Plant epsp synthases and methods of use

Abstract

Compositions and methods comprising polynucleotides and polypeptides having EPSP (5-enolpyruvylshikimate-3-phosphate) synthase (EPSPS) activity are provided. In specific embodiments, the sequence has an improved property, such as, but not limited to, improved catalytic capacity in the presence of the inhibitor, glyphosate. Further provided are nucleic acid constructs, plants, plant cells, explants, seeds and grain having the EPSPS sequences. Various methods of employing the EPSPS sequences are provided. Such methods include methods for producing a glyphosate tolerant plant, plant cell, explant or seed and methods of controlling weeds in a field containing a crop employing the plants and/or seeds disclosed herein.

What is claimed is: 
     
         1 - 52 . (canceled) 
     
     
         53 . A polynucleotide modification template comprising a partial EPSP synthase (EPSPS) sequence, wherein the polynucleotide modification template comprises one or more nucleotide mutations that correspond to G102A and to at least one or more amino acid mutations selected from the group consisting of:
 a) A2R,   b) A4W,   c) H54M,   d) A72Q,   e) K84R,   f) L98C,   g) K173R,   h) I208L,   i) K243E,   j) T279A,   k) E302S,   I) T361S,   m) E391P,   n) E391G,   o) D402G,   p) A416G,   q) V438R,   r) S440R,   s) T441Q, and   t) F442V,
 wherein each amino acid mutation position corresponds to the amino acid position set forth in SEQ ID NO: 1. 
   
     
     
         54 . A plant cell comprising the polynucleotide modification template of  claim 53 , a guide RNA, and CRISPR-Cas endonuclease, wherein the polynucleotide modification template, guide RNA, and CRISPR/Cas9 in combination targets an endogenous maize EPSPS sequence that encodes an EPSPS polypeptide that is at least 90% identical to SEQ ID NO: 2. 
     
     
         55 . A method of rapidly assaying catalytic efficiency of a plurality of enzyme variants for a substrate in the presence of an inhibitor, the method comprising:
 (a) providing the plurality of enzyme variants;   (b) providing the inhibitor;   (c) providing the substrate;   (d) performing a reaction involving the plurality of enzyme variants and the substrate, with two different inhibitor concentrations;   (e) measuring reaction rate at the two different inhibitor concentrations; and   (f) calculating (k cat /K M )*K I  of the plurality of enzyme variants.   
     
     
         56 . The method of  claim 55 , wherein one of the inhibitor concentrations is zero. 
     
     
         57 . The method of  claim 55 , wherein the substrate is at a concentration that is substantially similar to Michaelis-Menten constant (K M ) of a parental enzyme for the enzyme variant. 
     
     
         58 . The method of  claim 55 , wherein the enzyme is at a sufficient concentration to result in a substantially linear reaction rate at the two different inhibitor concentrations. 
     
     
         59 . The method of  claim 55 , wherein one of the inhibitor concentrations is sufficient to result in at least about 50% inhibition. 
     
     
         60 . The method of  claim 55 , wherein the method is performed in a high-throughput system. 
     
     
         61 . The method of  claim 55 , wherein catalytic capacity in the presence of the inhibitor is estimated by obtaining a numerical value for (k cat /K M )*K I , wherein k cat  is maximum enzyme turnover rate, K M  is Michaelis-Menten constant and K I  is inhibitor dissociation constant. 
     
     
         62 . The method of  claim 55 , wherein the substrate is PEP, the inhibitor is glyphosate, and the plurality of enzyme variants is EPSPS. 
     
     
         63 . The method of  claim 55 , wherein in step (d), no more than two different concentrations of the inhibitor are used. 
     
     
         64 . The method of  claim 55 , wherein the enzyme and the substrate concentrations are the same, at the two inhibitor concentrations.