Genetic manipulation of Escherichia coli influences the regulation of bacterial metabolism, which could be useful for the production of different targeted products. The RpoZ gene encodes for the ω subunit of the RNA polymerase (RNAP) and is involved in the regulation of the relA gene pathway. RelA is responsible for the production of guanosine pentaphosphate (ppGpp), which is a major alarmone in the stringent response. Expression of relA is reduced in the early hours of growth of RpoZ mutant E. coli. In the absence of the ω subunit, ppGpp affinity to RNAP is decreased; thus, rpoZ gene deleted E. coli strains show a modified stringent response. We used the E. coli K-12 MG1655 strain that lacks rpoZ (JEN202) to investigate the effect of the modified stringent response on recombinant protein production. However, the absence of the ω subunit results in diminished stability of the RNA polymerase at the promoter site. To avoid this, we used a deactivated CRISPR system that targets the ω subunit to upstream of the promoter site in the expression plasmid. The expression plasmid encodes for Chaetomium thermophilum formate dehydrogenase (CtFDH), a valuable enzyme for cofactor regeneration and CO₂ reduction. A higher amount of CtFDH from the soluble fraction was purified from the JEN202 strain compared to the traditional BL21(DE3) method, thus offering a new strategy for batch-based recombinant enzyme production.

大肠杆菌的遗传操作影响细菌代谢的调节，这可能有助于生产不同的目标产品。该RpoZ的RNA聚合酶（RNAP）的ω亚基基因编码，并参与所述的调节RELA基因通路。RelA负责生产五磷酸鸟苷 (ppGpp)，这是严格反应中的主要警报。在RpoZ突变型大肠杆菌生长的早期，relA 的表达降低。在没有 ω 亚基的情况下，ppGpp 对 RNAP 的亲和力降低；因此，rpoZ基因删除了大肠杆菌菌株显示出改良的严格反应。我们使用缺乏rpoZ (JEN202) 的大肠杆菌K-12 MG1655 菌株来研究改良的严格响应对重组蛋白生产的影响。然而，ω 亚基的缺失导致 RNA 聚合酶在启动子位点的稳定性降低。为了避免这种情况，我们使用了一种去活的 CRISPR 系统，该系统将 ω 亚基定位到表达质粒中启动子位点的上游。表达质粒编码嗜热毛壳菌甲酸脱氢酶 ( CtFDH )，这是一种用于辅助因子再生和 CO ₂还原的有价值的酶。更高的Ct与传统的 BL21(DE3) 方法相比，从 JEN202 菌株中纯化了可溶性部分的 FDH，从而为基于批次的重组酶生产提供了一种新策略。