Engineering of the Small Noncoding RNA (sRNA) DsrA Together with the sRNA Chaperone Hfq Enhances the Acid Tolerance of Escherichia coli
Engineering of the Small Noncoding RNA (sRNA) DsrA Together with the sRNA Chaperone Hfq Enhances the Acid Tolerance of Escherichia coli
复制标题
小非编码 RNA (sRNA) DsrA 与 sRNA 伴侣 Hfq 的工程改造增强了大肠杆菌的耐酸能力
DOI：
10.1128/aem.02923-20
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发表时间：
2021-05-01
影响因子：
4.4
通讯作者：
Yang, Xiaofeng
中科院分区：
文献类型：
--
作者：
Lin, Zhanglin;Li, Jiahui;Yang, Xiaofeng
微生物的酸耐受性是许多工业发酵应用的理想表型。在大肠杆菌中，应力响应Sigma因子RPO是耐酸表型的有希望的目标。但是，仅RPO的简单过表达不足以赋予这些表型。在这项研究中，我们表明，无编码的小RNA（SRNA）DSRA和SRNA伴侣HFQ的同时过表达，它们充当RPOS活化剂，在适度的酸性pH值下，细胞生长的酸耐受性显着提高了酸的耐受性，以及在极度酸性休克中的细胞存活。 DSRA-HFQ模块的定向演化进一步提高了酸的耐受性，与起始菌株MG1655相比，最好的突变体在pH 4.5时显示出51％至72％的增长。进一步的分析发现，这些DSRA-HFQ菌株的酸耐受性的提高与与质子消费酸的抗抗抗抗性系统2（AR2），蛋白伴侣HDEB和反应性氧（ROS）在指数相中的去除相吻合。这项研究表明，SRNA及其伴侣的微调可能是提高大肠杆菌耐受性的新型策略。重要的是，许多关于细菌酸耐受性的传统研究通常集中在极端pH条件下改善细胞存活，但是在较少的苛刻的酸性条件下，细胞生长与工业应用更为相关。在正常条件下，通过多种机制，在生长阶段，一般应力响应Sigma因子RPO保持在较低水平。这项研究表明，RPO可以在固定相之前通过工程激活剂SRNA DSRA和SRNA伴侣HFQ激活RPO，从而在适度的酸性pH值下显着改善了细胞生长。这项工作表明，可以通过操纵各自的调节性SRNA以及相应的SRNA伴侣（即HFQ）的充分供应来重新调整或重述Sigma因子和其他转录因子。这为微生物的应变工程提供了一种新颖的途径。
Acid tolerance of microorganisms is a desirable phenotype for many industrial fermentation applications. In Escherichia coli, the stress response sigma factor RpoS is a promising target for engineering acid-tolerant phenotypes. However, the simple overexpression of RpoS alone is insufficient to confer these phenotypes. In this study, we show that the simultaneous overexpression of the noncoding small RNA (sRNA) DsrA and the sRNA chaperone Hfq, which act as RpoS activators, significantly increased acid tolerance in terms of cell growth under modest acidic pH, as well as cell survival upon extreme acid shock. Directed evolution of the DsrA-Hfq module further improved the acid tolerance, with the best mutants showing a 51 to 72% increase in growth performance at pH 4.5 compared with the starting strain, MG1655. Further analyses found that the improved acid tolerance of these DsrA-Hfq strains coincided with activation of genes associated with proton-consuming acid resistance system 2 (AR2), protein chaperone HdeB, and reactive oxygen species (ROS) removal in the exponential phase. This study illustrated that the fine-tuning of sRNAs and their chaperones can be a novel strategy for improving the acid tolerance of E. coli.IMPORTANCE Many of the traditional studies on bacterial acid tolerance generally focused on improving cell survival under extreme-pH conditions, but cell growth under less harsh acidic conditions is more relevant to industrial applications. Under normal conditions, the general stress response sigma factor RpoS is maintained at low levels in the growth phase through a number of mechanisms. This study showed that RpoS can be activated prior to the stationary phase via engineering its activators, the sRNA DsrA and the sRNA chaperone Hfq, resulting in significantly improved cell growth at modest acidic pH. This work suggests that the sigma factors and likely other transcription factors can be retuned or retimed by manipulating the respective regulatory sRNAs along with the sufficient supply of the respective sRNA chaperones (i.e., Hfq). This provides a novel avenue for strain engineering of microbes.