TY - JOUR
T1 - Thailandenes, Cryptic Polyene Natural Products Isolated from Burkholderia thailandensis Using Phenotype-Guided Transposon Mutagenesis
AU - Miller, Cheryl
AU - Rose, Jessica
AU - Xu, Fei
AU - Ebmeier, Christopher C.
AU - Jacobsen, Jeremy R.
AU - Mao, Dainan
AU - Old, William M.
AU - Deshazer, David
AU - Seyedsayamdost, Mohammad R.
N1 - Funding Information:
This research was sponsored by the Defense Advanced Research Projects Agency (DARPA, cooperative agreement 13-34-RTA-FP-007 to W.M.O.), the Medical Biological Defense Research Program, U.S. Army Medical Research and Materiel Command (Project 02-4-5X-026 to D.D.), the National Academy of Science, the National Research Council fellowship funded through the Defense Threat Reduction Agency (Project 132141744 to C.M. and D.D.), and the National Institutes of Health (Grant DP2-AI-124786 to M.R.S.). We thank B. Curry, W. Nierman, J. Varga, and A. Yoshimura for advice and technical assistance. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.
Publisher Copyright:
Copyright © 2019 American Chemical Society.
PY - 2020/5/15
Y1 - 2020/5/15
N2 - Burkholderia thailandensis has emerged as a model organism for investigating the production and regulation of diverse secondary metabolites. Most of the biosynthetic gene clusters encoded in B. thailandensis are silent, motivating the development of new methods for accessing their products. In the current work, we add to the canon of available approaches using phenotype-guided transposon mutagenesis to characterize a silent biosynthetic gene cluster. Because secondary metabolite biosynthesis is often associated with phenotypic changes, we carried out random transposon mutagenesis followed by phenotypic inspection of the resulting colonies. Several mutants exhibited intense pigmentation and enhanced expression of an iterative type I polyketide synthase cluster that we term org. Disruptions of orgA, orgB, and orgC abolished the biosynthesis of the diffusible pigment, thus linking it to the org operon. Isolation and structural elucidation by HR-MS and 1D/2D NMR spectroscopy revealed three novel, cryptic metabolites, thailandene A-C. Thailandenes are linear formylated or acidic polyenes containing a combination of cis and trans double bonds. Variants A and B exhibited potent antibiotic activity against Staphylococcus aureus and Saccharomyces cerevisiae but not against Escherichia coli. One of the transposon mutants that exhibited an enhanced expression of org contained an insertion upstream of a σ54-dependent transcription factor. Closer inspection of the org operon uncovered a σ54 promoter consensus sequence upstream of orgA, providing clues regarding its regulation. Our results showcase the utility of phenotype-guided transposon mutagenesis in uncovering cryptic metabolites encoded in bacterial genomes.
AB - Burkholderia thailandensis has emerged as a model organism for investigating the production and regulation of diverse secondary metabolites. Most of the biosynthetic gene clusters encoded in B. thailandensis are silent, motivating the development of new methods for accessing their products. In the current work, we add to the canon of available approaches using phenotype-guided transposon mutagenesis to characterize a silent biosynthetic gene cluster. Because secondary metabolite biosynthesis is often associated with phenotypic changes, we carried out random transposon mutagenesis followed by phenotypic inspection of the resulting colonies. Several mutants exhibited intense pigmentation and enhanced expression of an iterative type I polyketide synthase cluster that we term org. Disruptions of orgA, orgB, and orgC abolished the biosynthesis of the diffusible pigment, thus linking it to the org operon. Isolation and structural elucidation by HR-MS and 1D/2D NMR spectroscopy revealed three novel, cryptic metabolites, thailandene A-C. Thailandenes are linear formylated or acidic polyenes containing a combination of cis and trans double bonds. Variants A and B exhibited potent antibiotic activity against Staphylococcus aureus and Saccharomyces cerevisiae but not against Escherichia coli. One of the transposon mutants that exhibited an enhanced expression of org contained an insertion upstream of a σ54-dependent transcription factor. Closer inspection of the org operon uncovered a σ54 promoter consensus sequence upstream of orgA, providing clues regarding its regulation. Our results showcase the utility of phenotype-guided transposon mutagenesis in uncovering cryptic metabolites encoded in bacterial genomes.
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U2 - 10.1021/acschembio.9b00883
DO - 10.1021/acschembio.9b00883
M3 - Article
C2 - 31816232
AN - SCOPUS:85084783384
SN - 1554-8929
VL - 15
SP - 1195
EP - 1203
JO - ACS chemical biology
JF - ACS chemical biology
IS - 5
ER -