A novel point mutation in RpoB improves osmotolerance and succinic acid production in Escherichia coli.
A novel point mutation in RpoB improves osmotolerance and succinic acid production in Escherichia coli.
复制标题
RpoB 中的一个新点突变可改善大肠杆菌的渗透压耐受性和琥珀酸产量。
DOI：
10.1186/s12896-017-0337-6
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发表时间：
2017-02-13
影响因子：
3.5
通讯作者：
Zhang X
中科院分区：
文献类型：
--
作者：
Xiao M;Zhu X;Xu H;Tang J;Liu R;Bi C;Fan F;Zhang X
大肠杆菌在琥珀酸（SA）生产过程中受到渗透胁迫，降低了这个微生物工厂的性能。在这里，我们报道了导致dna依赖性RNA聚合酶(RpoB) β-亚基内单个氨基酸变化（D654Y）的点突变显著提高了大肠杆菌的渗透耐受性。将RpoB的D654Y突变输入亲本菌株Suc-T110，在高葡萄糖渗透压条件下，细胞生长和SA产量比对照提高了40%以上。通过rna测序确定的转录组谱显示，突变的RpoB引发了两种不同的胁迫反应，抵消了渗透胁迫。在非胁迫条件下，即使没有渗透刺激，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上调，表明rpoB突变体可能形成了“预防御”机制。在渗透胁迫条件下，编码多种糖转运蛋白的基因在高渗透压下被下调，在rpoB突变体中被下调。另外的遗传实验表明，增加异常调控的表达，特别是编码糖铜蛋白LamB和麦芽糖转运蛋白的基因，有助于渗透耐受性表型。RpoB中的D654Y单氨基酸取代使大肠杆菌细胞抵抗渗透胁迫，可能是由于在胁迫条件下通过增强糖摄取来改善细胞生长和活力，并激活了非胁迫条件下潜在的“预防御”机制。本研究结果将有助于细菌宿主的改良，提高其对渗透胁迫的抵抗力，促进生物基有机酸的生产。本文的在线版本（doi:10.1186/s12896-017-0337-6）包含补充材料，可供授权用户使用。
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. The online version of this article (doi:10.1186/s12896-017-0337-6) contains supplementary material, which is available to authorized users.