A functional screen for recovery of 4'-phosphopantetheinyl transferase and associated natural product biosynthesis genes from metagenome libraries.
(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:
https://lup.lub.lu.se/record/2366384
- author
- Owen, J G ; Robins, K J ; Skorupa Parachin, Nadia LU and Ackerley, D F
- organization
- publishing date
- 2012
- 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
- pmid:22356582
- 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
- 2016-04-01 10:52:53
- date last changed
- 2022-01-26 03:23:30
@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}},
doi = {{10.1111/j.1462-2920.2012.02699.x}},
volume = {{12}},
year = {{2012}},
}