Reconstitution of Glyphosate Resistance from a Split 5-Enolpyruvyl Shikimate-3-Phosphate Synthase Gene in
            <i>Escherichia coli</i>
            and Transgenic Tobacco

Dun, Baoqing; Wang, Xujing; Lu, Wei; Zhao, Zhonglin; Hou, Songna; Zhang, Baoming; Li, Guiying; Evans, Thomas C.; Xu, Ming‐Qun; Lin, Min

doi:10.1128/aem.00956-07

Cited by 31 publications

(23 citation statements)

References 27 publications

(25 reference statements)

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“…In good agreement with results from other works, which showed that seeds were usually inhibited under glyphosate stress (Dun et al 2007), we observed that approximately 85 % of the seeds of VvEPSPS mutanttransgenic plants germinated well in 1000 lM glyphosate, whereas approximately 30 % seeds of CK and VvEPSPS-transgenic plants germinated at 200 lM glyphosate, although germination was completely inhibited at 1000 lM glyphosate (Fig. 3a).…”

Section: Assay For the Glyphosate Resistance Of Transgenic Arabidopsissupporting

confidence: 94%

Improved glyphosate resistance of 5-enolpyruvylshikimate-3-phosphate synthase from Vitis vinifera in transgenic Arabidopsis and rice by DNA shuffling

Tian

Xing

et al. 2015

Mol Breeding

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To date, only AroA variant derived from Agrobacterium tumefaciens CP4 has been used to generate the commercial glyphosate-resistant crops currently available in the market. This single source of the EPSPS gene may have caused the decrease in herbicide tolerance, which has become a major concern of those involved in field management programs. Therefore, it is of interest to increase aroA/EPSPS gene diversity and seek new glyphosate-tolerant genes for developing glyphosate-tolerant crops. In the current study, EPSPS gene from Vitis vinifera (VvEPSPS) was cloned using reverse transcription polymerase chain reaction. However, wild type VvEPSPS cannot be directly used for developing transgenic crops because of its extreme glyphosate sensitivity. Recent studies have demonstrated that DNA shuffling is an effective strategy in producing multi-mutated EPSPS resourced from plants (EPSPS plant ) with improved glyphosate resistance in bacteria and plants. After performing DNA shuffling on VvEPSPS gene, one highly glyphosateresistant mutant with seven amino acid variations was isolated after five rounds of shuffling and screening. The mutant showed seven amino acid changes in the EPSPS gene, namely, Q93R, T113A, P117L, G126A, C160Y, N239H, and V343A. The assay of glyphosate resistance further confirmed the potential of the VvEPSPS mutant in developing glyphosate-resistant crops.

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Section: Assay For the Glyphosate Resistance Of Transgenic Arabidopsissupporting

confidence: 94%

Improved glyphosate resistance of 5-enolpyruvylshikimate-3-phosphate synthase from Vitis vinifera in transgenic Arabidopsis and rice by DNA shuffling

Tian

Xing

et al. 2015

Mol Breeding

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“…Glyphosate can affect seed germination. Seeds are usually poorly developed under glyphosate stress (7). Figure 4 shows that AroA mutant -transgenic plants grew well in 750 M glyphosate, whereas AroA O.anthropi -transgenic plants did not grow at 500 M. The control plants grew only at 250 M.…”

Section: Resultsmentioning

confidence: 99%

Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Ochrobactrum 5-Enopyruvylshikimate-3-Phosphate Synthase

Tian

Xiong

et al. 2011

Appl Environ Microbiol

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A mutant of 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenic Arabidopsis was obtained to confirm the potential of the mutant in developing glyphosate-resistant crops.

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“…For example, the metabolic enzyme acetolactate synthase has been split and successfully reconstituted by PTS in E. coli (80). In a similar fashion, the bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was also reconstituted by PTS in the chloroplasts of Nicotiana tabacum to produce an herbicide-resistant transgenic crop (81,82). By splitting the gene between nuclear and chloroplast chromosomes, the potential for recombination and transfer to an undesired host is strongly inhibited.…”

Section: Gene Delivery and Control In Transgenic Organismsmentioning

confidence: 99%

Intein Applications: From Protein Purification and Labeling to Metabolic Control Methods

Wood

Camarero

2014

Journal of Biological Chemistry

121

102

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The discovery of inteins in the early 1990s opened the door to a wide variety of new technologies. Early engineered inteins from various sources allowed the development of self-cleaving affinity tags and new methods for joining protein segments through expressed protein ligation. Some applications were developed around native and engineered split inteins, which allow protein segments expressed separately to be spliced together in vitro. More recently, these early applications have been expanded and optimized through the discovery of highly efficient trans-splicing and trans-cleaving inteins. These new inteins have enabled a wide variety of applications in metabolic engineering, protein labeling, biomaterials construction, protein cyclization, and protein purification.The ability of inteins to form and cleave specific peptide bonds in a variety of contexts has enabled the development of powerful new tools in molecular biology. Initial applications focused on self-cleaving affinity tags and protein modification and labeling, and utilized mutations that altered the native splicing reactions described by Perler and co-workers (89) in this series. An important advantage of intein-based methods is that they are generally enzymatic in nature, and therefore exhibit highly specific activities under physiological conditions. Although thiol compounds are used in some applications, the chemistries involved in most intein methods are innocuous to their various target proteins. Intein-based methods have greatly expanded in subsequent years through the discovery of hundreds of additional inteins, and new applications in protein activity regulation and modification have followed. An important underlying theme in this work has been the discovery and development of highly efficient split inteins for advanced applications in vitro and in vivo. These trans-splicing and -cleaving inteins have been employed for applications ranging from the purification of recombinant products to the in vivo control of protein function and labeling. Protein PurificationOne of the first major applications of inteins was the development of self-cleaving affinity tags for the recovery of untagged target proteins in recombinant expression systems (1-3). In these applications, a modified intein is expressed in fusion to an affinity tag and target protein, and once the fusion is affinity-purified, the intein is induced to cleave the target protein from the intein and tag (Fig. 1). The first commercial intein system was released by New England Biolabs in 1997 and employed a modified Sce VMA1 3 intein that is triggered to cleave at its N terminus (IMPACT system), or both N and C termini (IMPACT-CN system), by the addition of thiol compounds (e.g. Refs. 4 -7) (Fig. 1A, left). Shortly thereafter, inteins with rapid C-terminal cleaving activity were published. These inteins exhibit suppressed cleavage activity at pH 8.5, allowing purification of the tagged target, and are then induced to cleave by a shift to pH 6.5. The cleavage activity of these inteins is als...

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Reconstitution of Glyphosate Resistance from a Split 5-Enolpyruvyl Shikimate-3-Phosphate Synthase Gene in Escherichia coli and Transgenic Tobacco

Cited by 31 publications

References 27 publications

Improved glyphosate resistance of 5-enolpyruvylshikimate-3-phosphate synthase from Vitis vinifera in transgenic Arabidopsis and rice by DNA shuffling

Improved glyphosate resistance of 5-enolpyruvylshikimate-3-phosphate synthase from Vitis vinifera in transgenic Arabidopsis and rice by DNA shuffling

Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Ochrobactrum 5-Enopyruvylshikimate-3-Phosphate Synthase

Intein Applications: From Protein Purification and Labeling to Metabolic Control Methods

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