Construction of Recombinant Escherichia coli Expressing Ammonia Assimilation Genes and Evaluation of Its Effect on Removing Ammonium Nitrogen (NH4+-N)

Treating wastewater with high ammonium nitrogen (NH₄⁺-N) is a public and environmental priority. Unlike nitrification-denitrification, ammonia assimilation channels NH₄⁺-N into the glutamate biosynthetic pathway, avoiding gaseous nitrogen species (NOx, N₂). Here we engineered <i>Escherichia coli</i> to enhance ammonia assimilation by co-expressing three key genes, <i>gdhA</i>, <i>glnA</i>, and <i>guaA</i>. The genes were synthesized, cloned into expression plasmids, and introduced into <i>E. coli</i> BL21 for IPTG-inducible expression. Expression of the target proteins at expected sizes was observed, and NH₄⁺-N removal was assessed in flask fermentations. Recombinant strains exhibited significantly higher NH₄⁺-N reduction than the empty vector control; among them, BL21(pET-<i>gdhA</i>-<i>glnA</i>-<i>guaA</i>) 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 NH₄⁺-N in culture and provide a basis for developing recombinant bacteria for practical sewage treatment.