Background

Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a powerful genetic element to mediate protein expression in different cells. Among all, Escherichia coli possess advantages of fast growth rate, easy for culture and comprehensive elements for genetic engineering. As E. coli W3110 owns the benefits of more heat shock proteins and higher tolerance to toxic chemicals, further execution of T7-based system in W3110 as cell factory is a conceivable strategy.

Results

Three novel W3110 strains, i.e., W3110:IL5, W3110::L5 and W3110::pI, were accomplished by chromosome-equipped T7RNAP. At first, the LacZ and T7RNAP with isopropyl-β-D-thiogalactopyranoside (IPTG) induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), carbonic anhydrase (CA) for carbon dioxide uptake and lysine decarboxylase (CadA) to produce a toxic chemical cadaverine (DAP). All the proteins showed better expression in W3110::L5 and W3110::pI, respectively. As a result, the highest cadaverine production of 36.9 g/L, lysine consumption of 43.8 g/L and up to 100% yield were obtained in W3110::pI(−) with plasmid pSU-T7-CadA constitutively.

Conclusion

Effect of IPTG and lacI/lacO regulator has been investigated in three chromosome-based T7RNAP E. coli strains. The newly engineered W3110 strains possessed similar protein expression compared to commercial BL21(DE3). Furthermore, W3110::pI displays higher production of sfGFP, CA and CadA, due to it having the highest sensitivity to IPTG, thus it represents the greatest potential as a cell factory.

中文翻译：

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作为细胞工厂的大肠杆菌W3110中基于染色体的T7 RNA聚合酶和正交T7启动子电路的开发

背景

正交T7 RNA聚合酶（T7RNAP）和T7启动子是介导不同细胞中蛋白质表达的强大遗传元件。其中，大肠杆菌具有生长速度快，易于培养和基因工程综合的优点。由于大肠杆菌W3110具有更多的热激蛋白和对有毒化学物质更高的耐受性的优点，因此可以设想在W3110中作为细胞工厂进一步执行基于T7的系统。

结果

通过配备染色体的T7RNAP完成了三种新型W3110菌株，即W3110：IL5，W3110 :: L5和W3110 :: pI。首先，用异丙基-β-D-硫代半乳糖吡喃糖苷（IPTG）诱导的LacZ和T7RNAP在W3110衍生物中的表达水平高于在BL21（DE3）中的表达水平。有和没有lac I / lac的质粒使用O抑制来研究超折叠绿色荧光蛋白（sfGFP），碳酸酐酶（CA）吸收二氧化碳和赖氨酸脱羧酶（CadA）的蛋白表达，以产生有毒的尸胺（DAP）。所有蛋白质分别在W3110 :: L5和W3110 :: pI中显示出更好的表达。结果，组成型为pSU-T7-CadA的W3110 :: pI（-）可获得最高的尸胺产量36.9 g / L，赖氨酸消耗量43.8 g / L和最高100％的产率。

结论

IPTG和lac I / lac O调节剂的作用已在三种基于染色体的T7RNAP大肠杆菌菌株中进行了研究。与商业BL21（DE3）相比，新设计的W3110菌株具有相似的蛋白质表达。此外，由于W3110 :: pI对IPTG的敏感性最高，因此它显示出更高的sfGFP，CA和CadA产量，因此，它代表着细胞工厂的最大潜力。