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KMID : 0903520050480040345
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Journal of the Korean Society of Agricultural Chemistry and Biotechnology
2005 Volume.48 No. 4 p.345 ~ p.351
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Screening and Isolation of a Gene Encoding 4-Hydroxyphenylpyruvate Dioxygenase from a Metagenomic Library of Soil DNA
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Yun Sang-Soon
Lee Jung-Han
Kim Soo-Jin
Kim Sam-Sun
Park In-Cheol
Lee Mi-Hye
Koo Bon-Sung
Yoon Sang-Hong
Yeo Yun-Soo
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Abstract
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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.
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KEYWORD
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Soil DNA, metagenomic library, HPPD(4-hydroxyphenylpyruvate dioxygenases)
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