Guidelines for metabolomics-guided transposon mutagenesis for microbial natural product discovery

Brett C. Covington, Mohammad R. Seyedsayamdost

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

There is a great discrepancy between the natural product output of cultured microorganisms and their bioinformatically predicted biosynthetic potential, such that most of the molecular diversity contained within microbial reservoirs has yet to be discovered. One of the primary reasons is insufficient expression of natural product biosynthetic gene clusters (BGCs) under standard laboratory conditions. Several methods have been developed to increase production from such “cryptic” BGCs. Among these, we recently implemented mass spectrometry-guided transposon mutagenesis, a forward genetic screen in which mutants that exhibit stimulated biosynthesis of cryptic metabolites, as read out by mass spectrometry, are selected from a transposon mutant library. Herein, we use Burkholderia gladioli as an example and provide guidelines for generating transposon mutant libraries, measuring metabolomic inventories through mass spectrometry, performing comparative metabolomics to prioritize cryptic natural products from the mutant library, and isolating and characterizing novel natural products elicited through mutagenesis. Application of this approach will be useful in both accessing novel natural products from cryptic BGCs and identifying genes involved in their global regulation.

Original languageEnglish (US)
Title of host publicationChemical Microbiology Part B
EditorsErin E. Carlson
PublisherAcademic Press Inc.
Pages305-323
Number of pages19
ISBN (Print)9780323952675
DOIs
StatePublished - Jan 2022

Publication series

NameMethods in Enzymology
Volume665
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Keywords

  • Burkholderia gladioli
  • Cryptic metabolite
  • Mass spectrometry
  • Natural product
  • Silent biosynthetic gene cluster
  • Transposon mutagenesis

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