IP Library Granted Patent US 9,080,178
Granted Patent B2
US 9,080,178 · App. 13/166,560 · Granted Jul 14, 2015

Patent

Inventors: Bernhard Palsson (San Diego, CA); Vasiliy A. Portnoy (San Diego, CA)
Assignee: The Regents Of The University Of California
C12N15/70C12N9/0053C12P7/18C12P7/56C12P13/04
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Quick Facts
Patent No.
US 9,080,178
App. No.
13/166,560
Granted
Jul 14, 2015
Kind
B2
Abstract

The invention provides transgenic Escherichia coli cells comprising a mutation in cydAB gene, and/or cyoABCD gene, and/or cbdAB gene, and/or ygiN gene, wherein the mutation reduces (preferably, but not necessarily, by 100%) the cytochrome oxydase activity of a protein encoded by cydAB gene, and/or cyoABCD gene, and/or cbdAB gene, and/or ygiN gene. In a preferred embodiment, the mutation is a deletion of the cydAB gene, cyoABCD gene, and cbdAB gene. In another embodiment, the mutation is a deletion of the cydAB gene, cyoABCD gene, cbdAB gene, and ygiN gene. In another embodiment, the transgenic Escherichia coli cell, which comprises a deletion of cydAB gene, and/or cyoABCD gene, and/or cbdAB gene, and/or ygiN gene a) has substantially the same level of growth in oxic conditions as the level of growth in anoxic conditions of Escherichia coli that lacks said deletion of cydAB gene, and/or cyoABCD gene, and/or cbdAB gene, and/or ygiN gene, and b) is capable of converting glucose to D-lactate and/or amino acid and/or 2,3-butanediol (2,3-BDO) under one or both of oxic conditions and anoxic conditions.

Claims (21)

1. A transgenic Escherichia coli cell comprising an additional copy of Escherichia coli alanine dehydrogenase (DadA) gene, and a deletion in the endogenous cydA gene (SEQ ID NO: 1), cydB gene (SEQ ID NO: 2), cyoA gene (SEQ ID NO: 3), cyoB gene (SEQ ID NO: 4), cyoC gene (SEQ ID NO: 5), cyoD gene (SEQ ID NO: 6), cbdA gene (SEQ ID NO: 7), cbdB gene (SEQ ID NO: 8), ygiN gene (SEQ ID NO: 9), and ldhA gene, wherein said transgenic Escherichia coli cell

a) has substantially the same level of growth in oxic conditions as the level of growth in anoxic conditions of the corresponding Escherichia coli cell that has no deletion of the endogenous cydA gene, endogenous cydB gene, endogenous cyoA gene, endogenous cyoB gene, endogenous cyoC gene, endogenous cyoD gene, endogenous cbdA gene, endogenous cbdB gene, and endogenous ygiN gene,

b) is capable of converting glucose to D-lactate under oxic conditions and/or anoxic conditions,

c) has substantially the same level of growth as said corresponding Escherichia coli in M9 minimal essential media that

(i) comprises a carbon source consisting of glucose, and

(ii) lacks amino acids Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val, and

d) is capable of producing an increased level of alanine compared to the corresponding Escherichia coli cell that comprises the endogenous ldhA gene and that does not comprise a heterologous DNA sequence encoding DadA.

2. The transgenic Escherichia coli cell of claim 1 , wherein said transgenic cell has reduced oxygen uptake in oxic conditions compared to said corresponding Escherichia coli.

3. The transgenic Escherichia coli cell of claim 1 , wherein said transgenic cell produces increased levels of D-lactic acid, when cultured in the presence of glucose as substrate, under oxic conditions and/or anoxic conditions compared to D-lactic acid levels produced by said corresponding Escherichia coli.

4. The transgenic Escherichia coli cell of claim 1 , wherein said transgenic cell produces altered levels of acetate, when cultured in the presence of glucose as substrate, under oxic conditions and/or anoxic conditions compared to acetate levels produced by said corresponding Escherichia coli.

5. The transgenic Escherichia coli cell of claim 1 , wherein said transgenic cell has increased expression of at least one of endogenous sodA gene (SEQ ID NO:10), and endogenous sodB gene (SEQ ID NO:11) compared to said corresponding Escherichia coli.

6. The transgenic Escherichia coli cell of claim 1 , wherein said transgenic cell further comprises a deletion of at least one of endogenous sodA gene (SEQ ID NO:10) and endogenous sodB gene (SEQ ID NO:11).

7. A method for producing a transgenic Escherichia coli cell comprising

a) introducing an additional copy of Escherichia coli alanine dehydrogenase (DadA) gene into an Escherichia coli cell,

b) deleting the endogenous cydA gene (SEQ ID NO: 1), cydB gene (SEQ ID NO: 2), cyoA gene (SEQ ID NO: 3), cyoB gene (SEQ ID NO: 4), cyoC gene (SEQ ID NO: 5), cyoD gene (SEQ ID NO: 6), cbdA gene (SEQ ID NO: 7), cbdB gene (SEQ ID NO: 8), ygiN gene (SEQ ID NO: 9), and ldhA gene from the genome of said Escherichia coli cell to produce a transgenic Escherichia coli cell, and

c) culturing said transgenic Escherichia coli cell to produce a cultured Escherichia coli cell, wherein said culturing is in M9 minimal media that

i) comprises a carbon source consisting of glucose, and

ii) lacks amino acids Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

8. The method of claim 7 , further comprising purifying said cultured Escherichia coli cell.

9. A method for producing an amino acid comprising culturing the transgenic Escherichia coli cell of claim 1 in M9 minimal essential medium that contains glucose, wherein said culturing is under conditions for production of an amino acid.

10. A method for producing 2,3-butanediol (2,3-BDO) comprising culturing the transgenic Escherichia coli cell of claim 1 in M9 minimal essential medium that contains glucose, wherein said culturing is under conditions for production of 2,3-BDO.

Assignments (2)
CONFIRMATORY LICENSE Recorded Oct 16, 2012
From: UNIVERSITY OF CALIFORNIA SAN DIEGO
To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT
Reel/Frame 029135/0128 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 24, 2011
From: PALSSON, BERNHARD; PORTNOY, VASILIY A.
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 026799/0228 →
Continuity (2)
Provisional Application 61363863 · Jul 13, 2010
Related Publication 20120064581A1 · Mar 15, 2012