Genetic Engineering of Stenotrophomonas Strain YC-1 To Possess a Broader Substrate Range for Organophosphates

摘要

In this work, Stenotrophomonas sp. strain YC-1, a native soil bacterium that produces methyl parathion hydrolase (MPH), was genetically engineered to possess a broader substrate range for organophosphates (OPs). A surface anchor system derived from the truncated ice nucleation protein (INPNC) from Pseudomonas syringae was used to target organophosphorus hydrolase (OPH) onto the cell surface of strain YC-1, reducing the potential substrate uptake limitation. The surface localization of INPNC-OPH was verified by cell fractionation, Western blot, proteinase accessibility, and immunofluorescence microscopy. No growth inhibition was observed for the engineered strain, and suspended cultures retained almost 100% activity over a period of 2 weeks. Concomitant expression of OPH in strain YC-1 resulted in a recombinant strain capable of simultaneously degrading diethyl and dimethyl OPs. A mixture of six OP pesticides (0.2 mM each) could be degraded completely within 5 h. The broader substrate specificity in combination with the rapid degradation rate makes this engineered strain a promising candidate for in situ remediation of OP-contaminated sites.