# US 11,299,519 B2 · Host cells and methods using a repressor polypeptide and an inducible promoter for gene expression

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- Number: US 11,299,519 B2
- Document ID: US11299519B2
- Status: Granted
- Kind: B2
- Application number: 16/831,649
- Filed: 2020-03-26
- Published: 2020-08-20
- Granted: 2022-04-12
- Earliest claimed: 2015-07-01
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Thomas L. Ruegg
- Web page: https://patentgrep.com/patent/US11299519B2

## CPC

- C07K 14/245: Escherichia (G)
- C07K 14/70567: {Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- 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)

## Abstract

The present invention provides for a system comprising (a) a first nucleic acid comprising a nucleotide sequence encoding a nucleotide sequence of interest operatively linked to a promoter comprising a repressor polypeptide binding site, and (b) a second nucleic acid comprising a nucleotide sequence encoding a repressor polypeptide having at least 70% amino acid identity with EilR, SmvR, KmrR, RcdA, or QacR; wherein expression of the nucleotide sequence of interest from the promoter is induced by the presence of a hydrophobic inducer, such as a hydrophobic cation inducer, such as a triarylmethane, acridine, phenazine, phenothiazine, or xanthene.

## Claims

### Claim 1

A genetically modified host cell comprising
(a) a first nucleic acid comprising a nucleotide sequence of interest operatively linked to a promoter which is modified to comprise an EilR binding site,
(b) a second nucleic acid comprising a nucleotide sequence encoding a repressor polypeptide comprising an amino acid sequence comprising a biologically active fragment of EilR having at least 70% amino acid identity with SEQ ID NO:1 wherein expression of the nucleotide sequence of interest from the promoter is induced by the presence of a hydrophobic inducer in that when the repressor polypeptide is bound to the hydrophobic inducer, the repressor polypeptide does not bind to the EilR binding site, wherein the nucleotide sequence of interest is heterologous to EilR or the biologically active fragment thereof, wherein when the repressor polypeptide has an amino acid sequence consisting of SEQ ID NO:1, the nucleotide sequence of interest does not encode a green fluorescence protein (GFP); and
(c) the hydrophobic inducer within the genetically modified host cell.

### Claim 2 (depends on 1)

The genetically modified host cell of claim 1, wherein the hydrophobic inducer is a hydrophobic cation inducer.

### Claim 3 (depends on 2)

The genetically modified host cell of claim 2, wherein the hydrophobic cation inducer is a triarylmethane, acridine, phenazine, phenothiazine, xanthene, imidazole, or pyrazole.

### Claim 4 (depends on 2)

The genetically modified host cell of claim 2, wherein the hydrophobic cation inducer is a malachite green, acridine orange, acridine yellow, proflavine, acriflavine, neutral red, rhodamine 6G, rhodamine B, tetraphenylarsonium, cetylpyridium, benzalkonium, dequelinium, methyl green, ethidium, 3′,3′-dipropyloxacarbocyanine (DiOC3), pyronin Y, chlorhexidine, amicarbalide, 4′,6′-diamidino-2-phenylindole (DAPI), phenamidine, propamidine, palmatine, berberine, nitidine/chelerythrrine, avicin, 1-ethyl-3-methylimidazolium acetate (EMIN Acetate), 1-ethyl-3-methylimidazolium chloride (EMIM Cl), 1-ethyl-3-methylimidazolium hydrogensulfate (EMIM HOSO3), 1-ethyl-3-methylimidazolium methylsulfate (EMIM MeOSO3), 1-ethyl-3-methylimidazolium ethylsulfate (EMIM EtOSO3), 1-ethyl-3-methylimidazolium methanesulfonate (EMIM MeSO3), 1-ethyl-3-methylimidazolium tetrachloroaluminate (EMIM AlCl4), 1-ethyl-3-methylimidazolium thiocyanate (EMIM SCN), 1-butyl-3-methylimidazolium acetate (BMIM Acetate), 1-butyl-3-methylimidazolium chloride (BMIM Cl), 1-butyl-3-methylimidazolium hydrogensulfate (BMIM HOSO3), 1-butyl-3-methylimidazolium methanesulfonate (BMIM MeSO3), 1-butyl-3-methylimidazolium methylsulfate (BMIM MeOSO3), 1-butyl-3-methylimidazolium tetrachloroaluminate (BMIM AlCl4), 1-butyl-3-methylimidazolium thiocyanate (BMIM SCN), 1-ethyl-2,3-dimethylimidazolium ethylsulfate (EDIM EtOSO3), Tris(2-hydroxyethyl)methylammonium methylsulfate (MTEOA MeOSO3), 1-methylimidazolium chloride (MIM Cl), 1-methylimidazolium hydrogensulfate (MIM HOSO3), or 1,2,4-trimethylpyrazolium methylsulfate.

### Claim 5 (depends on 1)

The genetically modified host cell of claim 1, wherein the hydrophobic inducer is a hydrophobic anion inducer.

### Claim 6 (depends on 5)

The genetically modified host cell of claim 5, wherein the hydrophobic anion inducer is a quinoline, triarylmethane, azo-dye, or xanthene.

### Claim 7 (depends on 5)

The genetically modified host cell of claim 5, wherein the hydrophobic anion inducer is an erythrosine, indigo carmine (E132), quinolone yellow WS (Yellow 13), erythrosine (Red 3), sunset yellow (Yellow 6), alura red (E129 Red 40), tartrazine (Yellow 5), brilliant blue FCF (Blue 1), patent blue V (Blue 5, E131), or green S (E142).

### Claim 8 (depends on 1)

The genetically modified host cell of claim 1, wherein the nucleotide sequence of interest encodes a polypeptide of interest.

### Claim 9 (depends on 1)

The genetically modified host cell of claim 1, wherein the genetically modified host cell is a yeast.

### Claim 10 (depends on 9)

The genetically modified host cell of claim 9, wherein the yeast is a Yarrowia, Candida, Bebaromyces, Saccharomyces, Schizosaccharomyces or Pichia cell.

### Claim 11 (depends on 1)

The genetically modified host cell of claim 1, wherein the genetically modified host cell is an Escherichia, Bacillus, Salmonella, Klebsiella, Enterobacter, Pseudomonas, Streptomyces, Cynechocystis, Cynechococcus, Sinorhizobium, or Caulobacter cell.

### Claim 12 (depends on 11)

The genetically modified host cell of claim 11, wherein the genetically modified host cell is Escherichia coli and the promoter is a lac promoter, trc promoter, tac promoter, T7 promoter, PL promoter, tetA promoter, araBAD promoter, rhaPBAD promoter, Lambda promoter, or T5 promoter.

### Claim 13 (depends on 1)

The genetically modified host cell of claim 1, wherein the repressor polypeptide comprises an amino acid sequence comprising a biologically active fragment of EilR having at least 90% amino acid identity with SEQ ID NO:1.

### Claim 14 (depends on 13)

The genetically modified host cell of claim 13, wherein the repressor polypeptide comprises an amino acid sequence comprising a biologically active fragment of EilR having at least 95% amino acid identity with SEQ ID NO:1.

### Claim 15 (depends on 14)

The genetically modified host cell of claim 14, wherein the repressor polypeptide comprises an amino acid sequence comprising a biologically active fragment of EilR having at least 99% amino acid identity with SEQ ID NO:1.

### Claim 16 (depends on 1)

The genetically modified host cell of claim 1, wherein the repressor polypeptide comprises an amino acid sequence comprising M at position 1, I at position 13, A at position 17, G at position 25, T at position 30, I at position 34, G at position 43, H at position 48, F at position 49, L at position 55, F at position 60, W at position 79, W at position 101, Q at position 105, L at position 120, T at position 121, I at position 132, G at position 136, G at position 140, E at position 141, I at position 151, R at position 154, I at position 156, L at position 158, and G at position 169 of SEQ ID NO:1.

### Claim 17 (depends on 1)

The genetically modified host cell of claim 1, wherein the EilR binding site comprises the nucleotide sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

### Claim 18

A method for expressing a nucleotide sequence of interest comprising:
(a) providing a genetically modified host cell comprising (i) a first nucleic acid comprising a nucleotide sequence of interest operatively linked to a promoter which is modified to comprise an EilR binding site, (ii) a second nucleic acid comprising a nucleotide sequence encoding a repressor polypeptide comprising an amino acid sequence comprising a biologically active fragment of EilR having at least 70% amino acid identity with SEQ ID NO:1 wherein expression of the nucleotide sequence of interest from the promoter is induced by the presence of a hydrophobic inducer in that when the repressor polypeptide is bound to the hydrophobic inducer, the repressor polypeptide does not bind to the EilR binding site, wherein the nucleotide sequence of interest is heterologous to EilR or the biologically active fragment thereof, wherein when the repressor polypeptide has an amino acid sequence consisting of SEQ ID NO:1, then the nucleotide sequence of interest does not encode a green fluorescence protein (GFP);
(b) introducing a hydrophobic cation inducer to the genetically modified host cell; and
(c) expressing the nucleotide sequence of interest, or culturing or growing the genetically modified host cell such that nucleotide sequence of interest is expressed, such that the nucleotide sequence of interest is expressed at a rate higher when the hydrophobic cation inducer is present than if the hydrophobic cation inducer is not present in the genetically modified host cell.

### Claim 19 (depends on 18)

The method of claim 18, wherein the hydrophobic inducer is a hydrophobic cation inducer.

### Claim 20 (depends on 19)

The method of claim 19, wherein the hydrophobic cation inducer is a triarylmethane, acridine, phenazine, phenothiazine, xanthene, imidazole, or pyrazole.

### Claim 21 (depends on 19)

The method of claim 19, wherein the hydrophobic cation inducer is a malachite green, acridine orange, acridine yellow, proflavine, acriflavine, neutral red, rhodamine 6G, rhodamine B, tetraphenylarsonium, cetylpyridium, benzalkonium, dequelinium, methyl green, ethidium, 3′,3′-dipropyloxacarbocyanine (DiOC3), pyronin Y, chlorhexidine, amicarbalide, 4′,6′-diamidino-2-phenylindole (DAPI), phenamidine, propamidine, palmatine, berberine, nitidine/chelerythrrine, avicin, 1-ethyl-3-methylimidazolium acetate (EMIN Acetate), 1-ethyl-3-methylimidazolium chloride (EMIM Cl), 1-ethyl-3-methylimidazolium hydrogensulfate (EMIM HOSO3), 1-ethyl-3-methylimidazolium methylsulfate MeOSO3), 1-ethyl-3-methylimidazolium ethylsulfate (EMIM EtOSO3), 1-ethyl-3-methylimidazolium methane sulfonate (EMIM MeSO3), 1-ethyl-3-methylimidazolium tetrachloroaluminate (EMIM AlCl4), 1-ethyl-3-methylimidazolium thiocyanate (EMIM SCN), 1-butyl-3-methylimidazolium acetate (BMIM Acetate), 1-butyl-3-methylimidazolium chloride (BMIM Cl), 1-butyl-3-methylimidazolium hydrogensulfate (BMIM HOSO3), 1-butyl-3-methylimidazolium methanesulfonate (BMIM MeSO3), 1-butyl-3-methylimidazolium methylsulfate (BMIM MeOSO3), 1-butyl-3-methylimidazolium tetrachloroaluminate (BMIM AlCl4), 1-butyl-3-methylimidazolium thiocyanate (BMIM SCN), 1-ethyl-2,3-dimethylimidazolium ethylsulfate EtOSO3), Tris(2-hydroxyethyl)methylammonium methylsulfate (MTEOA MeOSO3), 1-methylimidazolium chloride (MIM Cl), 1-methylimidazolium hydrogensulfate (MIM HOSO3), or 1,2,4-trimethylpyrazolium methylsulfate.

### Claim 22 (depends on 18)

The method of claim 18, wherein the hydrophobic inducer is a hydrophobic anion inducer.

### Claim 23 (depends on 22)

The method of claim 22, wherein the hydrophobic anion inducer is a quinoline, triarylmethane, azo-dye, or xanthene.

### Claim 24 (depends on 22)

The method of claim 22, wherein the hydrophobic anion inducer is an erythrosine, indigo carmine (E132), quinolone yellow WS (Yellow 13), erythrosine (Red 3), sunset yellow (Yellow 6), alura red (E129 Red 40), tartrazine (Yellow 5), brilliant blue FCF (Blue 1), patent blue V (Blue 5, E131), or green S (E142).

### Claim 25 (depends on 18)

The method of claim 18, wherein the nucleotide sequence of interest encodes a polypeptide of interest.

### Claim 26 (depends on 18)

The method of claim 18, wherein the genetically modified host cell is a yeast.

### Claim 27 (depends on 26)

The method of claim 26, wherein the yeast is a Yarrowia, Candida, Bebaromyces, Saccharomyces, Schizosaccharomyces or Pichia cell.

### Claim 28 (depends on 18)

The method of claim 18, wherein the genetically modified host cell is an Escherichia, Bacillus, Salmonella, Klebsiella, Enterobacter, Pseudomonas, Streptomyces, Cynechocystis, Cynechococcus, Sinorhizobium, or Caulobacter cell.

### Claim 29 (depends on 28)

The method of claim 28, wherein the genetically modified host cell is Escherichia coli and the promoter is a lac promoter, trc promoter, tac promoter, T7 promoter, PL, promoter, tetA promoter, araBAD promoter, rhaPBAD promoter, Lambda promoter, or T5 promoter.

### Claim 30 (depends on 18)

The method of claim 18, wherein the repressor polypeptide comprises an amino acid sequence comprising a biologically active fragment of EilR having at least 90% amino acid identity with SEQ ID NO:1.

### Claim 31 (depends on 30)

The method of claim 30, wherein the repressor polypeptide comprises an amino acid sequence comprising a biologically active fragment of EilR having at least 95% amino acid identity with SEQ ID NO:1.

### Claim 32 (depends on 31)

The method of claim 31, wherein the repressor polypeptide comprises an amino acid sequence comprising a biologically active fragment of EilR having at least 99% amino acid identity with SEQ ID NO:1.

### Claim 33 (depends on 18)

The method of claim 18, wherein the repressor polypeptide comprises an amino acid sequence comprising M at position 1, I at position 13, A at position 17, G at position 25, T at position 30, I at position 34, G at position 43, H at position 48, F at position 49, L at position 55, F at position 60, W at position 79, W at position 101, Q at position 105, L at position 120, T at position 121, I at position 132, G at position 136, G at position 140, E at position 141, I at position 151, R at position 154, I at position 156, L at position 158, and G at position 169 of SEQ ID NO:1.

### Claim 34 (depends on 18)

The method of claim 18, wherein the EilR binding site comprises the nucleotide sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.