Recombineering Pseudomonas protegens CHA0: An innovative approach that improves nitrogen fixation with impressive bactericidal potency.

Pseudomonas protegens CHA0 is a well-characterized, root-colonizing bacterium with broad-spectrum biocontrol ability. Therefore, it has a great potential to curb plant diseases and to partly replace synthetic chemical pesticides that are harmful to humans. Here, we obtained the multifunctional mutant CHA0-ΔretS-Nif via Red/ET recombineering technology. After deletion of the retS gene and integration of the nitrogen-fixing gene island (Nif) into the CHA0 genome, the resulting mutant, CHA0-ΔretS-Nif, manifested improved both bactericidal activity and biological nitrogen-fixation function. A pot experiment of Arabidopsis thaliana indicated that the strain CHA0-ΔretS-Nif promoted plant growth via expressing several secondary factors, such as the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) and nitrogenase. In order to grow this biocontrol strain at an industrial level, the growth conditions in a 1 L continuous-flow fermenter were optimized to 28 °C, pH of 7.0, and 600 rpm. Moreover, growth experiments in a 5 L fermenter with these optimal growth conditions yielded the maximum cell density, providing vital insights for the industrialization and large-scale fermentation of P. protegens CHA0 for further applications. CHA0-ΔretS-Nif possesses both bactericidal and nitrogen-fixation activities and thus could be used as a biological agent to enhance crop production.