Compositions and methods for biocatalytic engineering

• US 20070048793A1
• Filed: 07/12/2006
• Published: 03/01/2007
• Est. Priority Date: 07/12/2005
• Status: Abandoned Application

• Current Assignee: Codon Devices Incorporated
• Original Assignee: Codon Devices Incorporated
• Inventors: Baynes, Brian
• Application Number:
  US11/485,848
• Publication Number:
  US 20070048793A1
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C40B 30/06 by measuring effects on living organisms, tissues or cells

Abstract

Provided herein are compositions and methods for metabolic pathway engineering. The methods involve combining two or more cells expressing potential pathway proteins extracellularly in the presence of reactants. Also provided are libraries of cells expressing a plurality of pathway components and/or a plurality of variants of a given pathway component extracellularly.

Claims

1. A method for engineering a pathway that produces a desired product, comprising:

i) mixing two or more cells in a reaction mixture comprising a substrate for the pathway, wherein said cells extracellularly express potential pathway components;

ii) assaying the reaction mixture for production of the desired product.

2. The method of claim 1, wherein said cells express the potential pathway components on the cell surface.

3. The method of claim 1, wherein said cells secrete the potential pathway components into the extracellular environment.

4. The method of claim 1, wherein said cells are prokaryotic cells.

5. The method of claim 4, wherein said cells are bacterial cells.

6. The method of claim 5, wherein said cells are E. coli.

7. The method of claim 1, wherein said cells are eukaryotic cells.

8. The method of claim 7, wherein said cells are yeast cells.

9. The method of claim 1, wherein 3 or more cells are mixed in the reaction mixture.

10. The method of claim 1, wherein a plurality of cells are mixed in the reaction mixture.

11. The method of claim 1, wherein expression of the potential pathway components is dependent on the presence of an appropriate substrate in the reaction mixture.

12. The method of claim 1, wherein viability or proliferation of a cell expressing a potential pathway component is regulatable.

13. The method of claim 12, wherein viability or proliferation of a cell expressing a potential pathway component is dependent on the presence of a component in the reaction mixture.

14. The method of claim 1, wherein each cell expresses at least one potential pathway component.

15. The method of claim 1, wherein at least one cell expresses at least two potential pathway components.

16. A method for engineering a pathway for biodegradation of an input substance, comprising:

i) mixing two or more cells in a reaction mixture comprising an input substance for degradation by the pathway, wherein said cells extracellularly express potential pathway components;

ii) assaying the reaction mixture for degradation of the input substance.

17. The method of claim 16, wherein the reaction mixture is assayed for disappearance of the input substance.

18. The method of claim 16, wherein the reaction mixture is assayed for production of a breakdown product.

19. A library comprising a plurality of cells extracellularly expressing a plurality of potential pathway components.

20. The library of claim 19, wherein said cells express said potential pathway components on the cell surface.

21. The library of claim 19, wherein said cells secrete said potential pathway components into the extracellular environment.

22. The library of claim 19, wherein said plurality of potential pathway components comprise enzymes involved in a biodegradation pathway, or variants thereof.

23. The library of claim 19, wherein said plurality of potential pathway components comprise enzymes involved in a biosynthetic pathway, or variants thereof.

24. The library of claim 19, wherein said plurality of potential pathway components comprise two or more variants of at least one metabolic or catabolic enzyme.

25. The library of claim 24, wherein said plurality of potential pathway components comprise a plurality of variants of at least one metabolic or catabolic enzyme.

26. The library of claim 19, wherein each cell expresses at least one potential pathway component.

27. The library of claim 19, wherein at least one cell expresses at least two potential pathway components.

28. The library of claim 27, wherein a plurality of cells each express at least two potential pathway components.