3-HYDROXYPROPIONIC ACID AND OTHER ORGANIC COMPOUNDS

• US 20120077236A1
• Filed: 11/11/2011
• Published: 03/29/2012
• Est. Priority Date: 11/20/2000
• Status: Active Grant

• Current Assignee: Cargill Incorporated
• Original Assignee: Cargill Incorporated
• Inventors: Gokarn, Ravi R., Selifonova, Olga V., Jessen, Holly Jean, Gort, Steven John, Selmer, Thorsten, Buckel, Wolfgang
• Granted Patent: US 8,198,066 B2
• Time in Patent Office: Days
• 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. An isolated transformed cell, comprising:

a nucleic acid molecule encoding a protein comprising malonyl-CoA reductase activity, wherein the nucleic acid molecule encodes a protein comprising at least 90% sequence identity to the protein sequence of SEQ ID NO;

141; and

a nucleic acid molecule encoding a carboxylase carboxyl transferase alpha subunit (accA) of an enzyme having acetyl-CoA carboxylase activity, wherein the nucleic acid molecule 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;

a nucleic acid molecule encoding a biotin carboxyl carrier protein subunit (accB) of an enzyme having acetyl-CoA carboxylase activity;

wherein the nucleic acid molecule hybridizes to SEQ ID NO;

190 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;

190, washing at about 65°

C. in a wash solution comprising 0.2×

SSC and 0.1% sodium dodecyl sulfate;

a nucleic acid molecule encoding a biotin carboxylase subunit (accC) of an enzyme having acetyl-CoA carboxylase activity wherein the nucleic acid molecule 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; and

a nucleic acid molecule encoding a carboxylase carboxyl transferase beta subunit (accD) of an enzyme having acetyl-CoA carboxylase activity, wherein the nucleic acid molecule hybridizes to SEQ ID NO;

192 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;

192, washing at about 65°

C. in a wash solution comprising 0.2×

SSC and 0.1% sodium dodecyl sulfate;

or

a nucleic acid molecule encoding an enzyme having acetyl-CoA carboxylase activity, wherein the nucleic acid molecule hybridizes to SEQ ID NO;

193 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;

193, washing at about 65°

C. in a wash solution comprising 0.2×

SSC and 0.1% sodium dodecyl sulfate.

2. The isolated transformed cell of claim 1, wherein said cell produces 3-HP.

3. The isolated transformed cell of claim 1, wherein the cell is prokaryotic and comprises:

the nucleic acid molecule encoding a carboxylase carboxyl transferase alpha subunit (accA) of an enzyme having acetyl-CoA carboxylase activity, wherein the nucleic acid molecule 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;

the nucleic acid molecule encoding a biotin carboxyl carrier protein subunit (accB) of an enzyme having acetyl-CoA carboxylase activity;

wherein the nucleic acid molecule hybridizes to SEQ ID NO;

190 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;

190, washing at about 65°

C. in a wash solution comprising 0.2×

SSC and 0.1% sodium dodecyl sulfate;

the nucleic acid molecule encoding a biotin carboxylase subunit (accC) of an enzyme having acetyl-CoA carboxylase activity wherein the nucleic acid molecule 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; and

the nucleic acid molecule encoding a carboxylase carboxyl transferase beta subunit (accD) of an enzyme having acetyl-CoA carboxylase activity, wherein the nucleic acid molecule hybridizes to SEQ ID NO;

192 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;

192, washing at about 65°

C. in a wash solution comprising 0.2×

SSC and 0.1% sodium dodecyl sulfate.

4. The isolated transformed cell of claim 3, wherein the cell is a Lactobacillus, Lactococcus, Bacillus, or Escherichia cell.

5. The isolated transformed cell of claim 1, wherein the cell is a yeast cell and comprises the nucleic acid molecule encoding an enzyme having acetyl-CoA carboxylase activity, wherein the nucleic acid molecule hybridizes to SEQ ID NO:

193 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;

193, washing at about 65°

C. in a wash solution comprising 0.2×

SSC and 0.1% sodium dodecyl sulfate.

6. The isolated transformed cell of claim 1, wherein the cell is a yeast, Lactobacillus, Lactococcus, Bacillus, or Escherichia cell.

7. The isolated transformed cell of claim 1, wherein the nucleic acid molecule encoding a protein comprising malonyl-CoA reductase activity encodes a protein comprising at least 95% sequence identity to the protein sequence of SEQ ID NO:

141.

8. The isolated transformed cell of claim 1, wherein the nucleic acid molecule encoding a protein comprising malonyl-CoA reductase activity encodes a protein comprising at least 97% sequence identity to the protein sequence of SEQ ID NO:

141.

9. The isolated transformed cell of claim 1, wherein the nucleic acid molecule encoding a protein comprising malonyl-CoA reductase activity encodes a protein comprising at least 98% sequence identity to the protein sequence of SEQ ID NO:

141.

10. The isolated transformed cell of claim 1, wherein the nucleic acid molecule encoding a protein comprising malonyl-CoA reductase activity encodes a protein comprising at least 99% sequence identity to the protein sequence of SEQ ID NO:

141.

11. The isolated transformed cell of claim 1, wherein the nucleic acid molecule encoding a protein comprising malonyl-CoA reductase activity encodes a protein comprising the protein sequence of SEQ ID NO:

141.

12. The isolated transformed cell of claim 1, wherein the nucleic acid molecule encoding a protein comprising malonyl-CoA reductase activity comprises at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:

140.

13. The isolated transformed cell of claim 1, wherein the nucleic acid molecule encoding a protein comprising malonyl-CoA reductase activity comprises at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:

140.

14. The isolated transformed cell of claim 1, wherein the nucleic acid molecule encoding a protein comprising malonyl-CoA reductase activity comprises at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO:

140.

15. The isolated transformed cell of claim 1, wherein the nucleic acid molecule encoding a protein comprising malonyl-CoA reductase activity comprises at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO:

140.

16. The isolated transformed cell of claim 1, wherein the nucleic acid molecule encoding a protein comprising malonyl-CoA reductase activity comprises at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO:

140.

17. The isolated transformed cell of claim 1, wherein the nucleic acid molecule encoding a protein comprising malonyl-CoA reductase activity comprises the nucleic acid sequence of SEQ ID NO:

140.

18. The isolated transformed cell of claim 1, wherein the cell comprises:

a nucleic acid molecule encoding a protein comprising malonyl-CoA reductase activity, wherein the nucleic acid molecule encodes a protein comprising at least 95% sequence identity to the protein sequence of SEQ ID NO;

141; and

a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO;

189;

a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO;

190, a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO;

191, and the nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO;

192, or

a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO;

193.

19. The isolated transformed cell of claim 1, further comprising lipase activity.

20. The isolated transformed cell of claim 19, further comprising a nucleic acid molecule encoding an enzyme having lipase activity, wherein the nucleic acid molecule hybridizes to SEQ ID NO:

194 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;

194, washing at about 65°

C. in a wash solution comprising 0.2×

SSC and 0.1% sodium dodecyl sulfate

21. A method for making 3-hydroxypropionic acid (3-HP), comprising:

culturing the cell of claim 1 under conditions wherein said cell produces 3-HP.

22. The method of claim 21, wherein the cell is a yeast, Lactobacillus, Lactococcus, Bacillus, or Escherichia cell.

23. A method for making an ester of 3-hydroxypropionic acid (3-HP), comprising:

culturing the cell of claim 19 under conditions wherein said cell produces the ester of 3-HP.

24. The method of claim 23, wherein the cell is a yeast, Lactobacillus, Lactococcus, Bacillus, or Escherichia cell.