Cloning A New 5-enolpyruvylshikimate-3-phosphate Synthase Gene Conferring Increased Glyphosate Tolerance And Identification Of The Active Sites In Glyphosate N-acetyltransferase

The shikimate pathway is essential in plant and microorganism, because it serves to biosynthesize the aromatic amino acids for the survival of plant and microorganism. The enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS; 3-phosphoshikimate- 1-carboxyvinyltransferase; EC 2.5.1.19) is a key enzyme in the shikimate pathway, which catalyzes phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P) to form 5-enolpyruvylshikimate 3-phosphate (EPSP) . It is the target of the non-selective herbicide glyphosate. Glyphosate, an analog of PEP, is a competitive inhibitor of EPSPS, and will interrupt the aromatic amino amids synthesis when it was absorbed in plant. Some EPSPS from microorganism can not be inhibited by glyphosate. It is the passive way of glyphosate tolerance in microorganism and plant. On other hand, new research indicated that glyphosate N-acetyltransferase (GAT) is able to acetylate glyphosate, N-acetylglyphosate is not herbicidal and is not an effective inhibitor of EPSPS. It is the other glyphosate-tolerant way, which is initiative. Glyphosate 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 the search for glyphosate-tolerant enzymes for transgenic crops. There is no EPSPS used for culturing glyphosate-tolerant crops successfully but CP4 EPSPS, which attributes to the low tolerance or low affinity for PEP of other known EPSPS. In addition, there is no report about new commercial glyphosate-tolerant crops with N-acetyltransferase. Although China is main country for producing and exporting glyphosate, she has no ascendancy in international competition because of no EPSPS genes with independent intellectual property rights; in addition, China just starts the research on GAT. So, it is important to clone new EPSPS gene with high glyphosate-tolerance and catalytic efficiency to change the situation. Besides, identifying the active sites in GAT is helpful for recombination and utilizing GAT to culture new commercial glyphosate-tolerant crops.In this study, in order to solve the key problems in herbicide-tolerant gene engineering in China, such as lack of glyphosate tolerant gene, a metagenomic library was constructed using DNA which was extracted from glyphosate polluted soils by un-cultured technique and the conserved amino acids in GAT gene were inferred , the main results are as followes:1. Inactivating the aroA gene of E. coli BW25113EPSPS is encoded by the aroA gene, and the aroA mutant Escherichia coli (E. coli) strain is an aromatic amino acids auxotroph and fails to grow on minimal medium. However, if aroA gene is transferred into the mutant, the recombination strain will recover the ability of growing in minimal medium. It is efficient to clone EPSPS genes by this method and it is necessary to construct the aroA mutant E. coli for isolating glyphosate-tolerant EPSPS genes from library. An E. coli mutant AKM4188 was constructed with insertion a kanamycin resistant cassette within the aroA coding sequence through homologous recombination between linear DNA fragment AK and the E. coli genome.2. Construction and screening of the metagenomic libraryMetagenomic library was constructed using DNA which was extractedfrom glyphosate polluted soil by un-cultured technique and was made up of 5, 000 recombinants. One glyphosate-tolerant recombination strain was obtained by selection of recombinants on Mops solid medium containing 150 mM glyphosate, which was named RD. The open reading frame of insertion fragment in RD was 1290 bp, with a G + C content of 51. 1%, and encoded a protein of 430 amino acid residues, which was named RD EPSPS (sequence accession numbers in Genbank: EF155420). The deduced molecular mass was 45, 374 Da. Nucleotide sequence analysis indicated that the gene product shares more than 50% amino acid identity with class II EPSPS, but less than 30% amino acid identity with class I EPSPS, and the multiple sequence alignment showed that RD EPSPS had the same conserved residues and domains as other class II EPSPS. RD aroA was expressed in E. coli BL21 (DE3), and its Kinetic characterization was determined by in vitro enzyme assays (IC50 [glyphosate], 15.075 mM; Km [PEP], 0.0343 mM; and Ki [glyphosate], 0.121 mM).These results indicated RD EPSPS had high glyphosate tolerance and high level of affinity for PEP. It was suggested that gene encoding RD EPSPS may be used for the engineering of glyphosate tolerant crops.3. Identification of the active sites in glyphosate N-acetyltransferaseTo identification of the active sites in glyphosate N-acetyltransferase, we first use Rate4Site, a rigorous statistical method for inferring the level of amino acid conservation at each amino acid site in GAT, taking into account the polygenetic relations between the sequences as well as the stochastic process underling their evolution. Three conserved amino acids P¹³⁴(MTGAT1),S⁵²(MTGAT13-2),I⁵³ (MTGAT87) and three un-conserved amino acids S¹²⁴(MTGAT100),E⁸³(MTGAT6),K⁸⁵(MTGAT13-1) were chosen for further study. These six sites were site-mutated and expressed in E. coli BL21 (DE3). The protein was soluble except MTGATl. The glyphosate-tolerant assay indicated that the glyphosate-tolerance of MTGAT1,MTGAT13-2,MTGAT87 strains was almost lost, but MTGAT100,MTGAT6,MTGAT13-1 strains still keep the tolerant as pre-mutated. The Circular dichroism(CD) and fluorescence spectra of the mutants showed that MTGAT13-2,MTGAT87 were different from GAT, but MTGAT100,MTGAT6,MTGAT13-1 were as the same as the GAT. These results suggested that the mutation of P¹³⁴,S⁵²,I⁵³ may affect the structure and function of GAT by different mechanisms.