Cell-free protein synthesis driven metabolic engineering for the production of 1-butanol

Patent metadata

Field	Value
Publication number	US-10577632-B2
Application number	US-201615175497-A
Country	US
Kind code	B2
Filing date	Jun 7, 2016
Priority date	Jun 10, 2015
Publication date	Mar 3, 2020
Grant date	Mar 3, 2020

Abstract

Disclosed are cell-free systems for metabolic engineering, methods for cell-free metabolic engineering, kits for preparing the disclosed systems, and kits for performing the disclosed methods. The disclosed systems, methods, and kits may be utilized to prepare a chemical product and to optimize conditions for preparing a chemical product. The disclosed systems, methods, and kits also may be utilized for combinatorial cell-free metabolism engineering.

First claim

What is claimed:

1. A method for the enzymatic preparation of 1-butanol or an intermediate of 1-butanol in the 1-butanol synthetic pathway in vitro from a feedstock comprising glucose or a product of glycolysis that reacts with one or more enzymes to produce the 1-butanol or the intermediate of 1-butanol in the 1-butanol synthetic pathway, the method comprising:
(a) reacting a cell-free protein synthesis reaction mixture, the cell-free protein synthesis reaction mixture comprising a cellular extract from a host strain, a translation template encoding the one or more enzymes, and cell-free protein synthesis reagents,
(b) expressing the translation template in the cell-free protein synthesis reaction mixture to prepare the one or more enzymes,
(c) combining the cell-free protein synthesis reaction mixture and a metabolic reaction mixture, the metabolic reaction mixture comprising the feedstock,
wherein the feedstock reacts in the presence of the one or more enzymes to prepare the 1-butanol or the intermediate of 1-butanol in the 1-butanol synthetic pathway and wherein the cellular extract provides natural enzyme metabolism from the host strain;
wherein the one or more enzymes are selected from the group consisting of AtoB, Hbd, Crt, Ter, AdhE, and combinations thereof; and
wherein the intermediate of 1-butanol in the 1-butanol synthetic pathway is selected from acetoacetyl-CoA, 3-hydroxybutyryl-CoA, crotonyl-CoA, butyryl-CoA, and butyraldehyde.

2. The method of claim 1 , wherein the natural enzyme metabolism from the host strain (i) provides energy; (ii) provides cofactor regeneration; (iii) provides a cellular extract enzyme; or (iv) any combination thereof.

3. The method of claim 1 , the method further comprising providing a transcription template, a polymerase, ATP, GTP, CTP, and UTP to prepare the translation template.

4. The method of claim 1 , wherein the protein reaction vessel and the metabolic reaction vessel are different vessels.

5. The method of claim 1 , wherein the protein reaction vessel and the metabolic reaction vessel are the same vessel.

6. The method of claim 1 , wherein the cellular extract is a prokaryotic cellular extract.

7. The method of claim 1 , wherein the enzyme prepared in step (b) is heterologous relative to the cellular extract.

8. The method of claim 1 , wherein the cell-free protein synthesis reagents comprise a reaction buffer, amino acids, and a tRNA mixture.

9. The method of claim 1 , wherein the cell-free protein synthesis reagents comprise CoA, ATP, NAD, NADH, NADP, NADPH, FMN, SAM, potassium, magnesium, ammonium, glutamate, acetate, or any combination thereof.

10. The method of claim 1 , wherein the enzyme prepared in step (b) is selected from the group consisting of AtoB, Hbd, Crt, Ter, AdhE, and combinations thereof.

11. The method of claim 1 , wherein the feedstock comprises glucose.

12. The method of claim 1 , wherein the metabolic reaction mixture comprises CoA, malonyl-CoA, acetyl-CoA, 4′-phosphopantetheinyl transferase enzyme Sfp, ATP, NAD, NADH, NADP, NADPH, FMN, potassium, magnesium, ammonium, glutamate, acetate, any of the 20 amino acids, or any combinations thereof.

13. A method for the enzymatic preparation of 1-butanol in vitro from a feedstock that comprises glucose or products of glycolysis that reacts with one or more enzymes in the 1-butanol synthetic pathway to produce 1-butanol, the method comprising:
(a) reacting a cell-free protein synthesis reaction mixture, the cell-free protein synthesis reaction mixture comprising a cellular extract from a host strain of E. coli , a translation template encoding the one or more enzymes, and cell-free protein synthesis reagents,
(b) expressing the translation template in the cell-free protein synthesis reaction mixture to prepare the one or more enzymes in the 1-butanol synthetic pathway,
(c) combining the cell-free protein synthesis reaction mixture and a metabolic reaction mixture, the metabolic reaction mixture comprising the feedstock,
wherein the feedstock reacts in the presence of the one or more enzymes in the in the 1-butanol synthetic pathway to prepare the 1-butanol and wherein the cellular extract provides natural enzyme metabolism from the host strain; and
wherein the one or more enzymes are selected from the group consisting of AtoB, Hbd, Crt, Ter, AdhE, and combinations thereof.

14. The method of claim 13 , wherein the natural enzyme metabolism from the host strain (i) provides energy; (ii) provides cofactor regeneration; (iii) provides a cellular extract enzyme; or (iv) any combination thereof.

15. The method of claim 13 , the method further comprising providing a transcription template, a polymerase, ATP, GTP, CTP, and UTP to prepare the translation template.

16. The method of claim 13 , wherein the enzyme prepared in step (b) is heterologous relative to the cellular extract.

Assignees

• Univ Northwestern

Inventors

• Jewett Michael Christopher
• Karim Ashty Stephen

Classifications

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• C12P17/182

  Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system (alloxazine or isoalloxazine, e.g. riboflavine C12P25/00) · CPC title

• C12P7/16 Primary

  Butanols · CPC title

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C40B30/08

  by measuring catalytic activity · CPC title