Identification of a novel 4-hydroxyphenylpyruvate dioxygenase from the soil metagenome |
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Authors: | Lee Chang-Muk Yeo Yun-Soo Lee Jung-Han Kim Soo-Jin Kim Jung-Bong Han Nam Soo Koo Bon-Sung Yoon Sang-Hong |
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Affiliation: | a Microbial Genetics Division, National Institute of Agricultural Biotechnology, 224, Suinro, Rural Development Administration, Suwon 441-707, Republic of Korea b Department of Food Science and Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea |
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Abstract: | 4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a Fe(II)-dependent, non-heme oxygenase that converts 4-hydroxyphenylpyruvate to homogentisate. Essential cofactors, such as plastoquinone and tocopherol, are produced by HPPD-dependent anabolic pathways in plants. To isolate a novel hppd using culture-independent method, a cosmid metagenomic library was constructed from soil in Korea. Screening of Escherichia coli metagenomic libraries led to the identification of a positive clone, YS103B, producing dark brown pigment in Luria-Bertani medium supplemented with l-tyrosine. In vitro transposon mutagenesis of YS103B showed that the 1.3 kb insert was sufficient to produce the hemolytic brown pigment. Sequence analysis of YS103B disclosed one open reading frame encoding a 41.4 kDa protein with the well-conserved prokaryotic oxygenase motif of the HPPD family of enzymes. The HPPD-specific β-triketone herbicide, sulcotrione, inhibited YS103B pigmentation. The recombinant protein expressed in E. coli generated homogentisic acid. Thus, we present the successful heterologous expression of a previously uncharacterized hppd gene from an uncultured soil bacterium. |
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Keywords: | 4-Hydroxylphenylpyruvate dioxygenase Homogentisic acid Uncultured microorganisms Metagenomic library Hemolysis |
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