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.

假单胞菌蛋白CHA0是一种具有广谱生物防治能力的特性良好的根定殖细菌。因此，它具有遏制植物病害和部分替代对人类有害的合成化学农药的巨大潜力。在这里，我们通过Red / ET重组技术获得了多功能突变体CHA0- ΔretS-Nif。删除retS基因并将固氮基因岛（Nif）整合到CHA0基因组后，所得突变体CHA0- ΔretS-Nif表现出改善的杀菌活性和生物固氮功能。拟南芥的盆栽实验表明，菌株CHA0- ΔretS-Nif通过表达几种次生因子（如抗生素2,4-二乙酰基间苯三酚（2,4-DAPG）和固氮酶）促进植物生长。为了使这种生物控制菌株在工业水平上生长，将1 L连续流发酵罐中的生长条件优化至28°C，pH 7.0和600 rpm。此外，在具有这些最佳生长条件的5 L发酵罐中进行的生长实验产生了最大的细胞密度，这为蛋白质假单胞菌CHA0的工业化和大规模发酵提供了重要的见识，以供进一步应用。CHA0- ΔretS-Nif同时具有杀菌和固氮活性，因此可以用作提高作物产量的生物制剂。