Improvement in Salt Tolerance Ability of Pseudomonas putida KT2440

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.