HPPD variants and methods of use

Patent metadata

Field	Value
Publication number	US-10597674-B2
Application number	US-201615758429-A
Country	US
Kind code	B2
Filing date	Sep 8, 2016
Priority date	Sep 11, 2015
Publication date	Mar 24, 2020
Grant date	Mar 24, 2020

Abstract

In the present invention, HPPD polypeptides and plants containing them showing a full tolerance against one or more HPPD inhibitor herbicides belonging to various chemical classes are described. A set of mutant HPPD polypeptides have been designed which have either no or only a significantly reduced affinity to HPPD inhibitor herbicides and, at the same time, the rate of dissociation of the HPPD inhibitors of the mutant HPPD polypeptide is increased to such an extent that the HPPD inhibitors no longer act as slow-binding or slow, tight-binding inhibitors but, instead of this, have become fully reversible inhibitors. In particular, isolated polynucleotides encoding mutant HPPD polypeptides conferring tolerance to HPPD inhibitor herbicides belonging to various chemical classes are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed.

First claim

The invention claimed is:

1. A recombinant nucleic acid molecule encoding a 4-hydroxyphenylpyruvate dioxygenase (HPPD) polypeptide, wherein said HPPD polypeptide comprises an amino acid sequence comprising (a) a proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO:1, (b) a histidine or an aspartic acid at the position corresponding to amino acid position 336 of SEQ ID NO:1, and (c) a serine at the position corresponding to amino acid position 337 of SEQ ID NO:1, and wherein said HPPD polypeptide is tolerant to one or more HPPD inhibitor herbicide(s).

2. The recombinant nucleic acid molecule of claim 1 , wherein the amino acid sequence of said HPPD polypeptide further comprises:
i. a methionine, threonine, serine, or leucine at the amino acid position corresponding to amino acid position 204 of SEQ ID NO:1; and/or
ii. a lysine or leucine at the amino acid position corresponding to amino acid position 213 of SEQ ID NO:1; and/or
iii. an arginine, lysine, glutamine, or leucine at the amino acid position corresponding to amino acid position 264 of SEQ ID NO:1; and/or
iv. an arginine, glycine, or serine at the amino acid position corresponding to amino acid position 268 of SEQ ID NO:1; and/or
v. an arginine, leucine, glutamic acid, proline or serine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO:1; and/or
vi. a serine, histidine, or lysine at the amino acid position corresponding to amino acid position 310 of SEQ ID NO:1; and/or
vii. an arginine, methionine or histidine at the amino acid position corresponding to amino acid position 315 of SEQ ID NO:1; and/or
viii. a histidine, alanine, phenylalanine, valine, or glycine at the amino acid position corresponding to amino acid position 330 of SEQ ID NO:1; and/or
ix. a proline, histidine, serine, isoleucine, or leucine at the amino acid position corresponding to amino acid position 331 of SEQ ID NO:1; and/or
x. a valine at the amino acid position corresponding to amino acid position 338 of SEQ ID NO:1; and/or
xi. a glutamic acid, arginine, alanine, or threonine at the amino acid position corresponding to amino acid position 339 of SEQ ID NO:1; and/or
xii. an arginine, glutamine, methionine, glutamic acid, glycine, leucine, or valine at the amino acid position corresponding to amino acid position 340 of SEQ ID NO:1; and/or
xiii. a glutamine, proline, or arginine at the amino acid position corresponding to amino acid position 344 of SEQ ID NO:1; and/or
xiv. a lysine, arginine, methionine, alanine, or valine at the amino acid position corresponding to amino acid position 345 of SEQ ID NO:1.

3. The recombinant nucleic acid molecule of claim 1 , wherein the amino acid sequence of said HPPD polypeptide further comprises:
i. a leucine or lysine at the amino acid position corresponding to amino acid position 213 of SEQ ID NO:1; and/or
ii. an arginine or leucine at the amino acid position corresponding to amino acid position 264 of SEQ ID NO:1; and/or
iii. an arginine, glycine or serine at the amino acid position corresponding to amino acid position 268 of SEQ ID NO:1; and/or
iv. a glutamic acid or serine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO:1; and/or
v. an arginine or methionine at the amino acid position corresponding to amino acid position 315 of SEQ ID NO:1; and/or
vi. a histidine at the amino acid position corresponding to amino acid position 330 of SEQ ID NO:1; and/or
vii. a valine at the amino acid position corresponding to amino acid position 338 of SEQ ID NO:1; and/or
viii. an arginine, or valine at the amino acid position corresponding to amino acid position 340 of SEQ ID NO:1; and/or
ix. a glutamine at the amino acid position corresponding to amino acid position 344 of SEQ ID NO:1; and/or
x. a lysine, valine, or methionine at the amino acid position corresponding to amino acid position 345 of SEQ ID NO:1.

4. The recombinant nucleic acid molecule of claim 1 , wherein the amino acid sequence of said HPPD polypeptide further comprises:
i. a lysine at the amino acid position corresponding to amino acid position 213 of SEQ ID NO:1; and/or
ii. an arginine or leucine at the amino acid position corresponding to amino acid position 264 of SEQ ID NO:1; and/or
iii. a glycine or arginine at the amino acid position corresponding to amino acid position 268 of SEQ ID NO:1; and/or
iv. a glutamic acid at the amino acid position corresponding to amino acid position 270 of SEQ ID NO:1; and/or
v. an arginine at the amino acid position corresponding to amino acid position 315 of SEQ ID NO:1; and/or
vi. a histidine at the amino acid position corresponding to amino acid position 330 of SEQ ID NO:1; and/or
vii. a valine at the amino acid position corresponding to amino acid position 338 of SEQ ID NO:1; and/or
viii. an arginine or valine at the amino acid position corresponding to amino acid position 340 of SEQ ID NO:1; and/or
ix. a glutamine at the amino acid position corresponding to amino acid position 344 of SEQ ID NO:1; and/or
x. a lysine, valine, or methionine at the amino acid position corresponding to amino acid position 345 of SEQ ID NO:1.

5. The recombinant nucleic acid molecule of claim 1 , wherein said HPPD polypeptide comprises an amino acid sequence having at least 53% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

6. The recombinant nucleic acid molecule of claim 5 , wherein said HPPD polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

7. The recombinant nucleic acid molecule of claim 6 , wherein said HPPD polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

8. The recombinant nucleic acid molecule of claim 1 , wherein the recombinant nucleic acid molecule is a synthetic nucleic acid molecule that has been designed for expression in a plant.

9. The recombinant nucleic acid molecule of claim 1 , wherein the recombinant nucleic acid molecule is operably linked to a promoter, wherein said promoter is capable of directing expression of the HPPD polypeptide in a plant cell.

10. The recombinant nucleic acid molecule of claim 1 , wherein said HPPD inhibitor herbicide is selected from the group consisting of triketones, diketonitriles, isoxazoles, hydroxypyrazoles, N-(1,2,5-oxadiazol-3yl)benzamides, N-(1,3,4-oxadiazol-2-yl)benzamides, N-(tetrazol-5-yl)- or N-(triazol-5-yl)-arylcarboxamides, pyridazinone derivatives, oxoprazine derivatives, N-(triazol-2-yl)arylcarboxamides, triazinones, and pyrazolones.

11. The recombinant nucleic acid molecule of claim 10 , wherein said HPPD inhibitor herbicide is selected from the group consisting of benzobicyclon, sulcotrione, mesotrione, tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione, isoxaflutole, diketonitrile, pyrazoxyfen, benzofenap, pyrazolynate, pyrasulfotole, topramezone, tolpyralate, 2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide, 2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide, 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide, 2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide, and 2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide.

12. A host cell comprising the recombinant nucleic acid molecule of claim 1 .

13. The host cell of claim 12 , wherein the host cell is a bacterial host cell.

14. The host cell of claim 12 , wherein

Assignees

• BASF Agricultural Solutions Seed US LLC

Inventors

• Linka Marc
• Poree Fabien
• Laber Bernd
• Lange Gudrun
• Tebbe Jan
• Coco Wayne
• Strerath Michael
• Weber Ernst
• Pawlowski Nikolaus
• Geske Sandra
• Balven-Ross Heike
• Wobst Nina
• Thies Christina
• Dubald Manuel

Classifications

• C12N9/0069 Primary

  acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13) · CPC title

• C12N15/8274 Primary

  for herbicide resistance · CPC title

• C12Y113/11027

  4-Hydroxyphenylpyruvate dioxygenase (1.13.11.27) · CPC title