HPPD variants and methods of use

Patent metadata

Field	Value
Publication number	US-11180770-B2
Application number	US-201816489620-A
Country	US
Kind code	B2
Filing date	Mar 1, 2018
Priority date	Mar 7, 2017
Publication date	Nov 23, 2021
Grant date	Nov 23, 2021

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 an enzymatically active 4-hydroxyphenylpyruvate dioxygenase (HPPD) polypeptide whose amino acid sequence (I) comprises the substitutions (a) an alanine at the amino acid position corresponding to amino acid position 268 of SEQ ID NO:1, (b) a proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO:1, (c) a histidine or an aspartic acid at the position corresponding to amino acid position 336 of SEQ ID NO:1, and (d) a serine at the position corresponding to amino acid position 337 of SEQ ID NO:1; or
(II) comprises the substitutions of (I) and further comprises
i. a lysine or leucine at the amino acid position corresponding to amino acid position 213 of SEQ ID NO:1; and/or
ii. an alanine at the amino acid position corresponding to amino acid position 215 of SEQ ID NO:1; and/or
iii. an arginine, asparagine, leucine, glutamic acid, proline or serine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO:1; and/or
iv. an arginine, lysine, glutamine, methionine or histidine at the amino acid position corresponding to amino acid position 315 of SEQ ID NO:1; and/or
v. 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
vi. a glutamine, proline, or arginine at the amino acid position corresponding to amino acid position 344 of SEQ ID NO:1; and/or
vii. a lysine, arginine, methionine, alanine, or valine at the amino acid position corresponding to amino acid position 345 of SEQ ID NO:1; or

(III) comprises the substitutions of (I) and 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 alanine at the amino acid position corresponding to amino acid position 215 of SEQ ID NO:1; and/or
iii. an asparagine, glutamic acid or serine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO:1; and/or
iv. an arginine, lysine, glutamine or methionine at the amino acid position corresponding to amino acid position 315 of SEQ ID NO:1; and/or
v. an arginine, or valine at the amino acid position corresponding to amino acid position 340 of SEQ ID NO:1; and/or
vi. a glutamine at the amino acid position corresponding to amino acid position 344 of SEQ ID NO:1; and/or
vii. a lysine, valine, or methionine at the amino acid position corresponding to amino acid position 345 of SEQ ID NO:1; or

(IV) comprises the substitutions of (I) and 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 asparagine, glutamic acid or serine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO:1; and/or
iii. an arginine, lysine, glutamine at the amino acid position corresponding to amino acid position 315 of SEQ ID NO:1; and/or
iv. an arginine or valine at the amino acid position corresponding to amino acid position 340 of SEQ ID NO:1; and/or
v. a glutamine at the amino acid position corresponding to amino acid position 344 of SEQ ID NO:1; and/or
vi. a lysine, valine, or methionine at the amino acid position corresponding to amino acid position 345 of SEQ ID NO:1; or

(V) comprises the substitutions of any one of (I)-(IV) and has at least 53% sequence identity to the amino acid sequence set forth in SEQ ID NO:1;
and wherein said HPPD polypeptide provides to a host tolerance to one or more HPPD inhibitor herbicide(s).

2. The recombinant nucleic acid molecule of claim 1 , wherein its nucleotide sequence is a synthetic sequence that has been designed for expression in a plant.

3. The recombinant nucleic acid molecule of claim 1 , wherein its nucleotide sequence is operably linked to a promoter capable of directing expression of the nucleotide sequence in a plant cell.

4. 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.

5. The recombinant nucleic acid molecule of claim 4 , 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.

6. A host cell that contains the recombinant nucleic acid molecule of claim 1 .

7. The host cell of claim 6 that is a bacterial host cell.

8. The host cell of claim 6 that is a plant cell.

9. A transgenic plant comprising the recombinant nucleic acid molecule of claim 1 .

10. The plant of claim 9 , wherein said plant is selected from the group consisting of maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

11. A transgenic seed comprising the recombinant nucleic acid molecule of claim 1 .

12. A recombinant polypeptide comprising an enzymatically active HPPD polypeptide, wherein said HPPD polypeptide (I) comprises the substitutions (a) an alanine at the amino acid position corresponding to amino acid position 268 of SEQ ID NO: 1, (b) a proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1, (c) a histidine or an aspartic acid at the position corresponding to amino acid position 336 of SEQ ILS NO: 1, and (d) a serine at the position corresponding to amino acid position 337 of SEQ ID NO:1; or
(II) comprises the substitutions of (I) and further comprises
i. a lysine or leucine at the amino acid position corresponding to amino acid position 213 of SEQ ID NO:1; and/or
ii. an alanine at the amino acid position corresponding to amino acid position 215 of SEQ ID NO:1; and/or
iii. an arginine, asparagine, leucine, glutamic acid, proline or serine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO: 1; and/or
iv. an arginine, lysine, glutamine, methionine or histidine the amino acid position corresponding to amino acid position 315 of SEQ ID NO: 1; and/or
v. 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
a glutamine, proline, or arginine at the amino acid position corresponding to amino acid position 344 of SEQ ID NO:1; and/or vii. a lysine, arginine, methionine, alanine, or valine at the amino acid position corresponding to amino acid position 345 of SEQ ID NO:1; or
(III) comprises the substitutions of (I) and 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 alanine at the amino acid position corresponding to amino acid position 215 of SEQ ID NO:1; and/or
iii. an asparagine, glutamic acid or serine, at the amino a

Assignees

• BASF Agricultural Solutions Seed US LLC

Inventors

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

Classifications

• C07K14/415

  from plants · CPC title

• C07K14/195

  from bacteria · CPC title

• 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

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title