Microbial production of renewable glycolate

Patent metadata

Field	Value
Publication number	US-10294481-B2
Application number	US-201615281994-A
Country	US
Kind code	B2
Filing date	Sep 30, 2016
Priority date	Oct 2, 2015
Publication date	May 21, 2019
Grant date	May 21, 2019

Abstract

Some aspects provide engineered microbes for glycolate production. Methods for microbe engineering and culturing are also provided herein. Such engineered microbes exhibit greatly enhanced capabilities for glycolate production.

First claim

What is claimed is:

1. An Escherichia coli cell engineered to produce glycolate, wherein the cell comprises a deletion of a xylB gene, recombinantly expresses a dte gene from Pseudomonas cichorii that is codon-optimized for E. coli , recombinantly expresses a fucA gene from E. coli , recombinantly expresses a fucK gene from E. coli , recombinantly expresses an aldA gene from E. coli , and recombinantly expresses a ycdW gene from E. coli,
wherein one or more of the recombinantly expressed genes are under the control of one or more promoters,
wherein the one or more promoters is not a native promoter, and
wherein the cell has higher production of glycolate relative to a wild type cell.

2. An Escherichia coli cell engineered to produce glycolate,
wherein the cell comprises a deletion of a xylB gene, recombinantly expresses a dte gene from Pseudomonas cichorii that is codon-optimized for E. coli , recombinantly expresses a fucA gene from E. coli , recombinantly expresses a fucK gene from E. coli , and recombinantly expresses an aldA gene from E. coli,
wherein the cell further comprises a deletion of a fucO gene.

3. The Escherichia coli cell of claim 2 , wherein the cell further comprises a deletion of a glcD gene.

4. The Escherichia coli cell of claim 1 , wherein the cell further recombinantly expresses an aceA gene from E. coli.

5. The Escherichia coli cell of claim 1 , wherein the cell further recombinantly expresses an aceK gene from E. coli , or wherein the cell has reduced or eliminated activity of, or reduced or eliminated expression of isocitrate lyase regulator relative to a wild type cell.

6. The Escherichia coli cell of claim 2 , wherein the cell further comprises a deletion of a gcl gene and/or a aceB gene and/or a glcB gene.

7. The Escherichia coli cell of claim 3 , wherein the cell further comprises a deletion of a fucO gene.

8. The Escherichia coli cell of claim 6 , wherein the cell further comprises a deletion of an aceB gene.

9. The Escherichia coli cell of claim 8 , wherein the cell further comprises a deletion of a gcl gene.

10. The Escherichia coli cell of claim 1 , wherein the promoter is a modified promoter.

11. The Escherichia coli cell of claim 1 , wherein the promoter is a constitutive promoter.

12. The Escherichia coli cell of claim 11 , wherein the constitutive promoter is a CP1 promoter.

13. The Escherichia coli cell of claim 1 , wherein the promoter is a conditional or inducible promoter.

14. The Escherichia coli cell of claim 13 , wherein the inducible promoter is a T5 promoter.

15. The Escherichia coli cell of claim 1 , wherein one or more of the recombinantly expressed genes is expressed in a recombinant expression vector.

16. The Escherichia coli cell of claim 1 , wherein one or more of the recombinantly expressed genes is expressed from one or more chromosomally integrated genes.

17. The Escherichia coli cell of claim 2 , wherein the cell produces about 0.63 grams of glycolate per gram of substrate.

18. The Escherichia coli cell of claim 2 , wherein the dte gene from Pseudomonas cichorii comprises a sequence of SEQ ID NO: 39.

Assignees

• Massachusetts Inst Technology

Inventors

• Stephanopoulos Gregory
• Li zheng-jun
• Pereira Brian

Classifications

• C12P7/42

  Hydroxy-carboxylic acids · CPC title

• C12N15/70 Primary

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

• C12Y101/01026

  Glyoxylate reductase (1.1.1.26) · CPC title

• C12N9/90

  Isomerases (5.) · CPC title

• C12Y401/03001

  Isocitrate lyase (4.1.3.1) · CPC title