Abstract
BackgroundNext Generation DNA Sequencing (NGS) and genome mining of actinomycetes and other microorganisms is currently one of the most promising strategies for the discovery of novel bioactive natural products, potentially revealing novel chemistry and enzymology involved in their biosynthesis. This approach also allows rapid insights into the biosynthetic potential of microorganisms isolated from unexploited habitats and ecosystems, which in many cases may prove difficult to culture and manipulate in the laboratory. Streptomyces leeuwenhoekii (formerly Streptomyces sp. strain C34) was isolated from the hyper-arid high-altitude Atacama Desert in Chile and shown to produce novel polyketide antibiotics.ResultsHere we present the de novo sequencing of the S. leeuwenhoekii linear chromosome (8 Mb) and two extrachromosomal replicons, the circular pSLE1 (86 kb) and the linear pSLE2 (132 kb), all in single contigs, obtained by combining Pacific Biosciences SMRT (PacBio) and Illumina MiSeq technologies. We identified the biosynthetic gene clusters for chaxamycin, chaxalactin, hygromycin A and desferrioxamine E, metabolites all previously shown to be produced by this strain (J Nat Prod, 2011, 74:1965) and an additional 31 putative gene clusters for specialised metabolites. As well as gene clusters for polyketides and non-ribosomal peptides, we also identified three gene clusters encoding novel lasso-peptides.ConclusionsThe S. leeuwenhoekii genome contains 35 gene clusters apparently encoding the biosynthesis of specialised metabolites, most of them completely novel and uncharacterised. This project has served to evaluate the current state of NGS for efficient and effective genome mining of high GC actinomycetes. The PacBio technology now permits the assembly of actinomycete replicons into single contigs with >99 % accuracy. The assembled Illumina sequence permitted not only the correction of omissions found in GC homopolymers in the PacBio assembly (exacerbated by the high GC content of actinomycete DNA) but it also allowed us to obtain the sequences of the termini of the chromosome and of a linear plasmid that were not assembled by PacBio. We propose an experimental pipeline that uses the Illumina assembled contigs, in addition to just the reads, to complement the current limitations of the PacBio sequencing technology and assembly software.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1652-8) contains supplementary material, which is available to authorized users.
Highlights
Generation DNA Sequencing (NGS) and genome mining of actinomycetes and other microorganisms is currently one of the most promising strategies for the discovery of novel bioactive natural products, potentially revealing novel chemistry and enzymology involved in their biosynthesis
Sequencing and assembly of the S. leeuwenhoekii genome Two technologies were used to sequence genomic DNA isolated from S. leeuwenhoekii: Illumina MiSeq and Pacific Biosciences (PacBio) RSII
Benefits and challenges of sequencing technologies: a revised pipeline To our knowledge, this is the first report of the use of Next Generation DNA Sequencing (NGS) to produce a high quality and non-fragmented genome sequence of an actinomycete, an essential prerequisite for efficient genome mining for natural product discovery in these GC-rich bacteria
Summary
Generation DNA Sequencing (NGS) and genome mining of actinomycetes and other microorganisms is currently one of the most promising strategies for the discovery of novel bioactive natural products, potentially revealing novel chemistry and enzymology involved in their biosynthesis This approach allows rapid insights into the biosynthetic potential of microorganisms isolated from unexploited habitats and ecosystems, which in many cases may prove difficult to culture and manipulate in the laboratory. The pioneering sequencing and analysis of the genomes of Streptomyces coelicolor A3(2) [4] and Streptomyces avermitilis [5] revealed that actinomycetes possess an unexpected abundance of natural product biosynthetic gene clusters and that they have the potential to make many more compounds than previously thought This led to the “genome mining” approach to natural product discovery, where bioinformatic analysis is used to estimate the biosynthetic capacity, and potential metabolite novelty, of an organism before extensive analysis in the laboratory. It may be useful when analysing novel and possibly difficult to culture microorganisms isolated from unusual and unexploited habitats such as the oceans, deserts, and the surfaces of plants and animals
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