Transcriptional terminators allow leak-free chromosomal integration of genetic constructs in cyanobacteria
Transcriptional terminators allow leak-free chromosomal integration of genetic constructs in cyanobacteria
复制标题
转录终止子允许蓝细菌中基因构建体的无泄漏染色体整合
DOI：
10.1101/689281
复制
发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Kelly C
中科院分区：
文献类型：
--
作者：
Kelly C
蓝细菌是利用阳光和二氧化碳可持续生产化学品的有希望的候选者。然而，蓝藻的遗传学和代谢比模型异养生物更不容易理解，并且一套具有良好特征的蓝藻遗传工具和部件不太成熟和完整。转录终止子使用特定的RNA结构来停止转录，并且通常用于天然和重组环境中，以实现基因表达的独立控制并将基因和操纵子彼此“隔离”。当异源或合成的遗传构建体插入发生来自染色体启动子的转录通读的基因组位置时，绝缘基因表达可能特别重要，导致对引入基因的表达的不良控制。到目前为止，很少终止子已被描述和特点的蓝藻。在这项工作中，十九异源，合成或推定的本地Rho独立（内在）终止子进行了测试，在模型淡水蓝藻，集胞藻。PCC 6803，从中鉴定出11个强终止子。然后使用这些强终止子的子集成功地将染色体整合的、鼠李糖诱导的haBAD表达系统与邻近染色体启动子的假设“通读”隔离开来，从而大大改善了诱导型控制。在蓝藻工具包中添加经验证的强终止子将允许改进对引入基因的独立控制。
Cyanobacteria are promising candidates for sustainable bioproduction of chemicals from sunlight and carbon dioxide. However, the genetics and metabolism of cyanobacteria are less well understood than those of model heterotrophic organisms, and the suite of well-characterised cyanobacterial genetic tools and parts is less mature and complete. Transcriptional terminators use specific RNA structures to halt transcription and are routinely used in both natural and recombinant contexts to achieve independent control of gene expression and to ‘insulate’ genes and operons from one another. Insulating gene expression can be particularly important when heterologous or synthetic genetic constructs are inserted at genomic locations where transcriptional read-through from chromosomal promoters occurs, resulting in poor control of expression of the introduced genes. To date, few terminators have been described and characterised in cyanobacteria. In this work, nineteen heterologous, synthetic or putative native Rho-independent (intrinsic) terminators were tested in the model freshwater cyanobacterium,Synechocystissp. PCC 6803, from which eleven strong terminators were identified. A subset of these strong terminators was then used to successfully insulate a chromosomally–integrated, rhamnose-induciblerhaBADexpression system from hypothesised ‘read-through’ from a neighbouring chromosomal promoter, resulting in greatly improved inducible control. The addition of validated strong terminators to the cyanobacterial toolkit will allow improved independent control of introduced genes.