Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, 32 West 7th Ave, Tianjin Airport Economic Park, Tianjin, 300308, China.
BMC Biotechnol. 2017 Feb 13;17(1):10. doi: 10.1186/s12896-017-0337-6.
Escherichia coli suffer from osmotic stress during succinic acid (SA) production, which reduces the performance of this microbial factory.
Here, we report that a point mutation leading to a single amino acid change (D654Y) within the β-subunit of DNA-dependent RNA polymerase (RpoB) significantly improved the osmotolerance of E. coli. Importation of the D654Y mutation of RpoB into the parental strain, Suc-T110, increased cell growth and SA production by more than 40% compared to that of the control under high glucose osmolality. The transcriptome profile, determined by RNA-sequencing, showed two distinct stress responses elicited by the mutated RpoB that counterbalanced the osmotic stress. Under non-stressed conditions, genes involved in the synthesis and transport of compatible solutes such as glycine-betaine, glutamate or proline were upregulated even without osmotic stimulation, suggesting a "pre-defense" mechanism maybe formed in the rpoB mutant. Under osmotic stressed conditions, genes encoding diverse sugar transporters, which should be down-regulated in the presence of high osmotic pressure, were derepressed in the rpoB mutant. Additional genetic experiments showed that enhancing the expression of the mal regulon, especially for genes that encode the glycoporin LamB and maltose transporter, contributed to the osmotolerance phenotype.
The D654Y single amino acid substitution in RpoB rendered E. coli cells resistant to osmotic stress, probably due to improved cell growth and viability via enhanced sugar uptake under stressed conditions, and activated a potential "pre-defense" mechanism under non-stressed conditions. The findings of this work will be useful for bacterial host improvement to enhance its resistance to osmotic stress and facilitate bio-based organic acids production.
大肠杆菌在琥珀酸（SA）生产过程中会遭受渗透压胁迫，从而降低这种微生物工厂的性能。
在这里，我们报告称，DNA 依赖性 RNA 聚合酶（RpoB）β亚基中的一个点突变导致单个氨基酸变化（D654Y），可显著提高大肠杆菌的渗透压耐受性。与对照相比，将 RpoB 的 D654Y 突变导入亲本菌株 Suc-T110 中，在高葡萄糖渗透压下，细胞生长和 SA 产量增加了 40%以上。通过 RNA 测序确定的转录组谱显示，突变的 RpoB 引发了两种截然不同的应激反应，这两种反应抵消了渗透压胁迫。在非胁迫条件下，即使没有渗透压刺激，参与甘氨酸甜菜碱、谷氨酸或脯氨酸等相容溶质合成和运输的基因也被上调，这表明 rpoB 突变体可能形成了“预先防御”机制。在渗透压胁迫条件下，编码各种糖转运蛋白的基因被下调，但在 rpoB 突变体中被解除了抑制。额外的遗传实验表明，增强 mal 调控子的表达，特别是编码糖蛋白 LamB 和麦芽糖转运蛋白的基因，有助于渗透压耐受表型。
RpoB 中的 D654Y 单个氨基酸取代使大肠杆菌细胞对渗透压胁迫具有抗性，这可能是由于在应激条件下通过增强糖摄取来改善细胞生长和活力，并在非应激条件下激活了潜在的“预先防御”机制。这项工作的发现将有助于细菌宿主的改良，以提高其对渗透压胁迫的抗性并促进基于生物的有机酸生产。
