title: The Streptomyces leeuwenhoekii genome: de novo sequencing and assembly in single contigs of the chromosome, circular plasmid pSLE1 and linear plasmid pSLE2.
abstract: BACKGROUND
  Next 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.
  
  
  RESULTS
  Here 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, United Kingdom. Juan-Pablo.Gomez-Escribano@jic.ac.uk.
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Gomez-Escribano
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, United Kingdom. jcastrofigueroa@gmail.com.|Centre for Biotechnology and Bioengineering (CeBiB), Department of Chemical Engineering and Biotechnology, Universidad de Chile, Beauchef 850, Santiago, Chile. jcastrofigueroa@gmail.com.
authors.forename: Jean Franco
authors.initials: JF
authors.lastname: Castro
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, United Kingdom. vale.razmilic@gmail.com.|Centre for Biotechnology and Bioengineering (CeBiB), Department of Chemical Engineering and Biotechnology, Universidad de Chile, Beauchef 850, Santiago, Chile. vale.razmilic@gmail.com.
authors.forename: Valeria
authors.initials: V
authors.lastname: Razmilic
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, United Kingdom. govind.chandra@jic.ac.uk.
authors.forename: Govind
authors.initials: G
authors.lastname: Chandra
authors.affiliation: Centre for Biotechnology and Bioengineering (CeBiB), Department of Chemical Engineering and Biotechnology, Universidad de Chile, Beauchef 850, Santiago, Chile. bandrews@ing.uchile.cl.
authors.forename: Barbara
authors.initials: B
authors.lastname: Andrews
authors.affiliation: Centre for Biotechnology and Bioengineering (CeBiB), Department of Chemical Engineering and Biotechnology, Universidad de Chile, Beauchef 850, Santiago, Chile. juasenjo@ing.uchile.cl.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Asenjo
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, United Kingdom. mervyn.bibb@jic.ac.uk.
authors.forename: Mervyn J
authors.initials: MJ
authors.lastname: Bibb
mesh_terms: D020451:Contig Mapping; D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D055029:Inverted Repeat Sequences; D018942:Macrolides / Q000378:metabolism; D005810:Multigene Family; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000235:genetics*
chemical_list: D018942:Macrolides