Isolation of Antibiotics Turbomycin A and B from a Metagenomic Library of Soil Microbial DNA

Abstract

Three clones, P57G4, P89C8, and P214D2, produced colonies with a dark brown melanin-like color. We fractionated the culture supernatant of P57G4 to identify the pigmented compound or compounds. Methanol extracts of the acid precipitate from the culture supernatant contained a red and an orange pigment. Structural analysis revealed that these were triaryl cations, designated turbomycin A and turbomycin B, respectively; both exhibited broad-spectrum antibiotic activity against gram-negative and gram-positive organisms. Mutagenesis, subcloning, and sequence analysis of the 25-kb insert in P57G4 demonstrated that a single open reading frame was necessary and sufficient to confer production of the brown, orange, and red pigments on E. coli; the predicted product of this sequence shares extensive sequence similarity with members of the 4-hydroxyphenylpyruvate dioxygenase (4HPPD) family of enzymes. Another member of the same family of genes, lly, which is required for production of the hemolytic pigment in Legionella pneumophila, also conferred production of turbomycin A and B on E. coli. We further demonstrated that turbomycin A and turbomycin B are produced from the interaction of indole, normally secreted by E. coli, with homogentisic acid synthesized by the 4HPPD gene products. The results demonstrate successful heterologous expression of DNA extracted directly from soil as a means to access previously uncharacterized small organic compounds, serving as an example of a chimeric pathway for the generation of novel chemical structures.

Proteorhodopsin photosystem gene expression enables photophosphorylation in a heterologous host

“…The results presented here demonstrate the utility of functionally screening large-insert DNA ''metagenomic'' libraries for new phenotypes and activities directly and without subcloning, an approach pioneered by soil microbiologists (15,21,22). Although large-insert libraries increase the probability of capturing complete metabolic pathways in a single clone, their low copy number decreases the sensitivity of detecting heterologous gene expression.…”

Soil eukaryotic functional diversity, a metatranscriptomic approach

“…Such libraries have been generated for different purposes that encompass basic and applied research. The metagenome is considered as a treasure-trove for new enzymes (see, for example, Voget et al, 2003;Yun et al, 2004) and bioactive compounds whose biosynthetic pathways can be coded by full-length genes, operons or gene clusters present on single, long DNA inserts of the libraries (see, for example, Gillespie et al, 2002;Courtois et al, 2003;Schirmer et al, 2005). Analysis of metagenomic libraries also offers the opportunity to get insights into genome organization and gene content of new bacterial species belonging to phyla with no cultivable representative (see, for example, Tyson et al, 2004;Nesbø et al, 2005;García Martín et al, 2006;Strous et al, 2006).…”

Functional metagenomics reveals novel salt tolerance loci from the human gut microbiome

“…Since their inception, metagenomic strategies have led to the identification of numerous novel and diverse genes, enzymes and proteins from many diverse environments through sequence-based and/ or functional approaches (Beja et al, 2000;Gillespie et al, 2002;Lee et al, 2007;Banik and Brady, 2008;Heath et al, 2009;Meilleur et al, 2009). In principle, metagenomics can provide access to all of the genetic resources in a given environmental niche and as such is an extremely powerful tool to access the genomes of difficult-to-culture or unculturable microorganisms .…”