3-hydroxypropionic acid and other organic compounds

• US 8,076,120 B2
• Filed: 05/27/2008
• Issued: 12/13/2011
• Est. Priority Date: 11/20/2000
• Status: Expired due to Term

• 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
• Primary Examiner(s): Saidha, Tekchand
• Application Number:
  US12/127,700
• Publication Number:
  US 20090053783A1
• Time in Patent Office: 1,295 Days
• Field of Search: 435/69.1, 435/141, 435/183, 435/232, 435/252.3
• US Class Current: 435/252.3
• 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 comprising at least 90% sequence identity to SEQ ID NO;

9, encoding an E1 activator of an enzyme having lactyl-CoA dehydratase activity;

a nucleic acid molecule comprising at least 90% sequence identity to SEQ ID NO;

17, encoding an E2 α

subunit of an enzyme having lactyl-CoA dehydratase activity;

a nucleic acid molecule comprising at least 90% sequence identity to SEQ ID NO;

25, encoding an E2 β

subunit of an enzyme having lactyl-CoA dehydratase activity;

a nucleic acid molecule comprising at least 90% sequence identity to SEQ ID NO;

40, encoding an enzyme having 3-hydroxypropionyl-CoA dehydratase activity; and

a nucleic acid molecule encoding an enzyme having poly hydroxyacid synthase activity.

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

3. The isolated transformed cell of claim 1, wherein said cell is prokaryotic.

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

5. The isolated transformed cell of claim 1, wherein said poly hydroxyacid synthase activity is encoded by a nucleic acid sequence comprising a sequence as set forth in 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 1-15ng/mL SEQ ID NO;

189, washing at about 65°

C. in a wash solution comprising 0.2×

SSC and 0.1% sodium dodecyl sulfate.

6. The transformed isolated cell of claim 1, further comprising CoA synthetase activity.

7. The isolated transformed cell of claim 1, wherein the cell is a yeast cell.

8. The isolated transformed cell of claim 1, wherein:

the nucleic acid molecule encoding an E1 activator of an enzyme having lactyl-CoA dehydratase activity comprises at least 95% sequence identity to SEQ ID NO;

9;

the nucleic acid molecule encoding an E2 α

subunit of an enzyme having lactyl-CoA dehydratase activity comprises at least 95% sequence identity to SEQ ID NO;

17;

the nucleic acid molecule encoding an E2 β

subunit of an enzyme having lactyl-CoA dehydratase activity comprises at least 95% sequence identity to SEQ ID NO;

25;

the nucleic acid molecule encoding an enzyme having 3-hydroxypropionyl-CoA dehydratase activity comprises at least 95% sequence identity to SEQ ID NO;

40; and

the nucleic acid molecule encoding an enzyme having poly hydroxyacid synthase activity comprises 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.

9. The isolated transformed cell of claim 1, wherein:

the nucleic acid molecule encoding an E1 activator of an enzyme having lactyl-CoA dehydratase activity comprises at least 97% sequence identity to SEQ ID NO;

9;

the nucleic acid molecule encoding an E2 α

subunit of an enzyme having lactyl-CoA dehydratase activity comprises at least 97% sequence identity to SEQ ID NO;

17;

the nucleic acid molecule encoding an E2 β

subunit of an enzyme having lactyl-CoA dehydratase activity comprises at least 97% sequence identity to SEQ ID NO;

25;

the nucleic acid molecule encoding an enzyme having 3-hydroxypropionyl-CoA dehydratase activity comprises at least 97% sequence identity to SEQ ID NO;

40; and

the nucleic acid molecule encoding an enzyme having poly hydroxyacid synthase activity comprises 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.

10. The isolated transformed cell of claim 1, wherein:

the nucleic acid molecule encoding an E1 activator of an enzyme having lactyl-CoA dehydratase activity comprises at least 98% sequence identity to SEQ ID NO;

9;

the nucleic acid molecule encoding an E2 α

subunit of an enzyme having lactyl-CoA dehydratase activity comprises at least 98% sequence identity to SEQ ID NO;

17;

the nucleic acid molecule encoding an E2 β

subunit of an enzyme having lactyl-CoA dehydratase activity comprises at least 98% sequence identity to SEQ ID NO;

25;

the nucleic acid molecule encoding an enzyme having 3-hydroxypropionyl-CoA dehydratase activity comprises at least 98% sequence identity to SEQ ID NO;

40; and

the nucleic acid molecule encoding an enzyme having poly hydroxyacid synthase activity comprises 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, 50μ

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.

11. The isolated transformed cell of claim 1, wherein:

the nucleic acid molecule encoding an E1 activator of an enzyme having lactyl-CoA dehydratase activity comprises at least 99% sequence identity to SEQ ID NO;

9;

the nucleic acid molecule encoding an E2 α

subunit of an enzyme having lactyl-CoA dehydratase activity comprises at least 99% sequence identity to SEQ ID NO;

17;

the nucleic acid molecule encoding an E2 β

subunit of an enzyme having lactyl-CoA dehydratase activity comprises at least 99% sequence identity to SEQ ID NO;

25;

the nucleic acid molecule encoding an enzyme having 3-hydroxypropionyl-CoA dehydratase activity comprises at least 99% sequence identity to SEQ ID NO;

40; and

the nucleic acid molecule encoding an enzyme having poly hydroxyacid synthase activity comprises 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, 50μ

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.