Construction of Recombinant Escherichia coli Expressing Ammonia Assimilation Genes and Evaluation of Its Effect on Removing Ammonium Nitrogen (NH(4)(+)-N).

Treating wastewater with high ammonium nitrogen (NH(4)(+)-N) is a public and environmental priority. Unlike nitrification-denitrification, ammonia assimilation channels NH(4)(+)-N into the glutamate biosynthetic pathway, avoiding gaseous nitrogen species (NOx, N(2)). Here we engineered Escherichia coli to enhance ammonia assimilation by co-expressing three key genes, gdhA, glnA, and guaA. The genes were synthesized, cloned into expression plasmids, and introduced into E. coli BL21 for IPTG-inducible expression. Expression of the target proteins at expected sizes was observed, and NH(4)(+)-N removal was assessed in flask fermentations. Recombinant strains exhibited significantly higher NH(4)(+)-N reduction than the empty vector control; among them, BL21(pET-gdhA-glnA-guaA) performed best, achieving a maximum removal efficiency of 90.09% under the tested conditions. These results indicate that reinforcing the glutamate pathway through multi-gene co-expression can effectively lower NH(4)(+)-N in culture and provide a basis for developing recombinant bacteria for practical sewage treatment.