Forest Soil Metagenome Gene Cluster Involved in Antifungal Activity Expression in Escherichia coli

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 those of 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 organization to the putative antibiotic 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 ATP binding cassette 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 obtained from a soil metagenome using S. cerevisiae as a target fungus.

An uncultivated crenarchaeota contains functional bacteriochlorophyll a synthase

“…One of the most promising approaches, the metagenomic approach has been widely and successfully used in genome analysis of uncharacterized microbial taxa (Hallam et al, 2004(Hallam et al, , 2006Moreira et al, 2004;Nunoura et al, 2005;Xu et al, 2007), expression of novel genes from uncultured environmental microorganisms (Schloss and Handelsman, 2003;Chung et al, 2008;Xu et al, 2008), elucidation of community-specific metabolism and comparison of gene contents in different communities (Culley et al, 2006;Green and Keller, 2006;Martin-Cuadrado et al, 2007). By using the metagenomic studies, our understanding of the bacterial and archaeal phototroph based on rhodopsin has been revolutionized (Frigaard et al, 2006;Walter et al, 2007).…”

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

“…For example, in an antibacterial screen, one out of every 10,000 to 20,000 metagenomic cosmid clones exhibited antibacterial activity (Brady, Chao et al 2004). Another example reported one positive clone with antifungal activity out of 113,700 fosmid clones when E. coli was used as a host (Chung, Lim et al 2008). The quantification of screening sensitivity (number of positives/number of screened clones) in E. coli hosts was reviewed by Uchiyama and Miyazaki (Uchiyama and Miyazaki 2009).…”

Bioprospecting microbial metagenome for natural products

“…Overlay assay represents the most straightforward screen based on the detection of clones exhibiting inhibition zones around clones growing on bacterial (Rondon et al 2000;Wang et al 2000) or fungal (Chung et al 2008) test lawns (Fig. 1).…”