Genetically engineered microorganisms for the production of chorismate-derived products

Patent metadata

Field	Value
Publication number	US-10174303-B2
Application number	US-201615166224-A
Country	US
Kind code	B2
Filing date	May 26, 2016
Priority date	May 27, 2015
Publication date	Jan 8, 2019
Grant date	Jan 8, 2019

Abstract

The invention provides genetically engineered microorganisms and methods for producing chorismate-derived products, such as para-hydroxybenzoic acid, salicylate, 2-aminobenzoate, 2,3-dihydroxybenzoate, and 4-hydroxycyclohexane carboxylic acid. Typically, the microorganism comprises at least one of (a) an exogenous chorismate pyruvate lyase, (b) an exogenous isochorismate synthase, (c) an exogenous isochorismate pyruvate lyase, and (d) a prephenate synthase comprising a disruptive mutation.

First claim

The invention claimed is:

1. A genetically engineered C1-fixing bacterium capable of producing at least one chorismate-derived product, wherein the bacterium comprises at least one of:
a. an exogenous chorismate pyruvate lyase,
b. an exogenous isochorismate synthase,
c. an exogenous isochorismate pyruvate lyase, and
d. a prephenate synthase comprising a disruptive mutation that partially inactivates, fully inactivates, deletes, or knocks out the prephenate synthase.

2. The bacterium of claim 1 , wherein the bacterium is a Clostridium bacterium capable of producing at least one chorismate-derived product by fermentation of a gaseous substrate.

3. The bacterium of claim 1 , wherein the chorismate pyruvate lyase is ubiC.

4. The bacterium of claim 1 , wherein the isochorismate synthase is pchA.

5. The bacterium of claim 1 , wherein the isochorismate pyruvate lyase is pchB.

6. The bacterium of claim 1 , wherein the prephenate synthase is pheA.

7. The bacterium of claim 1 , wherein the disruptive mutation reduces or eliminates the expression or activity of the prephenate synthase.

8. The bacterium if claim 7 , wherein the bacterium produces a reduced amount of prephenate or prephenate-derived products compared to a parental bacterium.

9. The bacterium of claim 7 , wherein the bacterium produces substantially no tyrosine or phenylalanine.

10. The bacterium of claim 1 , wherein the bacterium comprises at least one nucleic acid encoding at least one of:
a. the exogenous chorismate pyruvate lyase,
b. the exogenous isochorismate synthase,
c. the exogenous isochorismate pyruvate lyase, and
d. the prephenate synthase comprising a disruptive mutation.

11. The bacterium of claim 10 , wherein the nucleic acid is codon optimized for expression in Clostridium.

12. The bacterium of claim 1 , wherein the chorismate-derived product comprises a 6-membered carbon ring substituted with a carboxyl group or carboxylate anion and further substituted with one or more OH groups and/or one or more NH 2 groups.

13. The bacterium of claim 1 , wherein the chorismate-derived product is selected from the group consisting of para-hydroxybenzoic acid, salicylate, 2-aminobenzoate, dihydroxybenzoate, 4-hydroxycyclohexane carboxylic acid, and salts and ions thereof.

14. The bacterium of claim 1 , wherein the bacterium expresses a chorismate pyruvate lyase of ubiC and produces a chorismate-derived product of para-hydroxybenzoic acid.

15. The bacterium of claim 1 , wherein the bacterium expresses an isochorismate synthase of pchA and an isochorismate pyruvate lyase of pchB and produces a chorismate-derived product of salicylate.

16. The bacterium of any one of claims 14 and 15 , wherein the bacterium further expresses a feedback-insensitive DAHP synthase.

17. The bacterium of claim 1 , wherein the bacterium comprises a prephenate synthase comprising a disruptive mutation and produces a chorismate-derived product of 2-aminobenzoate, 2,3-dihydroxybenzoate, or 4-hydroxycyclohexane carboxylic acid.

18. The bacterium of claim 1 , wherein the bacterium produces at least one chorismate-derived product not produced by a parental bacterium.

19. The bacterium of claim 1 , wherein the bacterium produces a greater amount of at least one chorismate-derived product than a parental bacterium.

20. The bacterium of claim 1 , wherein the bacterium is derived from a parental bacterium selected from the group consisting of Clostridium autoethanogenum, Clostridium ljungdahlii , and Clostridium ragsdalei.

21. The bacterium of claim 20 , wherein the Clostridium autoethanogenum is Clostridium autoethanogenum DSM23693.

22. The bacterium of claim 1 , wherein the gaseous substrate comprises at least one of CO, CO 2 , and H 2 .

23. A method of producing a fermentation product, comprising fermenting the bacterium of claim 1 in the presence of a gaseous substrate to produce a fermentation product.

24. The method of claim 23 , wherein the gaseous substrate comprises at least one of CO, CO 2 , and H 2 .

Assignees

• Lanzatech New Zealand Ltd

Inventors

• Behrendorff James Bruce Yarnton Haycock
• Koepke Michael
• Tran Loan Phuong
• Allen Wyatt Eric

Classifications

• C12Y401/0304

  Chorismate lyase (4.1.3.40) · CPC title

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C12Y402/99021

  Isochorismate lyase (4.2.99.21) · CPC title

• C12P1/04

  by using bacteria · CPC title

• C12P7/42

  Hydroxy-carboxylic acids · CPC title