• Journal of Plant Biotechnology
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• v.34 no.4
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• pp.375-380
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• 2007

This study was conducted to develop herbicide-resistance domestic maize plants by introducing the CP4 5-enol-pyruvylshikimate-3-phosphate synthase (CP4 EPSPS) gene using Agrobacterium tumefaciens-mediated immature embryo transformation. Immature embryos of five genotypes (HW1, KL103, HW3, HW4, HW7) were co-cultivated with strains Agrobacterium tumefaciens (strain C58C1) containing the binary vector (pCAMBIA2300) carrying Ubiquitin promoter-CP4 EPSPS gene and Cauliflower mosaic virus 35S (CaMV35S) promoter-nptll gene conferring resistance to paromomycin as a selective agent. The presence and expression of CP4 EPSPS transgene were confirmed by PCR, RT-PCR and Northern blot analysis, respectively. Also, the resistance to glyphosate in the transgenic maize ($T_1$) was analyzed by shikimate accumulation assay. The frequency (%) of paromomycin-resistance callus was 0.37, 0.03, 2.20, 2.37, and 0.81% in pure lines HW1, KL103, HW3, HW4 and HW7, respectively. EPSP transgene sequences were amplified in putative transgenic plants that regenerated from paromomycin-resistance calli of two inbred lines (HW3, HW4). Of them, RT-PCR and Northern blot analyses revealed that the transgene was only expressed in two transgenic events (M266, M104) of HW4 inbred line, and a mild glyphosate resistance of transgenic event (M266) was confirmed by the lower shikimate accumulation in leaf segments. These results demonstrate that transgenic maize with herbicide-resistance traits in Korean genotype can be genetically obtained.

• Horticultural Science & Technology
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• v.35 no.1
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• pp.59-68
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• 2017

Caffeoyl shikimate esterase (CSE) is a key enzyme in lignin synthesis in Arabidopsis thaliana. To determine the role of CSE in lignification of the endocarp in peach (Prunus persica L.) fruit, we cloned and characterized the P. persica CSE homolog, which we designated PpCSE. The 954 - bp PpCSE gene encoded a 317 - amino acid polypeptide. PpCSE expression patterns in the mesocarp and endocarp changed during peach fruit development. There was no significant difference between the expression levels of PpCSE in the mesocarp and endocarp at 39 and 44 days after full bloom (DAFB), but the expression level of PpCSE in the endocarp at 50 and 55 DAFB was 80.73 and 72.75 times higher, respectively, than that in the mesocarp. During peach fruit development, PpCSE expression in the endocarp increased rapidly; the relative PpCSE expression level at 50 DAFB was 122.70 times higher than that at 39 DAFB. At the protein level, CSE was detected in the peach fruit endocarp at 50 and 55 DAFB. Our study suggests that PpCSE expression during peach fruit development is closely related to the degree of endocarp lignification.

• Korean Journal of Microbiology
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• v.55 no.2
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• pp.164-166
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• 2019

The complete genomic information of Microbacterium aurum KACC $15219^T$ (= IFO $15204^T$ = DSM $8600^T$) is described. The genome of M. aurum KACC $15219^T$ contains 3,096 protein coding genes and an average G+C content of 69.9% in its chromosome (3.42 Mbp). This strain can use various carbon sources for growth, including quinic acid. Quinic acid is used as a substrate for the synthesis of aromatic amino acids via the shikimate pathway which are useful in the industry. M. aurum KACC $15219^T$ will provide basis to improve our understanding of this organism and allow more efficient application of the strain to industry.

• Journal of Microbiology and Biotechnology
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• v.32 no.8
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• pp.1011-1016
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• 2022

Bacillus subtilis is a useful bacterium in the food industry with applications as a starter strain for fermented food and as a probiotic. However, it is difficult to discriminate B. subtilis from other Bacillus species because of high phenotypic and genetic similarity. In this study, we employed five previously constructed multilocus sequence typing (MLST) methods for the discrimination of B. subtilis from other Bacillus species and all five MLST assays clearly distinguished B. subtilis. Additionally, the 17 housekeeping genes used in the five MLST assays also clearly distinguished B. subtilis. The pyruvate carboxylase (pyrA) and shikimate dehydrogenase (aroE) genes were selected for the discrimination of B. subtilis because of their high number of polymorphic sites and the fact that they displayed the lowest homology among the 17 housekeeping genes. Specific primer sets for the pyrA and aroE genes were designed and PCR products were specifically amplified from B. subtilis, demonstrating the high specificity of the two housekeeping genes for B. subtilis. This species-specific PCR method provides a quick, simple, powerful, and reliable alternative to conventional methods in the detection and identification of B. subtilis.

• Journal of Microbiology and Biotechnology
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• v.33 no.12
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• pp.1595-1605
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• 2023

Dehydroquinate dehydratase (DHQD) catalyzes the conversion of 3-dehydroquinic acid (DHQ) into 3-dehydroshikimic acid in the mid stage of the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids and folates. Here, we report two the crystal structures of type II DHQD (CgDHQD) derived from Corynebacterium glutamicum, which is a widely used industrial platform organism. We determined the structures for CgDHQD^WT with the citrate at a resolution of 1.80Å and CgDHQD^R19A with DHQ complexed forms at a resolution of 2.00 Å, respectively. The enzyme forms a homododecamer consisting of four trimers with three interfacial active sites. We identified the DHQ-binding site of CgDHQD and observed an unusual binding mode of citrate inhibitor in the site with a half-opened lid loop. A structural comparison of CgDHQD with a homolog derived from Streptomyces coelicolor revealed differences in the terminal regions, lid loop, and active site. Particularly, CgDHQD, including some Corynebacterium species, possesses a distinctive residue P105, which is not conserved in other DHQDs at the position near the 5-hydroxyl group of DHQ. Replacements of P105 with isoleucine and valine, conserved in other DHQDs, caused an approximately 70% decrease in the activity, but replacement of S103 with threonine (CgDHQD^S103T) caused a 10% increase in the activity. Our biochemical studies revealed the importance of key residues and enzyme kinetics for wild type and CgDHQD^S103T, explaining the effect of the variation. This structural and biochemical study provides valuable information for understanding the reaction efficiency that varies due to structural differences caused by the unique sequences of CgDHQD.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1385-1389
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• 2007

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the formation of EPSP and inorganic phosphate from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) in the biosynthesis of aromatic amino acids. To delineate the domain-specific function, we successfully isolated the discontinuous C-terminal domain (residues 1-21, linkers, 240-427) of EPSP synthase (427 residues) by site-directed mutagenesis. The engineered C-terminal domains containing no linker (CTD), or with gly-gly ($CTD^{GG}$) and gly-ser-ser-gly ($CTD^{GSSG}$) linkers were purified and characterized as having distinct native-like secondary and tertiary structures. However, isothermal titration calorimetry (ITC), $^{15}N-HSQC$,\;and\;^{31}P-NMR$ revealed that neither its substrate nor inhibitor binds the isolated domain. The isolated domain maintained structural integrity, but did not function as the half of the full-length protein.

• Journal of Microbiology and Biotechnology
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• v.28 no.8
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• pp.1384-1390
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• 2018

Glyphosate inhibits the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway. A mutant of EPSPS from Pantoea sp. was identified using site-directed mutagenesis. The mutant showed significantly improved glyphosate resistance. The mutant had mutations in three amino acids: Gly97 to Ala, Thr 98 to Ile, and Pro 102 to Ser. These mutation sites in Escherichia coli have been studied as significant active sites of glyphosate resistance. However, in our research, they were found to jointly contribute to the improvement of glyphosate tolerance. In addition, the level of glyphosate tolerance in transgenic Arabidopsis confirmed the potentiality of the mutant in breeding glyphosate-resistant plants.

• Horticultural Science & Technology
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• v.28 no.4
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• pp.648-655
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• 2010

This study is the first comprehensive report on the molecular cloning, structural characterization, sequence comparison between wild and mutant types, copy number in the genome, expression features and activities of a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in Korean lawn grass ($Zoysia$ $japonica$). The full length cDNA of the EPSPS from Korean lawn grass ($zj$EPSPS) obtained from a 3' and 5' RACE method was 1540 bp, containing a 1176 bp ORF, a 144 bp leader sequence (5' UTR) and a 220 bp 3' UTR, which was eventually decoded 391 amino acid residues with a molecular mass of 41.74 kDa. The Southern blot detection of the $zj$EPSPS showed that the gene exists as a single copy in the Korean lawn grass genome. Sequence comparison of the $zj$EPSPS gene demonstrated that the glyphosate-tolerant mutant (GT) having a Pro-53 to Ser substitution in the gene seems to have a preferred binding activity of the enzyme to phosphoenol pyruvate(PEP) over glyphosate, which allows the continuous synthesis of aromatic amino acids in the shikimate pathway. From the Northern blotting analysis, the $zj$EPSPS was found to be highly expressed, with continuous increase until 36 hours after 0.5% glyphosate treatment in both wild and mutant samples, but 1.5-fold higher EPSP synthase activity was observed in the tolerant mutant when exposed to the glyphosate treatment. The molecular information of the $zj$EPSPS gene obtained from this study needs to be further dissected to be more effectively applied to the development of gene-specific DNA markers and zoysiagrass cultivars; nevertheless, the glyphosate-tolerant mutant having the featured $zj$EPSPS gene can be provided to turfgrass managers for weed problems with timely adoptable management options.

• Applied Biological Chemistry
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• v.45 no.4
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• pp.185-189
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• 2002

Along with the worldwide rapid increase of the cultivation area and commercial production of genetically modified (GM) crops, the amount of GM grains imported to Korea has also been increasing. Roundup-Ready soybean (RRS) was introduced with 5-enolpyruvyl shikimate-3-photphate synthase (EPSPS) gene derived from Agrobacterium CP4 to confer the resistance to herbicide, glyphosate. In this study, we tried to develop PCR-based analytical method to detection the presence of RRS among non-GM soybeans. In order to detect RRS specifically, oligonucleotide primers were specifically designed based on the nucleotide sequence of EPSPS transgene. Qualitative PCR method was established and its specificity and accuracy were confirmed by analysing the nucleotide sequence of PCR DNA fragments. Bioassay was also conducted by spraying glyphosate at seedling stage. Survived individuals showed obvious resistance to Roundup Ready, however all of non-GM seedlings died in two weeks after spray. Conclusively, the highly selective detection systems for RRS were successfully established by both PCR using specific primers to EPSPS transgene and bioassay using the herbicide resistance of RRS. In addition to, the imported soybean showed to be mixed to several varieties regarding to 100-seed weight and hilum color.

• Weed & Turfgrass Science
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• v.3 no.3
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• pp.165-173
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• 2014

This review study was conducted to recommend the effective use of herbicide mixtures in Korea. The herbicide ingredients by Herbicide Resistancce Action Committee (HRAC) was classified into 23 groupes according to the mode of action (acetyl CoA carboxylase inhibitors, acetolactate synthase, photosystem I and II inhibitors, protoporphyrinogen oxidase inhibitors, carotenoid biosynthesis inhibitors, enolpyruvyl shikimate-3-phosphate synthase inhibitors, glutamine synthetase inhibitors, dihydropteroate synthetase inhibitors, mitosis inhibitors, cellulose inhibitors, oxidative phosphorylation uncouplers, fatty acid and lipid biosynthesis inhibitors, synthetic auxins, auxin transport inhibitors and potential nucleic acid inhibitors or non-descript mode of action). The rice herbicide mixtures registered in Korea were classified based on the guideline of HRAC. Accordingly, such a classification system for resistance management can help to avoid continuous use of the herbicide having the same mode of action in the same field.