# US 2010/0167371 A1 · Systems using cell culture for production of isoprene

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- Number: US 2010/0167371 A1
- Document ID: US20100167371A1
- Status: Published application
- Kind: A1
- Application number: 12/560,366
- Filed: 2009-09-15
- Published: 2010-07-01
- Earliest claimed: 2008-09-15
- Assignees: Danisco US Inc.; The Goodyear Tire & Rubber Company
- Inventors: Gopal K. Chotani; Joseph C. McAuliffe; Caroline M. Peres; Gregory M. Whited
- Web page: https://patentgrep.com/patent/US20100167371A1

## CPC

- C12P 5/007: {containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00)}
- C12P 5/026: {Unsaturated compounds, i.e. alkenes, alkynes or allenes}
- Y02E 50/30: Fuel from waste, e.g. synthetic alcohol or 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)}
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The invention features methods for producing isoprene from cultured cells. The invention also provides compositions that include these cultured cells. The invention provides isoprene compositions, such as compositions with increased amount of isoprene or increased purity. Additionally, the invention provides methods of producing isoprene by culturing cells under conditions suitable for isoprene production while maintaining cell viability and/or metabolic activity.

## Claims

### Claim 1

A method of producing isoprene, the method comprising:
a) culturing cells comprising one or more nucleic acids encoding for isoprene synthase polypeptide under suitable conditions for production of isoprene; and
b) producing isoprene, wherein the cells produce greater than about 400 nmole/gwcm/hour of isoprene, and wherein the carbon dioxide evolution rate of the cells is greater than about 1×10−18 mmol/L/hour.

### Claim 2 (depends on 1)

The method of claim 1, further comprising recovering the isoprene.

### Claim 3 (depends on 1)

The method of claim 1, wherein the cells further comprise one or more heterologous nucleic acids or one or more additional copies of an endogenous nucleic acid encoding an isoprene synthase polypeptide.

### Claim 4 (depends on 1)

The method of claim 1, wherein the cells further comprise one or more heterologous nucleic acids or one or more additional copies of an endogenous nucleic acid encoding an IDI polypeptide, an MVA pathway enzyme, or a DXP pathway enzyme.

### Claim 5 (depends on 4)

The method of claim 4 wherein the MVA pathway enzyme is mevalonate kinase.

### Claim 6 (depends on 1)

The method of claim 1, wherein the isoprene synthase polypeptide is from Pueraria or Populus or a hybrid, Populus alba×Populus tremula.

### Claim 7 (depends on 6)

The method of claim 6, wherein the isoprene synthase polypeptide is selected from the group consisting of Pueraria montana or Pueraria lobata, Populus tremuloides, Populus alba, Populus nigra, and Populus trichocarpa.

### Claim 8 (depends on 1)

The method of claim 1, wherein the cells are gram-positive bacterial cells, Streptomyces cells, gram-negative bacterial cells, Escherichia cells, Pantoea cells, fungal cells, filamentous fungal cells, Trichoderma cells, Aspergillus cells, or yeast cells.

### Claim 9 (depends on 8)

The method of claim 8, wherein the cells are selected from the group consisting of Bacillus subtilis, Streptomyces lividans, Streptomyces coelicolor, Streptomyces griseus, Escherichia coli, Pantoea citrea, Trichoderma reesei, Aspergillus oryzae and Aspergillus niger, Saccharomyces cerevisiae and Yarrowia lipolytica.

### Claim 10

A method of producing isoprene, the method comprising:
a) culturing cells comprising one or more nucleic acids encoding for isoprene synthase polypeptide under suitable conditions for production of isoprene; and
b) producing isoprene, wherein the liquid phase concentration of isoprene is less than about 1 g/L and the cells produce greater than about 400 nmole/gwcm/hour of isoprene.

### Claim 11 (depends on 10)

The method of claim 10, further comprising recovering the isoprene.

### Claim 12 (depends on 10)

The method of claim 10, wherein the cells further comprise one or more heterologous nucleic acids or one or more additional copies of an endogenous nucleic acid encoding an isoprene synthase polypeptide.

### Claim 13 (depends on 10)

The method of claim 10, wherein the cells further comprise one or more heterologous nucleic acids or one or more additional copies of an endogenous nucleic acid encoding an IDI polypeptide, an MVA pathway enzyme, or a DXP pathway enzyme.

### Claim 14 (depends on 13)

The method of claim 13 wherein the MVA pathway enzyme is mevalonate kinase.

### Claim 15 (depends on 10)

The method of claim 10, wherein the isoprene synthase polypeptide is from Pueraria or Populus or a hybrid, Populus alba×Populus tremula.

### Claim 16 (depends on 10)

The method of claim 10, wherein the cells are gram-positive bacterial cells, Streptomyces cells, gram-negative bacterial cells, Escherichia cells, Pantoea cells, fungal cells, filamentous fungal cells, Trichoderma cells, Aspergillus cells, or yeast cells.

### Claim 17

A method of producing a compound, wherein the compound has one or more characteristics selected from the group consisting of (a) a Henry's law coefficient of less than about 250 M/atm and (b) a solubility in water of less than about 100 g/L, the method comprising:
a) culturing cells comprising one or more nucleic acids encoding for isoprene synthase polypeptide under suitable conditions for production of the compound, wherein a gas is added at a gas sparging rate between about 0.01 vvm to about 2 vvm; and
b) producing the compound.

### Claim 18 (depends on 17)

The method of claim 17, further comprising recovering the compound.

### Claim 19 (depends on 17)

The method of claims 17, wherein the compound produced is ethylene.

### Claim 20 (depends on 17)

The method of claim 17, wherein the compound produced is isoprene.

### Claim 21

Cells in culture comprising a nucleic acid encoding an isoprene synthase polypeptide, wherein the cells produce greater than about 400 nmole/gwcm/hour of isoprene and carbon dioxide evolution rate of the cells is greater than about 1×10−18 mmol/L/hour.

### Claim 22 (depends on 21)

A system comprising a reaction chamber with cells of claim 21.

### Claim 23

A method of producing a compound, wherein the compound has one or more characteristics selected from the group consisting of (a) a Henry's law coefficient of less than about 250 M/atm and (b) a solubility in water of less than about 100 g/L, the method comprising:
a) culturing cells comprising one or more nucleic acids encoding for isoprene synthase polypeptide under suitable conditions for production of the compound, wherein the compound has one or more characteristics selected from the group consisting of (a) a Henry's law coefficient of less than about 250 M/atm and (b) a solubility in water of less than about 100 g/L;
b) producing the compound;
c) and recovering the compound from the gas phase.

### Claim 24 (depends on 23)

The method of claim 23, wherein the compound produced is ethylene.

### Claim 25 (depends on 23)

The method of claim 23, wherein the compound is isoprene.