Engineering Pseudomonas putida KT2440 for simultaneous degradation of carbofuran and chlorpyrifos

Abstract

SummaryCurrently, chlorpyrifos (CP) and carbofuran are often applied together to control major agricultural pests in many developing countries, in most cases, they are simultaneously detected in agricultural soils. Some cost‐effective techniques are required for the remediation of combined pollution caused by multiple pesticides. In this work, we aim at constructing a detectable recombinant microorganism with the capacity to simultaneously degrade CP and carbofuran. To achieve this purpose, CP/carbofuran hydrolase genes and gfp were integrated into the chromosome of a biosafety strain Pseudomonas putida KT2440 using a chromosomal scarless modification strategy with upp as a counter‐selectable marker. The toxicity of the hydrolysis products was significantly lower compared with the parent compounds. The recombinant strain could utilize CP or carbofuran as the sole source of carbon for growth. The inoculation of the recombinant strain to soils treated with carbofuran and CP resulted in a higher degradation rate than in noninoculated soils. Introduced green fluorescent protein can be employed as a biomarker to track the recombinant strain during bioremediation. Therefore, the recombinant strain has potential to be applied for in situ bioremediation of soil co‐contaminated with carbofuran and CP.

Nicotiana tabacum-associated bioengineered Pseudomonas putida can enhance rhizoremediation of soil containing 2,4-dinitrotoluene

“…Here, the maximum 2,4-DNT reduction (98.03%) was achieved with inoculated plants for 14 days in 1 mM of 2,4-DNT contaminated soil with no accumulation of intermediates because of the almost complete biotransformation of 2,4-DNT. The slightly (1%) initial decrease in the concentration of 2.4-DNT in boxes containing N. tabacum and N. tabacum plants inoculated with KT2440 probably caused by abiotic factors, such as photolysis of 2,4-DNT with sunlight or absorption in the container walls (Gong et al 2016;Podlipná et al 2015). The fact that the rate of biotransformation did not change when the incubation time is extended to 3 weeks may be due to the adsorption of the remaining 2,4-DNT by the adventitious root system in soil (Pandey and Souza-Alonso 2019).…”

Simultaneous hydrolysis of carbaryl and chlorpyrifos by Stenotrophomonas sp. strain YC-1 with surface-displayed carbaryl hydrolase

“…Strains bearing plasmids were grown in Luria-Bertani (LB) medium 44 or M9 minimal medium 45 supplemented with 50 μg/ml kanamycin at 30 °C. Transformation of plasmid into Stenotrophomonas was carried out using the electroporation method 46 .…”

“…Samples were incubated at 30 °C and 200 rpm and removed every 4 h for the quantitative analysis of carbaryl and chlorpyrifos using high performance liquid chromatography (HPLC). Pesticide extraction and HPLC analysis were carried out as described previously 45 .…”

Protocols for RecET‐based markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida

“…Although reduction of the genome size can be beneficial in some cases for better strain performance (Lieder et al ., ), only few cases of deleting large genomic fragment have been reported (Martinez‐Garcia et al ., ; Aparicio et al ., ). In addition, integration of heterologous biosynthetic gene clusters (BGCs) to P. putida chromosome for the production of heterologous secondary metabolites has relied on time‐consuming homologous recombination with selection markers (Wenzel et al ., ; Gross et al ., ; Cao et al ., ; Gong et al ., ) and unpredictable transposon‐mediated random insertion (Glandorf et al ., ; Chai et al ., ; Loeschcke et al ., ; Domrose et al ., , ), requiring the development of rapid and reliable integration system for programmed introduction of heterologous BGCs to P. putida chromosome.…”