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Screening and Isolation of a Gene Encoding 4-Hydroxyphenylpyruvate Dioxygenase from a Metagenomic Library of Soil DNA  

Yun, Sang-Soon (Microbial Function Team, National Institute of Agricultural Biotechnology)
Lee, Jung-Han (Microbial Function Team, National Institute of Agricultural Biotechnology)
Kim, Soo-Jin (Microbial Function Team, National Institute of Agricultural Biotechnology)
Kim, Sam-Sun (Microbial Function Team, National Institute of Agricultural Biotechnology)
Park, In-Cheol (Microbial Function Team, National Institute of Agricultural Biotechnology)
Lee, Mi-Hye (Microbial Function Team, National Institute of Agricultural Biotechnology)
Koo, Bon-Sung (Microbial Function Team, National Institute of Agricultural Biotechnology)
Yoon, Sang-Hong (Microbial Function Team, National Institute of Agricultural Biotechnology)
Yeo, Yun-Soo (Microbial Function Team, National Institute of Agricultural Biotechnology)
Publication Information
Applied Biological Chemistry / v.48, no.4, 2005 , pp. 345-351 More about this Journal
Abstract
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.
Keywords
Soil DNA; metagenomic library; HPPD(4-hydroxyphenylpyruvate dioxygenases);
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