title: A functional screen for recovery of 4'-phosphopantetheinyl transferase and associated natural product biosynthesis genes from metagenome libraries.
abstract: The single-module non-ribosomal peptide synthetase BpsA from Streptomyces lavendulae has the unique ability to autonomously synthesize a coloured product (indigoidine) from a single substrate (l-glutamine), conditional upon activation by a 4'-phosphopantetheinyl transferase (PPTase) partner. We show that bpsA can be expressed in an entD PPTase gene deleted mutant of Escherichia coli to yield a sensitive reporter strain for recovery of PPTase genes from metagenome libraries. We also show that recombinant bpsA constructs, generated by substitution of the native peptidyl carrier protein domain followed by directed evolution to restore function, can be used to increase the diversity of PPTase genes recovered from a sample. As PPTases are essential for activation of non-ribosomal peptide synthetase and polyketide synthase enzymes, they are frequently associated with secondary metabolite gene clusters. Nearly half of the PPTases recovered in our screening of two small-insert soil metagenome libraries were genetically linked to recognizable secondary metabolite biosynthetic genes, demonstrating that PPTase-targeting functional screens can be used for efficient recovery of natural product gene clusters from metagenome libraries. The plasticity and portability of bpsA reporter genes can potentially be exploited to maximize recovery and expression of PPTase-bearing clones in a wide range of hosts.
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington 6140, New Zealand.
authors.forename: J G
authors.initials: JG
authors.lastname: Owen
authors.forename: K J
authors.initials: KJ
authors.lastname: Robins
authors.forename: N S
authors.initials: NS
authors.lastname: Parachin
authors.forename: D F
authors.initials: DF
authors.lastname: Ackerley
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001688:Biological Products; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D005798:Genes, Bacterial / Q000235:genetics*; D057166:High-Throughput Screening Assays / Q000379:methods*; D054892:Metagenome; D005810:Multigene Family; D016366:Open Reading Frames / Q000235:genetics; D010453:Peptide Synthases / Q000235:genetics; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D012988:Soil Microbiology; D013302:Streptomyces / Q000235:genetics; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D001688:Biological Products; C082662:phosphopantetheinyl transferase; D048630:Polyketide Synthases; D017855:Transferases (Other Substituted Phosphate Groups); D010453:Peptide Synthases