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A functional screen for recovery of 4'-phosphopantetheinyl transferase and associated natural product biosynthesis genes from metagenome libraries.

Owen, J G; Robins, K J; Skorupa Parachin, Nadia LU and Ackerley, D F (2012) In Environmental Microbiology 12(5). p.1198-1209
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... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Environmental Microbiology
volume
12
issue
5
pages
1198 - 1209
publisher
Wiley-Blackwell
external identifiers
  • wos:000302934000008
  • pmid:22356582
  • scopus:84859988527
ISSN
1462-2920
DOI
10.1111/j.1462-2920.2012.02699.x
language
English
LU publication?
yes
id
f1e4138d-35d4-4055-8748-88cfdd4250a9 (old id 2366384)
date added to LUP
2012-03-06 16:42:04
date last changed
2017-11-05 03:20:23
@article{f1e4138d-35d4-4055-8748-88cfdd4250a9,
  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.},
  author       = {Owen, J G and Robins, K J and Skorupa Parachin, Nadia and Ackerley, D F},
  issn         = {1462-2920},
  language     = {eng},
  number       = {5},
  pages        = {1198--1209},
  publisher    = {Wiley-Blackwell},
  series       = {Environmental Microbiology},
  title        = {A functional screen for recovery of 4'-phosphopantetheinyl transferase and associated natural product biosynthesis genes from metagenome libraries.},
  url          = {http://dx.doi.org/10.1111/j.1462-2920.2012.02699.x},
  volume       = {12},
  year         = {2012},
}