Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases

Abstract

Genes encoding Class II EPSPS enzymes are disclosed. The genes are useful in producing transformed bacteria and plants which are tolerant to glyphosate herbicide. Class II EPSPS genes share little homology with known, Class I EPSPS genes, and do not hybridize to probes from Class I EPSPS's. The Class II EPSPS enzymes are characterized by being more kinetically efficient than Class I EPSPS's in the presence of glyphosate. Plants transformed with Class II EPSPS genes are also disclosed as well as a method for selectively controlling weeds in a planted transgenic crop field.

Description

[0001]This is a continuation-in-part of a U.S. patent application Ser. No. 07 / 749,611, filed Aug. 28, 1991 now abandoned, which is a continuation-in-part of U.S. patent application Ser. No. 07 / 576,537, filed Aug. 31, 1990, now abandoned.BACKGROUND OF THE INVENTION[0002]This invention relates in general to plant molecular biology and, more particularly, to a new class of glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases.[0003]Recent advances in genetic engineering have provided the requisite tools to transform plants to contain foreign genes. It is now possible to produce plants which have unique characteristics of agronomic importance. Certainly, one such advantageous trait is more cost effective, environmentally compatible weed control via herbicide tolerance. Herbicide-tolerant plants may reduce the need for tillage to control weeds thereby effectively reducing soil erosion.[0004]One herbicide which is the subject of much investigation in this regard is N-phosphonom...

AI Technical Summary

Benefits of technology

[0012]DNA molecules comprising DNA encoding kinetically efficient, glyphosate-tolerant EPSP synthases are disclosed. The EPSP synthases of the present invention reduce the amount of overproduction of the EPSPS enzyme in a transgenic plant necessary for the enzyme to maintain catalytic activity while still conferring glyphosate tolerance. The EPSP synthases described herein represent a new class of EPSPS enzymes, referred to hereinafter as Class II EPSPS enzymes. Class II EPSPS enzymes of the present invention usually share only between about 47% and 55% amino acid similarity or between about 22% and 30% amino acid identity to other known bacterial or plant EPSPS enzymes and exhibit tolerance to glyphosate while maintaining suitable Km (PEP) ranges. Suitable ranges of Km (PEP) for EPSPS for enzymes of the present invention are between 1-150 μM, with a more preferred range of between 1-35 μM, and a most preferred range between 2-25 μM. These kinetic constants are determined under the assay conditions specified hereinafter. An EPSPS of the present invention preferably has a Ki for glyphosate range of between 15-10000 μM. The Ki / Km ratio should be between about 2-500, and more preferably between 25-500. The Vmax of the purified enzyme should preferably be in the range of 2-100 units / mg (μmoles / minute.mg at 25° C.) and the Km for shikimate-3-phosphate should preferably be in the range of 0.1 to 50 μM.

[0043]c) a 3′ nontranslated region which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequencewhere the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the EPSP synthase polypeptide to enhance the glyphosate tolerance of a plant cell transformed with said DNA molecule.

Problems solved by technology

Again, the glyphosate-tolerant EPSPS was impaired due to an increase in the Km for PEP and a slight reduction of the Vmax of the native plant enzyme (Kishore and Shah, 1988) thereby lowering the catalytic efficiency (Vmax / Km) of the enzyme.

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