Engineering of Azotobacter chroococcum enables potential replacement of synthetic nitrogen fertilizer and mitigation of nitrogen pollutants release under medium-fertility field conditions

Engineered free-living diazotrophs with enhanced ammonium excretion have long been proposed as a promising biofertilizer to replace chemical nitrogen fertilizers synthesized via the Haber-Bosch process. Deletion of nifL has been widely used as a strategy to engineer nitrogen-fixing strains with enhanced NH₄⁺ excretion. However, the effects of nifL mutation on the global expression of genes and proteins in nitrogen-fixing strains, as well as their actual environmental effects under field conditions, remain not fully understood. We created an Azotobacter mutant (A4) through deletion of the central nifL gene region without introducing any additional promoters or other genetic modifications. The A4 exhibited excellent ammonium excretion and retained phenotypic stability for four years of subculturing. Transcriptomic and proteomic analyses revealed a significant upregulation of NifA-activated genes and their corresponding nitrogen-fixation proteins in A4 compared to the wild types. The high-level nitrogen fixation supports the ability of A4 to potentially replace the synthetic nitrogen fertilizers while maintaining normal yields in vegetable field cultivation under medium-fertility soil conditions. Notably, A4 application reduced nitrogen pollutant release by 87.4%, compared to conventional fertilization. Inoculation with A4 significantly enhanced the predicted nitrogen fixation-related functions of the rhizosphere microbial community without introducing potential ecological risks. This work offers a stable and field-effective strategy for sustainable agriculture.