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A multifunctional Pseudomonas putida X3 strain was successfully engineered by introducing methyl parathion (MP)-degrading gene and enhanced green fluorescent protein (EGFP) gene in P. putida X4 (CCTCC: 209319). In liquid cultures, the engineered X3 strain utilized MP as sole carbon source for growth and degraded 100 mg L of MP within 24 h; however, this strain did not further metabolize p-nitrophenol (PNP), an intermediate metabolite of MP. No discrepancy in minimum inhibitory concentrations (MICs) to cadmium (Cd), copper (Cu), zinc (Zn), and cobalt (Co) was observed between the engineered X3 strain and its host strain. The inoculated X3 strain accelerated MP degradation in different polluted soil microcosms with 100 mg MP kg dry soil and/or 5 mg Cd kg dry soil; MP was completely eliminated within 40 h. However, the presence of Cd in the early stage of remediation slightly delayed MP degradation. The application of X3 strain in Cd-contaminated soil strongly affected the distribution of Cd fractions and immobilized Cd by reducing bioavailable Cd concentrations with lower soluble/exchangeable Cd and organic-bound Cd. The inoculated X3 strain also colonized and proliferated in various contaminated microcosms. Our results suggested that the engineered X3 strain is a potential bioremediation agent showing competitive advantage in complex contaminated environments. [ABSTRACT FROM AUTHOR]

Whole-cell biocatalyst has the potential to become a cost-effective alternative to conventional enzyme methods for solving ecological and energy issues. However, cytosolic-expressing biocatalyst systems are critically disadvantaged due to the low permeability of the cell membrane. To overcome substrate transport barrier, periplasmic secretion and surface display biocatalysts were developed by expressing signal peptides or anchor proteins in Escherichia coli. In this work, six carriers were compared in regard to whole-cell activity of methyl parathion hydrolase (MPH). Our results indicate that the surface display systems yielded one to three times whole-cell activity than the periplasmic secretion systems. Although periplasmic secretion systems showed generally more stable than surface display systems, surface display appeared more suitable for whole-cell biocatalyst. It should note that the applicability of the DsbA/PhoA/AIDA-I leader to MPH expression is shown here for the first time. In addition, the result provided a useful reference for other whole-cell biocatalyst selection. [ABSTRACT FROM AUTHOR]

Pseudomonas sp. strain NyZ402, a native soil organism that grows on para-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of methyl parathion (MP) and ortho-nitrophenol (ONP) by integrating mph (methyl parathion hydrolase gene) from Pseudomonas sp. strain WBC-3 and onpAB (ONP 2-monooxygenase and ONP o-benzoquinone reductase genes) from Alcaligenes sp. strain NyZ215 into the genome of strain NyZ402. Methyl parathion hydrolase (MPH), ONP 2-monooxygenase (OnpA) and o-benzoquinone reductase (OnpB) were constitutively expressed in the engineered strain NyZ-MO. Strain NyZ-MO was free of exogenous antibiotic resistance gene markers and the introduced genes were genetically stable. Degradation experiments showed that strain NyZ-MO could utilize MP or ONP as the sole carbon and energy source, and mineralize 0.1 mM MP–0.1 mM ONP simultaneously. This method may serve as a useful strategy for the construction of engineered strains in the degradation of multiple environmental pollutants. [ABSTRACT FROM AUTHOR]