Development of chromosome-based T7 RNA polymerase and orthogonal T7 promoter circuit inEscherichia coliW3110 as a cell factory
Development of chromosome-based T7 RNA polymerase and orthogonal T7 promoter circuit inEscherichia coliW3110 as a cell factory
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DOI：
10.1186/s40643-020-00342-6
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发表时间：
2020-10-16
影响因子：
4.6
通讯作者：
Ng, I-Son
中科院分区：
文献类型：
--
作者：
Ting, Wan-Wen;Tan, Shih-, I;Ng, I-Son
背景：T7RNAP和T7启动子是调节不同细胞蛋白质表达的重要遗传元件。其中，大肠埃希菌具有生长速度快、易于培养、可进行基因工程的综合要素等优点。ASE.ColiW3110具有更多的热休克蛋白和更高的对有毒化学物质的耐受性，进一步在W3110作为细胞工厂执行基于T7的系统是一个可以想象的战略。结果T7RNAP获得了W3110：IL5、W3110：：L5和W3110：：PI 3株新菌株。首先，异丙基-β-D-硫代半乳糖苷(IPTG)诱导的LacZ和T7RNAP在W3110中的表达水平高于在BL21(DE3)中的表达水平。利用含lacI/LACO阻遏和不含lacI/LACO阻遏的重组表达载体，研究超倍体绿色荧光蛋白(SfGFP)、二氧化碳摄取碳酸酐酶(CA)和赖氨酸脱羧酶(CADA)的蛋白表达，以产生毒性化学物质身体碱(DAP)。所有蛋白分别在W3110：：L5和W3110：：PI中有较好的表达。结果表明，以pSU-T7-CADA为载体的W3110：：PI(-)菌株的身体碱产量最高，达36.9g/L，赖氨酸消耗量为43.8g/L，产率达100%。结论研究了IPTG和lacI/LACO调节基因在3个染色体T7RNAPE中的作用。科里斯特林。与商业菌株BL21(DE3)相比，新工程菌株W3110具有相似的蛋白质表达。此外，由于W3110：：PI对IPTG具有最高的敏感性，因此显示出较高的sfGFP、CA和CADA产量，因此它具有最大的电池工厂潜力。
Background Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a powerful genetic element to mediate protein expression in different cells. Among all,Escherichia colipossess advantages of fast growth rate, easy for culture and comprehensive elements for genetic engineering. AsE. coliW3110 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-beta-D-thiogalactopyranoside (IPTG) induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and withoutlacI/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 andlacI/lacO regulator has been investigated in three chromosome-based T7RNAPE. colistrains. 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.