Department Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19711, USA, Molecular Biotechnology Laboratory, Department of Chemical and Biomolecular Engineering, The Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA, Department of Biological Sciences, University of Delaware, Newark, DE 19711, USA and Department of Biology, Sultan Qaboos University, Muscat, 123, Oman.
Nucleic Acids Res. 2013 Oct;41(18):8726-37. doi: 10.1093/nar/gkt651. Epub 2013 Jul 27.
Synthetic acid tolerance, especially during active cell growth, is a desirable phenotype for many biotechnological applications. Natively, acid resistance in Escherichia coli is largely a stationary-phase phenotype attributable to mechanisms mostly under the control of the stationary-phase sigma factor RpoS. We show that simultaneous overexpression of noncoding small RNAs (sRNAs), DsrA, RprA and ArcZ, which are translational RpoS activators, increased acid tolerance (based on a low-pH survival assay) supra-additively up to 8500-fold during active cell growth, and provided protection against carboxylic acid and oxidative stress. Overexpression of rpoS without its regulatory 5'-UTR resulted in inferior acid tolerance. The supra-additive effect of overexpressing the three sRNAs results from the impact their expression has on RpoS-protein levels, and the beneficial perturbation of the interconnected RpoS and H-NS networks, thus leading to superior tolerance during active growth. Unlike the overexpression of proteins, overexpression of sRNAs imposes hardly any metabolic burden on cells, and constitutes a more effective strain engineering strategy.
合成酸耐受性，特别是在细胞活跃生长期间的酸耐受性，是许多生物技术应用中所需的理想表型。在大肠杆菌中，天然的酸抗性主要是一种静止期表型，归因于主要受静止期 sigma 因子 RpoS 控制的机制。我们表明，同时过表达非编码小 RNA（sRNA），DsrA、RprA 和 ArcZ，它们是翻译 RpoS 的激活剂，在细胞活跃生长期间，酸耐受性（基于低 pH 存活测定）以超加性方式增加了高达 8500 倍，并提供了对羧酸和氧化应激的保护。没有其调节 5'-UTR 的 rpoS 的过表达导致酸耐受性较差。这三个 sRNA 的过表达的超加性效应源自它们对 RpoS-蛋白水平的影响，以及相互连接的 RpoS 和 H-NS 网络的有益干扰，从而导致在活跃生长期间具有更好的耐受性。与蛋白质的过表达不同，sRNA 的过表达几乎不会给细胞带来代谢负担，并且是一种更有效的菌株工程策略。
