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

FEMS Microbiology Letters
2017.0

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.

Knowledge Graph

Similar Paper

Identification of biosynthetic gene clusters from metagenomic libraries using PPTase complementation in a Streptomyces host
FEMS Microbiology Letters 2017.0
A chromatogram-simplified Streptomyces albus host for heterologous production of natural products
Antonie van Leeuwenhoek 2020.0
Generation of a cluster-free Streptomyces albus chassis strains for improved heterologous expression of secondary metabolite clusters
Metabolic Engineering 2018.0
Fluostatins Produced by the Heterologous Expression of a TAR Reassembled Environmental DNA Derived Type II PKS Gene Cluster
Journal of the American Chemical Society 2010.0
Cloning and Characterization of an Environmental DNA-Derived Gene Cluster That Encodes the Biosynthesis of the Antitumor Substance BE-54017
Journal of the American Chemical Society 2011.0
Natural Product Discovery through Improved Functional Metagenomics in <i>Streptomyces</i>
Journal of the American Chemical Society 2016.0
Natural Product Discovery through Improved Functional Metagenomics in <i>Streptomyces</i>
Journal of the American Chemical Society 2016.0
Discovery of indolotryptoline antiproliferative agents by homology-guided metagenomic screening
Proceedings of the National Academy of Sciences 2013.0
Direct Pathway Cloning (DiPaC) to unlock natural product biosynthetic potential
Metabolic Engineering 2018.0
Cloning of the complete biosynthetic gene cluster for an aminonucleoside antibiotic, puromycin, and its regulated expression in heterologous hosts.
The EMBO Journal 1992.0