Christian Doppler Laboratory for Production of Next-level Biopharmaceuticals in E. coli, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190, Vienna, Austria.
Boehringer Ingelheim RCV GmbH & Co KG, Dr.-Boehringer-Gasse 5-11, 1120, Vienna, Austria.
Microb Cell Fact. 2020 Mar 5;19(1):58. doi: 10.1186/s12934-020-01311-6.
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 σ 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 σ 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, lacI, 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.
基因组整合的 T7 表达系统在生产力和产品质量方面具有显著优势，即使是从单个拷贝表达目的基因（GOI）也是如此。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担，并且在生产过程中不会改变 GOI 的剂量。然而，使用 T7 表达系统进行长期生产会导致非生产性群体迅速增长，因为 T7 RNA 聚合酶（RNAP）容易发生突变。本研究旨在探讨两种 σ 启动子是否适合基因组整合表达系统，这两种启动子被大肠杆菌宿主 RNAP 识别。我们应用了一种启动子工程策略，通过在组成型 T5 和 A1 启动子序列中引入 lac 操纵子（lacO）来控制模型蛋白 GFP 的表达。
我们表明，在使用 σ 启动子的基因组整合大肠杆菌表达系统中，lacO 位点的数量必须得到很好的平衡。含有三个和两个 lacO 位点的启动子表现出低基础表达，但在部分诱导培养物中导致重组蛋白生产完全停止。相比之下，由单个 lacO 位点和 lacI 组成的表达系统（lacI 是 lac 操纵子的阻遏物），在同一染色体上，其基础表达非常低，但在重组蛋白生产中效率很高，并且能够在细胞水平上精细调节基因表达水平。
基于我们的结果，我们假设这种现象与 lac 阻遏物蛋白 LacI 的自动调节有关。我们推断，LacI 与 GOI 的 lacO 位点的亲和力必须低于 LacI 与内源性 lac 操纵子的 lacO 位点的亲和力；否则，LacI 自动调节就不可能发生，而 LacI 自动调节的缺失将导致 lac 阻遏物介导的转录调节紊乱。通过利用 LacI 自动调节的机制，我们创建了一种新型大肠杆菌表达系统，用于重组蛋白生产、合成生物学和代谢工程应用。
