TY - JOUR
T1 - Reporter-Guided Transposon Mutant Selection for Activation of Silent Gene Clusters in Burkholderia thailandensis
AU - Mao, Dainan
AU - Yoshimura, Aya
AU - Wang, Rurun
AU - Seyedsayamdost, Mohammad R.
N1 - Publisher Copyright:
© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
PY - 2020/7/1
Y1 - 2020/7/1
N2 - 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.
AB - 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.
KW - Burkholderia thailandensis
KW - biosynthesis
KW - natural products
KW - silent gene cluster
KW - transposon mutagenesis
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U2 - 10.1002/cbic.201900748
DO - 10.1002/cbic.201900748
M3 - Article
C2 - 31984619
AN - SCOPUS:85081548145
SN - 1439-4227
VL - 21
SP - 1826
EP - 1831
JO - ChemBioChem
JF - ChemBioChem
IS - 13
ER -