A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase from Rahnella aquatilis with Significantly Reduced Glyphosate Sensitivity

Abstract

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroAR.aquatilis, was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroAE.coli), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroAR.aquatilis were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroAE.coli. To probe the sites contributing to increased tolerance to glyphosate, mutant R.aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R.aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious.

Simultaneous Expression of PDH45 with EPSPS Gene Improves Salinity and Herbicide Tolerance in Transgenic Tobacco Plants

“…This finding confirmed the role of EPSPS gene as an active target for glyphosate action. Previously reported studies also focused on isolation and characterization of R. aqualilis strain EPSPS in tobacco significantly reduced the glyphosate sensitivity (Peng et al, 2012). Besides that, Cao et al (2013) proved the role of five bacterial gene coding for EPSPS in the E. coli and transgenic tobacco against glyphosate.…”

Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Pantoea sp. 5-Enolpyruvylshikimate-3- Phosphate Synthase

“…In addition, the glyphosate resistance level of the transgenic Arabidopsis was 2-fold higher than that of the agricultural application level. We also compared our results with previous studies, which showed that the glyphosate tolerance level of our transgenic A. thaliana and tobacco is 10 mM and 5 mM [30,31], respectively. The 20 mM glyphosate resistance of our transgenic Arabidopsis is obviously higher.…”

Functional Characterization of aroA from Rhizobium leguminosarum with Significant Glyphosate Tolerance in Transgenic Arabidopsis

“…2). Compared with other EPSPSs [20,25], aroA R. leguminosarum has similar performance regarding glyphosate tolerance. However, transgenic plants with aroA R. leguminosarum showed excellent glyphosate resistance.…”