Zhou Shenghu, Ding Renpeng, Chen Jian, Du Guocheng, Li Huazhong, Zhou Jingwen
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, and ‡National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University , 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
ACS Synth Biol. 2017 Jun 16;6(6):1065-1075. doi: 10.1021/acssynbio.7b00006. Epub 2017 Mar 9.
A promoter is one of the most important and basic tools used to achieve diverse synthetic biology goals. Escherichia coli is one of the most commonly used model organisms in synthetic biology to produce useful target products and establish complicated regulation networks. During the fine-tuning of metabolic or regulation networks, the limited number of well-characterized inducible promoters has made implementing complicated strategies difficult. In this study, 104 native promoter-5'-UTR complexes (PUTR) from E. coli were screened and characterized based on a series of RNA-seq data. The strength of the 104 PUTRs varied from 0.007% to 4630% of that of the P promoter in the transcriptional level and from 0.1% to 137% in the translational level. To further upregulate gene expression, a series of combinatorial PUTRs and cascade PUTRs were constructed by integrating strong transcriptional promoters with strong translational 5'-UTRs. Finally, two combinatorial PUTRs (P-UTR and P-UTR) and two cascade PUTRs (PUTR-PUTR and PUTR-PUTR) were identified as having the highest activity, with expression outputs of 170%, 137%, 409%, and 203% of that of the P promoter, respectively. These engineered PUTRs are stable for the expression of different genes, such as the red fluorescence protein gene and the β-galactosidase gene. These results show that the PUTRs characterized and constructed in this study may be useful as a plug-and-play synthetic biology toolbox to achieve complicated metabolic engineering goals in fine-tuning metabolic networks to produce target products.
启动子是实现多种合成生物学目标所使用的最重要且最基本的工具之一。大肠杆菌是合成生物学中最常用的模式生物之一，用于生产有用的目标产物并建立复杂的调控网络。在对代谢或调控网络进行微调的过程中，经过充分表征的可诱导启动子数量有限，这使得实施复杂策略变得困难。在本研究中，基于一系列RNA测序数据，对来自大肠杆菌的104个天然启动子-5'-非翻译区复合物（PUTR）进行了筛选和表征。这104个PUTR的强度在转录水平上为P启动子的0.007%至4630%，在翻译水平上为0.1%至137%。为了进一步上调基因表达，通过将强转录启动子与强翻译5'-非翻译区整合，构建了一系列组合PUTR和级联PUTR。最后，鉴定出两个组合PUTR（P-UTR和P-UTR）和两个级联PUTR（PUTR-PUTR和PUTR-PUTR）具有最高活性，其表达输出分别为P启动子的170%、137%、409%和203%。这些工程化的PUTR对于不同基因（如红色荧光蛋白基因和β-半乳糖苷酶基因）的表达是稳定的。这些结果表明，本研究中表征和构建的PUTR可用作即插即用的合成生物学工具箱，以在微调代谢网络以生产目标产物时实现复杂的代谢工程目标。
