# US 8,465,953 B2 · Ethanol production in non-recombinant hosts

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- Number: US 8,465,953 B2
- Document ID: US8465953B2
- Status: Granted
- Kind: B2
- Application number: 12/298,216
- Filed: 2007-04-26
- Published: 2009-11-19
- Granted: 2013-06-18
- Earliest claimed: 2006-05-01
- Assignees: University of Florida Research Foundation, Inc.
- Applicants: Youngnyun Kim; Keelnatham Shanmugam; Lonnie O. Ingram
- Inventors: Youngnyun Kim; Keelnatham Shanmugam; Lonnie O. Ingram
- Web page: https://patentgrep.com/patent/US8465953B2

## CPC

- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- C12N 9/0051: {acting on a sulfur group of donors (1.8)}
- C12P 7/06: Ethanol, i.e. non-beverage
- 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)}
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

Non-recombinant bacteria that produce ethanol as the primary fermentation product, associated nucleic acids and polypeptides, methods for producing ethanol using the bacteria, and kits are disclosed.

## Claims

### Claim 1

A method for producing ethanol from an oligosaccharide source comprising contacting the oligosaccharide with an isolated non-recombinant bacterium comprising an lpd gene having a mutation, wherein the mutation is one that corresponds to the substitution of the His residue at amino acid position 322 relative to an E. coli LPD sequence and the mutation renders the non-recombinant bacterium capable of producing ethanol as the primary fermentation product under anaerobic conditions, thereby producing ethanol from an oligosaccharide source.

### Claim 2 (depends on 1)

The method of claim 1, wherein the ethanol produced comprises greater than 50% of total non-gaseous fermentation products under anaerobic conditions.

### Claim 3 (depends on 1)

The method of claim 1, wherein the bacterium, in the absence of the mutation, is non-ethanologenic.

### Claim 4 (depends on 3)

The method of claim 3, wherein ethanol is the minor fermentation product and comprises less than 40% of total non gaseous fermentation products.

### Claim 5 (depends on 1)

The method of claim 1, wherein the mutation provides a homoethanol fermentation pathway.

### Claim 6 (depends on 1)

The method of claim 1, wherein one or more alternative pathways for fermentation in the bacterium are inactivated.

### Claim 7 (depends on 6)

The method of claim 6, wherein the alternative pathways for fermentation include lactate production by lactate dehydrogenase (ldhA), acetate, ethanol, formate, H2 and CO2 starting with pyruvate formate-lyase (pfl) and succinate.

### Claim 8 (depends on 7)

The method of claim 7, wherein the alternative pathways for fermentation are inactivated by mutation.

### Claim 9 (depends on 8)

The method of claim 8, wherein the mutation is in the ldhA gene.

### Claim 10 (depends on 8)

The method of claim 8, wherein the mutation is in the pfl gene.

### Claim 11 (depends on 9)

The method of claim 9, wherein the mutation is in the ldhA or pflB genes.

### Claim 12 (depends on 10)

The method of claim 10, wherein the mutation is in the ldhA or pflB genes.

### Claim 13 (depends on 1)

The method of claim 1, wherein the oligosaccharide is selected from the group consisting of lignocellulose, hemicellulose, cellulose, pectin and any combination thereof.

### Claim 14 (depends on 1)

The method of claim 1, wherein mutation in the lpd gene causes NADH insensitivity.

### Claim 15

A method for producing ethanol from an oligosaccharide source comprising contacting the oligosaccharide with an isolated non-recombinant bacterium comprising an E. coli lpd gene having a mutation selected from the group consisting of H322Y and E354K, wherein the mutation renders the non-recombinant bacterium capable of producing ethanol as the primary fermentation product under anaerobic conditions, thereby producing ethanol from an oligosaccharide source.

### Claim 16 (depends on 15)

The method of claim 15, wherein the ethanol produced comprises greater than 50% of total non-gaseous fermentation products under anaerobic conditions.

### Claim 17 (depends on 15)

The method of claim 15, wherein the bacterium, in the absence of the mutation, is non-ethanologenic.

### Claim 18 (depends on 17)

The method of claim 17, wherein ethanol is the minor fermentation product and comprises less than 40% of total non gaseous fermentation products.

### Claim 19 (depends on 15)

The method of claim 15, wherein the mutation provides a homoethanol fermentation pathway.

### Claim 20 (depends on 15)

The method of claim 15, wherein one or more alternative pathways for fermentation in the bacterium are inactivated.

### Claim 21 (depends on 20)

The method of claim 20, wherein the alternative pathways for fermentation include lactate production by lactate dehydrogenase (ldhA), acetate, ethanol, formate, H2 and CO2 starting with pyruvate formate-lyase (pfl) and succinate.

### Claim 22 (depends on 21)

The method of claim 21, wherein the alternative pathways for fermentation are inactivated by mutation.

### Claim 23 (depends on 22)

The method of claim 22, wherein the mutation is in the ldhA gene.

### Claim 24 (depends on 22)

The method of claim 22, wherein the mutation is in the pfl gene.

### Claim 25 (depends on 23)

The method of claim 23, wherein the mutation is in the ldhA or pflB genes.

### Claim 26 (depends on 24)

The method of claim 24, wherein the mutation is in the ldhA or pflB genes.

### Claim 27 (depends on 15)

The method of claim 15, wherein the oligosaccharide is selected from the group consisting of lignocellulose, hemicellulose, cellulose, pectin and any combination thereof.

### Claim 28 (depends on 15)

The method of claim 15, wherein mutation in the lpd gene causes NADH insensitivity.