Metabolic engineering of Pseudomonas putida for mineralization of 1,2-dichloroethane and 2,4-dichlorophenoxyacetic acid and its application in wastewater bioremediation

In this study, to remediate 1,2-dichloroethane (DCA)- and 2,4-dichlorophenoxyacetic acid (2,4-D)-polluted wastewater, an autofluorescent engineered strain KTU-DDGV was constructed from a biosafety strain Pseudomonas putida KT2440 by the implantation of the DCA and 2,4-D mineralization pathways, green fluorescent protein (GFP) and Vitreoscilla hemoglobin (VHb). All exogenous genes stably existed in passage cultures of strain KTU-DDGV. The successful transcription of all fourteen exogenous genes in strain KTU-DDGV was confirmed by RT-PCR. Chromatographic analysis demonstrated that strain KTU-DDGV was able to degrade DCA, 2,4-D and their intermediates. The capacity of strain KTU-DDGV to mineralize DCA and 2,4-D was further confirmed by stable isotope assay. When strain KTU-DDGV was incubated with 13 C-labeled DCA and 2,4-D, the 13 C-labeled CO 2 was detected in the collected gases using an isotope mass spectrometer, demonstrating that strain KTU-DDGV was capable of converting 13 C 2 -DCA and 13 C 6 -2,4-D into 13 CO 2 , cell biomass and cellular components. Under oxygen-limited conditions, strain KTU-DDGV still possessed the capability to co-degrade DCA and 2,4-D. The strain KTU-DDGV exhibits visible green fluorescence with naked eyes under daylight, enabling real-time monitoring of strain KTU-DDGV in polluted environments. During wastewater remediation, strain KTU-DDGV maintained high viability and showed high mineralization efficiency for DCA and 2,4-D. This study highlights the potential of strain KTU-DDGV for in situ bioremediation of DCA- and 2,4- D -polluted wastewater. More importantly, constructing composite degradation circuits in a robust chassis may serve as a useful strategy to cope with multiple organic pollutants.