Enhancing Biological Denitrification Efficiency With a Novel Antibiotic-Free Plasmid Strategy

The increasing nitrogen emissions from industrial and agricultural activities have exacerbated pollution in aquatic ecosystems, highlighting a great need for effective nitrogen removal strategies. Genetic engineering has proven to be an effective strategy to improve the nitrogen removal capacity of denitrifying bacteria. However, the widespread use of antibiotics in the genetic modification of denitrifying bacteria poses additional environmental risks. Herein, we developed a versatile plasmid toolkit with optimized genetic elements to expand the functionality of a typical denitrifying bacterium, Paracoccus denitrificans. We subsequently introduced an antibiotic-free plasmid system based on auxotrophic marker complementation, ensuring gene expression levels comparable to those achieved by conventional plasmids. This system enhanced the microbial stability and environmental safety by eliminating dependence on antibiotics. Using this system, we reprogrammed genetic circuits related to acetate metabolism and nitrogen reduction in P. denitrificans, leading to accelerated electron transfer and increased activity of denitrification enzymes. Consequently, the engineered strain exhibited a 1.95-fold increase in nitrogen removal efficiency under aerobic conditions and a 11.07-fold increase under anaerobic conditions. The engineered strain also demonstrated improved aerobic denitrification in eutrophic water bodies. Moreover, in a continuous-flow wastewater treatment reactor, the engineered strain maintained stable and efficient anaerobic denitrification. This study provides a stable microbial genetic engineering platform and advances the application of high-performance denitrifying bacteria for actual wastewater treatment.