Claims
- 1. A metabolically engineered cell that overexpresses pyruvate carboxylase.
- 2. The metabolically engineered cell of claim 1 which is a bacterial cell.
- 3. The metabolically engineered cell of claim 1 which is a gram-negative bacterial cell.
- 4. The bacterial cell of claim 3 which is selected from the group consisting of a Corynebacterium glutamicum cell, an Escherichia coli cell, a Salmonella typhimurium cell, a Brevibacterium flavum cell and a Brevibacterium lactofermentum cell.
- 5. The bacterial cell of claim 4 which is a C. glutamicum cell.
- 6. The C. glutamicum cell of claim 5 having at least one of the mutations selected from the group consisting of alanine−, valine− and acetate−.
- 7. The bacterial cell of claim 4 which is an E. coli cell.
- 8. The bacterial cell of claim 4 which is a S. typhimurium cell.
- 9. The metabolically engineered cell of claim 1 wherein a comparable wild-type of the engineered cell does not express a pyruvate carboxylase.
- 10. The metabolically engineered cell of claim 1 which expresses a pyruvate carboxylase derived from Rhizobium etli.
- 11. The metabolically engineered cell of claim 1 which expresses a pyruvate carboxylase derived from Pseudomonas fluorescens.
- 12. The metabolically engineered cell of claim 1 comprising a heterologous nucleic acid sequence encoding the pyruvate carboxylase.
- 13. The metabolically engineered cell of claim 12 wherein the heterologous nucleic acid sequence is chromosomally integrated.
- 14. The metabolically engineered cell of claim 1 that further overexpresses PEP carboxylase.
- 15. The metabolically engineered cell of claim 1 that further expresses PEP carboxykinase at a level lower than the level of PEP carboxykinase expressed in a comparable wild-type of the engineered cell.
- 16. The metabolically engineered cell of claim 15 that does not express a detectable level of PEP carboxykinase.
- 17. A metabolically engineered cell that expresses a heterologous pyruvate carboxylase.
- 18. The metabolically engineered cell of claim 17 which is a bacterial cell.
- 19. The bacterial cell of claim 18 which is selected from the group consisting of a C. glutamicum cell, an E. coli cell, an S. typhimurium cell, a B. flavum cell and a B. lactofermentum cell.
- 20. The bacterial cell of claim 19 which is selected from the group consisting of a C. glutamicum cell, an S. typhimurium cell and an E. coli cell.
- 21. The metabolically engineered cell of claim 17 that expresses a pyruvate carboxylase derived from an organism selected from the group consisting of R. etli and P. fluorescens.
- 22. The metabolically engineered cell of claim 17 comprising a nucleic acid sequence encoding the heterologous pyruvate carboxylase, wherein the nucleic acid sequence is chromosomally integrated.
- 23. The metabolically engineered cell of claim 17 wherein a comparable wild-type of the engineered cell does not express a pyruvate carboxylase.
- 24. The metabolically engineered cell of claim 17 that further overexpresses PEP carboxylase.
- 25. The metabolically engineered cell of claim 17 that further expresses PEP carboxykinase at a level lower than the level of PEP carboxykinase expressed in a comparable wild-type of the engineered cell.
- 26. The metabolically engineered cell of claim 25 that does not express a detectable level of PEP carboxykinase.
- 27. A metabolically engineered gram-negative bacterial cell that overexpresses pyruvate carboxylase.
- 28. A metabolically engineered cell that expresses pyruvate carboxylase, wherein a comparable wild-type of the engineered cell does not express a pyruvate carboxylase.
- 29. A metabolically engineered E. coli cell that expresses pyruvate carboxylase.
- 30. A metabolically engineered S. typhimurium cell that expresses pyruvate carboxylase.
- 31. A method for making a metabolically engineered cell comprising transforming a cell with a nucleic acid fragment comprising a heterologous nucleotide sequence encoding an enzyme having pyruvate carboxylase activity to yield a metabolically engineered cell that overexpresses pyruvate carboxylase.
- 32. The method of claim 31 comprising transforming a bacterial cell.
- 33. The method of claim 31 comprising transforming a gram-negative bacterial cell.
- 34. The method of claim 31 comprising transforming a bacterial cell selected from the group consisting of a C. glutamicum cell, an E. coli cell, an S. typhimurium cell, a B. flavum cell and a B. lactofermentum cell.
- 35. The method of claim 31 comprising transforming a C. glutamicum cell.
- 36. The method of claim 31 comprising transforming an E. coli cell.
- 37. The method of claim 31 comprising transforming an S. typhimurium cell.
- 38. The method of claim 31 comprising transforming a cell with a nucleic acid fragment comprising a nucleotide sequence selected from the group consisting of a R. etli gene encoding pyruvate carboxylase and a P. fluorescens gene encoding pyruvate carboxylase.
- 39. The method of claim 31 further comprising transforming the cell with a nucleic acid fragment comprising a nucleotide sequence encoding PEP carboxylase such that metabolically engineered cell overexpresses PEP carboxylase.
- 40. The method of claim 31 comprising transforming a metabolically engineered cell that does not express a detectable level of PEP carboxykinase.
- 41. A method for making a metabolically engineered cell comprising increasing the intracellular activity of an endogenous pyruvate carboxylase enzyme in a cell to yield a metabolically engineered cell that overexpresses pyruvate carboxylase.
- 42. The method of claim 41 wherein increasing the intracellular activity of an endogenous pyruvate carboxylase enzyme comprises transforming the cell with a nucleic acid fragment comprising a nucleotide sequence encoding the endogenous pyruvate carboxylase enzyme.
- 43. The method of claim 41 wherein increasing the intracellular activity of an endogenous pyruvate carboxylase enzyme comprises mutating a gene of the cell, wherein the gene encodes the endogenous pyruvate carboxylase enzyme.
- 44. The method of claim 41 comprising increasing the intracellular activity of an endogenous pyruvate carboxylase enzyme in a bacterial cell.
- 45. The method of claim 41 comprising increasing the intracellular activity of an endogenous pyruvate carboxylase enzyme in a C. glutamicum cell.
- 46. A method for making an oxaloacetate-derived biochemical comprising:
(a) providing a cell that produces the biochemical; (b) transforming the cell with a nucleic acid fragment comprising a heterologous nucleotide sequence encoding an enzyme having pyruvate carboxylase activity; (c) expressing the enzyme in the cell to cause increased production of the biochemical; and (d) isolating the biochemical produced by the cell.
- 47. The method of claim 46 wherein step (a) comprises providing a bacterial cell.
- 48. The method of claim 46 wherein step (a) comprises providing a gram-negative bacterial cell.
- 49. The method of claim 46 wherein step (a) comprises providing a bacterial cell selected from the group consisting of a C. glutamicum cell, an E. coli cell, an S. typhimurium cell, a B. flavum cell and a B. lactofermentum.
- 50. The method of claim 46 wherein step (a) comprises providing an E. coli cell.
- 51. The method of claim 46 wherein step (a) comprises providing a C. glutamicum cell.
- 52. The method of claim 46 wherein step (a) comprises providing an S. typhimurium cell.
- 53. The method of claim 46 wherein step (b) comprises transforming the cell with a nucleic acid fragment comprising a heterologous nucleotide sequence selected from the group consisting of an R. etli gene encoding pyruvate carboxylase and a P. fluorescens gene encoding pyruvate carboxylase.
- 54. The method of claim 46 wherein step (c) comprises expressing the enzyme in the cell to cause increased production of a biochemical selected from the group consisting of an organic acid, an amino acid, a porphyrin and a pyrimidine nucleotide.
- 55. The method of claim 46 wherein step (c) comprises expressing the enzyme in the cell to cause increased production of a biochemical selected from the group consisting of arginine, asparagine, aspartate, glutamate, glutamine, proline, isoleucine, malate, fumarate, citrate, isocitrate, α-ketoglutarate and succinyl-CoA.
- 56. The method of claim 46 wherein step (c) comprises expressing the enzyme in the cell to cause increased production of lysine.
- 57. The method of claim 46 wherein step (c) comprises expressing the enzyme in the cell to cause increased production of succinate.
- 58. The method of claim 46 wherein step (c) comprises expressing the enzyme in the cell to cause increased production of threonine.
- 59. The method of claim 46 wherein step (c) comprises expressing the enzyme in the cell to cause increased production of methionine.
- 60. A method for making an oxaloacetate-derived biochemical comprising:
(a) providing a cell that produces the biochemical, wherein the cell expresses an endogenous pyruvate carboxylase; (b) metabolically engineering the cell to yield a metabolically engineered cell that overexpresses endogenous pyruvate carboxylase; (c) overexpressing the pyruvate carboxylase to cause increased production of the biochemical; and (d) isolating the biochemical produced by the cell.
- 61. The method of claim 60 wherein step (b) comprises mutating a gene of a cell, said gene encoding the pyruvate carboxylase.
- 62. The method of claim 60 wherein step (b) comprises transforming the cell with a nucleic acid fragment comprising a nucleotide sequence encoding the pyruvate carboxylase.
- 63. The method of claim 60 wherein step (a) comprises providing a bacterial cell.
- 64. The method of claim 60 wherein step (a) comprises providing a C. glutamicum cell.
- 65. The method of claim 60 wherein step (c) comprises overexpressing the pyruvate carboxylase to cause increased production of a biochemical selected from the group consisting of an organic acid, an amino acid, a porphyrin and a pyrimidine nucleotide.
- 66. The method of claim 60 wherein step (c) comprises overexpressing the pyruvate carboxylase to cause increased production of a biochemical selected from the group consisting of arginine, asparagine, aspartate, glutamate, glutamine, proline, isoleucine, malate, fumarate, citrate, isocitrate, α-ketoglutarate and succinyl-CoA.
- 67. The method of claim 60 wherein step (c) comprises overexpressing the pyruvate carboxylase to cause increased production of lysine.
- 68. The method of claim 60 wherein step (c) comprises overexpressing the pyruvate carboxylase to cause increased production of succinate.
- 69. The method of claim 60 wherein step (c) comprises overexpressing the pyruvate carboxylase to cause increased production of threonine.
- 70. The method of claim 60 wherein step (c) comprises overexpressing the pyruvate carboxylase to cause increased production of methionine.
- 71. A method for making an oxaloacetate-derived biochemical comprising:
(a) providing a metabolically engineered cell that produces the biochemical, wherein the metabolically engineered cell overexpresses pyruvate carboxylase; (b) anaerobically culturing the metabolically engineered cell under conditions that permit overexpression of the pyruvate carboxylase to cause increased production of the biochemical; and (c) isolating the biochemical produced by the cell.
- 72. The method of claim 71 wherein step (a) comprises providing a metabolically engineered bacterial cell.
- 73. The method of step 71 wherein step (a) comprises providing a metabolically engineered gram-negative bacterial cell.
- 74. The method of claim 71 wherein step (a) comprises providing a metabolically engineered E. coli cell.
- 75. The method of claim 71 wherein step (a) comprises providing a metabolically engineered S. typhimurium cell.
- 76. The method of claim 71 wherein step (b) comprises anaerobically culturing the metabolically engineered cell to cause increased production of a biochemical selected from the group consisting of an organic acid, an amino acid, a porphyrin and a pyrimidine nucleotide.
- 77. The method of claim 71 wherein step (b) comprises anaerobically culturing the metabolically engineered cell to cause increased production of a biochemical selected from the group consisting of arginine, asparagine, aspartate, glutamate, glutamine, proline, isoleucine, malate, fumarate, citrate, isocitrate, α-ketoglutarate and succinyl-CoA.
- 78. The method of claim 71 wherein step (b) comprises anaerobically culturing the metabolically engineered cell to cause increased production of lysine.
- 79. The method of claim 71 wherein step (b) comprises anaerobically culturing the metabolically engineered cell to cause increased production of succinate.
- 80. The method of claim 71 wherein step (b) comprises anaerobically culturing the metabolically engineered cell to cause increased production of threonine.
- 81. The method of claim 71 wherein step (b) comprises anaerobically culturing the metabolically engineered cell to cause increased production of methionine.
- 82. A method for making an oxaloacetate-derived biochemical comprising:
(a) providing a metabolically engineered cell that produces the biochemical, wherein the metabolically engineered cell expresses a heterologous pyruvate carboxylase; (b) culturing the metabolically engineered cell under conditions that permit overexpression of pyruvate carboxylase to cause increased production of the biochemical; and (c) isolating the biochemical produced by the cell.
- 83. The method of claim 82 wherein step (a) comprises providing a metabolically engineered bacterial cell.
- 84. The method of claim 82 wherein step (a) comprises providing a metabolically engineered gram-negative bacterial cell.
- 85. The method of claim 82 wherein step (a) comprises providing a metabolically engineered cell selected from the group consisting of an E. coli cell, an S. typhimurium cell and a C. glutamicum cell.
- 86. The method of claim 82 wherein step (b) comprises culturing the metabolically engineered cell to cause increased production of a biochemical selected from the group consisting of an organic acid, an amino acid, a porphyrin and a pyrimidine nucleotide.
- 87. The method of claim 82 wherein step (b) comprises culturing the metabolically engineered cell to cause increased production of a biochemical selected from the group consisting of arginine, asparagine, aspartate, glutamate, glutamine, proline, isoleucine, malate, fumarate, citrate, isocitrate, α-ketoglutarate and succinyl-CoA.
- 88. The method of claim 82 wherein step (b) comprises culturing the metabolically engineered cell to cause increased production of lysine.
- 89. The method of claim 82 wherein step (b) comprises culturing the metabolically engineered cell to cause increased production of succinate.
- 90. The method of claim 82 wherein step (b) comprises culturing the metabolically engineered cell to cause increased production of threonine.
- 91. The method of claim 82 wherein step (b) comprises culturing the metabolically engineered cell to cause increased production of methionine.
- 92. The method of claim 82 wherein, prior to step (b), the metabolically engineered cell is cultured aerobically to increase biomass.
- 93. A method for making an oxaloacetate-derived biochemical comprising:
(a) providing a metabolically engineered cell that produces the biochemical, wherein the metabolically engineered cell overexpresses an endogenous pyruvate carboxylase; (b) culturing the metabolically engineered cell under conditions that permit overexpression of the endogenous pyruvate carboxylase to cause increased production of the biochemical; and (c) isolating the biochemical produced by the cell.
- 94. The method of claim 93 wherein step (a) comprises providing a metabolically engineered bacterial cell.
- 95. The method of claim 93 wherein step (a) comprises providing a metabolically engineered C. glutamicum cell.
- 96. The method of claim 93 wherein step (b) comprises culturing the metabolically engineered cell to cause increased production of a biochemical is selected from the group consisting of an organic acid, an amino acid, a porphyrin and a pyrimidine nucleotide.
- 97. The method of claim 93 wherein step (b) comprises culturing the metabolically engineered cell to cause increased production of a biochemical is selected from the group consisting of arginine, asparagine, aspartate, glutamate, glutamine, proline, isoleucine, malate, fumarate, citrate, isocitrate, α-ketoglutarate and succinyl-CoA.
- 98. The method of claim 93 wherein step (b) comprises culturing the metabolically engineered cell to cause increased production of lysine.
- 99. The method of claim 93 wherein step (b) comprises culturing the metabolically engineered cell to cause increased production of succinate.
- 100. The method of claim 93 wherein step (b) comprises culturing the metabolically engineered cell to cause increased production of threonine.
- 101. The method of claim 93 wherein step (b) comprises culturing the metabolically engineered cell to cause increased production of methionine.
- 102. A method for making succinate comprising:
(a) providing a metabolically engineered cell that produces succinate, wherein the metabolically engineered cell overexpresses pyruvate carboxylase; (b) culturing the metabolically engineered cell under conditions that permit overexpression of the pyruvate carboxylase to cause increased production of succinate; and (c) isolating the succinate produced by the cell.
- 103. The method of claim 102 wherein step (a) comprises providing a metabolically engineered bacterial cell.
- 104. The method of claim 102 wherein step (a) comprises providing a metabolically engineered gram-negative bacterial cell.
- 105. The method of claim 102 wherein step (a) comprises providing a metabolically engineered cell selected from the group consisting of an E. coli cell, an S. typhimurium cell and a C. glutamicum cell.
- 106. The method of claim 102 wherein step (a) comprises providing a metabolically engineered cell that overexpresses a heterologous pyruvate carboxylase.
- 107. The method of claim 102 further comprising metabolically engineering a cell to yield the metabolically engineered cell of step (a) that overexpresses pyruvate carboxylase
- 108. The method of claim 107 wherein metabolically engineering the cell comprises mutating a gene of the cell, said gene encoding the pyruvate carboxylase.
- 109. The method of claim 107 wherein metabolically engineering the cell comprises transforming the cell with a nucleic acid fragment comprising a nucleotide sequence encoding the pyruvate carboxylase.
Parent Case Info
[0001] This application is a continuation-in-part application of U.S. application Ser. No. 09/417,557, filed Oct. 13, 1999, which is a continuation-in-part of International Application PCT/US99/08014, with an international filing date of Apr. 13, 1999, which in turn claims the benefit of U.S. Provisional Application No. 60/081,598, filed Apr. 13, 1998, and U.S. Provisional Application No. 60/082,850, filed Apr. 23, 1998, each of which is incorporated herein by reference in its entirety.
Provisional Applications (2)
|
Number |
Date |
Country |
|
60081598 |
Apr 1998 |
US |
|
60082850 |
Apr 1998 |
US |
Continuation in Parts (2)
|
Number |
Date |
Country |
Parent |
09417557 |
Oct 1999 |
US |
Child |
10215440 |
Aug 2002 |
US |
Parent |
PCT/US99/08014 |
Apr 1999 |
US |
Child |
09417557 |
Oct 1999 |
US |