An engineered Pseudomonas putida can simultaneously degrade organophosphates, pyrethroids and carbamates

Highlights: • P. putida was engineered for simultaneous degradation of three classes of pesticides. • This GFP-marked strain can be easily tracked by fluorescence during bioremediation. • VHb enhances the capacity of P. putida to sequester oxygen in hypoxic environments. • This engineered strain is a promising candidate for in situ bioremediation of soil. • Synthetic biology can serve as a powerful tool to create novel degraders. Agricultural soils are often polluted with a variety of pesticides. Unfortunately, natural microorganisms lack the capacity to simultaneously degrade different types of pesticides. Currently, synthetic biology provides powerful approaches to create versatile degraders. In this work, a biosafety strain Pseudomonas putida KT2440 was engineered for simultaneous degradation of organophosphates, pyrethroids, and carbamates, enhanced oxygen-sequestering capability, and real-time monitoring by targeted insertion of four pesticide-degrading genes, vgb, and gfp into the chromosome using a scarless genome-editing method. The resulting recombinant strain, designated as P. putida KTUe, could completely degrade 50 mg/L methyl parathion, chlorpyrifos, fenpropathrin, cypermethrin, carbofuran and carbaryl within 30 h when incubated in M9 minimal medium supplemented with 20 g/L glucose. In soil remediation studies, all the tested six pesticides (50 mg/kg soil each) were completely removed in soils inoculated with P. putida KTUe within 15 days. Moreover, Vitreoscilla hemoglobin (VHb)-expressing P. putida KTUe grew faster than P. putida KTUd without VHb expression under oxygen-limited conditions, suggesting that VHb may enhance the capability of this recombinant strain to sequester oxygen. Furthermore, the green fluorescence was observed on the P. putida KTUe cells, suggesting that this green fluorescent protein (GFP)-marked strain may be tracked by fluorescence during bioremediation. Therefore, this recombinant strain may serve as a promising candidate for in situ bioremediation of soil contaminated with multiple pesticides. This work not only underscores the value of P. putida KT2440 as an ideal host for bioremediation but also highlights the power of synthetic biology for expanding the degradation capability of natural degraders.