# US 9,752,161 B2 · Methods of producing isoprene and a co-product

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- Number: US 9,752,161 B2
- Document ID: US9752161B2
- Status: Granted
- Kind: B2
- Application number: 14/148,332
- Filed: 2014-01-06
- Published: 2014-05-08
- Granted: 2017-09-05
- Earliest claimed: 2008-12-30
- Assignees: Danisco US Inc.; The Goodyear Tire & Rubber Company
- Applicants: Danisco US Inc.; The Goodyear Tire & Rubber Company
- Inventors: Gopal K. Chotani; Joseph C. McAuliffe; Caroline M. Peres; Karl J. Sanford; Derek H. Wells
- Web page: https://patentgrep.com/patent/US9752161B2

## CPC

- C12P 5/007: {containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00)}
- C12N 9/88: Lyases (4.)
- C12P 7/06: Ethanol, i.e. non-beverage
- C12P 7/065: {with microorganisms other than yeasts}
- C12P 7/18: polyhydric
- Y02E 50/10: Biofuels, e.g. bio-diesel
- 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)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The invention features methods producing isoprene and a co-product, such as ethanol, 1,3-propanediol, or hydrogen from cultured cells. The invention also provides compositions that include these cultured cells. The invention provides compositions comprising isoprene and ethanol, isoprene and 1,3-propanediol, and isoprene and hydrogen. Additionally, the invention provides methods of co-producing isoprene and ethanol, isoprene and 1,3-propanediol, and isoprene and hydrogen by culturing cells under conditions suitable for co-production of isoprene and ethanol, isoprene and 1,3-propanediol, and isoprene and hydrogen.

## Claims

### Claim 1

Recombinant host cells comprising (a) a heterologous nucleic acid encoding an isoprene synthase polypeptide, (b) a nucleic acid encoding an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide, (c) nucleic acid(s) encoding a 1-Deoxyxylulose-5-phosphate synthase (DXS) polypeptide and/or one or more mevalonate (MVA) pathway polypeptides and (d) heterologous nucleic acid(s) encoding (i) one or more polypeptide involved in ethanol fermentation, wherein the polypeptide involved in ethanol fermentation is an alcohol dehydrogenase from yeast or bacteria, or (ii) a pyruvate decarboxylase (pdc) polypeptide,
wherein said cells co-produce isoprene and ethanol under oxygen-limited culture conditions, wherein the oxygen transfer rate (OTR) is less than the oxygen uptake rate (OUR).

### Claim 2 (depends on 1)

The cells of claim 1, wherein the heterologous nucleic acid encoding an isoprene synthase polypeptide is operably linked to a promoter.

### Claim 3 (depends on 2)

The cells of claim 2, wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

### Claim 4 (depends on 3)

The cells of claim 3, wherein the plant isoprene synthase polypeptide is from Populus alba.

### Claim 5 (depends on 1)

The cells of claim 1, wherein the cells further comprise one or more heterologous nucleic acid encoding a deoxyxylulose-5-phosphate (DXP) pathway polypeptide.

### Claim 6 (depends on 1)

The cells of claim 1, wherein the cells comprise a DXP pathway polypeptide and a MVA pathway polypeptide.

### Claim 7 (depends on 1)

The cells of claim 1, wherein the cells comprise one or more MVA pathway polypeptides selected from the group consisting of: (i) an acetoacetyl-Coenzyme A synthase (thiolase) polypeptide; (ii) a 3-hydroxy-3-methylglutaryl-Coenzyme A synthase polypeptide (iii) a 3-hydroxy-3-methylglutaryl-Coenzyme A reductase polypeptide, (iv) mevalonate kinase (MVK); (v) phosphomevalonate kinase (PMK); and (vi) diphosphomevalonate decarboxylase (MVD).

### Claim 8 (depends on 1)

The cells of claim 1, wherein the cells are fungal cells.

### Claim 9 (depends on 8)

The cells of claim 8, wherein the fungal cells are yeast cells.

### Claim 10 (depends on 9)

The cells of claim 9, wherein the yeast cells are Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida sp.

### Claim 11 (depends on 10)

The cells of claim 10, wherein the yeast cells are Saccharomyces cerevisiae.

### Claim 12 (depends on 1)

The cells of claim 1, wherein the cells are bacterial cells.

### Claim 13 (depends on 12)

The cells of claim 12, wherein the bacterial cells are Bacillus sp., Streptomyces sp., Escherichia sp., Rhodopseudomonas sp., Pseudomonas sp., Pantoea sp., or Zymomonas sp.

### Claim 14 (depends on 13)

The cells of claim 13, wherein the cells are E. coli cells.

### Claim 15 (depends on 1)

The cells of claim 1, wherein the oxygen-limited conditions are anaerobic conditions.

### Claim 16 (depends on 1)

The recombinant host cells of claim 1, wherein the alcohol dehydrogenase is an alcohol dehydrogenase E (adhE) from E. coli or Zymomonas.

### Claim 17 (depends on 1)

The recombinant host cells of claim 1, wherein the alcohol dehydrogenase is an alcohol dehydrogenase B (adhB) from Zymomonas.

### Claim 18

A method of co-producing isoprene and ethanol, the method comprising: (a) culturing recombinant host cells capable of co-producing isoprene and ethanol under oxygen limited culture conditions wherein, the oxygen transfer rate (OTR) is less than the oxygen uptake rate (OUR), and wherein the cells comprise (a) a heterologous nucleic acid encoding an isoprene synthase polypeptide (b) a nucleic acid encoding an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide, (c) nucleic acid(s) encoding a 1-Deoxyxylulose-5-phosphate synthase (DXS) polypeptide and/or one or more mevalonate (MVA) pathway polypeptides and (d) heterologous nucleic acid(s) encoding (i) one or more polypeptide involved in ethanol fermentation, wherein the polypeptide involved in ethanol fermentation is an alcohol dehydrogenase from yeast or bacteria, or (ii) a pyruvate decarboxylase (pdc) polypeptide; and
(b) co-producing isoprene and ethanol.

### Claim 19 (depends on 18)

The method of claim 18, wherein the heterologous nucleic acid encoding an isoprene synthase polypeptide is operably linked to a promoter.

### Claim 20 (depends on 19)

The method of claim 19, wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

### Claim 21 (depends on 20)

The method of claim 20, wherein the plant isoprene synthase polypeptide is from Populus alba.

### Claim 22 (depends on 18)

The method of claim 18, wherein the cells further comprise one or more heterologous nucleic acid encoding a deoxyxylulose-5-phosphate (DXP) pathway polypeptide.

### Claim 23 (depends on 18)

The method of claim 18, wherein the cells comprise a DXP pathway polypeptide and a MVA pathway polypeptide.

### Claim 24 (depends on 18)

The method of claim 18, wherein the cells comprise one or more MVA pathway polypeptides selected from the group consisting of: (i) an acetoacetyl-Coenzyme A synthase (thiolase) polypeptide; (ii) a 3-hydroxy-3-methylglutaryl-Coenzyme A synthase polypeptide (iii) a 3-hydroxy-3-methylglutaryl-Coenzyme A reductase polypeptide, (iv) mevalonate kinase (MVK); (v) phosphomevalonate kinase (PMK); and (vi) diphosphomevalonate decarboxylase (MVD).

### Claim 25 (depends on 18)

The method of claim 18, wherein the cells are fungal cells.

### Claim 26 (depends on 25)

The method of claim 25, wherein the fungal cells are yeast cells.

### Claim 27 (depends on 26)

The method of claim 26, wherein the yeast cells are Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida sp.

### Claim 28 (depends on 27)

The method of claim 27, wherein the yeast cells are Saccharomyces cerevisiae.

### Claim 29 (depends on 18)

The method of claim 18, wherein the cells are bacterial cells.

### Claim 30 (depends on 29)

The method of claim 29, wherein the bacterial cells are Bacillus sp., Streptomyces sp., Escherichia sp., Rhodopseudomonas sp., Pseudomonas sp., Pantoea sp., or Zymomonas sp.

### Claim 31 (depends on 30)

The method of claim 30, wherein the cells are E. coli cells.

### Claim 32 (depends on 18)

The method of claim 18, wherein the oxygen-limited conditions are anaerobic conditions.