Metabolic Engineering of Halomonas cupida for Efficient Mineralization of an Organochlorine Herbicide 2,4-Dichlorophenoxyacetic Acid in High Saline Wastewater

Abstract

The treatment performance of high-salinity wastewater containing 2,4-dichlorophenoxyacetic acid (2,4-D) is severely impaired by high osmotic pressure and toxic substances. In this study, a heterologous biodegradation pathway comprising the six genes tfdABCDEF, responsible for the bioconversion of 2,4-D into 3-oxoadipate, and the genes encoding Vitreoscilla hemoglobin (VHb) and green fluorescent protein (GFP) were integrated into the genome of the salt-tolerant chassis Halomonas cupida J9 to generate a halotolerant degrader J9U_2,4-D. The successful transcription of the eight exogenous genes in J9U_2,4-D was demonstrated by RT-PCR. The catalytic activity of the tfdABC genes was directly demonstrated by incubating each intermediate strain with a specific substrate. Stable isotope analysis indicated that J9U_2,4-D efficiently converted ¹³C₆-2,4-D into ¹³CO₂, demonstrating the complete mineralization of 2,4-D in high salt media. Under oxygen-limited conditions, 25 mg/L 2,4-D was completely degraded by J9U_2,4-D within 8 h, suggesting the strain’s applicability in groundwater bioremediation. The strong green fluorescence emitted by J9U_2,4-D is visible in sunlight and provides a reliable tracking system during bioremediation. The removal efficiency of 2,4-D in high-saline wastewater containing 100 mg/L 2,4-D and 100 g/L NaCl reached 90% within 15 h, and ¹³C₆-2,4-D can be converted by J9U_2,4-D into ¹³CO₂ in high-saline wastewater containing 100 g/L NaCl. The high 2,4-D-mineralizing activity of J9U_2,4-D in high-salt environments highlights the potential of this strain for the in situ bioaugmentation of high-salinity organic wastewater. Our strategy of combining extremophiles with synthetic biology may be utilized to create stress-resistant degraders for the bioremediation of polluted extreme environments.