# US 10,370,418 B2 · Engineered microorganisms having resistance to ionic liquids

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- Number: US 10,370,418 B2
- Document ID: US10370418B2
- Status: Granted
- Kind: B2
- Application number: 14/717,962
- Filed: 2015-05-20
- Published: 2016-03-31
- Granted: 2019-08-06
- Earliest claimed: 2014-05-20
- Assignees: Lawrence Livermore National Security, LLC
- Applicants: Lawrence Livermore National Security, LLC
- Inventors: Michael P. Thelen; Douglas A. Higgins; Thomas L. Ruegg
- Web page: https://patentgrep.com/patent/US10370418B2

## CPC

- C07K 14/255: Salmonella (G)
- C07K 14/395: from Saccharomyces
- C12N 1/14: Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor
- C12N 1/16: Yeasts; Culture media therefor
- C12N 1/20: Bacteria; Culture media therefor
- C12P 7/10: substrate containing cellulosic material
- C12P 2203/00: Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
- Y02E 50/10: Biofuels, e.g. bio-diesel
- 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)
- 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 present invention provides for a method of genetically modifying microorganisms to enhance resistance to ionic liquids using a yeast Major Facilitator Superfamily (MFS), or a Salmonella MFS SmvA Pump or SmvR Regulator, a Small Multidrug Resistance Family (SMR), or Saccharomyces cerevisiae YDR090C, polypeptide, host cells genetically modified in accordance with the methods, and methods of using the host cells in a reaction comprising biomass that has been pretreated with ionic liquids.

## Claims

### Claim 1

A culture medium comprising an ionic liquid and a recombinant microorganism, wherein the microorganism comprises a heterologous gene encoding a polypeptide operably linked to a promoter, wherein the polypeptide has at least 95% identity to one amino acid sequence selected from the group consisting of SEQ ID NOs:1 and 21, and the expression of the polypeptide confers to the microorganism resistance to the concentration of the ionic liquid in the culture medium.

### Claim 2 (depends on 1)

The culture medium of claim 1, wherein the polypeptide has at least 99% identity to one amino acid sequence selected from the group consisting of SEQ ID NOs:1 and 21.

### Claim 3 (depends on 1)

The culture medium of claim 1, wherein the polypeptide comprises at least 95% identity to SEQ ID NO:21 and the polypeptide comprises a proline at a position corresponding to position 282 of SEQ ID NO:21, a leucine at a position corresponding to position 284 of SEQ ID NO:21, or both.

### Claim 4 (depends on 1)

The culture medium of claim 1, wherein the heterologous gene is operably linked to a tet repressor sequence.

### Claim 5 (depends on 1)

The culture medium of claim 1, wherein the microorganism is a bacteria.

### Claim 6 (depends on 5)

The culture medium of claim 5, wherein the bacteria is Escherichia coli.

### Claim 7 (depends on 1)

The culture medium of claim 1, wherein the microorganism is a yeast or filamentous fungi.

### Claim 8 (depends on 1)

The culture medium of claim 1, wherein the anion of the ionic liquid is Cl− or acetate.

### Claim 9 (depends on 1)

The culture medium of claim 1, wherein the heterologous gene encoding the polypeptide is on a vector or is integrated into the genome of the microorganism.

### Claim 10 (depends on 1)

The culture medium of claim 1, wherein the culture medium further comprises a biomass.

### Claim 11 (depends on 1)

The culture medium of claim 1, wherein the culture medium comprises about 1% to about 20% of the ionic liquid.