School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, 2 Xuelin Road, Qixia District, Nanjing, People's Republic of China.
Nanjing Foreign Language School, Nanjing, People's Republic of China.
Microb Cell Fact. 2021 Sep 26;20(1):189. doi: 10.1186/s12934-021-01680-6.
Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (P), which is leakier and more active than wild-type lac promoter (P) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced P with other inducible promoters respectively, including arabinose promoter (P), rhamnose promoter (P), tetracycline promoter (P), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.
大肠杆菌是原核表达系统中最广泛用于生产重组蛋白的细菌。在 BL21（DE3）中，编码 T7 RNA 聚合酶（T7 RNAP）的基因受强 lacUV5 启动子（P）的控制，在某些生长条件下，该启动子比野生型 lac 启动子（P）漏出性更强且更活跃。这些特性不利于生产那些具有毒性或生长负担的重组蛋白。一方面，T7 RNAP 的渗漏表达导致在非诱导期快速产生目标蛋白，从而从细胞生长中吸取资源。此外，在非诱导期或诱导期，T7 RNAP 大量表达会导致难以表达的蛋白大量产生，这可能导致表达质粒丢失或表达基因发生突变。因此，应该开发更多具有严格表达和不同 T7 RNAP 表达水平的 BL21（DE3）衍生的变体菌株。因此，我们分别用其他诱导启动子代替 P，包括阿拉伯糖启动子（P）、鼠李糖启动子（P）、四环素启动子（P），以通过调节 T7 RNAP 的转录水平和渗漏水平来优化重组蛋白的生产。与 BL21（DE3）相比，构建的工程菌株对诱导剂的敏感性更高，其中鼠李糖和四环素启动子的渗漏能力最低。在生产葡萄糖脱氢酶（GDH）时，该蛋白会导致宿主自溶，工程菌株 BL21（DE3::ara）的生物量、细胞存活率和外源蛋白表达水平均高于 BL21（DE3）。此外，这些工程菌株已成功应用于提高膜蛋白的产量，包括大肠杆菌胞嘧啶转运蛋白（CodB）、大肠杆菌膜蛋白插入酶/折叠酶（YidC）和大肠杆菌 F-ATP 酶亚基 b（Ecb）。本文构建的工程菌株为生产重组蛋白提供了更多的宿主选择。
