物的巨大潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Molecular Systems Biology
Molecular Systems Biology 生物-生化与分子生物学
CiteScore
18.50
自引率
1.00%
发文量
62
审稿时长
6-12 weeks
期刊介绍: Systems biology is a field that aims to understand complex biological systems by studying their components and how they interact. It is an integrative discipline that seeks to explain the properties and behavior of these systems. Molecular Systems Biology is a scholarly journal that publishes top-notch research in the areas of systems biology, synthetic biology, and systems medicine. It is an open access journal, meaning that its content is freely available to readers, and it is peer-reviewed to ensure the quality of the published work.
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