Methods for control of flux in metabolic pathways

• US 9,637,746 B2
• Filed: 12/14/2009
• Issued: 05/02/2017
• Est. Priority Date: 12/15/2008
• Status: Active Grant

• Current Assignee: GreenLight Biosciences, Inc.
• Original Assignee: GreenLight Biosciences, Inc.
• Inventors: Klein-Marcuschamer, Daniel
• Primary Examiner(s): Nashed, Nashaat
• Assistant Examiner(s): Moore, William W
• Application Number:
  US13/132,721
• Publication Number:
  US 20110269198A1
• Time in Patent Office: 2,696 Days
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/1022 transferring aldehyde or ketonic groups (2.2)
• CPC Class Codes: C12N 9/1085 transferring alkyl or aryl groups other than methyl groups (2.5)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/48 acting on peptide bonds (3.4)
• CPC Class Codes: C12P 7/42 Hydroxy-carboxylic acids
• CPC Class Codes: C12Y 101/01025 Shikimate dehydrogenase (1.1.1.25)
• CPC Class Codes: C12Y 202/01001 Transketolase (2.2.1.1)
• CPC Class Codes: C12Y 205/01054 3-Deoxy-7-phosphoheptulonate synthase (2.5.1.54)
• CPC Class Codes: C12Y 301/27 Endoribonucleases producing 3'-phosphomonoesters (3.1.27)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The invention pertains to a method for preparing cells that can be used as biocatalysts by inducing in them a growth-decoupled state, in which interferase inhibits the expression of genes except the ones that code for the pathway enzymes of interest. mRNAs that code for interferase-resistant products are overexpressed in the background of a metabolically-frozen cell. Enzymes that compete for a substrate or product of the pathway of interest may be altered such that the enzyme is sensitive to a site-specific protease, which protease is inducible in the host cell.

Claims

1. A cell lysate of bacterial cells genetically manipulated to comprise:

(a) a coding sequence for a protease that cleaves proteins at a specific sequence motif that comprises at least 4 amino acid residues, wherein the coding sequence for the protease is operably linked to an inducible promoter; and

(b) a coding sequence for at least one competing enzyme selected from the group consisting of enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, and glucokinase, wherein the coding sequence for the at least one competing enzyme is modified to encode the specific sequence motif cleaved by the protease.

2. The cell lysate of claim 1, wherein the coding sequence for the protease is present on either an episomal vector or a chromosome.

3. The cell lysate of claim 1, wherein the protease is a tobacco etch virus protease, a yellow fever virus protease, thrombin, or Factor Xa.

4. The cell lysate of claim 1, wherein the specific sequence motif comprises at least 5 amino acid residues.

5. The cell lysate of claim 1, wherein the specific sequence motif comprises at least 6 amino acid residues.

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

7. The cell lysate of claim 1, further comprising one or more of substrates, nutrients, cofactors, buffers, reducing agents, and ATP generating systems.

8. The cell lysate of claim 1, wherein the at least one competing enzyme is glucose-6-phosphate isomerase.

9. The cell lysate of claim 1, wherein the at least one competing enzyme is glyceraldehyde-3-phosphate dehydrogenase.

10. The cell lysate of claim 1, wherein the at least one competing enzyme is phosphofructokinase.

11. The cell lysate of claim 1, wherein the at least one competing enzyme is 3-phosphoglycerate mutase.

12. The cell lysate of claim 1, wherein the at least one competing enzyme is pyruvate kinase.

13. The cell lysate of claim 1, wherein the at least one competing enzyme is triosephosphate isomerase.

14. The cell lysate of claim 1, wherein the at least one competing enzyme is glucokinase.