# US 2014/0193869 A1 · Cell-free system for converting methane into fuel and chemical compounds

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- Number: US 2014/0193869 A1
- Document ID: US20140193869A1
- Status: Published application
- Kind: A1
- Application number: 14/137,524
- Filed: 2013-12-20
- Published: 2014-07-10
- Earliest claimed: 2012-12-21
- Assignees: GreenLight Biosciences, Inc.
- Applicants: GreenLight Biosciences, Inc.
- Inventors: William Jeremy Blake; James R. Swartz
- Web page: https://patentgrep.com/patent/US20140193869A1

## CPC

- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 9/0008: {acting on the aldehyde or oxo group of donors (1.2)}
- C12N 9/0073: {with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13}
- C12N 9/1022: {transferring aldehyde or ketonic groups (2.2)}
- C12N 9/1205: {Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases}
- C12N 9/1217: {Phosphotransferases with a carboxyl group as acceptor (2.7.2)}
- C12N 9/88: Lyases (4.)
- C12N 9/90: Isomerases (5.)
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12P 7/16: Butanols
- C12P 7/40: containing a carboxyl group {including Peroxycarboxylic acids}
- C12Y 101/01244: Methanol dehydrogenase (1.1.1.244)
- C12Y 102/01012: Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (1.2.1.12)
- C12Y 114/13025: Methane monooxygenase (1.14.13.25)
- C12Y 202/01001: Transketolase (2.2.1.1)
- C12Y 207/01011: 6-Phosphofructokinase (2.7.1.11)
- C12Y 207/0104: Pyruvate kinase (2.7.1.40)
- C12Y 207/02003: Phosphoglycerate kinase (2.7.2.3)
- C12Y 401/02013: Fructose-bisphosphate aldolase (4.1.2.13)
- C12Y 401/02043: 3-Hexulose-6-phosphate synthase (4.1.2.43)
- C12Y 402/01011: Phosphopyruvate hydratase (4.2.1.11), i.e. enolase
- C12Y 501/03001: Ribulose-phosphate 3-epimerase (5.1.3.1)
- C12Y 503/01001: Triose-phosphate isomerase (5.3.1.1)
- C12Y 503/01027: 6-Phospho-3-hexuloisomerase (5.3.1.27)
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present disclosure relates, in some aspects, to cell-free methods and systems for large-scale conversion of methane to isobutanol, comprising combining, in a bioreactor at elevated pressure, methane, oxygen, and cell lysates containing methane monooxygenase, methanol dehydrogenase, and enzymes that catalyze the conversion of formaldehyde to isobutanol, to form a cell-free reaction mixture, and incubating under suitable conditions the cell-free reaction to convert methane to isobutanol.

## Claims

### Claim 1

A cell-free method for large-scale conversion of methane to isobutanol, comprising:
combining, in a bioreactor at elevated pressure, methane, oxygen, and cell lysates containing methane monooxygenase, methanol dehydrogenase, and enzymes that catalyze the conversion of formaldehyde to isobutanol, to form a cell-free reaction mixture; and
incubating under suitable conditions the cell-free reaction to convert methane to isobutanol.

### Claim 2 (depends on 1)

The cell-free method of claim 1, wherein the methane monooxygenase is obtained from a methanotroph.

### Claim 3 (depends on 1)

The cell-free method of claim 1, wherein the methanol dehydrogenase is a heterologous NAD-linked methanol dehydrogenase.

### Claim 4 (depends on 1)

The cell-free method of claim 1, wherein the enzymes that catalyze the conversion of formaldehyde to isobutanol are derived from recombinant bacteria.

### Claim 5 (depends on 4)

The cell-free method of claim 4, wherein the recombinant bacteria are recombinant Escherichia coli.

### Claim 6 (depends on 1)

The cell-free method of claim 1, wherein the enzymes that catalyze the conversion of formaldehyde to isobutanol comprise one or more of hexulose-6-phosphate synthase, 6-phospho-3-hexuloisomerase, 6-phosphofructokinase, fructose biphosphate aldolase, triose phosphate isomerase, transketolase, ribose-5-phosphate isomerase, ribose-5-phosphate 3-epimerase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, acetolactate synthase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, alpha-ketoisovalerate decarboxylase, and isobutanol dehydrogenase.

### Claim 7 (depends on 1)

The cell-free method of claim 1, wherein the pressure is at least 1 bar.

### Claim 8 (depends on 7)

The cell-free method of claim 7, wherein the pressure is at least 2 bar.

### Claim 9

(canceled)

### Claim 10 (depends on 1)

The cell-free method of claim 1, wherein the isobutanol is produced at a production rate of at least 1 g/L-h.

### Claim 11 (depends on 10)

The cell-free method of claim 10, wherein the isobutanol is produced at a production rate of at least 10 g/L-h.

### Claim 12

(canceled)

### Claim 13 (depends on 1)

The cell-free method of claim 1, wherein the bioreactor comprises a gas phase and an aqueous phase.

### Claim 14 (depends on 13)

The cell-free method of claim 13, wherein the bioreactor further comprises an organic solvent.

### Claim 15 (depends on 14)

The cell-free method of claim 14, wherein the organic solvent is an alkane.

### Claim 16

(canceled)

### Claim 17

A cell-free method for large-scale conversion of methane to pyruvate, comprising:
combining, in a bioreactor at elevated pressure, methane, oxygen, and cell lysates containing methane monooxygenase, methanol dehydrogenase, and enzymes that catalyze the conversion of formaldehyde to pyruvate, to form a cell-free reaction mixture; and
incubating under suitable conditions the cell-free reaction to convert methane to pyruvate.

### Claim at position 18

18-32. (canceled)

### Claim 33

A cell-free method for large-scale conversion of methane to a biofuel or other chemical compound, comprising:
combining, in a bioreactor at elevated pressure, methane, oxygen, and cell lysates containing methane monooxygenase, methanol dehydrogenase, enzymes that catalyze the conversion of formaldehyde to pyruvate, and enzymes that catalyze the conversion of pyruvate to a biofuel or other chemical compound, to form a cell-free reaction mixture; and
incubating under suitable conditions the cell-free reaction to convert methane to the biofuel or other chemical compound.

### Claim at position 20

34-37. (canceled)

### Claim 38 (depends on 33)

The cell-free method of claim 33, wherein the enzymes that catalyze the conversion of formaldehyde to pyruvate comprise one or more of hexulose-6-phosphate synthase, 6-phospho-3-hexuloisomerase, 6-phosphofructokinase, fructose biphosphate aldolase, triose phosphate isomerase, transketolase, ribose-5-phosphate isomerase, ribose-5-phosphate 3-epimerase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase.

### Claim at position 22

39-48. (canceled)

### Claim 49

A cell-free system for large-scale conversion of methane to isobutanol, the system comprising a bioreactor that comprises:
a gas phase comprising methane and oxygen; and
an aqueous phase comprising a cell lysate containing methane monooxygenase, methanol dehydrogenase, and enzymes that catalyze the conversion of formaldehyde to isobutanol.

### Claim at position 24

50-57. (canceled)

### Claim 58

A cell-free system for large-scale conversion of methane to pyruvate, the system comprising a bioreactor that comprises:
a gas phase comprising methane and oxygen; and
an aqueous phase comprising a cell lysate containing methane monooxygenase, methanol dehydrogenase, and enzymes that catalyze the conversion of formaldehyde to pyruvate.

### Claim at position 26

59-66. (canceled)

### Claim 67

A cell-free system for large-scale conversion of methane to pyruvate, the system comprising a bioreactor that comprises:
a gas phase comprising methane and oxygen; and
an aqueous phase comprising a cell lysate containing methane monooxygenase, methanol dehydrogenase, enzymes that catalyze the conversion of formaldehyde to pyruvate, and enzymes that catalyze the conversion of pyruvate to a biofuel or other chemical compound, to form a cell-free reaction mixture.

### Claim at position 28

68-75. (canceled)

### Claim 76 (depends on 1)

A cell lysate of claim 1.