Ribosomal RNA (rRNA) operons have recently been identified as promising sites for chromosomal integration of genetic elements in Pseudomonas putida, a bacterium that has gained considerable popularity as a microbial cell factory. We have developed a tool for targeted integration of recombinant genes into the rRNA operons of various Pseudomonas strains, where the native context of the rRNA clusters enables effective transcription of heterologous genes. However, a sufficient translation of foreign mRNA transcriptionally fused to rRNA required optimization of RNA secondary structures, which was achieved utilizing synthetic ribozymes and a bicistronic design. The generated tool further enabled the characterization of the six rRNA promoter units of P. putida S12 under different growth conditions. The presence of multiple, almost identical rRNA operons in Pseudomonas also allowed the integration of multiple copies of heterologous genetic elements. The integration of two expression cassettes and the resulting disruption of rRNA units only moderately affects growth rates, and the constructs were highly stable over more than 160 generations.
Keywords: bicistronic design; rRNA; recombinant gene expression; ribozymes; synthetic biology.