Development of Promoter and Plasmid Tools in <i>Paracoccus denitrificans</i> DYTN-1 for Enhanced Nitrogen Pollutant Removal

Paracoccus denitrificans plays a pivotal role in the nitrogen removal process. Synthetic biology techniques hold the potential for enhancing these nitrogen removal properties. Nonetheless, the absence of specialized gene expression tools in P. denitrificans has hindered their implementation. In this study, we have engineered a collection of plasmids and an array of nitrogen-inducible promoters to augment the nitrogen degradation efficiency of the P. denitrificans strain DYTN-1. The plasmids have been optimized to an approximate size of 3800 bp and incorporate various replication origins and antibiotic resistance markers, ensuring robust genetic stability. These promoters are responsive to nitrite, nitrate, and ammonium, resulting in an induction range spanning 1.30 (±0.23) to 34.63 (±4.49)-fold. Employing these genetic constructs, we established a dynamic regulatory network sensitive to nitrate and ammonium concentrations, which orchestrates a balance between cellular proliferation and nitrogen elimination. This is achieved by modulating the expression of both the novel Dirmmox pathway (dnfABC) and the critical nirS gene involved in the denitrification process. The modified P. denitrificans strain exhibits enhanced growth rates and improved efficiencies in removing nitrate and ammonium. This study established a new gene expression system in P. denitrificans DYTN-1, marking a significant milestone in the application of synthetic biology to bolster nitrogen pollutant degradation.