School of Biology and Biological Engineering, South China University of Technology, Guangzhou, Guangdong, China
School of Biology and Biological Engineering, South China University of Technology, Guangzhou, Guangdong, China.
Appl Environ Microbiol. 2021 Apr 27;87(10). doi: 10.1128/AEM.02923-20.
Acid tolerance of microorganisms is a desirable phenotype for many industrial fermentation applications. In , 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 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.
微生物的耐酸能力是许多工业发酵应用中理想的表型。在 中，应激反应σ因子 RpoS 是工程耐酸表型的有前途的靶标。然而，单独过表达 RpoS 本身不足以赋予这些表型。在这项研究中，我们表明，同时过表达非编码小 RNA (sRNA) DsrA 和 sRNA 伴侣 Hfq，它们作为 RpoS 激活剂，可显著提高适度酸性 pH 下细胞生长的耐酸性，以及极端酸冲击下细胞的存活率。DsrA-Hfq 模块的定向进化进一步提高了耐酸性，最佳突变体在 pH 4.5 下的生长性能比起始菌株 MG1655 提高了 51%至 72%。进一步分析发现，这些 DsrA-Hfq 菌株的耐酸性提高与质子消耗型酸抗性系统 2 (AR2) 相关基因的激活、蛋白质伴侣 HdeB 和活性氧 (ROS) 去除有关。在指数期。这项研究表明，精细调节 sRNA 及其伴侣可以成为提高许多传统细菌耐酸性研究通常侧重于改善极端 pH 条件下的细胞存活率，但在不那么苛刻的酸性条件下的细胞生长与工业应用更相关。在正常条件下，通过多种机制，生长阶段的通用应激反应σ因子 RpoS 保持在低水平。本研究表明，通过工程其激活剂 sRNA DsrA 和 sRNA 伴侣 Hfq，RpoS 可以在静止期之前被激活，从而导致适度酸性 pH 下细胞生长显著改善。这项工作表明，通过操纵各自的调节 sRNA 以及足够供应各自的 sRNA 伴侣（即 Hfq），可以重新调整或重新调整 sigma 因子和可能的其他转录因子。这为微生物菌株工程提供了一条新途径。
