Improvement in Salt Tolerance Ability of Pseudomonas putida KT2440.

Simple Summary: Pseudomonas putida KT2440 is an attractive platform for bioremediation with versatile growth substrate and tolerance capabilities. The limited research on the salt tolerance of strain KT2440 hinders its application in bioremediation in high-salinity environments, where the composition of pollutants becomes increasingly complex and wastewater salinity often remains high. Therefore, the salt tolerance of strain KT2440 was explored in this work. By analyzing transcriptomic data from the high-salinity minimal salts medium, we identified key salt-tolerance genes and used them to improve the salt tolerance of strain KT2440 under 4% w/v NaCl. With co-expression of salt-tolerant genes, the maximum salt tolerance of strain KT2440 increased to 5% w/v NaCl. Further addition of compatible solutes increased the salt tolerance of the strain to 6% w/v NaCl. The engineered strain KT2440 could degrade common pollutants under 4% w/v NaCl in 48 h, and could be a promising platform for bioremediation in high-salinity environments. Pseudomonas putida KT2440 is a popular platform for bioremediation due to its robust tolerance to environmental stress and strong biodegradation capacity. Limited research on the salt tolerance of P. putida KT2440 has hindered its application. In this study, the strain KT2440 was tested to tolerate a maximum of 4% w/v NaCl cultured with minimal salts medium. Transcriptomic data in a high-salinity environment showed significant expression changes in genes in membrane components, redox processes, chemotaxis, and cellular catabolic processes. betB-encoding betaine-aldehyde dehydrogenase was identified from the transcriptome data to overexpress and enhance growth profile of the strain KT2440 in minimal salts medium containing 4% w/v NaCl. Meanwhile, screening for exogenous salt-tolerant genes revealed that the Na+/H+ antiporter EcnhaA from Escherichia coli significantly increased the growth of the strain KT2440 in 4% w/v NaCl. Then, co-expression of EcnhaA and betB (KT2440-EcnhaA-betB) increased the maximum salt tolerance of strain KT2440 to 5% w/v NaCl. Further addition of betaine and proline improved the salt tolerance of the engineered strain to 6% w/v NaCl. Finally, the engineered strain KT2440-EcnhaA-betB was able to degrade 56.70% of benzoic acid and 95.64% of protocatechuic acid in minimal salt medium containing 4% w/v NaCl in 48 h, while no biodegradation was observed in the normal strain KT2440 in the same conditions. However, the strain KT2440-EcnhaA-betB failed to degrade catechol in minimal salt medium containing 3% w/v NaCl. This study illustrated the improvement in the salt tolerance performance of Pseudomonas putida KT2440 and the feasibility of engineered strain KT2440 as a potential salt-tolerant bioremediation platform. [ABSTRACT FROM AUTHOR]