Recombinant cellular iysate system for producing a product of interest

• US 10,036,001 B2
• Filed: 11/14/2014
• Issued: 07/31/2018
• Est. Priority Date: 08/31/2010
• Status: Active Grant

• Current Assignee: GreenLight Biosciences, Inc., Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: GreenLight Biosciences, Inc., Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Swartz, James R.
• Primary Examiner(s): Swope, Sheridan
• Application Number:
  US14/542,074
• Publication Number:
  US 20150064751A1
• Time in Patent Office: 1,355 Days
• Field of Search: None
• 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 bacterial cell lysate obtained from bacterial cells genetically modified to comprise:

nucleic acids that encode

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

(b) a recombinant 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 recombinant 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,

wherein the bacterial cell lysate comprises a substrate and supports the production of the product of interest from said substrate.

2. The bacterial cell lysate of claim 1, wherein the periplasmic targeting sequence is selected from the group consisting of:

3. The bacterial cell lysate of claim 1, wherein the site-specific protease is selected from the group consisting of:

TEV NIa, HRV 3C, Enterokinase, Factor Xa, and Thrombin.

4. The bacterial cell lysate of claim 1, wherein the nucleic acid encoding the site-specific protease recognition sequence is operably linked to an inducible promoter.

5. The bacterial cell lysate of claim 1, wherein the targeted enzyme competes with an enzyme that increases the rate of precursor supplied to the pathway of interest.

6. The bacterial cell lysate of claim 1, wherein the targeted enzyme competes with a key pathway entry enzyme.

7. The bacterial cell lysate of claim 1, wherein the gene coding for the native counterpart of the targeted enzyme is replaced with a gene coding for the targeted enzyme.

8. The bacterial cell lysate of claim 1, wherein the gene encoding the native counterpart of the targeted enzyme is knocked out.

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

10. The bacterial cell lysate of claim 1, wherein the targeted enzyme is present on either an episomal vector or a chromosome.

11. The bacterial cell lysate of claim 1, wherein the cell growth medium has been modified by the addition or enhancement of a factor that increases or preserves the activity of the targeted enzyme.

12. The bacterial cell lysate of claim 1, wherein the product is a biopolymer.

13. The bacterial cell lysate 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.

14. The bacterial cell lysate claim 13, wherein the biological fuel is isobutanol or 1-butanol.

15. The bacterial cell lysate of claim 1, wherein the bacterial cell is an Escherichia coli cell.

16. The bacterial cell lysate of claim 1, wherein the activity of the targeted enzyme is reduced by about 50% upon cleavage by the protease.

17. The bacterial cell lysate of claim 16, wherein the activity of the targeted enzyme is reduced by about 90% upon cleavage by the protease.

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