Pseudomonas putida is a widely used host for metabolic engineering and synthetic biology. However, the use of P. putida has been hampered by the availability of a limited set of expression vectors for producing heterologous proteins. To widen the scope of expression vectors for gene co-expression studies, a previously established dual-inducible expression vector pRG_Duet2 developed for Corynebacterium glutamicum has been modified for use in P. putida. This expression vector, named pRGPDuo2, harbors two origins of replication, colE1 for replication in E. coli and pRO1600 for replication in P. putida. Two multiple cloning sites (MCS1 and MCS2) in pRGPDuo2 are individually controlled by inducible promoters P_tac or P_tetR/tetA. Functional validation of pRGPDuo2 was confirmed by the co-expression of genes for the fluorescent proteins namely, superfolder green fluorescent protein (sfGFP), and red fluorescent protein (RFP). Moreover, the strength of the fluorescence signal was dependent on the inducer concentrations present in the culture medium. The expression vector pRGPDuo2 is an attractive addition to the existing repertoire of expression plasmids for expression profiling and adds to the tools available for P. putida metabolic engineering.

恶臭假单胞菌是代谢工程和合成生物学中广泛使用的宿主。但是，恶臭假单胞菌的使用已被用于生产异源蛋白的有限表达载体的可用性所阻碍。为了扩大用于基因共表达研究的表达载体的范围，已经对先前为谷氨酸棒杆菌开发的双重诱导型表达载体pRG_Duet2进行了修饰，以用于恶臭假单胞菌。该表达载体名为pRGPDuo2，具有两个复制起点，可在大肠杆菌中复制的colE1和可在恶臭假单胞菌中复制的pRO1600 。。pRGPDuo2中的两个多个克隆位点（MCS1和MCS2）分别由诱导型启动子P _tac或P _{tetR / tetA控制}。pRGPDuo2的功能验证通过荧光蛋白基因的共表达得到证实，这些基因分别是超文件夹绿色荧光蛋白（sfGFP）和红色荧光蛋白（RFP）。此外，荧光信号的强度取决于培养基中存在的诱导剂浓度。表达载体pRGPDuo2是用于表达谱分析的现有表达质粒库的一个有吸引力的补充，并添加到恶臭假单胞菌代谢工程可用的工具中。