Expanding the genetic toolset: using serine recombinases to integrate riboregulatory elements into industrially...

To realize the full potential of biomanufacturing, the breadth of industrial microbes used to consume diverse feedstock and generate bioproducts needs to expand. As such, portable tools are required that can be used by multiple hosts for straightforward genomic manipulation and precise gene expression. Here, we demonstrate the co-utilization of two synthetic biology tools to achieve these goals: cis-repressors (CRs) and serine recombinase-assisted genome engineering (SAGE). CRs are small, noncoding RNAs that are placed upstream of the target gene to modulate bacterial translation rates at varying, discrete levels. SAGE uses site-specific serine recombinases to catalyze highly efficient, unidirectional insertion of DNA into the chromosome of diverse organisms. We used SAGE to integrate a suite of CRs into the industrially relevant hosts Pseudomonas putida, Corynebacterium glutamicum, and Cupriavidus necator. Using a fluorescent reporter as a readout of CR functionality, we found that CR performance across these backgrounds was similar-providing a range of translational repression up to 100-fold. Overall, these results demonstrate the high portability of CRs across bacterial genetic backgrounds, which ideally can be used in future microbial engineering efforts pertinent to biomanufacturing. One-Sentence Summary: Fine tuning translation across phylogenetically diverse microorganisms using riboregulators and serine recombinase-assisted genome engineering.