Development of a novel promoter engineering-based strategy for creating an efficient para-nitrophenol-mineralizing bacterium.

A toxic and persistent pollutant para-nitrophenol (PNP) enters into the environment through improper industrial waste treatment and agricultural usage of chemical pesticides, leading to a potential risk to humans. Although a variety of PNP-degrading bacteria have been isolated, their application in bioremediation has been precluded due to unknown biosafety, poor PNP-mineralizing capacity, and lack of genome editing tools. In this study, a novel promoter engineering-based strategy is developed for creating efficient PNP-mineralizing bacteria. Initially, a complete PNP biodegradation pathway from Pseudomonas sp. strain WBC-3 was introduced into the genome of a biosafety and soil-dwelling bacterium Pseudomonas putida KT2440. Subsequently, five strong promoters were identified from P. putida KT2440 by transcriptome analysis and strength characterization, and each of the five promoters was independently inserted into upstream of the pnp operon in the KT2440 genome. Consequently, a P8 promoter-substituted mutant strain showed the highest PNP degradation rate and strong tolerance against high concentrations of PNP. Furthermore, when using P8 promoter to regulate the transcription of all PNP degradation genes pnpABCDEF, the complete and efficient PNP mineralization was demonstrated by stable isotope C-labeled PNP transformation assay. Additionally, the finally constructed KTU-P8pnp can be monitored using integrated GFP on chromosome. This strategy of a combination of pathway construction and promoter engineering should open new avenues for creating efficient degraders for bioremediation.