# US 10,308,964 B2 · Activity of Fe—S cluster requiring proteins

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- Number: US 10,308,964 B2
- Document ID: US10308964B2
- Status: Granted
- Kind: B2
- Application number: 15/340,084
- Filed: 2016-11-01
- Published: 2017-04-13
- Granted: 2019-06-04
- Earliest claimed: 2010-02-17
- Assignees: Butamax Advanced Biofuels LLC
- Applicants: Butamax Advanced Biofuels LLC
- Inventors: Dennis Flint; Brian James Paul; Rick W. Ye
- Web page: https://patentgrep.com/patent/US10308964B2

## CPC

- C12P 7/16: Butanols
- C07K 14/395: from Saccharomyces
- C12N 9/88: Lyases (4.)
- C12N 15/81: for yeasts
- C12P 7/40: containing a carboxyl group {including Peroxycarboxylic acids}
- C12P 13/02: Amides, e.g. chloramphenicol {or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes (peptides C12P21/00 or C07K)}
- C12P 13/04: Alpha- or beta- amino acids {(other amino acids C12P13/005)}
- C12Q 1/527: involving lyase
- C12Y 402/01009: Dihydroxy-acid dehydratase (4.2.1.9), i.e. acetohydroxyacid dehydratase
- G01N 33/573: for enzymes or isoenzymes
- Y02E 50/10: Biofuels, e.g. bio-diesel
- Y02P 20/52: using catalysts, e.g. selective catalysts
- 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)}
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

The present invention is related to a recombinant host cell, in particular a yeast cell, comprising a dihydroxy-acid dehydratase polypeptide. The invention is also related to a recombinant host cell having increased specific activity of the dihydroxy-acid dehydratase polypeptide as a result of increased expression of the polypeptide, modulation of the Fe—S cluster biosynthesis of the cell, or a combination thereof. The present invention also includes methods of using the host cells, as well as, methods for identifying polypeptides that increase the flux in an Fe—S cluster biosynthesis pathway in a host cell.

## Claims

### Claim 1

A method of making isobutanol comprising:
(a) providing a recombinant yeast host cell comprising:
(i)at least one heterologous polynucleotide encoding a dihydroxy-acid dehydratase enzyme, wherein the dihydroxy-acid dehydratase enzyme has an amino acid sequence with at least 95% identity to SEQ ID NO: 958, and
(ii) at least one deletion in an endogenous gene encoding a polypeptide affecting Fe—S cluster biosynthesis, wherein the gene encoding the polypeptide affecting Fe—S cluster biosynthesis is selected from Fe Repressor of Activation-2 (FRA2), glutaredoxin-3 (GRX3), and CCC1,
wherein the recombinant yeast host cell produces isobutanol; and
(b) contacting the recombinant yeast host cell of (a) with a fermentable carbon substrate in a fermentation medium under conditions wherein isobutanol is produced.

### Claim 2 (depends on 1)

The method of claim 1, wherein the polypeptide affecting Fe-S cluster biosynthesis is FRA2.

### Claim 3 (depends on 1)

The method of claim 1, wherein the recombinant yeast host cell is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Issatchenkia, and Pichia.

### Claim 4 (depends on 1)

The method of claim 1, wherein the dihydroxy-acid dehydratase enzyme is expressed in the cytosol of the recombinant yeast host cell.

### Claim 5 (depends on 1)

The method of claim 1, wherein the recombinant yeast host cell comprises an isobutanol biosynthetic pathway.

### Claim 6 (depends on 1)

The method of claim 1, wherein the isobutanol is isolated from the fermentation medium by distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation.

### Claim 7 (depends on 1)

The method of claim 1, wherein solids are removed from the fermentation medium by centrifugation, filtration, or decantation.