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EP-4735588-A2 - METHODS AND COMPOSITIONS FOR GLYPHOSATE TOLERANCE IN PLANTS

EP4735588A2EP 4735588 A2EP4735588 A2EP 4735588A2EP-4735588-A2

Abstract

The present disclosure relates to novel methods and compositions for conferring tolerance to glyphosate to plants. The present disclosure also provides glyphosate-tolerant plants, seeds, tissues, cells, and plant parts comprising modified EPSP synthases and recombinant DNA molecules encoding modified EPSP synthases, as well as methods of producing the same and the use thereof.

Inventors

  • ALPER, HAL
  • GUO, Shirley, X.
  • IVASHUTA, SERGEY
  • LARUE, Clayton, T.
  • REED, Kevin, B.

Assignees

  • Monsanto Technology LLC
  • Board of Regents, The University of Texas System

Dates

Publication Date
20260506
Application Date
20240529

Claims (20)

  1. CLAIMS 1. A recombinant DNA molecule encoding a glyphosate-tolerant 5-enol-pyruvyl-shikimate- 3-phosphate synthase (EPSPS), wherein the EPSPS comprises at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-P126S-M217L-K296R, T102I-P106S- V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I- P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the position of the amino acid substitution is relative to the position of the amino acid sequence provided as SEQ ID NO:6.
  2. 2. The recombinant DNA molecule of claim 1, wherein the EPSPS is a maize EPSPS.
  3. 3. The recombinant DNA molecule of claim 1, wherein the EPSPS confers increased tolerance to glyphosate or increased enzymatic efficiency as compared to an EPSPS lacking said combination.
  4. 4. The recombinant DNA molecule of claim 1, wherein the EPSPS comprises at least two of said substitution combinations.
  5. 5. A glyphosate-tolerant EPSPS comprising at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S- P126S, T102I-P106S-K296R, T102I-P106S-P126S-M217L-K296R, T102I-P106S-V66L- M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I- P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the position of the amino acid substitution is relative to the position of the amino acid sequence provided as SEQ ID NO:6.
  6. 6. The glyphosate-tolerant EPSPS of claim 5, wherein the EPSPS confers increased tolerance to glyphosate or increased enzymatic efficiency as compared to an EPSPS lacking said combination, or as compared to an EPSPS containing only T102I-P106S.
  7. 7. The glyphosate-tolerant EPSPS of claim 5, wherein the EPSPS comprises at least two of said substitution combinations.
  8. 8. A plant, seed, cell, plant part, or commodity product comprising the recombinant DNA molecule of claim 1.
  9. 9. The plant of claim 8, wherein the plant exhibits increased glyphosate tolerance when compared to a plant lacking said combination.
  10. 10. The plant of claim 9, wherein the plant is a corn, soy, cotton, canola, wheat, rice, alfalfa, sugar beet, oilseed rape or sugar cane plant.
  11. 11. A transgenic maize plant comprising a recombinant DNA molecule encoding a glyphosate- tolerant 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS), wherein the EPSPS comprises at least a first amino acid substitution combination selected from the group consisting of: T102I- P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-P126S- M217L-K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V- H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V- I163K, wherein the position of the amino acid substitution is relative to the position of the amino acid sequence provided as SEQ ID NO:6, and wherein said EPSPS confers glyphosate tolerance to said transgenic maize plant.
  12. 12. The transgenic maize plant of claim 11, wherein said glyphosate tolerance is greater than the glyphosate tolerance conferred by said EPSPS that comprises only a T102I-P106S substitution.
  13. 13. A seed, cell or plant part of the plant of claim 11, wherein the seed, cell or plant part comprises said recombinant DNA molecule.
  14. 14. A method for conferring glyphosate tolerance to a plant comprising expressing in the plant the glyphosate-tolerant EPSPS of claim 5.
  15. 15. The method of claim 14, comprising introducing the recombinant DNA molecule of claim 1 into the genome of said plant or a progenitor thereof.
  16. 16. A method for producing a glyphosate-tolerant EPSPS comprising introducing into a nucleic acid molecule encoding a plant EPSPS at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I- P106S-K296R, T102I-P106S-P126S-M217L-K296R, T102I-P106S-V66L-M104L, T102I- P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I- P106S-I177V, and P106S-A109V-I163K, wherein the position of the amino acid substitution is relative to the position of the amino acid sequence provided as SEQ ID NO:6.
  17. 17. The method of claim 16, wherein said introducing is carried out in vitro.
  18. 18. The method of claim 16, wherein said introducing is carried out in planta.
  19. 19. The method of claim 16, wherein said introducing comprises use of at least a first site- specific endonuclease.
  20. 20. The method of claim 16, wherein the method comprises introducing at least two of said substitution combinations into said EPSPS.

Description

TITLE OF THE INVENTION METHODS AND COMPOSITIONS FOR GLYPHOSATE TOLERANCE IN PLANTS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit under 35 U.S.C. §119(e) of U.S. provisional application Serial No. 63/511,073, filed on June 29, 2023, which is herein incorporated by reference in its entirety. FIELD OF THE INVENTION [0002] The present disclosure relates to the field of biotechnology. More specifically, the disclosure relates to recombinant DNA molecules encoding engineered 5-enolpyruvylshikimate- 3-phosphate synthase (EPSPS) enzymes that provide tolerance to the herbicide glyphosate. The present disclosure also provides methods of engineering enzymes with improved herbicide tolerance and enzymatic efficiency. INCORPORATION OF SEQUENCE LISTING [0003] The sequence listing that is contained in the file named MONS540WO_ST26.xml, which is 41.3 kilobytes (measured in MS-WINDOWS) and created on May 22, 2024, is filed herewith by electronic submission and incorporated herein by reference. BACKGROUND OF THE INVENTION [0004] Glyphosate, or N-phosphonomethylglycine, is a broad-spectrum, foliar-applied herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase or EPSPS) in plants. EPSPS is part of the shikimate pathway used in plants for the biosynthesis of folates and aromatic amino acids. EPSP synthases from different organisms have been divided into two classes based on glyphosate sensitivity. All plants have class I EPSP synthases, which are glyphosate-sensitive. Glyphosate tolerant crops have been primarily produced using the glyphosate-insensitive class II EPSPS from Agrobacterium sp. strain CP4. Glyphosate tolerance in crops permits the use of glyphosate to control weeds while maintaining crop yield. The T102I-P106S double mutation of the class I EPSPS from maize has been shown to confer insensitivity to glyphosate; however, these resistance-conferring mutations also reduce the catalytic efficiency of this essential enzyme and thus lead to reduced plant growth under native expression. Thus, there is a need for novel plant EPSPS variants having improved tolerance to glyphosate and high catalytic efficiency as well as methods of producing and using the same. SUMMARY OF THE INVENTION [0005] The present disclosure provides a recombinant DNA molecule encoding a glyphosate- tolerant 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS), wherein the EPSPS comprises at least a first amino acid substitution combination selected from the group consisting of: T102I- P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-P126S- M217L-K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V- H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V- I163K, wherein the position of the amino acid substitution is relative to the position of the amino acid sequence provided as SEQ ID NO:6. In certain embodiments, the EPSPS is a maize EPSPS. In some embodiments, the EPSPS confers increased tolerance to glyphosate or increased enzymatic efficiency as compared to an EPSPS lacking the combination. In other embodiments, the EPSPS comprises at least two of the substitution combinations. [0006] The present disclosure also provides a glyphosate-tolerant EPSPS comprising at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S- P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-P126S-M217L- K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I- P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the position of the amino acid substitution is relative to the position of the amino acid sequence provided as SEQ ID NO:6. In particular embodiments, the EPSPS confers increased tolerance to glyphosate or increased enzymatic efficiency as compared to an EPSPS lacking the combination, or as compared to an EPSPS containing only T102I-P106S. In further embodiments, the EPSPS comprises at least two of the substitution combinations. [0007] The present disclosure additionally provides a plant, seed, cell, plant part, or commodity product comprising a recombinant DNA molecule encoding a glyphosate-tolerant 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS), wherein the EPSPS comprises at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S- P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-P126S-M217L- K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I- P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the position of the amino acid substitution is relative to the position of the amino acid sequence provided as SEQ ID NO:6. In certain embodiments, the plant exhibits increased glyphosate tolerance when compared to a plant lacking the combination. In some embodiments, the plant is a corn, soy, cotton, canola, w