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Unlocking Cryptic Metabolites with Mass Spectrometry-Guided Transposon Mutant Selection.

ABSTRACT: The products of most secondary metabolite biosynthetic gene clusters (BGCs) have yet to be discovered, in part due to low expression levels in laboratory cultures. Reporter-guided mutant selection (RGMS) has recently been developed for this purpose: a mutant library is generated and screened, using genetic reporters to a chosen BGC, to select transcriptionally active mutants that then enable the characterization of the "cryptic" metabolite. The requirement for genetic reporters limits the approach to a single pathway within genetically tractable microorganisms. Herein, we utilize untargeted metabolomics in conjunction with transposon mutagenesis to provide a global read-out of secondary metabolism across large numbers of mutants. We employ self-organizing map analytics and imaging mass spectrometry to identify and characterize seven cryptic metabolites from mutant libraries of two different Burkholderia species. Applications of the methodologies reported can expand our understanding of the products and regulation of cryptic BGCs across phylogenetically diverse bacteria.

SUBMITTER: Yoshimura A

PROVIDER: S-EPMC7572862 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Unlocking Cryptic Metabolites with Mass Spectrometry-Guided Transposon Mutant Selection.

Yoshimura Aya A Covington Brett C BC Gallant Étienne É Zhang Chen C Li Anran A Seyedsayamdost Mohammad R MR

ACS chemical biology 20200914 10

The products of most secondary metabolite biosynthetic gene clusters (BGCs) have yet to be discovered, in part due to low expression levels in laboratory cultures. Reporter-guided mutant selection (RGMS) has recently been developed for this purpose: a mutant library is generated and screened, using genetic reporters to a chosen BGC, to select transcriptionally active mutants that then enable the characterization of the "cryptic" metabolite. The requirement for genetic reporters limits the approa ...[more]

PMID: 32808751

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Project description:Few articles are reported for the simultaneous separation and sensitive detection of the kynurenine pathway (KP) metabolites. This work describes a capillary electrochromatography-mass spectrometry (CEC-MS) method using acrylamido-2-methyl-1-propanesulfonic acid (AMPS) functionalized stationary phase. The AMPS column was prepared by first performing silanization of bare silica with gamma-maps, followed by polymerization with AMPS. The CEC-MS/MS methods were established for six upstream and three downstream KP metabolites. The simultaneous separation of all nine KP metabolites is achieved without derivatization for the first time in the open literature. Numerous parameters such as pH and the concentration of background electrolyte, the concentration of the polymerizable AMPS monomer, column length, field strength, and internal pressure were all tested to optimize the separation of multiple KP metabolites. A baseline separation of six upstream metabolites, namely tryptophan (TRP), kynurenine (KYN), 3-hydroxykynurenine (HKYN), kynurenic acid (KA), anthranilic acid (AA), and xanthurenic acid (XA), was possible at pH 9.25 within 26 min. Separation of six downstream and related metabolites, namely: tryptamine (TRPM), hydroxy‑tryptophan (HTRP), hydroxyindole-3 acetic acid (HIAA), 3-hydroxyanthranilic acid (3-HAA), picolinic acid (PA), and quinolinic acid (QA), was achieved at pH 9.75 in 30 min. However, the challenging simultaneous separation of all nine KP metabolites was only accomplished by increasing the column length and simultaneous application of internal pressure and voltage in 114 min. Quantitation of KP metabolites in commercial human plasma was carried out, and endogenous concentration of five KP metabolites was validated. The experimental limit of quantitation ranges from 100 to 10,000 nM (S/N = 8-832, respectively), whereas the experimental limit of detection ranges from 31 to 1000 nM (S/N = 2-16, respectively). Levels of five major KP metabolites, namely TRP, KYN, KA, AA, and QA, and their ratios in patient plasma samples previously screened for inflammatory biomarkers [C-reactive protein (CRP) and tumor necrosis factor-alpha (TNF-α)] was measured. Pairs of the level of metabolites with significant positive correlation were statistically evaluated.

Dataset Information

Unlocking Cryptic Metabolites with Mass Spectrometry-Guided Transposon Mutant Selection.

Publications

Unlocking Cryptic Metabolites with Mass Spectrometry-Guided Transposon Mutant Selection.

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