3-hydroxypropionic acid and other organic compounds

• US 7,638,316 B2
• Filed: 11/19/2007
• Issued: 12/29/2009
• Est. Priority Date: 11/20/2000
• Status: Active Grant

• Current Assignee: Cargill, Inc. (Agricultural Services Division)
• Original Assignee: Cargill, Inc. (Agricultural Services Division)
• Inventors: Selmer, Thorsten, Buckel, Wolfgang, Gokarn, Ravi R., Gort, Steven John, Selifonova, Olga V., Jessen, Holly Jean
• Primary Examiner(s): Saidha; Tekchand
• Application Number:
  US11/942,669
• Publication Number:
  US 20080076167A1
• Time in Patent Office: 771 Days
• Field of Search: 435/135, 435/232, 435/252.3, 435/320.1, 536/23.2
• US Class Current: 435/135
• 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 method of making 3-hydroxypropionic acid (3-HP), comprising:

culturing at least one cell comprising at least one nucleic acid molecule encoding at least one polypeptide that is capable of producing said 3-HP from phosphoenolpyruvate (PEP) or pyruvate, under conditions such that said 3-HP is produced, wherein the at least one polypeptide comprises;

a Clostridium acetobutycilicum 4-aminobutyrate aminotransferase; and

a Pseudomonas aeruginosa 3-hydroxyisobutyrate dehydrogenase,

wherein at least one of the nucleic acid molecules is exogenous.

2. The method of claim 1, wherein 3-HP is made from PEP.

3. The method of claim 1, wherein 3-HP is made by a biosynthetic route that utilizes a β

-alanine intermediate, an acetyl-CoA intermediate, a malonyl-CoA intermediate or a malonate semialdehyde intermediate.

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

-alanine intermediate.

5. The method of claim 1, wherein the at least one polypeptide further comprises:

a PEP carboxylase;

an aspartate aminotransferase that can convert oxaloacetate to aspartate; and

an aspartate decarboxylase that can convert the aspartate to β

-alanine.

6. The method of claim 1, further comprising contacting the 3-HP with a peptide having lipase activity to make an ester of 3-HP.

7. The method of claim 1, further comprising contacting the 3-HP with a polypeptide comprising oxidoreductase activity, a polypeptide comprising reductase activity, a polypeptide comprising aldehyde dehydrogenase activity, and/or a polypeptide comprising alcohol dehydrogenase activity to make 1,3-propanediol.

8. The method of claim 1, wherein 3-HP is made from pyruvate, and the at least one polypeptide that is capable of producing said 3-HP from pyruvate comprises:

a pyruvate carboxylase;

an aspartate aminotransferase; and

an aspartate decarboxylase.

9. The method of claim 1, wherein the cell is prokaryotic.

10. The method of claim 1, wherein the cell is a yeast cell, Lactobacillus cell, Lactococcus cell, Bacillus cell, or Escherichia cell.

11. The method of claim 1, wherein 3-HP is made by a biosynthetic route that utilizes a malonate semialdehyde intermediate.

15. The method of claim 1, wherein the at least one polypeptide further comprises:

a PEP carboxylase;

an aspartate aminotransferase that can convert oxaloacetate to aspartate; and

an aspartate decarboxylase that can convert the aspartate to β

-alanine.

12. A method of making 3-hydroxypropionic acid (3-HP), comprising:

culturing at least one cell comprising at least one nucleic acid molecule encoding at least one polypeptide that is capable of producing said 3-HP from lactate under conditions such that said 3-HP is produced, wherein the polypeptide comprises

an Escherichia coli, Rhodobacter sphaeroides, Saccharomyces cervisiae, or Salmonella enteric CoA synthetase or a Megasphaera elsdenii, Clostridium propionicum, Clostridium kluyveri, or Escherichia coli CoA transferase that can convert lactate to lactyl-CoA; and

one or more enzymes that can convert the lactyl-CoA to 3-HP,

wherein at least one of the nucleic acid molecules is exogenous.

13. The method of claim 12, wherein the one or more enzymes that can convert the lactyl-CoA to 3-HP comprises:

a Megasphaera elsdenii or Clostridium propionicum lactyl-CoA dehydratase that can convert the lactyl-CoA to acrylyl-CoA.

14. The method of claim 13, wherein the one or more enzymes that can convert the lactyl-CoA to 3-HP further comprises:

a Chloroflexus aurantiacus, Candida rugosa, Rhodosprillium rubrum, or Rhodobacter capsulates 3-hydroxypropionyl-CoA dehydratase that can convert the acrylyl-CoA to 3-HP-CoA; and

a Megasphaera elsdenii, Clostridium propionicum, Clostridium kluyveri, or Escherichia coli CoA transferase, a Candida rugosa 3-hydroxypropionyl-CoA-hydrolase, or a Pseudomonas fluorescens, Rattus, or Homo sapiens 3-hydroxyisobutryl-CoA hydrolase that can convert the 3-HP-CoA to 3-HP.

16. A method of making 3-hydroxypropionic acid (3-HP), comprising:

culturing at least one cell comprising at least one exogenous nucleic acid molecule encoding at least one polypeptide that is capable of producing said 3-HP from phosphoenolpyruvate (PEP) or pyruvate, under conditions such that said 3-HP is produced, wherein the at least one polypeptide comprises;

a 4-aminobutyrate aminotransferase polypeptide encoded by SEQ ID NO;

189 or a sequence that hybridizes to SEQ ID NO;

189 under highly stringent hybridization conditions, wherein the highly stringent hybridization conditions comprise hybridization at about 42°

C. in a hybridization solution comprising 25 mM KPO4 (pH 7.4), 5×

SSC, 5×

Denhart'"'"'s solution, 50 μ

g/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/mL SEQ ID NO;

189, washing at about 65°

C. in a wash solution comprising 0.2×

SSC and 0.1% sodium dodecyl sulfate; and

a 3-hydroxyisobutyrate dehydrogenase polypeptide encoded by SEQ ID NO;

191 or a sequence that hybridizes to SEQ ID NO;

191 under highly stringent hybridization conditions, wherein the highly stringent hybridization conditions comprise hybridization at about 42°

C. in a hybridization solution comprising 25 mM KPO4 (pH 7.4), 5×

SSC, 5×

Denhart'"'"'s solution, 50 μ

g/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/mL SEQ ID NO;

191, washing at about 65°

C. in a wash solution comprising 0.2×

SSC and 0.1% sodium dodecyl sulfate;

wherein at least one of the nucleic acid molecules is exogenous.

17. The method of claim 16, wherein 3-HP is made from PEP.

18. The method of claim 16, wherein 3-HP is made by a biosynthetic route that utilizes a β

-alanine intermediate, an acetyl-CoA intermediate, a malonyl-CoA intermediate or a malonate semialdehyde intermediate.

19. The method of claim 18, wherein 3-HP is made by a biosynthetic route that utilizes a β

-alanine intermediate.

20. The method of claim 16, further comprising contacting the 3-HP with a peptide having lipase activity to make an ester of 3-HP.

21. The method of claim 16, further comprising contacting the 3-HP with a polypeptide comprising oxidoreductase activity, a polypeptide comprising reductase activity, a polypeptide comprising aldehyde dehydrogenase activity, and/or a polypeptide comprising alcohol dehydrogenase activity to make 1,3-propanediol.

22. The method of claim 16, wherein 3-HP is made from pyruvate, and the at least one polypeptide that is capable of producing said 3-HP from pyruvate comprises:

a pyruvate carboxylase;

an aspartate aminotransferase; and

an aspartate decarboxylase.

23. The method of claim 16, wherein the cell is prokaryotic.

24. The method of claim 16, wherein the cell is a yeast cell, Lactobacillus cell, Lactococcus cell, Bacillus cell, or Escherichia cell.