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Appl. Environ. Microbiol. doi:10.1128/AEM.01911-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A Forest Soil Metagenome Gene Cluster Encoding Antifungal Activity in Escherichia coli

Division of Applied Biology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea; Division of Applied Life Sciences, Gyeongsang National University, Jinju 660-701, Republic of Korea; Biological Function Research Team, Korea Research Institute of Chemical Technology, 100 Jang-Dong, Yuseong-Gu, Daejeon 305-343, Republic of Korea; Department of Medical Bioscience, Dong-A University, Busan 604-714, Republic of Korea

^* To whom correspondence should be addressed. Email: seonlee{at}dau.ac.kr.

	Abstract

Using two forest soils, we previously constructed two fosmid libraries containing 113,700 members in total. The libraries were screened to select active antifungal clones using Saccharomyces cerevisiae as a target fungus. One clone from the Yuseong pine tree rhizosphere soil library, pEAF66, showed S. cerevisiae growth inhibition. Despite an intensive effort, active chemicals were not isolated. DNA sequence analysis and transposon mutagenesis of pEAF66 revealed 39 open reading frames (ORFs) and indicated that eight ORFs, probably in one transcriptional unit, might be directly involved in the expression of antifungal activity in Escherichia coli. The deduced amino acid sequences of eight ORFs were similar to the core genes encoding type II family polyketide synthases such as the acyl carrier protein (ACP), ACP synthases, aminotransferase, and ACP reductase. The gene cluster involved in antifungal activity was similar in its organization to the putative antibiotics production locus of Pseudomonas putida KT2440, although we could not select a similar active clone from the KT2440 genomic DNA library in E. coli. ORFs encoding ABC transporters and membrane proteins were located at both ends of the antifungal gene cluster. Upstream ORFs encoding an IclR family response regulator and a LysR family response regulator were involved in the positive regulation of antifungal gene expression. Our results suggested the metagenomic approach as an alternative to search for novel antifungal antibiotics from unculturable soil bacteria. This is the first report of an antifungal gene cluster from a soil metagenome using S. cerevisiase as a target fungus.