Acetyl-coA carboxylases

Patent metadata

Field	Value
Publication number	US-9447438-B2
Application number	US-201414215379-A
Country	US
Kind code	B2
Filing date	Mar 17, 2014
Priority date	Mar 15, 2013
Publication date	Sep 20, 2016
Grant date	Sep 20, 2016

Abstract

The present invention provides various combinations of genetic modifications to a transformed host cell that provide increase conversion of carbon to a chemical product. The present invention also provides methods of fermentation and methods of making various chemical products.

First claim

We claim:

1. A genetically modified microorganism comprising a heterologous gene encoding an acetyl-CoA carboxylase (ACCase) polypeptide fusion wherein the polypeptide fusion comprises at least two functional subunits of an ACCase enzyme selected from the group consisting of accA-accB, accA-accC, accA-accD, accB-accC, accB-accD, accC-accD, accA-accB-accC, accA-accB-accD, accA-accC-accD, accB-accC-accD and accA-accB-accC-accD.

2. The genetically modified microorganism of claim 1 , wherein said microorganism is capable of converting a renewable carbon source to at least 60 grams/liter 3-hydroxypropionic acid (3-HP).

3. The genetically modified microorganism of claim 1 , wherein the microorganism encodes an active ACCase enzyme comprising two or more polypeptide fusions wherein at least two of the polypeptide fusions are encoded by genes expressed on a same plasmid.

4. The genetically modified microorganism of claim 1 , wherein the microorganism encodes an active ACCase enzyme comprising two or more polypeptide fusions wherein at least two of the polypeptide fusions are encoded for by genes expressed on different plasmids.

5. The genetically modified microorganism of claim 1 , wherein the microorganism encodes an active ACCase enzyme and wherein at least one subunit of the enzyme is not fused to another subunit.

6. The genetically modified microorganism of claim 5 , wherein the subunit that is not fused to another subunit is expressed by a gene on the same plasmid as the gene encoding the polypeptide fusion.

7. The genetically modified microorganism of claim 5 , wherein the gene that encodes the subunit that is not fused to another subunit is expressed on a different plasmid than the gene that encodes the fusion.

8. The genetically modified microorganism of claim 1 , wherein the gene encoding an acetyl-CoA carboxylase polypeptide encodes an accA-accD polypeptide fusion, and further comprises nucleotide sequence encoding for an accB polypeptide subunit, and a nucleotide sequence encoding for an accC subunit.

9. The genetically modified microorganism of claim 1 , wherein the microorganism encodes an active ACCase enzyme comprising two or more polypeptide fusions wherein the stoichiometric ratio of the polypeptide fusions relative to one another is between 1 to 10.

10. The genetically modified microorganism of claim 9 , wherein the stoichiometric ratio of the polypeptide fusions relative to one another is between about 1 to about 2 or between about 7 to about 9.

11. The genetically modified microorganism of claim 1 , wherein the microorganism encodes an active ACCase enzyme and wherein the ACCase enzyme comprises an accA-accD polypeptide fusion, an accB polypeptide subunit that is not fused to another polypeptide subunit, and an accC polypeptide subunit that is not fused to another polypeptide subunit.

12. The genetically modified microorganism of claim 1 , wherein the polypeptide fusion comprises two, three, or four different ACCase subunits in a single polypeptide.

13. The genetically modified microorganism of claim 1 , wherein the microorganism comprises a stoichiometric ratio of
a) accB and accC fusion to accA and accD fusion;
b) accA and accD fusion to accB and accC fusion;
c) accC and accD fusion to accA and accB fusion;
d) accA and accB fusionto accC and accD fusion;
e) accA subunit to accB subunit;
f) accB subunit to accA subunit;
g) accA subunit to accC subunit;
h) accC subunit to accA subunit;
i) accA subunit to accD subunit;
j) accD subunit to accA subunit;
k) accB subunit to accC subunit;
l) accC subunit to accB subunit;
m) accB subunit to accD subunit;
n) accD subunit to accB subunit;
o) accC subunit to accD subunit; or
p) acct) subunit to accC subunit;

of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, 4:5, 4:6, 4:7, 4:8, 5:1, 5:3, 5:4, 5:5, 5:6, 5:7, 5:8, 6:1, 6:3, 6:4, 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5, 7:6, 7:7 7:8, 8:1, 8:3, 8:4, 8:5, 8:6, 8:7, or 8:8 in low, medium, high or inducible expression.

14. The genetically modified microorganism of claim 1 , wherein the microorganism comprises a polypeptide fusion of accA-aaD, a subunit of accB that is not fused to another subunit, and an accC subunit that is not fused to another subunit wherein the molar ratios for accA:accB:accC:accD are about 1:2:1:1.

15. The genetically modified microorganism of claim 1 , wherein the microorganism comprises an active ACCase enzyme that comprises a polypeptide fusion of accA-aaD, a subunit of accB that is not fused to another subunit, and an accC subunit that is not fused to another subunit wherein the molar ratios for accDAfusion:accB:accC are about 1:2:1.

16. A method of producing chemical products comprising culturing the genetically modified microorganism of claim 1 in a suitable medium and under suitable conditions to produce a desired chemical product selected from the group consisting of 3-hydroxypropionic acid (3-HP) or a derivative of, 1,4-butanediol, butanol, isobutanol, polyketide chemical products and C4-C18 fatty acid chains.

17. The method of claim 16 , wherein said chemical is converted to acrylic acid, acrylates, 1,3-propanediol, malonic acid, ethyl-3-hydroxypropionate, ethyl ethoxy propionate, propiolactone, acrylamide, or acrylonitrile to make consumer products.

18. The method of claim 16 , wherein said chemical is oligomerized or polymerized to form polyacrylic acid, methyl acrylate, acrylamide, acrylonitrile, propiolactone, ethyl 3-HP, ethyl acrylate, n-butyl acrylate, hydroxypropyl acrylate, hydroxyethyl acrylate, isobutyl acrylate, 2-ethylhexyl acrylate, and acrylic acid ester to which an alkyl or aryl addition may be made, and/or to which halogens, aromatic amines or amides, and aromatic hydrocarbons may be added to make consumer products.

Assignees

• Opx Biotechnologies Inc
• Cargill Inc

Inventors

• Liao Hans
• Mercogliano Christopher Patrick
• Wolter Travis Robert
• Louie Michael Tai Man
• Ribble Wendy Kathleen
• Lipscomb Tanya
• Spindler Eileen Colie
• Lynch Michael D

Classifications

• C12Y604/01002

  Acetyl-CoA carboxylase (6.4.1.2) · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title

• C12N9/0008

  acting on the aldehyde or oxo group of donors (1.2) · CPC title