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首页> 外文期刊>BMC Biotechnology >A novel point mutation in RpoB improves osmotolerance and succinic acid production in Escherichia coli
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A novel point mutation in RpoB improves osmotolerance and succinic acid production in Escherichia coli

机译：RpoB中的新型点突变可提高大肠杆菌中的渗透压和琥珀酸生成

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Background Escherichia coli suffer from osmotic stress during succinic acid (SA) production, which reduces the performance of this microbial factory. ResultsHere, 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. ConclusionsThe 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.
机译：背景技术大肠杆菌在生产琥珀酸（SA）时会遭受渗透压，从而降低了该微生物工厂的性能。结果在这里，我们报道了导致DNA依赖的RNA聚合酶（RpoB）的β亚基内单个氨基酸改变（D654Y）的点突变显着改善了大肠杆菌的渗透耐受性。将RpoB的D654Y突变导入亲本菌株Suc-T110，与在高葡萄糖渗透压下的对照相比，将细胞生长和SA产生增加了超过40％。通过RNA测序确定的转录组图谱显示，由突变的RpoB引起的两种独特的应激反应可抵消渗透压。在非胁迫条件下，即使没有渗透刺激，参与相容性溶质（如甘氨酸-甜菜碱，谷氨酸或脯氨酸）的合成和运输的基因也被上调，这表明在rpoB突变体中可能形成了“防御”机制。在渗透胁迫下，rpoB突变体会抑制编码多种糖转运蛋白的基因，这些基因在高渗透压下应下调。额外的基因实验表明，增强麦芽糖调节子的表达，特别是对于编码糖蛋白LamB和麦芽糖转运蛋白的基因，是渗透压表现型的原因。结论RpoB中的D654Y单氨基酸取代使大肠杆菌细胞对渗透胁迫具有抵抗力，这可能是由于在压力条件下增加了糖的摄取，从而改善了细胞的生长和生存能力，并在非压力条件下激活了潜在的“防御”机制。这项工作的发现将有助于细菌宿主的改良，以增强其对渗透胁迫的抵抗力，并促进生物基有机酸的生产。

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