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Multiple-Site Diversification of Regulatory Sequences Enables Interspecies Operability of Genetic Devices.
ACS Synthetic Biology ( IF 4.7 ) Pub Date : 2019-12-18 , DOI: 10.1021/acssynbio.9b00375
Angeles Hueso-Gil 1 , Ákos Nyerges 2 , Csaba Pál 2 , Belén Calles 1 , Víctor de Lorenzo 1
Affiliation  

The features of the light-responsive cyanobacterial CcaSR regulatory module that determine interoperability of this optogenetic device between Escherichia coli and Pseudomonas putida have been examined. For this, all structural parts (i.e., ho1 and pcyA genes for synthesis of phycocyanobilin, the ccaS/ccaR system from Synechocystis, and its cognate downstream promoter) were maintained but their expression levels and stoichiometry diversified by (i) reassembling them together in a single broad host range, standardized vector and (ii) subjecting the noncoding regulatory sequences to multiple cycles of directed evolution with random genomic mutations (DIvERGE), a recombineering method that intensifies mutation rates within discrete DNA segments. Once passed to P. putida, various clones displayed a wide dynamic range, insignificant leakiness, and excellent capacity in response to green light. Inspection of the evolutionary intermediates pinpointed translational control as the main bottleneck for interoperability and suggested a general approach for easing the exchange of genetic cargoes between different species, i.e., optimization of relative expression levels and upturning of subcomplex stoichiometry.

中文翻译:

调控序列的多站点多样化实现了遗传设备的种间可操作性。

已经研究了确定该光遗传学设备在大肠杆菌和恶臭假单胞菌之间的互操作性的光响应性蓝细菌CcaSR调节模块的功能。为此,所有结构部分(即用于合成藻蓝素的ho1和pcyA基因,来自集胞藻的ccaS / ccaR系统及其同源下游启动子)均得以保留,但它们的表达水平和化学计量因(i)在一个单一的广泛宿主范围,标准化载体,以及(ii)使非编码调控序列经历具有随机基因组突变(DIvERGE)的定向进化的多个循环,这是一种重组方法,可增强离散DNA片段内的突变率。传到恶臭假单胞菌后,各种克隆都表现出宽广的动态范围,微不足道的泄漏,并具有出色的响应绿光的能力。对进化中间体的检查指出,翻译控制是互操作性的主要瓶颈,并提出了一种简化不同物种之间遗传物质交换的通用方法,即优化相对表达水平和提高亚复杂化学计量比。
更新日期:2019-12-19
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