Functional Assembly of a Microbial Consortium with Autofluorescent and Mineralizing Activity for the Biodegradation of Organophosphates

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Abstract
Organophosphorus pesticides (OPs) cause serious environmental problems, and bioremediation using bacterial enzymes may provide an efficient and economical method for their detoxification. Green fluorescent protein (GFP) is a stable and easily detectable marker in monitoring genetically engineered microorganisms (GEMs) in the environment. In our research, the methyl parathion hydrolase gene (mpd) and enhanced green fluorescent protein gene (egfp) were successfully coexpressed using pETDuet vector in E. coli BL21 (DE3). The coexpression of methyl parathion hydrolase (MPH) and enhanced green fluorescent protein (EGFP) were confirmed by determining MPH activity and fluorescence intensity. The recombinant protein MPH showed high enzymatic degradative activity of several widely used OP residues on vegetables determined by GC analysis. Subsequently, a dual-species consortium comprising engineered E. coli and a natural p-nitrophenol (PNP) degrader Ochrobactrum sp. strain LL-1 for complete mineralization of dimethyl OPs was studied. It could completely mineralize methyl parathion (MP) via MP through PNP and hydroquinone and eventually through the TCA cycle as determined by HPLC analysis. The accumulation of PNP in suspended culture was prevented. The consortium could be further utilized for complete mineralization of PNP-substituted OPs in a laboratory-scale bioreactor and easily monitored by fluorescence of EGFP for its activity and fate.

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