Engineering a Root-Associated Bacterium Pseudomonas stutzeri  A1501 with Nitrogen Fixation and Phosphate Solubilization Activities for Enhanced Growth of Host Rice

Aims Both nitrogen (N) and phosphorus (P) are essential nutrients for plant growth but is often limiting in high yielding agricultural production systems. This study investigated the synergistic effect of nitrogen fixation and organic phosphate mineralization by rhizospheric microorganisms, thereby promoting the growth of host plants. Methods After assessing phosphate-solubilizing activities of five Pseudomonas strains and identifying putative phosphatase genes in silico , selected genes were expressed in nitrogen-fixing P. stutzeri A1501, and the resulting recombinants were evaluated for growth, nitrogenase activity, organic P solubilization (pure and rice co-culture), root colonization, and rice growth-promotion under different N regimes. Results Four phosphatase genes were introduced into A1501 and the resulted recombinant strains displayed significantly elevated extracellular phosphatase activity. Three recombinant strains, i.e. A15PAALP1, A15PAALP2, and A15NapA, exhibited enhanced nitrogenase activity, while six recombinant strains displayed reduced nitrogenase activity. Notably, nitrogenase activity of A15NapA was approximately 7500 nmol ethylene mg ^− 1 protein h ^− 1 , representing an obvious increase of 17%. Lecithin solubilization assay indicated that A1510ACP increased the concentration of available phosphorus by 10.98% relative to A1501. In a rice co-culture system, A1510ACP displayed superior root colonization ability, while enhanced hydrolysis of organic phosphorus increased available phosphorus levels were observed compared to A1501. Pot experiments demonstrated that inoculation with A1510ACP significantly promoted rice growth, particularly under nitrogen-supplemented conditions. Conclusion This study presents the first successful example of engineering a phosphate-solubilizing and nitrogen-fixing strain for enhanced growth of host rice, highlighting the potential of multifunctional engineered strains as the new generation of biological fertilizers.