# US 9,518,273 B2 · Modified microorganisms and methods of making butadiene using same

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- Number: US 9,518,273 B2
- Document ID: US9518273B2
- Status: Granted
- Kind: B2
- Application number: 14/365,441
- Filed: 2012-12-17
- Published: 2014-12-18
- Granted: 2016-12-13
- Earliest claimed: 2011-12-16
- Applicants: Braskem S/A
- Inventors: Mateus Schreiner Garcez Lopes; Avram Michael Slovic; Iuri Estrada Gouvea; Johana Rincones Perez; Lucas Pedersen Parizzi
- Web page: https://patentgrep.com/patent/US9518273B2

## CPC

- C12P 5/026: {Unsaturated compounds, i.e. alkenes, alkynes or allenes}
- C12N 9/88: Lyases (4.)
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12P 5/02: acyclic {(C12P5/007 takes precedence)}
- C12Y 402/01127: Linalool dehydratase (4.2.1.127)
- 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)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present disclosure generally relates to microorganisms that comprise one or more polynucleotides coding for enzymes in one or more pathways that catalyze a conversion of a fermentable carbon source to butadiene. Also provided are methods of using the microorganisms in industrial processes including, for use in the production of butadiene and products derived therefrom.

## Claims

### Claim 1

A method of producing butadiene from a fermentable carbon source, the method comprising:
a.) providing a fermentable carbon source;
b.) contacting the fermentable carbon source with a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the production of butadiene, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to butadiene in a fermentation media; and
c.) expressing the one or more polynucleotides coding for the enzymes in the pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the production of butadiene and the one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to butadiene in the microorganism to produce butadiene,
wherein the one or more intermediates in the pathway for the production of butadiene is crotonyl-CoA, and
wherein all of the enzymes are required from either (i) crotonyl-CoA reductase (bifunctional) (E.C. 1.1.1), and crotonyl alcohol dehydratase (E.C. 4.2.1, 4.2.1.127) or (ii) crotonaldehyde dehydrogenase (E.C. 1.2.1), crotonyl alchol dehydrogenase (E.C. 1.1.1, 1.1.1.1), and crotonyl alcohol dehydratase (E.C. 4.2.1, 4.2.1.127).

### Claim 2 (depends on 1)

The method of claim 1, wherein the microorganism is a bacterium is selected from the genera consisting of: Burkholderia, Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillus.

### Claim 3 (depends on 1)

The method of claim 1, wherein the microorganism is a eukaryote is a yeast, filamentous fungi, protozoa, or algae.

### Claim 4 (depends on 3)

The method of claim 3, wherein the yeast is Saccharomyces cerevisiae, Zymomonas mobilis, or Pichia pastoris.

### Claim 5 (depends on 1)

The method of claim 1, wherein the carbon source is sugarcane juice, sugarcane molasses, hydrolyzed starch, hydrolyzed lignocellulosic materials, glucose, sucrose, fructose, lactate, lactose, xylose, pyruvate, or glycerol in any form or mixture thereof.

### Claim 6 (depends on 1)

The method of claim 1, wherein the carbon source is a monosaccharide, oligosaccharide, or polysaccharide.

### Claim 7 (depends on 1)

The method of claim 1, wherein the butadiene is secreted by the microorganism into the fermentation media.

### Claim 8 (depends on 7)

The method of claim 7 further comprising recovering the butadiene from the fermentation media.

### Claim 9 (depends on 1)

The method of claim 1, wherein the conversion of the fermentable carbon source to butadiene is performed under anaerobic conditions.

### Claim 10

A genetically modified microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of a fermentable carbon source to one or more intermediates in a pathway for the production of butadiene and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to butadiene, wherein the one or more intermediates in the pathway for the production of butadiene is crotonyl-CoA, and wherein all of the enzymes are required from either (i) crotonyl-CoA reductase (bifunctional) (E.C. 1.1.1), and crotonyl alcohol dehydratase (E.C. 4.2.1, 4.2.1.127) or (ii) crotonaldehyde dehydrogenase (E.C. 1.2.1), crotonyl alcohol dehydrogenase (E.C. 1.1.1, 1.1.1.1), and crotonyl alcohol dehydratase (E.C. 4.2.1, 4.2.1.127).

### Claim 11 (depends on 10)

The microorganism of claim 10, wherein the microorganism is a bacterium is selected from the genera consisting of: Burkholderia, Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillus.

### Claim 12 (depends on 10)

The microorganism of claim 10, wherein the microorganism is a eukaryote is a yeast, filamentous fungi, protozoa, or algae.

### Claim 13 (depends on 11)

The microorganism of claim 11, wherein the yeast is Saccharomyces cerevisiae, Zymomonas mobilis, or Pichia pastoris.

### Claim 14 (depends on 1)

The method of claim 1, wherein the enzymes are crotonyl-CoA reductase (bifuncional) (E.C. 1.1.1), and crotonyl alcohol dehydratase (E.C. 4.2.1, 4.2.1.127).

### Claim 15 (depends on 1)

The method of claim 1, wherein the enzymes are crotonaldehyde dehydrogenase (E.C. 1.2.1), crotonyl alcohol dehydrogenase (E.C. 1.1.1, 1.1.1.1), crotonyl alcohol dehydratase (E.C. 4.2.1, 4.2.1.127).

### Claim 16 (depends on 10)

The method of claim 10, wherein the enzymes are crotonyl-CoA reductase (bifuncional) (E.C. 1.1.1), and crotonyl alcohol dehydratase (E.C. 4.2.1, 4.2.1.127).

### Claim 17 (depends on 10)

The method of claim 10, wherein the enzymes are crotonaldehyde dehydrogenase (E.C. 1.2.1), crotonyl alcohol dehydrogenase (E.C. 1.1.1, 1.1.1.1), crotonyl alcohol dehydratase (E.C. 4.2.1, 4.2.1.127).

### Claim 18 (depends on 1, 10)

The method of claim 1 or claim 10, wherein said crotonyl alcohol dehydratase is a linalool dehydratase (4.2.1.127).