Non-mevalonate isoprenoid pathway

Abstract

The present invention is directed to enzymes and intermediates of the non-mevalonate isoprenoid pathway downstream of 2C-methyl-D-erythritol 2,4-cyclopyrophosphate and upstream of isopentenyl pyrophosphate or dimethylallyl pyrophosphate. These are used as a basis for a screening method for inhibitors of these enzymes, and a method for identifying inhibitor-resistant variants thereof. Further disclosures refer to DNA coding for said enzymes and for inhibitor-resistant variants thereof, vectors containing said DNA, cells containing said vector, and plant seeds comprising cells containing said vector. This invention is useful for the inhibition of the biosynthesis of isoprenoids in plants, bacteria and protozoa, for conferring herbicide-resistance to plants, as well as for weed control in agriculture using a crop containing a herbicide-resistant gene and an effective amount of a suitable herbicide.

1 - 31 . (canceled) 
     
     
         32 . Optionally isotope-labelled intermediate in the non-mevalonate isoprenoid pathway characterized in that
 (a) it is producible by the reaction of 2C-methyl-D-erythritol 2,4-cyclopyrophosphate with an aqueous suspension of intact or lysed plastids or bacterial cells;   (b) it is separable from the aqueous supernatant of the reaction mixture obtainable according to item (a) by reversed-phase, ion pair, high performance liquid chromatography using a 4.6×250 mm C 18  reversed-phase column (crystalline silica gel of particle size 5 μm, average pore size 12 nm), equilibrated with aqueous 10 mM tetra-n-butylammonium hydrogen sulfate pH 6.0 and developed with 10 mM tetra-n-butylammonium hydrogen sulfate pH 6.0 and a linear gradient of 0 to 42% (v/v) methanol in 10 mM tetra-n-butyl ammonium hydrogen sulfate at pH 6.0 and ambient temperature;   (c) it exhibits under the conditions of item (b) a retention volume downstream from the retention volume of 2C-methyl-D-erythritol 2,4-cyclopyrophosphate and upstream from the retention volume of isopentenyl pyrophosphate; and   (d) its increase in the reaction mixture of item (a) is correlated with the decrease of 2C-methyl-D-erythritol 2,4-cyclopyrophosphate as detectable in the detection method of item (c).   
     
     
         33 . The intermediate according to  claim 32  characterized in that it is producible in a partially radiolabelled form from a partially  14 C-labelled 2C-methyl-D-erythritol 2,4-cyclopyrophosphate. 
     
     
         34 . The intermediate according to  claim 32  characterized in that it is producible as a [U— 13 C]-compound from [U— 13 C 5 ]-2C-methyl-D-erythritol 2,4-cyclopyrophosphate. 
     
     
         35 . The intermediate according to  claim 32  characterized by a retention volume
 of 1.27±0.15 times that of 2C-methyl-D-erythritol 2,4-cyclopyrophosphate and 0.68±0.06 times that of isopentenyl pyrophosphate or   of 1.50±0.16 times that of 2C-methyl-D-erythritol 2,4-cyclopyrophosphate and 0.80±0.06 times that of isopentenyl pyrophosphate   under the conditions described in claim  1  item b.   
     
     
         36 . The intermediate according to  claim 35  characterized by a retention volume
 of about 1.27 times that of 2C-methyl-D-erythritol 2,4-cyclopyrophosphate and about 0.68 times that of isopentenyl pyrophosphate or   of about 1.50 times that of 2C-methyl-D-erythritol 2,4-cyclopyrophosphate and about 0.80 times that of isopentenyl pyrophosphate   under the conditions described in claim  1  item b.   
     
     
         37 . A process for producing an intermediate in the non-mevalonate isoprenoid pathway by
 (a) adding 2C-methyl-D-erythritol 2,4-cyclopyrophosphate to an aqueous suspension of intact or lysed plastids or bacterial cells or of a protein fraction thereof;   (b) incubating the obtained mixture for a predetermined period of time at a predetermined temperature;   (c) optionally disrupting the plastids or cells;   (d) separating insoluble components and polymer components from the mixture;   (e) subjecting the obtained aqueous solution, optionally after concentration, to a reversed phase ion pair high performance liquid chromatography;   (f) separating a fraction containing a metabolite having a retention volume downstream from the retention volume of 2C-methyl-D-erythritol 2,4-cyclopyrophosphate and upstream from the retention volume of isopentenyl pyrophosphate; and   (g) isolating the intermediate from said fraction.   
     
     
         38 . The process according to  claim 37 , wherein a radioactively labelled starting material is used and the detection in step (f) is carried out with a radio detector. 
     
     
         39 . The process according to  claim 37  characterized in that a cobalt (II) salt is added in step (a). 
     
     
         40 . Use of the intermediate according to  claim 32  for screening for enzymes of the non-mevalonate pathway
 (a) either between 2C-methyl-D-erythritol 2,4-cyclopyrophosphate and said intermediate; or   (b) between said intermediate and isopentenyl pyrophosphate or dimethylallyl pyrophosphate.   
     
     
         41 . An enzyme in a form that is functional in the biosynthetic conversion of 2C-methyl-D-erythritol 2,4-cyclopyrophosphate into the intermediate according to  claim 32  within the non-mevalonate isoprenoid pathway selected from the group of enzymes coded by
 (a) gcpE of  E. coli  or by genes orthologous thereto;   (b) lytB of  E. coli  or by genes orthologous thereto;   (c) yjeE of  E. coli  or by genes orthologous thereto;   (d) ybeB of  E. coli  or by genes orthologous thereto.   
     
     
         42 . An isolated, purified nucleic acid coding for an enzyme according to  claim 41 . 
     
     
         43 . A DNA vector containing the sequence of the nucleic acid according to  claim 42 . 
     
     
         44 . Recombinant cell containing the vector according to  claim 43 , wherein said cell is selected from the group consisting of bacterial, protozoal, fungal, plant, insect and mammalian cells. 
     
     
         45 . A seed comprising a plant cell as defined in  claim 44 .