Methods for control of flux in metabolic pathways through protease manipulation

• US 8,916,358 B2
• Filed: 08/31/2011
• Issued: 12/23/2014
• Est. Priority Date: 08/31/2010
• Status: Active Grant

• Current Assignee: GreenLight Biosciences, Inc., Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: GreenLight Biosciences, Inc., Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Swartz, James R.
• Primary Examiner(s): Swope, Sheridan
• Application Number:
  US13/223,042
• Publication Number:
  US 20120052547A1
• Time in Patent Office: 1,210 Days
• Field of Search: None
• US Class Current: 435/41
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 9/50 Proteinases , e.g. Endopeptidases (3.4.21-3.4.25)
• CPC Class Codes: C12P 19/305 Pyrimidine nucleotides
• CPC Class Codes: C12P 7/42 Hydroxy-carboxylic acids
• CPC Class Codes: C12Y 304/21009 Enteropeptidase (3.4.21.9), i.e. enterokinase
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The embodiments described herein pertain to cells, and methods for preparing cells, that can be used as biocatalysts by altering enzymes that compete for a substrate or product of a pathway of interest such that the targeted enzyme is sensitive to a site-specific protease, which protease is expressed but relocated in the cell to a site where it is not in contact with the targeted enzyme in the intact cell. Upon cell lysis, the protease contacts the target enzyme, which is then inactivated by protease cleavage.

Claims

1. A method of producing a product of a biosynthetic pathway of interest, the method comprising:

(i) growing genetically modified bacterial cells that comprise nucleic acids that encode;

(a) enzymes of the biosynthetic pathway of interest that catalyze production of the product,

(b) a targeted enzyme that competes for substrates or cofactors with an enzyme in the biosynthetic pathway of interest, decreases the overall yield and/or the rate of production of the product, and includes a site-specific protease recognition sequence, and

(c) a site-specific protease that cleaves the site-specific protease recognition sequence of the targeted enzyme and is genetically modified to include a periplasmic targeting sequence;

(ii) lysing at least a portion of the bacterial cells to produce a lysate, thereby bringing the site-specific protease into contact with the targeted enzyme to cleave and inactivate or reduce the activity of the targeted enzyme;

(iii) adding substrate to the lysate; and

(iv) incubating the lysate and substrate, thereby producing the product of the biosynthetic pathway of interest.

2. The method of claim 1, wherein the method further comprises combining the cell lysate with one or more substrates, enzymes, nutrients, co-factors, buffers, reducing agents, or ATP generating systems.

3. The method of claim 1, wherein the periplasmic targeting sequence is selected from the group consisting of:

4. The method of claim 1, wherein the site-specific protease is selected from the group consisting of:

TEV NIa, HRV 3C, enterokinase, Factor Xa, and thrombin.

5. The method of claim 1, wherein the targeted enzyme is genetically modified to include the site-specific protease recognition sequence.

6. The method of claim 1, wherein the nucleic acid encoding the site-specific protease is operably linked to an inducible promoter.

7. The method of claim 1, wherein the enzyme in the biosynthetic pathway of interest that competes with the targeted enzyme increases the rate of precursor supplied to the biosynthetic pathway of interest.

8. The method of claim 7, wherein the enzyme in the biosynthetic pathway of interest that competes with the targeted enzyme is a key pathway entry enzyme of the biosynthetic pathway of interest.

9. The method of claim 1, wherein the enzyme in the biosynthetic pathway of interest that competes with the targeted enzyme supplies a substrate or cofactor of the biosynthetic pathway of interest.

10. The method of claim 1, wherein a gene encoding the native counterpart of the targeted enzyme is replaced with a gene coding for the targeted enzyme.

11. The method of claim 1, wherein the gene encoding the native counterpart enzyme is knocked out.

12. The method of claim 1, wherein the targeted enzyme is over-expressed in the bacterial cell.

13. The method of claim 1, wherein the targeted enzyme is present on either an episomal vector or a chromosome.

14. The method of claim 1, wherein the product is shikimic acid, shikimate, poly-3-hydroxybutyrate, isobutanol, or 1-butanol.

15. The method of claim 1, wherein the activity of the targeted enzyme is reduced by about 50% upon cleavage by the protease.

16. The method of claim 15, wherein the activity of the targeted enzyme is reduced by about 90% upon cleavage by the protease.

17. The method of claim 1, wherein the targeted enzyme is essential for growth of the bacterial cell.

18. The method of claim 1, wherein the bacterial cell is an Escherichia coli cell.

19. The method of claim 1, wherein the product is a polymer.

20. The method of claim 1, wherein the product is selected from the group consisting of antibiotics, biosurfactants, biological fuels, amino acids, organic acids, fatty acids, alcohols, polyols, flavors, fragrances, nucleotides, vitamins, pigments, sugars, polysaccharides, plastics, isoprenoids, and terpenes.

21. The method of claim 15, wherein the activity of the targeted enzyme is reduced by about 60% upon cleavage by the protease.

22. The method of claim 15, wherein the activity of the targeted enzyme is reduced by about 70% upon cleavage by the protease.

23. The method of claim 15, wherein the activity of the targeted enzyme is reduced by about 80% upon cleavage by the protease.

24. The method of claim 15, wherein the activity of the targeted enzyme is reduced by about 90% upon cleavage by the protease.