• Applied Biological Chemistry
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• v.48 no.4
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• pp.345-351
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• 2005

To access the natural products of uncultured microorganisms, we constructed and screened the metagenomic DNA libraries by using a cosmid vector and DNA inserts isolated directly from soil. Initial screening of the libraries in Escherichia coli resulted in the isolation of several clones that produce a dark brown color when grown in LB medium. One of the positive clones, designed pYS85C, was transposon mutagenized and the DNA surrounding the transposon insertions in cosmids that no longer conferred the production of brown pigment to E. coli was sequenced. Annotation of the pYS85C sequence obtained from the transposon mutagenesis experiment indicated a single 393 amino acid open reading frame (ORF) with a molecular mass of about 44.5 kDa, predicted to be a 4-hydroxyphenylpyruvate dioxygenases (HPPDs), was responsible for the observed brown pigment. In a BLAST search against deposited sequence, the translated protein from this ORF showed moderate-level identity (>60%) to the other known HPPDs and was most conserved in the C-terminal region of the protein. These results show that genes involved in natural product synthesis can be cloned directly from soil DNA and expressed in a heterologous host, supporting the idea that this technology has the potential to provide novel natural products from the wealth of environmental microbial diversity and is a potentially important new tool for drug discovery.

• Weed & Turfgrass Science
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• v.5 no.4
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• pp.191-195
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• 2016

Tefuryltrione is a new hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor, which has been recently registered for the use for paddy rice, Korea. Dose-response studies were conducted to compare rice safety and weed control efficacy of tefuryltrione against Eleocharis kuroguwai. When rice and E. kuroguwai were applied at a range of doses of tefuryltrione, $GR_{90}$ values (the dose required to inhibit weed growth by 90%) of E. kuroguwai were $82.38-93.39g\;a.i.\;ha^{-1}$ in two independent experiments. The $GR_{10}$ values (the dose required to inhibit rice growth by 10%) of tefuryltrione for rice were $297.77-471.54g\;a.i.\;ha^{-1}$. As a result, the selectivity indices ($GR_{10}$ for $rice/GR_{90}$ for E. kuroguwai) of tefuryltrione were 3.19-5.72. Therefore, these results demonstrate that tefuryltrione has a relatively high selectivity between rice and E. kuroguwai with a high herbicidal activity against E. kuroguwai and a good rice safety.

• Journal of Microbiology and Biotechnology
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• v.16 no.12
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• pp.1995-1999
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• 2006

Pseudomonas sp. K82 cultured in p-hydroxybenzoate induces protocatechuate 4,5-dioxygenase (PCD 4,5) for p-hydroxybenzoate degradation. In this study, a 6.0-kbp EcoR1 fragment containing p-hydroxybenzoate degradation genes was cloned from the genome of Pseudomonas sp. K82. Sequence analysis identified four genes, namely, pcaD, pcaA, pcaB, and pcaC genes known to be involved in p-hydroxybenzoate degradation. Two putative 4-hydroxyphenylpyruvate dioxygenases and one putative oxidoreductase were closely located by the p-hydroxybenzoate degradation genes. The gene arrangement and sequences of these p-hydroxybenzoate degradation genes were similar to those of Comamonas testosteroni and Pseudomonas ochraceae. PcaAB (PCD4,5) was overexpressed in the expression vector pGEX-4T-3, purified using a GST column, and confirmed to have protocatechuate 4,5-dioxygenase activity. The N-terminal amino acid sequences of overexpressed PCD4,5 were identical with those of purified PCD4,5 from Pseudomonas sp. K82.

• Bulletin of the Korean Chemical Society
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• v.34 no.10
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• pp.2909-2914
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• 2013

p-Hydroxyphenylpyruvate dioxygenase (HPPD) is a potent herbicide target that is in current use. In this study, we developed a predictive pharmacophore model that uses known HPPD inhibitors based on a theoretically constructed HPPD homology model. The pharmacophore model derived from the three-dimensional (3D) structure of a target protein provides helpful information for analyzing protein-ligand interactions, leading to further improvement of the ligand binding affinity.

• Journal of Microbiology and Biotechnology
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• v.27 no.9
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• pp.1692-1700
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• 2017

Ralstonia solanacearum causes bacterial wilt in a wide variety of host plant species and produces a melanin-like blackish-brown pigment in stationary phase when grown in minimal medium supplemented with tyrosine. To study melanin production regulation in R. solanacearum, five mutants exhibiting overproduction of melanin-like pigments were selected from a transposon (Tn) insertion mutant library of R. solanacearum SL341. Most of the mutants, except one (SL341T), were not complemented by the original gene or overproduced melanins. SL341T showed Tn insertion in a gene containing a conserved domain of eukaryotic transcription factor. The gene was annotated as a hypothetical protein, given its weak similarity to any known proteins. Upon complementation with its original gene, the mutant strains reverted to their wild-type phenotype. SL341T produced 3-folds more melanin at 72 h post-incubation compared with wild-type SL341 when grown in minimal medium supplemented with tyrosine. The chemical analysis of SL341T cultural filtrate revealed the accumulation of a higher amount of homogentisate, a major precursor of pyomelanin, and a lower amount of dihydroxyphenylalanine, an intermediate of eumelanin, compared with SL341. The expression study showed a relatively higher expression of hppD (encoding hydroxyphenylpyruvate dioxygenase) and lower expression of hmgA (encoding homogentisate dioxygenase) and nagL (encoding maleylacetoacetate isomerase) in SL341T than in SL341. SL341 showed a significantly higher expression of tyrosinase gene compared with SL341T at 48 h post-incubation. These results indicated that R. solanacearum produced both pyomelanin and eumelanin, and the novel hypothetical protein is involved in the negative regulation of melanin production.

• Weed & Turfgrass Science
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• v.5 no.4
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• pp.187-190
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• 2016

This study was conducted to determine phytotoxicity of domestic rice varieties and characteristic of sensitive gene involved in herbicide reaction of 4-HPPD (4-hydroxy phenylpyruvate dioxygenase) inhibiting herbicides. The five rice varieties were grown for 2 to 3 leaf stage on seedling trays and then transplanted into plastic pot: 4-Japonica type (Sangjubyeo, Sambaekbyeo, Sanduljinmi and Kumyoung) varieties and 1-Indica type variety (IR8). We trialled standard (14 g a.i. $10a^{-1}$) and double fold (28 g a.i. $10a^{-1}$) dose of benzobicyclon treatment at 10 days after transplanting in order to investigate phytoxicity. The Japonica-type Sangjubyeo showed no rice injury but Indica-type IR8 show 4-5 (standard) and 5-6 (double fold) phytoxicity levels. In spite of Japonica-type, Sanduljinmi and kumyoung showed 3-4 and 4-5 levels. Target resistant gene, Hydroxyphenylpyruvate dioxygenase inhibitor sensitive gene No.1 (HIS1) have been isolated from five domestic rice cultivars (Sangjubyeo, Sambaekbyeo, Sanduljinmi, Kumyoung and IR8). Results of the sequence through PCR, all five tested rice cultivars had HIS1 gene regardless to rice cultivars. And the difference between rice varieties from sequence of HIS1 were identified some variation in genes.

• Korean Journal of Weed Science
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• v.31 no.4
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• pp.384-393
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• 2011

Sulfonylurea herbicide-resistant weeds are spreading widely and distributed about 106,951 hectare in paddy rice fields in Korea. Morever all biotype of Scirpus juncoides which were collected at 69 spots all over paddy rice fields in 2008 were identified biotype of sulfonylurea herbicide-resistant. Benzobicyclon is a p-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor, which is absorbed through root and basal stem of weeds so cause bleaching of newly developing leaves. Benzobicyclon was very effective to control Scirpus juncoides, Monochoria vaginalis, sedges and broadleaves weeds, so it have been developed various formulation like a suspension concentrate (SC), a water dispersible granule (WG), a granule (GR) and a DT (tablet for Direct application). During recently 6 years, benzobicyclon-mixtures herbicides have been registered over than 54 products in paddy fields. Herbicidal efficacy by formulations of benzobicyclon and its mixture herbicides were highest in DT, followed by SC and GR. Herbicidal efficacy of the kaolin and $CaCO_3$ carrier of GR was better and stable than that of talc and bentonite carrier. Growth and yield of rice were not affected much by formulations, application rates and rice cultivation methods.