Identification Of Glyphosate-tolerant Psedomonas Fluorescens Strain G2 From Extremely Polluted Environment And Cloning Of Its EPSP Synthase Gene

The enzyme 5-enolpyuvyl-3-phosphoshikimic acid synthase (EPSP synthase; EC2.5.1.19) , encoded by anoA locus , is a key enzyme present in microorganisms and plants where it has a function in the biosynthesis of aromatic amino acids. Glyphosate (N-phosphonomethyl-glycine) is an effective non-selective, broad spectrum, postemergence herbicide, which has been shown to inhibit EPSP synthase activity in a competitive manner. Glyphosate tolerant plants can be mediated by either overproduction of the target enzyme or by the presence of an altered enzyme.In this research, a bacteria strain that can tolerant glyphosate up to 140mM has been isolated from soil polluted by glyphosate in a long term. This strain was identified as psedomonas fluorescens based on phenotypic identification and phylogenetic analysis of 16S rDNA sequencing. This strain is suggested to be named as psedomonas fluorescens G2 temporarily.A gene library of Psedomonas fluorescens G2 was constructed in the cosmid vector pLA2917 using E. coli JM109 as the host strain. Two recombinants, pGR3 and pGR7, which can confer glyphosate resistance ofE.coli JM109 were identified from the selective medium containing 10mM glyphosate. The insert DNA fragments are 7kb and llkb, respectively. Two subclones that were designated pGR3H1 and pGR7H1 and can increase glyphosate resistance of E. coli JM109 up to 150mM glyphosate were constructed by subcloning the 2.4kb and 3.2 kb Hind?/PstI fragments of pGR3 and pGR7 into the corresponding sites of pGEM-3zf and pBluescript. Sequence analysis of these two subclones revealed a completely identical 1323bp open reading frame that encodes an EPSP synthase. The predicted protein contained 441 amino acids.The activity of the EPSP synthase encoded by these two subclones was shown by complementation of the aroA mutation in E. coli strain ER2799, indicating two subclones possess a intact activity of the enzyme 5-enolpyuvyl-3-phosphoshikimic acid synthase.The deduced amino acid sequence of the EPSP synthase from Psedomonas fluorescens G2 was compared to EPSP synthase from various species. It shares 30% homology with the EPSP synthases from E. coli and Salmonella typhimurium in Class I and is different from patent-protected mutational sites. It shares less than 25% homology with the EPSP synthases in Class II. It also shares 24.5% homology with the EPSP synthase isolated by Monsanto company and does not contain any conservative sequences and mutational sitesprotected by patents. All of the above shows great potential for future development.