3-hydroxypropionic acid and other organic compounds

• US 20040076982A1
• Filed: 10/20/2003
• Published: 04/22/2004
• Est. Priority Date: 11/20/2000
• Status: Active Grant

• Current Assignee: Cargill Incorporated
• Original Assignee: Cargill Incorporated
• Inventors: Gokarn, Ravi R., Gort, Steven John, Selmer, Thorsten, Jessen, Holly Jean, Buckel, Wolfgang, Selifonova, Olga V
• Granted Patent: US 7,186,541 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0004 Oxidoreductases (1.)
• CPC Class Codes: C12P 7/18 polyhydric
• CPC Class Codes: C12P 7/40 containing a carboxyl group including Peroxycarboxylic acids
• CPC Class Codes: C12P 7/42 Hydroxy-carboxylic acids
• CPC Class Codes: C12P 7/52 Propionic acid; Butyric acids
• CPC Class Codes: C12P 7/625 Polyesters of hydroxy carboxylic acids
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

Methods and materials related to producing 3-HP as well as other organic compounds are disclosed. Specifically, isolated nucleic acids, polypeptides, host cells, and methods and materials for producing 3-HP and other organic compounds are disclosed.

Claims

1. A cell comprising lactyl-CoA dehydratase activity and 3-hydroxypropionyl-CoA dehydratase activity.

2. The cell of claim 1, wherein said cell comprises an activity selected from the group consisting of E1 activator activity, E2 α

activity, and E2 β

activity.

3. The cell of claim 1, wherein said cell comprises 3-hydroxypropionyl-CoA dehydratase activity.

4. The cell of claim 1 wherein said cell comprises CoA transferase activity.

5. The cell of claim 1, wherein said cell comprises an exogenous nucleic acid comprising:

(a) a sequence set forth in SEQ ID NO;

1, 9, 17, 25, 33, 34, 36, 38, 40, 42, 129, 140, 142, 162, or 163;

or

(b) a nucleic acid sequence that shares at least 65 percent sequence identity with a sequence set forth in SEQ ID NO;

1, 9, 17, 25, 33, 34, 36, 38, 40, 42, 129,140, 142, 162, or 163.

6. The cell of claim 1, wherein said cell comprises 3-hydroxypropionyl-CoA hydrolase activity or 3-hydroxyisobutryl-CoA hydrolase activity.

7. The cell of claim 1, wherein said cell comprises lipase activity.

8. The cell of claim 1, wherein said cell produces 3-HP.

9. The cell of claim 1, wherein said cell produces an ester of 3-HP.

10. The cell of claim 9, wherein said ester is selected from the group consisting of methyl 3-hydroxypropionate, ethyl 3-hydroxypropionate, propyl 3-hydroxypropionate, butyl 3-hydroxypropionate, and 2-ethylhexyl 3-hydroxypropionate.

11. The cell of claim 1, wherein said cell comprises CoA synthetase activity.

12. The cell of claim 1, wherein said cell comprises poly hydroxyacid synthase activity.

13. The cell of claim 1, wherein said cell produces polymerized 3-HP.

14. The cell of claim 1, wherein said cell is prokaryotic.

15. The cell of claim 1, wherein said cell is selected from the group consisting of yeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

91. The cell of claim 1, 8, 29, or 30, wherein the cell produces 1,3-propanediol.

92. A method for making 1,3-propanediol, comprising culturing the cell of claim 1, 8, 29, or 30 under conditions wherein the cell produces the 1,3-propanediol.

95. A method for making 1,3-propanediol, comprising:

transfecting the cell of claim 1, 8, 29, or 30 with a nucleic acid encoding a polypeptide comprising oxidoreductase activity or with a nucleic acid encoding a polypeptide comprising reductase activity; and

culturing the transfected cell to allow the transfected cell to make 1,3-propanediol.

96. A method for making 1,3-propanediol, comprising:

transfecting the cell of claim 1, 8, 29, or 30 with a nucleic acid encoding a polypeptide comprising aldyhyde dehydrogenase activity and/or with a nucleic acid encoding a polypeptide comprising alcohol dehydrogenase activity; and

culturing the transfected cell to allow the transfected cell to make 1,3-propanediol.

16. A cell comprising CoA synthetase activity, lactyl-CoA dehydratase activity, and poly hydroxyacid synthase activity.

17. The cell of claim 16, wherein said cell comprises an activity selected from the group consisting of E1 activator activity, E2 α

activity, and E2 β

activity.

18. The cell of claim 16, wherein the cell produces polymerized acrylate.

19. The cell of claim 16, wherein said cell is prokaryotic.

20. The cell of claim 16, wherein said cell is selected from the group consisting of yeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

21. A cell comprising CoA transferase activity, lactyl-CoA dehydratase activity, and lipase activity.

22. The cell of claim 21, wherein said cell comprises an activity selected from the group consisting of E1 activator activity, E2 α

activity, and E2 β

activity.

23. The cell of claim 21, wherein said cell produces an ester of acrylate.

24. The cell of claim 23, wherein said ester is selected from the group consisting of methyl acrylate, ethyl acrylate, propyl acrylate, and butyl acrylate.

25. The cell of claim 21, wherein said cell is prokaryotic.

26. The cell of claim 21, wherein said cell is selected from the group consisting of yeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

27. An polypeptide comprising an amino acid sequence selected from the group consisting of.

(a) a sequence set forth in SEQ ID NO:

2, 10, 18, 26, 35, 37, 39, 41, 141, 160, or 161;

(b) a sequence having at least 10 contiguous amino acid residues of a sequence set forth in SEQ ID NO;

2, 10, 18, 26, 35, 37, 39, 41, 141, 160, or 161;

(c) a sequence that has at least 65 percent sequence identity with a sequence set forth in SEQ ID NO;

2, 10, 18, 26, 35, 37, 39, 41, 141, 160, or 161;

(d) a sequence that has at least65 percent sequence identity with at least 10 contiguous amino acid residues of a sequence set forth in SEQ ID NO;

2, 10, 18, 26, 35, 37, 39, 41, 141, 160, or 161; and

(e) a sequence set forth in SEQ ID NO;

2, 10, 18, 26, 35, 37, 39, 41, 141, 160, or 161 that contains at least one conservative substitution.

28. A nucleic acid molecule comprising a nucleic acid sequence that encodes the polypeptide of claim 27.

29. A transformed cell comprising at least one exogenous nucleic acid molecule, wherein said molecule comprises a nucleic acid sequence that encodes the polypeptide of claim 27.

30. The cell of claim 29, wherein the cell produces 3-HP.

31. The cell of claim 29, wherein said exogenous nucleic acid molecule encodes an E2 α

polypeptide of an enzyme having lactyl-CoA dehydratase activity.

32. The cell of claim 29, wherein said exogenous nucleic acid molecule encodes an E2 β

polypeptide of an enzyme having said lactyl-CoA dehydratase activity.

33. The cell of claim 29, wherein said exogenous nucleic acid molecule encodes a polypeptide having 3-hydroxypropionyl-CoA dehydratase activity or CoA transferase activity.

34. The cell of claim 29, wherein said exogenous nucleic acid molecule encodes a polypeptide having 3-hydroxypropionyl-CoA hydrolase activity or 3-hydroxyisobutryl-CoA hydrolase activity.

35. The cell of claim 29, wherein the cell comprises lipase activity.

36. The cell of claim 29, wherein the cell produces an ester of 3-HP.

37. The cell of claim 36, wherein said ester is selected from the group consisting of methyl 3-hydroxypropionate, ethyl 3-hydroxypropionate, propyl 3-hydroxypropionate, butyl 3-hydroxypropionate, and 2-ethylhexyl 3-hydroxypropionate.

38. The cell of claim 29, wherein said cell comprises CoA synthetase activity.

39. The cell of claim 29, wherein said cell produces polymerized 3-HP.

40. The cell of claim 29, wherein said cell is prokaryotic.

41. The cell of claim 29, wherein said cell is selected from the group consisting of Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

42. The cell of claim 29, wherein the cell is a yeast cell.

43. A specific binding agent that specifically binds to the polypeptide of claim 27.

44. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:

(a) a sequence set forth in SEQ ID NO;

1, 9, 17, 25, 33, 34, 36, 38, 40, 42, 129, 140, 142, 162, or 163;

(b) a sequence having at least 10 contiguous nucleotides of a sequence set forth in SEQ ID NO;

1, 9, 17, 25, 33, 34, 36, 38, 40, 42, 129, 140, 142, 162, or 163;

(c) a sequence that has at least 65 percent sequence identity with a sequence set forth in SEQ ID NO;

1, 9, 17, 25, 33, 34, 36, 38, 40, 42, 129, 140, 142, 162, or 163;

(d) a sequence that has at least 65 percent sequence identity with at least 10 contiguous nucleotides of a sequence set forth in SEQ ID NO;

1, 9, 17, 25, 33, 34, 36, 38, 40, 42, 129, 140, 142, 162, or 163; and

(e) a sequence that hybridize under moderately stringent conditions a sequence set forth in SEQ ID NO;

1, 9, 17, 25, 33, 34, 36, 38, 40, 42, 129, 140, 142, 162, or 163.

45. A production cell comprising an isolated nucleic acid molecule of claim 44 that is exogenous to said production cell.

46. The cell of claim 45, wherein said isolated nucleic acid molecule encodes a polypeptide having an enzymatic activity selected from the group consisting of CoA transferase activity, lactyl-CoA dehydratase activity, CoA synthase activity, CoA dehydratase activity, dehydrogenase activity, malonyl-CoA reductase activity, and 3-hydroxypropionyl-CoA dehydratase activity.

47. A method of producing a polypeptide, comprising culturing the cell of claim 45 under conditions that allow said cell to produce said polypeptide, wherein said polypeptide is produced.

48. A method for making 3-HP, said method comprising culturing at least one cell comprising at least one exogenous nucleic acid molecule that encodes at least one polypeptide that is capable of producing said 3-HP from PEP under conditions such that said 3-HP is produced.

49. The method of claim 48, wherein said cell is selected from the group consisting of yeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

50. The method of claim 48, wherein 3-HP is made by a biosynthetic route that utilizes a β

-alanine intermediate.

51. The method of claim 48, wherein 3-HP is made by a biosynthetic route that utilizes a malonyl-CoA intermediate.

52. The method of claim 48, wherein 3-HP is made by a biosynthetic route that utilizes a lactate intermediate.

93. A method for making 1,3-propanediol from 3-HP, comprising:

making 3-HP using the method of any of claims 48-59 or 78-90;

contacting the 3-HP with a polypeptide comprising oxidoreductase activity or a polypeptide comprising reductase activity.

94. A method for making 1,3-propanediol from 3-HP, comprising:

making 3-HP using the method of any of claims 48-59 or 78-90;

contacting the 3-HP with a polypeptide comprising aldyhyde dehydrogenase activity and/or a polypeptide comprising alcohol dehydrogenase activity.

53. A method for making 3-HP, said method comprising culturing at least one cell comprising at least one exogenous nucleic acid molecule that encodes at least one polypeptide that is capable of producing said 3-HP from lactate under conditions such that said 3-HP is produced.

54. The method of claim 53, wherein said cells are selected from the group consisting of yeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

55. A method for making 3-HP, said method comprising culturing at least one cell under conditions wherein said cell produces said 3-HP, said cell comprising lactyl-CoA dehydratase activity and 3-hydroxypropionyl-CoA dehydratase activity.

56. The method of claim 55, wherein said cell is selected from the group consisting of yeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

57. The method of claim 55, wherein said cell comprises CoA transferase activity.

58. The method of claim 55, wherein said cell comprises 3-hydroxypropionyl-CoA hydrolase activity or 3-hydroxyisobutryl-CoA hydrolase activity.

59. A method for making 3-HP, said method comprising:

a) contacting lactate with a first polypeptide having CoA transferase activity to form lactyl-CoA,

b) contacting said lactyl-CoA with a second polypeptide having lactyl-CoA dehydratase activity to form acrylyl-CoA,

c) contacting said acrylyl-CoA with a third polypeptide having 3-hydroxypropionyl-CoA dehydratase activity to form 3-HP-CoA, and

d) contacting said 3-HP-CoA with said first polypeptide to form said 3-HP or with a fourth polypeptide having 3-hydroxypropionyl-CoA hydrolase activity or 3-hydroxyisobutryl-CoA hydrolase activity to form said 3-HP.

60. A method for making polymerized 3-HP, said method comprising culturing a cell under conditions wherein said cell produces said polymerized 3-HP, said cell comprising lactyl-CoA dehydratase activity and 3-hydroxypropionyl-CoA dehydratase activity.

61. The method of claim 60, wherein said cell is selected from the group consisting of yeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

62. The method of claim 60, wherein said cell comprises CoA synthetase activity.

63. The method of claim 60, wherein said cell comprises poly hydroxyacid synthase activity.

64. A method for making polymerized 3-HP, said method comprising:

a) contacting lactate with a first polypeptide having CoA synthetase activity to form lactyl-CoA,

b) contacting said lactyl-CoA with a second polypeptide having lactyl-CoA dehydratase activity to form acrylyl-CoA,

c) contacting said acrylyl-CoA with a third polypeptide having 3-hydroxypropionyl-CoA dehydratase activity to form 3-hydroxypropionic acid-CoA, and

d) contacting said 3-hydroxypropionic acid-CoA with a fourth polypeptide having poly hydroxyacid synthase activity to form said polymerized 3-HP.

65. A method for making an ester of 3-HP, said method comprising culturing a cell under conditions wherein said cell produces said ester, said cell comprising lactyl-CoA dehydratase activity and 3-hydroxypropionyl-CoA dehydratase activity.

66. The method of claim 65, wherein said cell is selected from the group consisting of yeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

67. The method of claim 65, wherein said cell comprises CoA transferase activity.

68. The method of claim 65, wherein said cell comprises 3-hydroxypropionyl-CoA hydrolase activity or 3-hydroxyisobutryl-CoA hydrolase activity.

69. A method for making an ester of 3-HP, said method comprising:

a) contacting lactate with a first polypeptide having CoA transferase activity to form lactyl-CoA,

b) contacting said lactyl-CoA with a second polypeptide having lactyl-CoA dehydratase activity to form acrylyl-CoA,

c) contacting said acrylyl-CoA with a third polypeptide having 3-hydroxypropionyl-CoA dehydratase activity to form 3-hydroxypropionic acid-CoA,

d) contacting said 3-hydroxypropionic acid-CoA with said first polypeptide to form 3-HP or a fourth polypeptide having 3-hydroxypropionyl-CoA hydrolase activity or 3-hydroxyisobutryl-CoA hydrolase activity to form 3-HP, and

e) contacting said 3-HP with a fifth polypeptide having lipase activity to form said ester.

70. A method for making polymerized acrylate, said method comprising culturing a cell under conditions wherein said cell produces said polymerized acrylate, said cell comprising CoA synthetase activity and lactyl-CoA dehydratase activity.

71. The method of claim 70, wherein said cell is selected from the group consisting of yeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

72. The method of claim 70, wherein said cell comprises poly hydroxyacid synthase activity.

73. A method for making polymerized acrylate, said method comprising:

a) contacting lactate with a first polypeptide having CoA synthetase activity to form lactyl-CoA,

b) contacting said lactyl-CoA with a second polypeptide having lactyl-CoA dehydratase activity to form acrylyl-CoA, and

c) contacting said acrylyl-CoA with a third polypeptide having poly hydroxyacid synthase activity to form said polymerized acrylate.

74. A method for making an ester of acrylate, said method comprising culturing a cell under conditions wherein said cell produces said ester, said cell comprising CoA transferase activity and lactyl-CoA dehydratase activity.

75. The method of claim 74, wherein said cell is selected from the group consisting of yeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

76. The method of claim 74, wherein said cell comprises lipase activity.

77. A method for making an ester of acrylate, said method comprising:

a) contacting lactate with a first polypeptide having CoA transferase activity to form lactyl-CoA,

b) contacting said lactyl-CoA with a second polypeptide having lactyl-CoA dehydratase activity to form acrylyl-CoA,

c) contacting said acrylyl-CoA with said first polypeptide to form acrylate, and

d) contacting said acrylate with a third polypeptide having lipase activity to form said ester.

78. A method for making 3-HP, said method comprising culturing a cell under conditions wherein said cell produces said 3-HP, said cell comprising at least one exogenous nucleic acid that encodes at least one polypeptide such that said 3-HP is produced from acetyl-CoA and under conditions such that said 3-HP is produced.

79. The method of claim 78, wherein said cell is selected from the group consisting of yeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

80. A method for making 3-HP, said method comprising culturing a cell under conditions wherein said cell produces said 3-HP, said cell comprising at least one exogenous nucleic acid that encodes at least one polypeptide such that said 3-HP is produced from malonyl-CoA and under conditions such that said 3-HP is produced.

81. The method of claim 80, wherein said cell is selected from the group consisting of yeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

82. A method for making 3-HP, said method comprising culturing a cell under conditions wherein said cell produces said 3-HP, said cell comprising at least one exogenous nucleic acid that encodes at least one polypeptide such that said 3-HP is produced from P-alanine and under conditions such that said 3-HP is produced.

83. The method of claim 82, wherein said cell is selected from the group consisting of yeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

84. A method for making 3-HP, said method comprising culturing cells comprising an exogenous nucleic acid that encodes polypeptides that are capable of producing 3-HP from acetyl-CoA under conditions such that said 3-HP is produced.

85. The method of claim 84, wherein said cells are selected from the group consisting of yeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

86. A method for making 3-HP, said method comprising culturing cells comprising at least one exogenous nucleic acid that encodes polypeptides that are capable of producing said 3-HP from malonyl-CoA, and under conditions such that said 3-HP is produced.

87. The method of claim 86, wherein said cells are selected from the group consisting of yeast, Lactobacillus, Lactococcus, Bacillus, and Escherichia cells.

88. A method for making 3-HP, said method comprising:

a) contacting acetyl-CoA with a first polypeptide having acetyl-CoA carboxylase activity to form malonyl-CoA, and

b) contacting said malonyl-CoA with a second polypeptide having malonyl-CoA reductase activity to form said 3-HP.

89. A method for making 3-HP, said method comprising contacting malonyl-CoA with a polypeptide having malonyl-CoA reductase activity to form said 3-HP.

90. A method for making 3-HP, said method comprising:

a) contacting β

-alanine CoA with a first polypeptide having β

-alanyl-CoA ammonia lyase activity to form acrylyl-CoA;

b) contacting said acrylyl-CoA with a second polypeptide having 3-HP-CoA dehydratase activity to form said 3-HP-CoA; and

c) contacting 3-HP-CoA with a third polypeptide having glutamate dehydrogenase to make 3-HP.

a) contacting β

-alanine CoA with a first polypeptide having β

-alanyl-CoA ammonia lyase activity to form acrylyl-CoA;

b) contacting said acrylyl-CoA with a second polypeptide having 3HP-CoA dehydratase activity to form said 3-HP-CoA; and

c) contacting 3-HP-CoA with a third polypeptide having glutamate dehydrogenase to make 3-HP