Systems and methods for untargeted metabolomic screening

Patent metadata

Field	Value
Publication number	US-10808256-B2
Application number	US-201715602917-A
Country	US
Kind code	B2
Filing date	May 23, 2017
Priority date	May 23, 2016
Publication date	Oct 20, 2020
Grant date	Oct 20, 2020

Abstract

Provided herein are systems and methods for untargeted screening of metabolite products of biosynthetic gene clusters. In particular, systems and methods are provided for the detection of secondary metabolites and the correlation off such metabolites to the biosynthetic gene clusters responsible for the biosynthesis thereof.

First claim

The invention claimed is:

1. A method of screening for metabolites comprising:
(a) expressing a test putative biosynthetic gene cluster (pBGC) in a host system;
(b) untargeted screening of metabolites produced by the host system by one or more bioanalytical techniques to identify metabolite features produced by the test pBGC;
(c) scoring a metabolite feature based on a combination of uniqueness and abundance, wherein a score is calculated by:
(i) comparing the abundance of the metabolite feature in a test sample from the host system expressing the test pBGC to the abundance of the metabolite feature in multiple samples from the host system lacking the test pBGC to determine the uniqueness of the metabolite feature among samples, and
(ii) combining the uniqueness of the metabolite feature with the relative abundance of the metabolite feature in the test sample to generate a compound score; and

(d) identifying:
(i) the test pBGC as a biosynthetic gene cluster (BGC), and/or
(ii) a particular metabolite as being produced by the test pBGC, if the compound score identifies the metabolite feature as being unique and abundant relative to other scored metabolite features

(e) validating the identification made in step (d) by repeating steps (a) and (b) with a validation pBGC comprising a deletion within the test pBGC, wherein the validation confirms that the test pBGC is a BGC that produces the metabolite if the deletion reduces or eliminates production of the metabolite by the host system.

2. The method of claim 1 , wherein the test pBGC comprises a sequence derived from genomic DNA of a fungus of interest.

3. The method of claim 2 , wherein the test pBGC has been inserted into a fungal artificial chromosome (FAC).

4. The method of claim 1 , wherein the host system is a fungal cell.

5. The method of claim 4 , wherein the fungal cell is selected from the group consisting of Ashbya gossypii, Aspergillus nidulans, Coprinus cinereus, Cryptococcus neoformans, Neurospora crassa, Saccharomyces cerevisiae, Schizophyllum commune, Schizosaccharomyces pombe , and Ustilago maydis.

6. The method of claim 1 , wherein the host system is a fungal cell lysate.

7. The method of claim 1 , wherein the host system is an in vitro expression system.

8. The method of claim 1 , wherein screening comprises subjecting the host system or test sample derived therefrom to the one or more bioanalytical techniques to identify bioanalytical features that correlate to metabolites produced by the host system expressing the test pBGC.

9. The method of claim 8 , wherein scoring comprises calculating a numerical score or otherwise ranking the features obtained in the screening based on a combination of uniqueness and abundance.

10. The method of claim 9 , wherein abundance is a measure of the size of a feature and/or amount of a metabolite, relative to other features and/or metabolites produced by the host system expressing the test pBGC.

11. The method of claim 10 , wherein uniqueness is a measure of the relative rarity of a feature and/or metabolite, relative to other features and/or metabolites produced by similar host systems expressing other BGCs or pBGCs.

12. The method of claim 1 , wherein the one or more bioanalytical techniques are selected from the group consisting of mass spectrometry (MS), tandem mass spectrometry (MS2), high performance liquid chromatography (HPLC), gas chromatography, ultra performance liquid chromatography (UPLC), supercritical fluid chromatography, nuclear magnetic resonance (NMR), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-diode array detection (LC-DAD), capillary electrophoresis-mass spectrometry (CE-MS), and liquid chromatography-tandem mass spectrometry (LC-MS2).

13. The method of claim 1 , further comprising isolating the particular metabolite.

14. The method of claim 1 , further comprising identifying the particular metabolite.

15. The method of claim 1 , wherein comparing the abundance of the metabolite feature in a test sample to abundance of the metabolite feature in one or more control samples comprises dividing the abundance of the metabolite feature in the test sample by the average abundance of the metabolite feature in the one or more control samples.

16. The method of claim 15 , wherein combining the uniqueness of the metabolite feature with the relative abundance of the metabolite feature comprises multiplying the uniqueness of the metabolite feature among samples tested by the abundance of the metabolite feature in the test sample.

17. The method of claim 1 , wherein samples comprising the host system lacking the test pBGC include host systems lacking a pBGC and/or host systems expressing other pBGCs.

Assignees

• Univ Northwestern

Inventors

• Kelleher Neil L
• Thomas Paul M
• Clevenger Kenneth D
• Robey Matthew Thomas

Classifications

• C12N15/52 Primary

  Genes encoding for enzymes or proenzymes · CPC title

• C07K14/195

  from bacteria · CPC title

• C12N15/00

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title

• C12N15/80 Primary

  for fungi · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title