Combinatorial metabolic libraries

• US 6,268,140 B1
• Filed: 05/12/1999
• Issued: 07/31/2001
• Est. Priority Date: 02/03/1994
• Status: Expired due to Term

• Current Assignee: Intrexon Corporation
• Original Assignee: Neugenesis Corporation
• Inventors: Stuart, W. Dorsey
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/310,398
• Time in Patent Office: 811 Days
• Field of Search: 435/6, 435/254.11, 435/254.21, 435/254.3, 435/254.4, 435/254.5, 435/320.1, 435/440, 435/171
• US Class Current: 435/6.13
• CPC Class Codes: C07K 14/59 Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
• CPC Class Codes: C12N 15/04 Fungi
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Abstract

The present invention provides methods and compositions for producing and screening combinatorial metabolic libraries of multimeric proteins. The present invention relies on the use of filamentous fingal heterokaryons that are produced using two or more parent strains into which a population of heterologous nucleic acids encoding components of a metabolic pathway have been introduced.

Claims

1. A combinatorial metabolic library comprising a plurality of heterologous nucleic acids from one or more species of source organism wherein the nucleic acids are capable of expressing all or part of a metabolic pathway in a host cell.

2. A panel comprising two or more cells wherein each cell contains a portion of the library of claim 1.

3. A panel of claim 2 wherein the cells are fungal cells.

4. A panel comprising two or more heterokaryonic cells wherein each of said heterokaryonic cells contains a portion of the library of claim 1.

5. A panel of claim 3 or 4 wherein the cells are selected from the groups Phycomycetes, Ascomycetes, Basidiomycetes, and Deuteromycetes.

6. A panel of claim 5 wherein the cells are filamentous fungal cells.

7. A panel of claim 6 wherein the cells are selected from the group consisting of N. intermedia, N. crassa, N. sitopula, N. tetraspora, A. nidulans, A. niger, A. terreus, and A. fumegatus.

8. A panel of claim 7 wherein the filamentous fungal cells are Neurospora crassa cells.

9. A panel of claim 4 wherein each heterokaryonic cell is formed by fusing two or more parental fungal strains, wherein said heterokaryon requires the presence of each parent fungal nuclei for survival, each parental fungal strain containing a heterologous nucleic acid present in one or more cells of the panel.

10. A panel of claim 4 wherein one or more heterokaryonic cell secrets a metabolite produced by the expressible metabolic pathway.

11. A kit comprising one or more containers that contains the heterokaryon panel of claim 4.

16. A method for screening a heterokaryon panel for expression of a desired metabolite comprising culturing the heterokaryon panel of claim 4 under conditions in which the heterologous nucleic acids are expressed;

and screening the panel to identify a heterokaryon which produces the metabolite.

17. The identified heterokaryon of claim 16.

18. A method for producing a desired metabolite, said method comprising the step of culturing the heterokaryon of claim 17 under conditions which produces the metabolite.

19. A method for screening a heterokaryon panel for expression of a desired catabolic activity comprising culturing the heterokaryon panel of claim 4 under conditions in which the heterologous nucleic acids are expressed;

and screening the panel to identify a heterokaryon which expresses the catabolic activity.

20. The identified heterokaryon of claim 19.

21. A method for degrading a substance with a catabolic activity expressed in a heterokaryon panel member, said method comprising combining the substance with the identified heterokaryon of claim 20;

and culturing under conditions which expresses the catabolic activity.

22. A method for identifying nucleic acids encoding components of a metabolic pathway comprising isolating heterologous nucleic acids from the identified heterokaryon of claim 17;

and screening them for nucleic acids encoding components of the pathway.

12. A method for producing a panel of heterokaryons comprising two or more heterokaryons wherein each of said heterokaryons contains at least two heterologous nucleic acids encoding a component of a metabolic pathway, comprising the steps of:

fusing two or more parental fungal strains, each strain containing a heterologous nucleic acid molecule that encodes a component of a metabolic pathway and where said parental strains are homozygous for all heterokaryon compatibility alleles and

selecting two or more heterokaryons thus formed to generate said panel,

wherein said heterokaryons requires the presence of each parental fungal nuclei for survival.

13. The method of claim 12 wherein at least one heterokaryon in the panel expresses all or part of a metabolic pathway requiring the expression of at least one heterologous nucleic acid.

14. The method of claim 13 wherein said metabolic pathway produces a secreted metabolite.

15. The method of claim 12 wherein said parental fungal strains are fused using a pulsed electric field.