REAL TIME MONITORING OF MICROBIAL ENZYMATIC PATHWAYS

• US 20080293086A1
• Filed: 09/11/2007
• Published: 11/27/2008
• Est. Priority Date: 09/18/2006
• Status: Abandoned Application

• Current Assignee: Cobalt Technologies, Inc.
• Original Assignee: Cobalt Technologies, Inc.
• Inventors: Contag, Pamela Reilly
• Application Number:
  US11/853,681
• Publication Number:
  US 20080293086A1
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12P 7/28 Acetone-containing products
• CPC Class Codes: C12P 7/52 Propionic acid; Butyric acids
• CPC Class Codes: C12P 7/54 Acetic acid vinegar C12J
• CPC Class Codes: C12P 7/56 Lactic acid
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: G06Q 99/00 Subject matter not provided for in other groups of this subclass
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

This invention provides compositions and methods for monitoring and regulating the production of a target product of a biochemical pathway in an organism, such as butanol. A gene encoding a light-emitting reporter molecule, such as luciferase, is operatively linked with a transcription regulatory nucleotide sequence that regulates transcription of an enzyme in the pathway that signals the rate of production of the target product, such as butanol dehyrogenase. When a microorganism is transfected with such a reporter construct and cultured, the reporter is expressed contemporaneously with the enzyme. The amount of light produced by the reporter indicates amount of enzyme being produced which, in turn, signals the amount of target product being produced. When the reporter is measured in real time, it provides information that can be used to regulate culture conditions and to optimize production of the target product.

Claims

1. A recombinant nucleic acid molecule comprising a transcription regulatory nucleotide sequence operatively linked with a nucleotide sequence encoding a self-contained light-emitting reporter, wherein the transcription regulatory nucleotide sequence regulates expression of a gene that signals production of a target product of a fermentative or synthetic pathway in a cell.

2. The molecule of claim 1 wherein the transcription regulatory nucleotide sequence is a bacterial transcription regulatory nucleotide sequence.

3. The molecule of claim 1 wherein the transcription regulatory nucleotide sequence regulates expression of a gene encoding an enzyme along the pathway and changes in expression of the reporter are positively correlated with changes in production of the target product.

4. The molecule of claim 1 wherein the transcription regulatory nucleotide sequence regulates expression of a gene encoding an enzyme along a branch off of the pathway and changes in expression of the reporter are negatively correlated with changes in production of the target product.

5. The molecule of claim 1 wherein expression of the reporter increases or decreases with increasing production of target product.

6. The molecule of claim 1 wherein expression of the reporter increases or decreases with decreasing production of target product.

7. The molecule of claim 1 wherein the pathway is a fermentation pathway.

8. The molecule of claim 1 wherein the target product is an end product.

10. The molecule of claim 1 wherein the target product is an acid intermediate.

11. The molecule of claim 1 wherein the acid intermediate is acetate, butyrate, or lactate.

12. The molecule of claim 1 wherein the pathway is a substrate utilization pathway selected from gluconeogenesis, glycolysis, Entner-Doudoroff pathway or non-oxidative pentose phosphate pathway.

13. The molecule of claim 1 wherein the gene encodes an enzyme along a pathway leading from acetyl CoA to butanol or a branch of that pathway.

14. The molecule of claim 1 wherein the pathway is an anaerobic pathway.

15. The molecule of claim 1 wherein the bacterium converts hexoses, pentoses or amino acids into acids or alcohols.

16. The molecule of claim 1 wherein the transcription regulatory nucleotide sequence is from Clostridium, E. coli, Z. mobilis, or S. cerevisiae.

17. The molecule of claim 1 wherein the gene encodes butanol dehydrogenase, butyraldehyde dehydrogenase, ethanol dehydrogenase, acid aldehyde dehydrogenase, acetoacetate decarboxylase, butyrate kinase, phosphobutyryltransferase, phosphotransacetylase, acetate kinase, acyl CoA transferase, lactate dehydrogenase, butyl CoA transferase.

18. The molecule of claim 1 wherein the self-contained light-emitting reporter is luminescent.

19. The molecule of claim 18 wherein the luminescent reporter comprises luciferase.

20. The molecule of claim 19 wherein the luciferase is from Coleoptera, Photorhabdus, Vibrio, Gaussia, Diptera, Renilla.

21. The molecule of claim 18 wherein the self-contained light-emitting reporter comprises a fluorescent reporter.

22. The molecule of claim 21 wherein the fluorescent reporter comprises green fluorescent protein (“

GFP”

).

23. The molecule of claim 18 wherein the self-contained light-emitting reporter comprises a phosphorescent reporter.

9. The molecule of claim 9 wherein the end product is acetone, ethanol, or butanol.

24. A cell comprising a self-contained reporter construct that indicates when a synthetic or fermentative pathway has been induced or inhibited so as to affect the concentration of an target product of the pathway.

25. A cell comprising a recombinant nucleic acid molecule comprising a transcription regulatory nucleotide sequence operatively linked with a nucleotide sequence encoding a self-contained light-emitting reporter, wherein the transcription regulatory nucleotide sequence regulates expression of a gene that signals production of a target product of a fermentative or synthetic pathway in the cell.

26. The cell of claim 25 that is a bacterial cell.

27. The cell of claim 25 that is Clostridium, E. coli, Z. mobilis or S. cerevisiae.

28. The cell of claim 25 wherein the target product of the pathway in the cell is an end product.

29. The cell of claim 28 wherein the end product of the pathway in the cell is butanol.

30. The cell of claim 25 wherein the gene encodes butanol dehydrogenase, butyraldehyde dehydrogenase, ethanol dehydrogenase, acid aldehyde dehydrogenase, acetoacetate decarboxylase, butyrate kinase, phosphobutyryltransferase, phosphotransacetylase, acetate kinase, acyl CoA transferase, lactate dehydrogenase, or butyl CoA transferase.

31. The cell of claim 30 further comprising a transcription regulatory nucleotide sequence operatively linked with a nucleotide sequence encoding a self-contained light-emitting reporter, wherein the transcription regulatory nucleotide sequence regulates expression of butyraldehyde dehydrogenase

32. A culture comprising cells that produce a target product of a synthetic or fermentative pathway in commercially valuable quantities and a light emitting reporter.

33. A method comprising:

(a) culturing cells that comprise a recombinant nucleic acid molecule comprising a transcription regulatory nucleotide sequence operatively linked to a nucleotide sequence encoding a light-emitting reporter, wherein the transcription regulatory nucleotide sequence regulates expression of a gene that signals the production of a target product of a fermentative or synthetic pathway in the cell, whereby emission of light by the reporter signals production of the target product;

(b) measuring the light emitted from the reporter in the culture; and

(c) changing culture conditions to adjust production of the target product based on the production signaled by the emitted light.

34. The method of claim 33 wherein the light-emitting reporter is self-contained.

35. The method of claim 33 wherein the target product is an end product.

36. The method of claim 33 wherein the target product is an acid intermediate.

37. The method of claim 33 comprising measuring emitted light in real time.

38. The method of claim 33 wherein the emitted light increases or decreases with increasing production of target product.

39. The method of claim 33 wherein the emitted light increases or decreases with decreasing production of target product.

40. The method of claim 33 wherein the cells are cultured in a culture container comprising a window and the light is measured through the window.

41. The method of claim 33 wherein the cells are cultured in a culture container comprising at least one light sensor within the culture that can sense the emitted light and directly or remotely signal a detector.

42. The method of claim 33 wherein the cells are cultured in a culture container comprising a device that continuously flows culture fluid over a light sensor that senses the emitted light in the flow.

43. The method of claim 33 wherein, if target product production decreases, changing culture conditions comprises remove target product, add nutrients, dilute the culture, remove cells. (synthetic pathways are catabolic, fermentation are metabolic or anabolic)

44. A method comprising:

(a) culturing a recombinant cell under culture conditions to produce a target product, wherein the cell comprises a reporter construct that produces a light-based signal, the intensity of which indicates the level of production of the target product;

(b) monitoring continuously over time the intensity of the signal in the culture at a plurality of different times to indicate the level of production of the target product at those times; and

(c) altering the culture conditions in response to changes in target product production to set target product production to a desired level.

45. Software comprising:

code that receives information about the state of a cell or a cell culture, code that determines whether and how culture conditions should be changed to optimize target production and code that transmits instructions on changing the culture conditions

46. The software of claim 45 wherein the code determines the state of the cell or cell culture.

47. A system comprising:

a) a container for culturing cells,

b) a photon detector for detecting light in a cell culture in the container; and

c) a computer controlled apparatus changes culture conditions in response to light detected by the detector.

48. The system of claim 47 further comprising a device that converts photons to electrons and electrons to photons.

49. The system of claim 47 further comprising the fermentation chamber comprises at least one window, or at least one light sensor within the culture that can directly or remotely signal a detector, or comprising sampling the culture, a continuous flow detector, whereby the culture fluid is passed over a detector/sensor that measures light.

50. The system of claim 47 further comprising a computer controlled apparatus that removes a target product from the container in response to signal from the computer indicating an amount of production of the target product.

51. A composition comprising substantially of butanol, and containing trace components from amaranth, or sweet sorghum, or both, and substantially free of petroleum by-products.

52. A business method comprising:

a) creating a joint venture between at least a first company that produces bioengineered cells that make a biofuel and a second company engaged in oil refining

b) running the joint venture wherein;

i) the first company provides a license to proprietary bioengineered bacterial strains that produce a biofuel;

ii) the second company sponsors research and development at the joint venture directed to biofuel production; and

iii) the second company purchases biofuel produced by the joint venture.