集胞藻PCC6803高效基因表达平台的构建与评价

Construction and evaluation of efficient gene expression platforms in Synechocystis sp. strain PCC6803

针对蓝细菌代谢工程改造的需求,成功构建了可以在模式蓝细菌菌株集胞藻PCC6803中高效表达外源基因的3个基因组整合表达平台,以及1个可以在多株蓝细菌中表达的广宿主穿梭表达平台。该表达平台通过选用集胞藻PCC6803中1,5-二磷酸核酮糖缩化酶/氧化酶的启动子驱动外源基因的表达,应用"SD-AUG"翻译融合的策略提高外源蛋白翻译效率,以及加入终止子序列Trbc以提高转录终止效率,实现了对外源基因的高效表达。利用lacZ作为报告基因,检测了所构建表达平台pFQ20在集胞藻中的基因表达效率,结果显示β-半乳糖苷酶的活性为109 Miller。同时,基于pFQ20表达平台在集胞藻PCC6803中表达了来自大肠杆菌的硫酯酶基因tesA’,蛋白印迹实验结果显示了硫酯酶的成功表达。该表达平台为在蓝细菌中开展遗传研究及基因工程改造提供了有用的遗传工具,其构建策略为在蓝细菌中构建高效稳定的外源基因表达元件提供了借鉴。

For metabolic engineering of cyanobacteria,there is an urgent need to construct a group of efficient heterologous gene expression platforms and to evaluate their expression efficiencies.Here we constructed three integrative vectors,the pKW1188-derived pFQ9F,pFQ9R and pFQ20,for integration of heterologous genes into the genome of the model cyanobacteria strain Synechocystis sp.strain PCC6803.The pFQ16,an RSF1010-derived broad host range shuttle vector,was constructed for conjugative transfer of genes to various cyanobacteria strains.All the four platforms constructed here applied the rbc(encodes Ribulose-1,5-bisphosphate carboxylase/oxygenase) and the rbc terminator to promote and terminate the gene transcription.Besides,a 'Shine-Dalgarno-AUG' fusion translation strategy was used to keep the high protein translation efficiency.Using lacZ as a reporter gene,the expression efficiency of pFQ20 was evaluated and showed a strong β-galactosidase expression(109 Miller).Furthermore,the platform pFQ20 was used to express the E.coli tesA' gene and showed significant protein bands through the Western Blot test.The expression platforms constructed in this study offer useful molecular tools for metabolic engineering of cyanobacteria in the future.