Modification of nifL-nifA operon of Klebsiella oxytoca improves N2-fixation efficiency, ammonium-excretion and plant-growth promotion ability

Klebsiella oxytoca is a N₂-fixing bacterium whose nif (nitrogen fixation) gene expression is controlled by the two antagonistic regulatory proteins NifA and NifL encoded by the nifLA operon. NifA is a transcriptional activator, while NifL inhibits the transcriptional activity of NifA. In order to develop an improved K. oxytoca strain with high N₂-fixation efficiency, ammonium-excretion and plant-growth promotion, the negative regulatory gene nifL was deleted and accompanied by insertion of a constitutive Tc promoter upstream of the positive regulatory gene nifA. Engineered strain (designated as K. oxytoca TcP15) exhibited high nitrogenase activity and increased ammonium-excretion, both in the absence and presence of different NH₄⁺ concentrations. Greenhouse experiments showed that engineered K. oxytoca TcP15 promoted growth of cucumber, tomato and rice more efficiently than wild-type K. oxytoca M5al did. This study demonstrates that engineered K. oxytoca TcP15 has the potential to be utilized as biofertilizer for non-legumes in nitrogen-poor and -rich fields.