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Construction, Model-Based Analysis, and Characterization of a Promoter Library for Fine-Tuned Gene Expression in Bacillus subtilis
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-06-26 00:00:00 , DOI: 10.1021/acssynbio.8b00115 Dingyu Liu , Zhitao Mao 1, 2 , Jiaxin Guo , Leyi Wei , Hongwu Ma 1, 2 , Yajie Tang 3 , Tao Chen , Zhiwen Wang , Xueming Zhao
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-06-26 00:00:00 , DOI: 10.1021/acssynbio.8b00115 Dingyu Liu , Zhitao Mao 1, 2 , Jiaxin Guo , Leyi Wei , Hongwu Ma 1, 2 , Yajie Tang 3 , Tao Chen , Zhiwen Wang , Xueming Zhao
Affiliation
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Promoters are among the most-important and most-basic tools for the control of metabolic pathways. However, previous research mainly focused on the screening and characterization of some native promoters in Bacillus subtilis. To develop a broadly applicable promoter system for this important platform organism, we created a de novo synthetic promoter library (SPL) based on consensus sequences by analyzing the microarray transcriptome data of B. subtilis 168. A total of 214 potential promoters spanning a gradient of strengths was isolated and characterized by a green fluorescence assay. Among these, a detailed intensity analysis was conducted on nine promoters with different strengths by reverse-transcription polymerase chain reaction (RT-PCR) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Furthermore, reconstructed promoters and promoter cassettes (tandem promoter cluster) were designed via statistical model-based analysis and tandem dual promoters, which showed strength that was increased 1.2- and 2.77-fold compared to that of promoter P43, respectively. Finally, the SPL was employed in the production of inosine and acetoin by repressing and over-expressing the relevant metabolic pathways, yielding a 700% and 44% increase relative to the respective control strains. This is the first report of a de novo synthetic promoter library for B. subtilis, which is independent of any native promoter. The strategy of improving and fine-tuning promoter strengths will contribute to future metabolic engineering and synthetic biology projects in B. subtilis.
中文翻译：
枯草芽孢杆菌微调基因表达的启动子文库的构建，基于模型的分析和表征
启动子是控制代谢途径的最重要和最基本的工具之一。然而，先前的研究主要集中在枯草芽孢杆菌中一些天然启动子的筛选和表征。为了为该重要平台生物开发广泛适用的启动子系统，我们通过分析枯草芽孢杆菌的微阵列转录组数据，基于共有序列创建了从头合成启动子文库（SPL）。168.分离出总共214个跨越强度梯度的潜在启动子，并通过绿色荧光分析对其进行了表征。其中，通过逆转录聚合酶链反应（RT-PCR）和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）对9个强度不同的启动子进行了详细的强度分析。此外，通过基于统计模型的分析和串联双启动子设计了重构的启动子和启动子盒（串联启动子簇），与启动子P43相比，串联双启动子显示出分别增加了1.2倍和2.77倍的强度。最后，通过抑制和过表达相关的代谢途径，将SPL用于肌苷和乙醛的生产，相对于相应的对照菌株，其分别增加了700％和44％。枯草芽孢杆菌的从头合成启动子文库，它独立于任何天然启动子。改善和微调启动子强度的策略将为枯草芽孢杆菌的未来代谢工程和合成生物学项目做出贡献。
更新日期：2018-06-26
中文翻译：
枯草芽孢杆菌微调基因表达的启动子文库的构建，基于模型的分析和表征
启动子是控制代谢途径的最重要和最基本的工具之一。然而，先前的研究主要集中在枯草芽孢杆菌中一些天然启动子的筛选和表征。为了为该重要平台生物开发广泛适用的启动子系统，我们通过分析枯草芽孢杆菌的微阵列转录组数据，基于共有序列创建了从头合成启动子文库（SPL）。168.分离出总共214个跨越强度梯度的潜在启动子，并通过绿色荧光分析对其进行了表征。其中，通过逆转录聚合酶链反应（RT-PCR）和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）对9个强度不同的启动子进行了详细的强度分析。此外，通过基于统计模型的分析和串联双启动子设计了重构的启动子和启动子盒（串联启动子簇），与启动子P43相比，串联双启动子显示出分别增加了1.2倍和2.77倍的强度。最后，通过抑制和过表达相关的代谢途径，将SPL用于肌苷和乙醛的生产，相对于相应的对照菌株，其分别增加了700％和44％。枯草芽孢杆菌的从头合成启动子文库，它独立于任何天然启动子。改善和微调启动子强度的策略将为枯草芽孢杆菌的未来代谢工程和合成生物学项目做出贡献。
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