Cloning And Expression Of The EPSPS In Pseudomonas Stutzeri A1501

The enzyme 5-enolpyuvyl-3-phosphoshikimic acid synthase (EPSP synthase; EC2.5.1.19) , encoded by aroA 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 study, in order to solve the key problems in herbicide-tolerant gene engineering in China, such as lack of glyphosate tolerant gene, the glyphosate-resistant strain and its EPSPS were studied, the main results are as follows:Analysis of the complete genome of Pseudomonas stutzeri strain A1501 showed that it has a putative ORF (aroA) of 1323 bp, encoding the EPSPS of 440 amino acid residues. This aroA gene enabled the E. coli aroA mutant strain AKM4188 to grow well on M9 minimal medium supplemented with 150 mM glyphosate. The deduced amino acid sequence of the EPSPS was aligned with those of class I and class II EPSPS. Phylogenetic analyses result showed that the wild type EPSPS is more similar to class II than class I EPSPS. The domains that are reportedly important for glyphosate tolerance and maintenance of productive PEP binding in class II AroA such as GDKS, SAQVK, NPTR, and RDHTE were all found in the EPSPS amino acid sequence. It was suggested that gene encoding aroAA1501 may be used for the engineering of glyphosate tolerant crops.