Methods for control of flux in metabolic pathways through enzyme relocation

• US 10,006,062 B2
• Filed: 02/11/2015
• Issued: 06/26/2018
• Est. Priority Date: 05/07/2010
• Status: Expired due to Fees

• 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): Saidha, Tekchand
• Application Number:
  US14/620,054
• Publication Number:
  US 20150191753A1
• Time in Patent Office: 1,231 Days
• Field of Search: 435 41, 435 698, 435183, 4352523
• CPC Class Codes: C07K 2319/034 containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved
• CPC Class Codes: C12N 15/625 containing a sequence coding for a signal sequence
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 23/00 Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes containing heterorings C12P17/00
• CPC Class Codes: C12P 5/007 containing one or more isoprene units, i.e. terpenes carotenes C12P23/00
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12P 7/42 Hydroxy-carboxylic acids
• CPC Class Codes: C12P 7/625 Polyesters of hydroxy carboxylic acids
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Genetically manipulated cells, lysates of such cells, systems, and methods of use thereof are provided, where one or more enzymes in a pathway of interest are genetically modified to incorporate a peptide sequence that provides for relocation of the protein, e.g., to the periplasm, so as to sequester the enzyme, and where the enzyme controls flux in the pathway of interest.

Claims

1. A bacterial cell that expresses enzymes of a biosynthetic pathway for production of a product, wherein at least one of the enzymes:

(i) controls metabolic flux in the biosynthetic pathway;

(ii) is modified by genetic means to attach a periplasmic-targeting sequence; and

(iii) is selected from the group consisting of amidophosphoribosyl transferase, orotate phosphoribosyltransferase, 2-dehydro-3-deoxyphosphoheptonate aldolase, phosphoribosyltransferase HisG, acetolactate/acetohydroxybutanoate synthase, UDP-N-acetylglucosamine acyltransferase, aspartate aminotransferase, arginine decarboxylase, GTP cyclohydrolase I, acetyl-CoA carboxylase, 2-keto-acid decarboxylase, acetyl-CoA acetyltransferase, and beta-ketothiolase,

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, biopolymers, plastics, isoprenoids, terpenes, and cell metabolites.

2. The bacterial cell of claim 1, wherein in the bacterial cell at least one gene encoding the at least one modified enzyme is present on an episomal vector or a chromosome.

3. The bacterial cell of claim 1, wherein in the bacterial cell at least two enzymes in the biosynthetic pathway are modified by genetic means to include a periplasmic targeting sequence.

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

5. A bacterial cell lysate of the bacterial cell of claim 1.

6. A method of producing a cell lysate composition for producing a product of a biosynthetic pathway of interest, the method comprising:

(a) culturing bacterial cells of claim 1; and

(b) lysing the cultured bacterial cells.

7. The method of claim 6, wherein in the bacterial cell at least two enzymes in the biosynthetic pathway are modified by genetic means to include a periplasmic targeting sequence.

8. The method of claim 6, wherein the step of culturing cells comprises culturing multiple cultures of cells.

9. The method of claim 6, wherein the step of lysing the cultured cells produces two or more lysates.

10. The method of claim 9 further comprising combining the two or more lysates.

11. The method of claim 6, wherein the step of culturing the cells comprises culturing the cells in cell growth medium that has been modified by the addition of a factor that increases or preserves the activity of the enzymes of the biosynthetic pathway.

12. The method of claim 6 further comprising combining the cell lysate with at least one substance selected from the group consisting of substrates, enzymes, nutrients, co-factors, buffers, reducing agents, and ATP generating systems;

and

incubating the cell lysate and the one or more substance for a period of time and under conditions sufficient to produce the product.

13. The method of claim 12, wherein in the bacterial cell at least two enzymes in the biosynthetic pathway are modified by genetic means to include a periplasmic targeting sequence.

14. The method of claim 12, wherein the step of culturing cells comprises culturing multiple cultures of cells.

15. The method of claim 12, wherein the step of lysing the cultured cells produces two or more lysates.

16. The method of claim 15 further comprising combining the two or more lysates.

17. The method of claim 12, wherein the step of culturing the cells comprises culturing the cells in cell growth medium that has been modified by the addition of a factor that increases or preserves the activity of the enzymes of the biosynthetic pathway.