Back to results
US 2023/0323410 A1Published application
Bacterial cells and methods for production of 2-fluoro-cis,cis-muconate
AssigneeDanmarks Tekniske Universitet
InventorsNicolas Thilo Wirth, Pablo Ivän Nikel
Life of this patent
Estimating the term…
Abstract
Herein are disclosed bacterial cells useful for production of 2-fluoro-cis,cis-muconate and derivatives thereof. The disclosure also provides methods and nucleic acid constructs therefor.
Claims25
A bacterial cell capable of producing 2-fluoro-cis,cis-muconate, in the presence of 3-fluorobenzoate or a salt thereof, said bacterial cell expressing:
- benzoate 1,2-dioxygenase (EC:
- 1.14.12.10), wherein the benzoate 1,2-dioxygenase is capable of converting 3-fluorobenzoate to 3-fluorocyclohexadiene-cis, cis-1,2-diol-1-carboxylate;
- a benzoate-1,2-dihydrodiol dehydrogenase (EC:
- 1.3.1.25), wherein the benzoate-1,2-dihydrodiol dehydrogenase is capable of converting 3-fluorocyclohexadiene-cis,cis-1,2-diol-1-carboxylate to 3-fluorocatechol;
- and
- a first catechol 1,2-dioxygenases (EC:
- 1.13.11.1), wherein the first catechol 1,2-dioxygenases is capable of converting 3-fluorocatechol to 2-fluoro-cis,cis-muconate,
- or
- a benzoate 1,2-dioxygenase (EC:
- 1.14.12.10), wherein the benzoate 1,2-dioxygenase is capable of converting 3-fluorobenzoate to 3-fluorocyclohexadiene-cis,cis-1,2-diol-1-carboxylate;
- a benzoate-1,2-dihydrodiol dehydrogenase (EC:
- 1.3.1.25), wherein the benzoate-1,2-dihydrodiol dehydrogenase is capable of converting 3-fluorocyclohexadiene-cis,cis-1,2-diol-1-carboxylate to 3-fluorocatechol;
- and
- a first and a second catechol 1,2-dioxygenases (EC:
- 1.13.11.1), wherein the first and second catechol 1,2-dioxygenases are each capable of converting 3-fluorocatechol to 2-fluoro-cis,cis-muconate,
- wherein:
- the expression of said benzoate 1,2-dioxygenase is under the control of a medium-strong constitutive promoter, whereby the benzoate 1,2-dioxygenase is constitutively overexpressed;
- the expression of said first catechol 1,2-dioxygenase is under the control of a first expression module comprising a first promoter, said first expression module further comprising a first strong translation initiation sequence such as a first strong translational coupler, and
- the expression of said second catechol 1,2-dioxygenase is under the control of a second expression module comprising a second promoter, said second expression module further comprising a second strong translational initiation sequence such as a second strong translational coupler,
- whereby the bacterial cell is capable of converting 3-fluorobenzoate to 2-fluoro-cis,cis-muconate.
17 more elements
(canceled)
7-9. (canceled)
(canceled)
A method of producing 2-fluoro-cis,cis-muconate, in a bacterial cell, said method comprising the steps of providing the bacterial cell and incubating said bacterial cell in a medium in the presence of 3-fluorobenzoate or a salt thereof, said bacterial cell expressing:
- a benzoate 1,2-dioxygenase (EC:
- 1.14.12.10);
- a benzoate-1,2-dihydrodiol dehydrogenase (EC:
- 1.3.1.25);
- and a first catechol 1,2-dioxygenase (EC:
- 1.13.11.1),
- or
- a benzoate 1,2-dioxygenase (EC:
- 1.14.12.10):
- a benzoate-1,2-dihydrodiol dehydrogenase (EC:
- 1.3.1.25);
- and a first and a second catechol 1,2-dioxygenase (EC:
- 1.13.11.1),
- wherein:
- the expression of said benzoate 1,2-dioxygenase is under the control of a medium-strong constitutive promoter;
- the expression of said first catechol 1,2-dioxygenase is under the control of a first expression module comprising a first promoter, said first expression module further comprising a first strong translation initiation sequence such as a first strong translational coupler;
- and
- the expression of said second catechol 1,2-dioxygenase is under the control of a second expression module comprising a second promoter, said second expression module further comprising a second strong translational initiation sequence such as a second strong translational coupler.
15 more elements
The method according to claim 120, wherein said bacterial cell expresses:
- a benzoate 1,2-dioxygenase (EC:
- 1.14.12.10);
- a benzoate-1,2-dihydrodiol dehydrogenase (EC:
- 1.3.1.25);
- and
- a first catechol 1,2-dioxygenase (EC:
- 1.13.11.1),
- wherein:
- the expression of said benzoate 1,2-dioxygenase is under the control of a medium-strong constitutive promoter;
- the expression of said first catechol 1,2-dioxygenase is under the control of a first expression module comprising a first medium-to-weak constitutive promoter, said first expression module further comprising a first strong translational coupler,
- or
- the expression of said first catechol 1,2-dioxygenase is under the control of a first expression module comprising a first inducible promoter coupled with a sequence encoding an associated activator protein, said first expression module further comprising a first strong translational coupler.
9 more elements
14-16. (canceled)
19-25. (canceled)
Dependencies are inferred from the claim text.