Cloning And Characterization Of High Glyphosate - Resistant AroA Gene From Streptococcus

EPSP synthase(5-enolpyruvylshikimate-3-phosphate synthase(EC 2.5.1.19)), the sixth enzyme on the shikimate pathway, catalyzes the transfer of the carboxyvinyl moiety from PEP to S3 P to form 5-enolpyruvylshikimate-3-phosphate(EPSP) and inorganic phosphate. The enzyme is essential for the synthasis of aromatic amino acids and many aromatic metabolites in higher plants, algae, bacteria, and microbes,including apicomplexan parasites. Also it is the target of the nonselective herbicide glyphosate. Glyphosate, an analogue of PEP, inhibits the binding site for PEP to form steady ternary complex with the enzyme and S3P(Aro A-S3P-glyphosate) and has been extensively used for broad-spectrum control of weeds in the world. However, glyphosate kills all plants, including food crops, and much attention has been paid to identify glyphosate-tolerant EPSPS that could be introduced into crops to provide herbicide resistance. Two major types of EPSPSs have been classified: Class ⅠEPSPS included those found in plants and bacteria such as Escherichia coli, Pseudomonas putida, Salmonella typhimurium and Petunia hybrid and Class Ⅱ EPSPS had found in Agrobacterium tumefaciens strain CP4, Halothermothrix orenii, Streptococcus pneumoniae and Staphylococcus aureus. And sequence analysis manifests that Class Ⅱ EPSPS share less than 30% amino acid identity with ClassⅠ. However, classⅡ enzymes not only have higher affinity for PEP but also sustain more efficient catalysis in the presence of high glyphosate concentrations than ClassⅠ. Apart from these, a novel EPSPS gene isolated from Pseudomonas putida 4G-1 was classified as a new type EPSPS.Although EPSPS has been received considerable attention since 1970 s, several disadvantages have limited their use in developing glyphosate-tolerant crops such as the low affinity for PEP, only Aro A variants derived from the A. tumefaciens strain CP4 have been successfully used commercially. In our previous study, a highly glyphosate-tolerant and high affinity for PEP EPSPS enzyme gene, was identified from extreme thermophile bacterium Sinorhizobium fredii NGR234 with high tolerance to glyphosate(400 m M). The aro A S. australis gene isolated from Streptococcus australis, which were able to grow on media with relatively high concentrations of Na Cl or KCl(up to 500 m M). Due to coming from a extremophilic strain Streptococcus australis, the enzyme appeared some different properties from other EPSP synthases. Optimal p H 7.5-8.0 is a relatively higher value than other EPSP synthases from E. coli(p H 5.5–6.0), Klebsiella pneumonia(p H 5.4 and 6.8), and Sclerotinia sclerotiorum(p H 7.2),whereas it is nearly same to the EPSP synthases from thermophilic Sinorhizobium Fredii(p H 8.0). Besides, the enzyme maintains activity over a broad p H range and for prolonged periods at elevated temperatures, illustrating the enzyme’s stability under harsh environmental conditions, especially in alkaline soil. The enzyme showed optimum activity at temperature 30 °C, which was much lower than other EPSP synthases from Sinorhizobium Fredii(50 °C). It seems like a advantage because plants grow at a moderate temperature generally. The metal-chelating reagent EDTA had little influence on enzyme activity, which suggested that the enzyme should be a non-metalloenzyme. However, many EPSP synthases appear to be dependence of the catalytic efficiency on univalent cations.Meanwhile, Aro A S. australis presented high tolerance to glyphosate, proving it has the potential usage for the generation of transgenic crops resistant to the glyphosate.