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在基因组规模上绘制温度敏感突变,以设计大肠杆菌的生长开关

Thorben Schramm^1,2, Paul Lubrano^1,2, Vanessa Pahl^1,2

¹Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany.

Molecular systems biology

|August 29, 2023

概括

我们在大肠杆菌中设计了15120个温度敏感 (TS) 突变体,以研究细胞代谢. 许多TS突变使非生长细胞中可调节的代谢物产生,提供了新的工程策略.

科学领域:

微生物学微生物学
合成生物学合成生物学
代谢工程是代谢工程.

背景情况:

温度敏感 (TS) 突变物是扰乱和工程细胞功能的宝贵工具.
了解遗传干扰如何影响细胞代谢,特别是在非生长状态下,对于生物工程至关重要.

研究的目的:

开发一种高通量方法,用于产生和选大肠杆菌中温度敏感 (TS) 突变体.
研究非生长细胞中特定突变的代谢后果,并探索它们对代谢产生的潜力.

主要方法:

构建一个基于CRISPR的大规模库,其中包含15,120个单氨基酸替代突变体在E. coli必需蛋白质中.
时间解决的竞争试验用于识别表现出温度敏感生长的TS突变.
在高温 (42°C) 下停止生长的精选TS突变体的代谢分析.

主要成果:

在15120个构造突变物中,有1269个突变物表现出对温度敏感的生长.
代谢分析显示了非生长细胞中强烈的突变特异性代谢变化.
特定的TS突变,例如影响同类氨酸激酶 (ThrB^F267D) 的突变,证明了它们的直接基质 (同类氨酸) 的可调节生产.
用DNA聚合酶III (DnaX^L289Q) 的一个TS突变来将细胞生长与阿金氨酸过度生产脱.

结论:

该研究提出了一种有效的战略,用于大规模识别TS突变.
转基因突变提供了一个强大的平台,用于精确控制非生长的细菌细胞中的细胞代谢和代谢物生产.
这种方法在代谢工程和合成生物学中具有很大的应用潜力.

关键词:

通过CRISPR-Cas9进行基因组编辑.增长开关是指增长开关.代谢门代谢门代谢生物组的代谢生物组对温度敏感的突变.

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