The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators (lacO) into the constitutive T5 and A1 promoter sequences. We showed that, in genome-integrated E. coli expression systems that used σ70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacIQ, on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level. Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

中文翻译：

---

大肠杆菌σ70启动子可在基因组整合的表达系统中控制细胞水平的表达速率。

即使在单拷贝中表达目的基因（GOI）时，与基因组整合的T7表达系统在生产率和产品质量方面也具有显着优势。与基于质粒的表达系统相比，该系统不会产生质粒介导的代谢负荷，并且在生产过程中不会改变GOI的剂量。但是，由于T7 RNA聚合酶（RNAP）容易发生突变，因此T7表达系统的长期生产导致非生产人群的迅速增长。本研究旨在调查被大肠杆菌宿主RNAP识别的两个σ70启动子是否适合基因组整合表达系统。我们应用了启动子工程学策略，可以控制表达模型蛋白GFP，通过将lac操纵子（lacO）引入组成型T5和A1启动子序列。我们表明，在使用σ70启动子的基因组整合大肠杆菌表达系统中，lacO位点的数量必须保持平衡。包含三个和两个lacO位点的启动子表现出较低的基础表达，但导致部分诱导的培养物中重组蛋白生产的完全停止。相反，在同一条染色体上由单个lacO位点和lac阻遏元件lacIQ调控的表达系统引起非常低的基础表达，在重组蛋白生产中非常有效，并能够在细胞水平上微调基因表达水平。根据我们的结果，我们假设此现象与lac阻遏蛋白LacI的自动调节有关。我们认为，LacI对GOI的lacO位点的亲和力必须低于LacI对内源性lac操纵子的lacO位点的亲和力。否则，LacI自动调节将无法进行，缺少LacI自动调节功能将导致lac阻遏物介导的转录调节受到干扰。通过利用LacI自动调节的机制，我们创建了一种新型的大肠杆菌表达系统，用于重组蛋白生产，合成生物学和代谢工程应用。