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Construction and application of a dual promoter system for efficient protein production and metabolic pathway enhancement in Bacillus licheniformis.
Journal of Biotechnology ( IF 4.1 ) Pub Date : 2020-02-28 , DOI: 10.1016/j.jbiotec.2020.02.015
Yi Rao 1 , Dongbo Cai 1 , Hao Wang 1 , Yuxiang Xu 1 , Shijie Xiong 1 , Lin Gao 2 , Min Xiong 1 , Zhi Wang 3 , Shouwen Chen 1 , Xin Ma 1
Affiliation  

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 σB-type promoters (PylB, PgsiB, PykzA) were active at stationary phase. Then, several dual promoters were constructed by coupling these σB-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.

中文翻译:

双启动子系统的构建和应用,用于地衣芽孢杆菌中高效的蛋白质生产和代谢途径增强。

启动子在调控基因转录中起着至关重要的作用,而双启动子由于其高效性和连续性而受到了广泛的关注,在此,我们希望构建一个高效的双启动子,用于蛋白质生产和代谢途径的增强。首先,我们的结果表明,P43启动子在对数期有效转录,而σB型启动子(PylB,PgsiB,PykzA)在固定期具有活性。然后,通过将这些σB型启动子与P43偶联,构建了几个双启动子,并且获得的双启动子PykzA-P43显示出最佳性能,从而导致绿色荧光强度,红色,绿色分别增加了1.72、3.46和1.85倍。荧光强度和α-淀粉酶活性,分别与公认的强启动子P43相比。此外,通过优化PykzA-P43的sigma因子结合位点(-10和-35盒),α-淀粉酶活性进一步提高到389.65 U / mL,提高了32.20%,从而获得了优化的双启动子Pdual3。最终,将Pdual3应用于代谢途径的增强,聚γ-谷氨酸,丙酮和2,3-丁二醇的收率分别提高了82.01%,17.09%和99.39%。我们的结果表明,双启动子显着增强了基因表达,这项研究为地衣芽孢杆菌的高效蛋白质生产和代谢途径增强提供了一个充满活力的双启动子Pdual3。聚γ-谷氨酸,丙酮酸和2,3-丁二醇的收率分别提高了82.01%,17.09%和99.39%。我们的结果表明,双启动子显着增强了基因表达,这项研究为地衣芽孢杆菌的高效蛋白质生产和代谢途径增强提供了一个充满活力的双启动子Pdual3。聚γ-谷氨酸,丙酮酸和2,3-丁二醇的收率分别提高了82.01%,17.09%和99.39%。我们的结果表明,双启动子显着增强了基因表达,这项研究为地衣芽孢杆菌的高效蛋白质生产和代谢途径增强提供了一个充满活力的双启动子Pdual3。
更新日期:2020-03-07
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