Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications.
Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications.
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不同条件下枯草芽孢杆菌启动子的筛选寻找合成生物学和工业应用
DOI：
10.1371/journal.pone.0158447
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Zhang D
中科院分区：
文献类型：
--
作者：
Song Y;Nikoloff JM;Fu G;Chen J;Li Q;Xie N;Zheng P;Sun J;Zhang D
枯草芽孢杆菌在合成生物学和代谢工程中的应用是非常理想的，以利用这种生物体中存在的独特代谢途径。为此，需要对B进行评估。枯草芽孢杆菌的内在生物学部分是确定精确调节代谢回路和靶蛋白表达的最佳策略所必需的。通过测量整合到B中的绿色荧光蛋白报道基因的相对荧光单位来评估启动子候选物的强度。枯草染色体共84个预测的启动子序列位于上游的不同类别的蛋白质，包括热休克蛋白，细胞包膜蛋白质，对有毒金属的蛋白质（基于相似性）和其他类型的基因进行了测试。测量的表达水平范围为0.0023至4.53倍的活性的充分表征的强启动子P43。没有观察到显着的变化时，菌株，携带不同的启动子候选人，培养在高温下或在培养基中与乙醇，但一些菌株表现出增加的活动时，培养在高渗透压。随机选择的启动子候选者进行了测试，并发现以相似的水平激活热稳定性β-半乳糖苷酶（bgaB）的转录，这意味着这些序列在多种遗传背景下作为启动子元件发挥作用的能力。此外，选择的启动子将细胞质bgaB和分泌蛋白α-淀粉酶的最终产量分别提高至约4倍和2倍。生成的数据允许更深入地理解B。枯草杆菌的代谢，并将促进未来的工作，以开发这种生物体的合成生物学。
The use of Bacillus subtilis in synthetic biology and metabolic engineering is highly desirable to take advantage of the unique metabolic pathways present in this organism. To do this, an evaluation of B. subtilis’ intrinsic biological parts is required to determine the best strategies to accurately regulate metabolic circuits and expression of target proteins. The strengths of promoter candidates were evaluated by measuring relative fluorescence units of a green fluorescent protein reporter, integrated into B. subtilis’ chromosome. A total of 84 predicted promoter sequences located upstream of different classes of proteins including heat shock proteins, cell-envelope proteins, and proteins resistant against toxic metals (based on similarity) and other kinds of genes were tested. The expression levels measured ranged from 0.0023 to 4.53-fold of the activity of the well-characterized strong promoter P43. No significant shifts were observed when strains, carrying different promoter candidates, were cultured at high temperature or in media with ethanol, but some strains showed increased activity when cultured under high osmotic pressure. Randomly selected promoter candidates were tested and found to activate transcription of thermostable β-galactosidase (bgaB) at a similar level, implying the ability of these sequences to function as promoter elements in multiple genetic contexts. In addition, selected promoters elevated the final production of both cytoplasmic bgaB and secreted protein α-amylase to about fourfold and twofold, respectively. The generated data allows a deeper understanding of B. subtilis’ metabolism and will facilitate future work to develop this organism for synthetic biology.