The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
J Ind Microbiol Biotechnol. 2022 Jul 30;49(4). doi: 10.1093/jimb/kuac016.
D, D-carboxypeptidase DacA plays an important role in the synthesis and stabilization of Escherichia coli cell wall peptidoglycan. The production level of extracellular recombinant proteins in E. coli can be enhanced by high D, D-carboxypeptidase activity. Construction of expression systems under optimal promoters is one of the main strategies to realize high protein production in E. coli. In this study, the promoter PdacA-3 from DacA on the genome of E. coli BL21 (DE3) was verified to be efficient for recombinant green fluorescent protein using the plasmid mutant pET28a-PdacA with PdacA-3. Meanwhile, the promoter PdacA-3 was engineered to increase the production level of proteins via inserting one or two Shine-Dalgarno (SD) sequences between the promoter PdacA-3 and the target genes. The expression level of dacA on the genome was increased by the improved transcription of the engineered promoters (especially after inserting one additional SD sequence). The engineered promoters increased cell membrane permeabilities to significantly enhance the secretion production of extracellular recombinant proteins in E. coli. Among them, the extracellular recombinant amylase activities in E. coli BL21::1SD-pET28a-amyK and E. coli BL21::2SD-pET28a-amyK were increased by 2.0- and 1.6-fold that of the control (E. coli BL21-pET28a-amyK), respectively. Promoter engineering also affected the morphology and growth of the E. coli mutants. It was indicated that the engineered promoters enhanced the expression of dacA on the genome to disturb the synthesis and structural stability of cell wall peptidoglycans.
D，D-羧肽酶 DacA 在大肠杆菌细胞壁肽聚糖的合成和稳定中发挥着重要作用。D，D-羧肽酶活性高可提高大肠杆菌中外源重组蛋白的产量。在最优启动子下构建表达系统是实现大肠杆菌中高蛋白质产量的主要策略之一。在本研究中，通过使用带有 PdacA-3 的质粒突变体 pET28a-PdacA 验证了来自大肠杆菌 BL21 (DE3) 基因组上 DacA 的启动子 PdacA-3 对重组绿色荧光蛋白的高效性。同时，通过在启动子 PdacA-3 和靶基因之间插入一个或两个 Shine-Dalgarno (SD) 序列来工程化启动子 PdacA-3，以提高蛋白质的产量水平。通过改进工程化启动子的转录，提高了基因组上 dacA 的表达水平（特别是在插入一个额外的 SD 序列后）。工程化的启动子增加了细胞膜的通透性，显著增强了大肠杆菌中外源重组蛋白的分泌产量。其中，大肠杆菌 BL21::1SD-pET28a-amyK 和大肠杆菌 BL21::2SD-pET28a-amyK 的细胞外重组淀粉酶活性分别比对照（大肠杆菌 BL21-pET28a-amyK）提高了 2.0 倍和 1.6 倍。启动子工程化也影响了大肠杆菌突变体的形态和生长。这表明，工程化的启动子增强了基因组上 dacA 的表达，从而干扰了细胞壁肽聚糖的合成和结构稳定性。
