Reporter-Guided Transposon Mutant Selection for Activation of Silent Gene Clusters in Burkholderia thailandensis.
Chembiochem
; 21(13): 1826-1831, 2020 07 01.
Article
in En
| MEDLINE
| ID: mdl-31984619
ABSTRACT
Most natural product biosynthetic gene clusters that can be observed bioinformatically are silent. This insight has prompted the development of several methodologies for inducing their expression. One of the more recent methods, termed reporter-guided mutant selection (RGMS), entails creation of a library of mutants that is then screened for the desired phenotype via reporter gene expression. Herein, we apply a similar approach to Burkholderia thailandensis and, using transposon mutagenesis, mutagenize three strains, each carrying a fluorescent reporter in the malleilactone (mal), capistruin (cap), or an unidentified ribosomal peptide (tomm) gene cluster. We show that even a small library of <500 mutants can be used to induce expression of each cluster. We also explore the mechanism of activation and find that inhibition of pyrimidine biosynthesis is linked to the induction of the mal cluster. Both a transposon insertion into pyrF as well as small-molecule-mediated inhibition of PyrF trigger malleilactone biosynthesis. Our results pave the way toward the broad application of RGMS and related approaches to Burkholderia spp.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Bacterial Proteins
/
DNA Transposable Elements
/
Burkholderia
Language:
En
Journal:
Chembiochem
Journal subject:
BIOQUIMICA
Year:
2020
Type:
Article
Affiliation country:
United States