State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, College of Life Sciences, Hubei University, Wuhan, 430062, China.
State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, College of Life Sciences, Hubei University, Wuhan, 430062, China; Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao, China.
J Biotechnol. 2020 Mar 20;312:1-10. doi: 10.1016/j.jbiotec.2020.02.015. Epub 2020 Feb 28.
Promoter plays the critical role in regulating gene transcription, and dual-promoter has received the widespread attentions due to its high efficiency and continuity, here, we want to construct an efficient dual-promoter for protein production and metabolic pathway enhancement. Firstly, our results indicated that P43 promoter efficiently transcribed at logarithmic period, while the σ-type promoters (PylB, PgsiB, PykzA) were active at stationary phase. Then, several dual promoters were constructed by coupling these σ-type promoters with P43, and the attained dual-promoter PykzA-P43 showed the best performance, which led to 1.72-, 3.46- and 1.85-fold increases of green fluorescence intensity, red fluorescence intensity and α-amylase activity, compared with those of the recognized strong promoter P43, respectively. Furthermore, α-amylase activity was further increased to 389.65 U/mL by 32.20 % via optimizing sigma factor binding sites (-10 and -35 boxes) of PykzA-P43, attaining the optimized dual promoter Pdual3. Finally, Pdual3 was applied in metabolic pathway enhancement, and the yields of Poly γ-glutamic acid, acetoin and 2, 3-butanediol were respectively improved by 82.01 %, 17.09 % and 99.39 %. Our results indicated that dual-promoter significantly enhanced gene expression, and this study provided an energetic dual-promoter Pdual3 for efficient protein production and metabolic pathway enhancement in Bacillus licheniformis.
启动子在基因转录调控中起着关键作用，双启动子由于其高效性和连续性而受到广泛关注，在这里，我们希望构建一种用于蛋白质生产和代谢途径增强的高效双启动子。首先，我们的结果表明 P43 启动子在对数生长期高效转录，而 σ 型启动子（PylB、PgsiB、PykzA）在静止期活跃。然后，通过将这些 σ 型启动子与 P43 偶联，构建了几种双启动子，所获得的双启动子 PykzA-P43 表现出最佳性能，与公认的强启动子 P43 相比，绿色荧光强度、红色荧光强度和 α-淀粉酶活性分别提高了 1.72 倍、3.46 倍和 1.85 倍。此外，通过优化 PykzA-P43 的 σ 因子结合位点（-10 和 -35 盒），α-淀粉酶活性进一步提高了 32.20%，达到 389.65U/mL，获得了优化的双启动子 Pdual3。最后，将 Pdual3 应用于代谢途径增强，聚γ-谷氨酸、乙酰醇和 2,3-丁二醇的产量分别提高了 82.01%、17.09%和 99.39%。我们的结果表明，双启动子显著增强了基因表达，本研究为地衣芽孢杆菌中高效蛋白质生产和代谢途径增强提供了一种有活力的双启动子 Pdual3。
