Identification of biosynthetic gene clusters from metagenomic libraries using PPTase complementation in a Streptomyces host

FEMS Microbiol Lett. 2017 Sep 1;364(16):fnx155. doi: 10.1093/femsle/fnx155.

Abstract

The majority of environmental bacteria are not readily cultured in the lab, leaving the natural products they make inaccessible using culture-dependent discovery methods. Cloning and heterologous expression of DNA extracted from environmental samples (environmental DNA, eDNA) provides a means of circumventing this discovery bottleneck. To facilitate the identification of clones containing biosynthetic gene clusters, we developed a model heterologous expression reporter strain Streptomyces albus::bpsA ΔPPTase. This strain carries a 4΄-phosphopantetheinyl transferase (PPTase)-dependent blue pigment synthase A gene, bpsA, in a PPTase deletion background. eDNA clones that express a functional PPTase restore production of the blue pigment, indigoidine. As PPTase genes often occur in biosynthetic gene clusters (BGCs), indigoidine production can be used to identify eDNA clones containing BGCs. We screened a soil eDNA library hosted in S. albus::bpsA ΔPPTase and identified clones containing non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS) and mixed NRPS/PKS biosynthetic gene clusters. One NRPS gene cluster was shown to confer the production of myxochelin A to S. albus::bpsA ΔPPTase.

Keywords: PPTase; Streptomyces albus; eDNA; environmental DNA; functional metagenomics; myxochelin.

MeSH terms

  • Bacterial Proteins / classification*
  • Bacterial Proteins / genetics*
  • Biological Products
  • Cloning, Molecular / methods*
  • Cosmids
  • Escherichia coli / genetics
  • Genes, Bacterial*
  • Genetic Complementation Test
  • Genomic Library
  • Metagenome / genetics*
  • Metagenomics
  • Multigene Family*
  • Peptide Synthases / genetics
  • Phylogeny
  • Piperidones / metabolism
  • Soil Microbiology
  • Streptomyces / genetics*
  • Transferases (Other Substituted Phosphate Groups) / classification*
  • Transferases (Other Substituted Phosphate Groups) / genetics*

Substances

  • Bacterial Proteins
  • Biological Products
  • Piperidones
  • phosphopantetheinyl transferase
  • indigoidine
  • Transferases (Other Substituted Phosphate Groups)
  • Peptide Synthases
  • non-ribosomal peptide synthase