# US 10,767,196 B2 · Engineering an increase in ethanol production by altering cofactor specificity

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- Number: US 10,767,196 B2
- Document ID: US10767196B2
- Status: Granted
- Kind: B2
- Application number: 14/361,948
- Filed: 2012-11-30
- Published: 2014-10-30
- Granted: 2020-09-08
- Earliest claimed: 2011-11-30
- Assignees: Enchi Corporation; Dartmouth College; UT-Battelle, LLC
- Applicants: Enchi Corporation; Dartmouth College; Ut-Battelle, LLC
- Inventors: Jonathan Lo; Adam M. Guss; Johannes P. Van Dijken; Arthur J. Shaw, IV; Daniel G. Olson; Christopher D. Herring; D. Aaron Argyros; Nicky Caiazza
- Web page: https://patentgrep.com/patent/US10767196B2

## CPC

- C12P 7/06: Ethanol, i.e. non-beverage
- 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 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12Y 101/01001: Alcohol dehydrogenase (1.1.1.1)
- C12Y 101/01002: Alcohol dehydrogenase (NADP+) (1.1.1.2), i.e. aldehyde reductase
- 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)
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present invention provides for the manipulation of cofactor usage in a recombinant host cell to increase the formation of desirable products. In some embodiments, the invention provides for a recombinant microorganism comprising a mutation in one or more native enzymes such that their cofactor specificity is altered in such a way that overall cofactor usage in the cell is balanced for a specified pathway and there is an increase in a specific product formation within the cell. In some embodiments, endogenous enzymes are replaced by enzymes with an alternate cofactor specificity from a different species.

## Claims

### Claim 1

A recombinant cellulolytic Clostridium host cell comprising at least one heterologous nucleic acid sequence encoding a bifunctional alcohol-acetaldehyde dehydrogenase, wherein the bifunctional alcohol-acetaldehyde dehydrogenase is AdhE;
wherein the bifunctional AdhE is encoded by a heterologous nucleic acid identical to a polynucleotide that encodes the polypeptide of SEQ ID NO:21, wherein the bifunctional AdhE uses NADPH as a cofactor instead of NADH;
wherein the recombinant Clostridium host cell further comprises a genetic modification that leads to the down-regulation of the native enzyme lactate dehydrogenase;
and wherein the recombinant Clostridium host cell has an increased production of ethanol compared to a wildtype cellulolytic Clostridium cell without the heterologous nucleic acid and the down-regulation of the native enzyme lactate dehydrogenase.

### Claim 2 (depends on 1)

The recombinant cellulolytic Clostridium host cell of claim 1, wherein the recombinant host cell further comprises a genetic modification that leads to the down-regulation of the native alcohol dehydrogenase and/or acetaldehyde dehydrogenase.

### Claim 3 (depends on 1)

The recombinant cellulolytic Clostridium host cell of claim 1, wherein the host cell is selected from the group consisting of Clostridium thermocellum, Clostridium cellulolyticum, Clostridium clariflavum, and Clostridium phytofermentans.

### Claim 4 (depends on 1)

The recombinant cellulolytic Clostridium host cell of claim 1, wherein the heterologous nucleic acid encoding the bifunctional alcohol-acetaldehyde dehydrogenase is at least about 99% identical to the polynucleotide of SEQ ID NO 20.

### Claim 5 (depends on 1)

The recombinant cellulolytic Clostridium host cell of claim 1, wherein the heterologous nucleic acid encoding the bifunctional alcohol-acetaldehyde dehydrogenase is identical to SEQ ID NO: 20.

### Claim 6 (depends on 1)

The recombinant cellulolytic Clostridium host cell of claim 1, wherein the host cell is Clostridium thermocellum.

### Claim 7 (depends on 1)

A composition comprising the host cell from claim 1 and a carbon-containing feedstock, wherein the feedstock is selected from the group consisting of woody biomass, grasses, sugar-processing residues, municipal waste, agricultural wastes and any combination thereof.

### Claim 8 (depends on 7)

The composition of claim 7, wherein the feedstock comprises recycled wood pulp fiber, sawdust, hardwood, softwood, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, succulents, agave, cane bagasse, switchgrass, miscanthus, paper sludge, municipal waste or any combination thereof.

### Claim 9 (depends on 1)

A method of producing ethanol comprising: (a) providing the host cell of claim 1; (b) culturing the host cell in the presence of a carbon containing feedstock for sufficient time to produce ethanol; and, optionally (c) extracting the ethanol.