Abstract
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 Ptac or PtetR/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.
Highlights
Pseudomonas putida is a Gram-negative, rod-shaped, soil bacterium that has been widely employed for bio-industrial applications (Tiso et al, 2014)
We have developed a dual-inducible duet expression vector pRGPDuo2 for the expression of heterologous proteins in P. putida
While vectors for gene co-expression have been developed in E. coli, C. glutamicum, and P. fluorescens, they have not been developed for P. putida
Summary
Pseudomonas putida is a Gram-negative, rod-shaped, soil bacterium that has been widely employed for bio-industrial applications (Tiso et al, 2014). P. putida is considered to be a favorable host for the production of heterologous proteins due to its advantageous traits such as low nutritional requirements and diverse aerobic metabolism (Timmis, 2002). Physiological features like its ability to generate high biomass yield, rapid growth rate, and minimal maintenance requirements allow P. putida to be developed as an industrial producer for the production of targeted recombinant proteins (PobleteCastro et al, 2012). For P. putida to achieve its full potential and to be used as a major microbial host in genetic and biotechnological studies, the key requirement is to develop robust synthetic biology tools for genetic manipulations including plasmid vectors for expression of heterologous proteins and gene knockouts or repression. It is noteworthy that there is still a limited number of vectors available for this organism that allows the tightly controlled or tunable expression of genes, and that in turn makes rewiring of metabolic pathways difficult (Volke et al, 2020)
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