Reporter‐Guided Transposon Mutant Selection for Activation of Silent Gene Clusters in Burkholderia thailandensis

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.

Unlocking Cryptic Metabolites with Mass Spectrometry-Guided Transposon Mutant Selection

“…19 In B. thailandensis, 17 Tn insertions from a total of 960 Tn mutants were found to induce three different silent BGCs, a frequency of 1.8%. 23 The 1−2% frequency from these studies, with which Tn mutants give rise to metabolically active strains, suggests that even subsaturating Tn libraries will be sufficient for inducing silent BGCs. Moreover, as phenotypic changes are often linked and some mutations can globally enhance secondary metabolism, both SOM and IMS are ideal methods for surveying secondary metabolomes on a global level and detecting linked metabolic changes, as in the case of haereoplantin and burrioplantin.…”

“…In a comprehensive study in S. coelicolor , 724 Tn insertions (from a library of 51,443 Tn mutants) were found to increase undecylprodigiosin production, a frequency of 1.4% . In B. thailandensis , 17 Tn insertions from a total of 960 Tn mutants were found to induce three different silent BGCs, a frequency of 1.8% . The 1–2% frequency from these studies, with which Tn mutants give rise to metabolically active strains, suggests that even subsaturating Tn libraries will be sufficient for inducing silent BGCs.…”

“…We recently extended RGMS to Burkholderia thailandensis , a saprophytic β-proteobacterium that is genetically tractable and contains more than a dozen silent BGCs. ,− In one study, we mutagenized wild-type B. thailandensis and inspected the resulting mutants for phenotypic changes, ultimately leading to the characterization of three new cryptic antibiotics, thailandenes A–C . In another study, we used transcriptional and translational fluorescent protein fusions as cluster-specific reporters and identified disruption of nucleotide biogenesis as a trigger for induction of the silent malleicyprol BGC. − One drawback of this approach is that two rounds of genetic manipulations are necessary, the first to generate the reporter constructs and the second for creation of the library of Tn mutants. In the current work, we explore the idea of replacing genetic reporters with mass spectrometry-based methods.…”

Modern Trends in Natural Antibiotic Discovery

“…Using improved RGMS, several cryptic metabolites from mutant libraries of various Burkholderia species were identified. The authors used transposon mutagenesis instead of UV [ 156 ] and MS-based metabolomics instead of a reporter construct [ 157 , 158 ].…”

Double‐reporter‐guided targeted activation of the oxytetracycline silent gene cluster in Streptomyces rimosus M527

“…This method can be used to relieve the complex regulatory network that usually regulate secondary metabolism, and to directly select mutant harboring the desired phenotype based on reporter gene. For example, activation of cryptic biosynthetic gene clusters encoding pyrimidine and gaudimycin D/E was successful carried out in Burkholderia thailandensis (Mao et al, 2020) and Streptomyces sp. PGA64 (P. Li et al, 2018) by using RGMS strategy, respectively.…”