Establishment of a halotolerant bioremediation platform from Halomonas cupida using synthetic biology approaches

At present, the vast majority of organic pollutants-degrading microbes are unable to grow and lose their degradation capability under high-salt conditions owing to lack of resistance to high osmotic pressure imposed by salt stress. To overcome the bottleneck, in this study, a halotolerant bioremediation platform is established from a halophilic chassis Halomonas cupida J9. As a proof of concept, we chose a typical environmental pollutant p-nitrophenol (PNP) to validate the feasibility of engineering H. cupida J9 for efficient mineralization of environmental pollutants in high saline environments. Firstly, four strong promoters including P3, P15, P16 and P22 were screened from H. cupida J9 by transcriptome analysis and promoter strength characterization. Subsequently, the constructed strain J9U-P15-AB and J9U-P8KT-AB with a chromosome-borne P15- and P8KT-pnpAB expression cassette could rapidly degrade and tolerate 200 mg/L PNP. Furthermore, a pathway for PNP biodegradation was functionally assembled in the genome of H. cupida J9 and optimized by enhanced expression of all PNP degradation genes with P15 and P8KT promoter, resulting in two stable genetically engineered strains J9U-P15PNP and J9U-P8KTPNP capable of metabolizing the only carbon source PNP to support cell growth in high-salinity media. Eventually, stable isotope analysis for elucidating the degradation pathway of 13C6-PNP indicated that the finally constructed strains were capable of converting PNP into CO2 in high saline media. These results suggest that promoter engineering may serve as a useful strategy for significantly improving the mineralization efficiency of organic pollutants. The engineered strains could degrade 25 mg/L PNP in seawater within 6 h, suggesting good potential of these degraders for in situ bioremediation. The gfp-labeled degraders also can be tracked during bioremediation. More importantly, this study underscores the value of H. cupida J9 as a promising chassis for the establishment of a halotolerant bioremediation platform using synthetic biology approaches.