Mutant prenyl diphosphate synthase

Abstract

A mutant prenyl diphosphate synthase capable of synthesizing prenyl diphosphates, shorter than those synthesized by the original enzyme, by modifying the amino acid sequence in and upstream of the aspartic acid-rich domain DDXX (XX)D (X denotes any amino acid, and XX in the parentheses may not be present) present in region II of the prenyl diphosphate synthase.

1. A mutant prenyl diphosphate synthase having a modified the amino acid sequence, wherein
 said mutant prenyl diphosphate synthase comprises an aspartic acid-rich domain having the sequence, D 1 D 2 X 1 X 2 (X 3 X 4 )D 3 , in region II of said mutant prenyl diphosphate synthase, 
 wherein each of D 1 , D 2  and D 3  denote an aspartic acid residue; X 1 , X 2 , X 3  and X 4  are each independently any amino acid and X 3  and X 4  are each optionally independently present in the aspartic acid rich domain, and wherein 
 said mutant prenyl diphosphate synthase comprises (1) at least one amino acid substitution, said at least one amino acid substitution located at at least one ammo acid position selected from (a) an amino acid between D 1  and the amino acid residue at the fifth position upstream of D 1  and (b) the amino acid residue located one amino acid position upstream of D 3 ; (2) at least one additional amino acid inserted between D 3  and the first amino acid upstream of D 3 ; or a combination of (2) and (3); 
 wherein said mutant prenyl diphosphate synthase synthesizes prenyl diphosphate which is shorter than prenyl diphosphate synthesized by a corresponding wild-type enzyme of SEQ ID NO:1, except that: 
 threonine at position 78 of SEQ ID NO:1 is replaced with phenylalanine and histidine at position 81 of SEQ ID NO:1 is replaced with alanine; 
 threonine at position 78 of SEQ ID NO:1 is replaced with phenylalanine and histidine at position 81 of SEQ ID NO:1 is replaced with leucine; 
 phenylalanine at position 77 of SEQ ID NO:1 is replaced with tyrosine, threonine at position 78 of SEQ ID NO:1 is replaced with phenylalanine, and histidine at position 81 of SEQ ID NO:1 is replaced with leucine; 
 phenylalanine at position 77 of SEQ ID NO:1 is replaced with tyrosine, threonine at position 78 of SEQ ID NO:1 is replaced with phenylalanine, and histidine at position 81 of SEQ ID NO:1 is replaced with alanine; or 
 phenylalanine at position 77 of SEQ ID NO:1 is replaced with tyrosine, threonine at position 78 of SEQ ID NO:1 is replaced with serine, valine at position 80 of SEQ ID NO:1 is replaced with isoleucine, isoleucine at position 84 of SEQ ID NO:1 is replaced with leucine, and proline and serine are inserted sequentially between position 84 and position 85 of SEQ ID NO:1. 
 
     
     
       2. A mutant prenyl diphosphate synthase according to  claim 1  wherein said mutant has the enzymatic activities and thermo stability of wild type prenyl diphosphate synthase. 
     
     
       3. A mutant enzyme prenyl diphosphate synthase according to  claim 1  wherein the a reaction product of the mutant prenyl diphosphate synthase is farnesyl diphosphate. 
     
     
       4. A mutant enzyme prenyl diphosphate synthase according to  claim 1  wherein the mutant prenyl diphosphate synthase is of the homodimer-type forms a homodimer. 
     
     
       5. A mutant enzyme according to  claim 1  wherein the prenyl diphosphate synthase is derived from archaea. 
     
     
       6. A mutant enzyme prenyl diphosphate synthase according to  claim 1  wherein the mutant prenyl diphosphate synthase is derived from a mutant of a Sulfolobus acidocaldarius prenyl diphosphate synthase. 
     
     
       7. A mutant enzyme prenyl diphosphate synthase according to  claim 1  wherein the mutant prenyl diphosphate synthase is a more thermostable enzyme at 70° C. or 80° C. than the wild-type geranylgeranyl diphosphate synthase of Sulfolobus acidocaldarius. 
     
     
       8. A mutant prenyl diphosphate synthase according to  claim 1 , wherein at least one amino acid selected from phenylalanine at position 77, threonine at position 78, valine at position 80, histidine at position 81, and isoleucine at position 84 has been substituted by another amino acid, or one or more amino acids have been inserted in between isoleucine at position 84 and methionine at position 85 in the geranylgeranyl diphosphate synthase as set forth in SEQ ID No: 1. 
     
     
       9. A mutant prenyl diphosphate synthase according to  claim 1  wherein at least one amino acid selected from phenylalanine at position 77, threonine at position 78, valine at position 80, histidine at position 81, and isoleucine at position 84 has been substituted by another amino acid, and/or two amino acids have been inserted in between isoleucine at position 84 and methionine at position 85 in the geranylgeranyl diphosphate synthase as set forth in SEQ ID NO: 1, wherein the phenyl alanine at position 77 has been replaced with tyrosine, the threonine at position 78 has been replaced with phenylalanine or serine, the valine at position 80 has been replaced with isoleucine, the histidine at position 81 has been replaced with leucine or alanine, or the isoleucine at position 84 has been replaced with leucine; or proline and serine have been inserted in between the isoleucine at position 84 and the methionine at position 85. 
     
     
       10. A mutant prenyl diphosphate synthase according to  claim 1 , wherein the mutant prenyl diphosphate synthase is derived from a native geranylgeranyl diphosphate synthase of an organism selected from the group consisting of Arabidopsis thaliana, Lupinas albus, Capsicum annuum, Sulfolobus acidocaldarius, Rhodobactor sphaeroides, Rhodobactor capsulatus, Erwinia herbicola, Myxococcus thaliana and Neurospora crassa. 
     
     
       11. A DNA encoding an enzyme the mutant prenyl diphosphate synthase according to  claim 1 . 
     
     
       12. An RNA transcribed from a DNA according to  claim 11  encoding the mutant prenyl diphosphate synthase according to claim 1. 
     
     
       13. A recombinant vector comprising a the DNA according to  claim 11 . 
     
     
       14. A An isolated host organism cell transformed with a the recombinant vector according to  claim 13 . 
     
     
       15. A process for producing a mutant enzyme prenyl diphosphate synthase according to  claim 1 , said method comprising the steps of culturing a an isolated host cell transformed with an expression vector comprising a DNA coding for encoding the mutant enzyme prenyl diphosphate synthase and of harvesting the expression product mutant prenyl diphosphate synthase according to claim 1 from the culture, wherein the mutant prenyl diphosphate synthase is produced by expression of the expression vector. 
     
     
       16. A process for producing a prenyl diphosphate having not more than 15 carbons comprising the step of bringing an enzyme the mutant prenyl diphosphate synthase according to claim any one of claims  1  or any of  claims 2  to  10  3, 4, 6 and 7 or an enzyme the mutant prenyl diphosphate synthase produced by the method according to  claim 15  into contact with a substrate selected from the group consisting of isopentenyl diphosphate, dimethylallyl diphosphate, and geranyl diphosphate. 
     
     
       17. The mutant prenyl diphosphate synthase of claim 1 having the amino acid sequence of SEQ ID NO:1 except that threonine at position 78 of SEQ ID NO:1 is replaced with phenylalanine and histidine at position 81 of SEQ ID NO:1 is replaced with alanine. 
     
     
       18. The mutant prenyl diphosphate synthase of claim 1 having the amino acid sequence of SEQ ID NO:1 except that threonine at position 78 of SEQ ID NO:1 is replaced with phenylalanine and histidine at position 81 of SEQ ID NO:1 is replaced with leucine. 
     
     
       19. The mutant prenyl diphosphate synthase of claim 1 having the amino acid sequence of SEQ ID NO:1 except that phenylalanine at position 77 of SEQ ID NO:1 is replaced with tyrosine, threonine at position 78 of SEQ ID NO:1 is replaced with phenylalanine, and histidine at position 81 of SEQ ID NO:1 is replaced with leucine. 
     
     
       20. The mutant prenyl diphosphate synthase of claim 1 having the amino acid sequence of SEQ ID NO:1 except that phenylalanine at position 77 of SEQ ID NO:1 is replaced with tyrosine, threonine at position 78 of SEQ ID NO:1 is replaced with phenylalanine, and histidine at position 81 of SEQ ID NO:1 is replaced with alanine. 
     
     
       21. The mutant prenyl diphosphate synthase of claim 1 having the amino acid sequence of SEQ ID NO:1 except that phenylalanine at position 77 of SEQ ID NO:1 is replaced with tyrosine, threonine at position 78 of SEQ ID NO:1 is replaced with serine, valine at position 80 of SEQ ID NO:1 is replaced with isoleucine, isoleucine at position 84 of SEQ ID NO:1 is replaced with leucine, and proline and serine are inserted sequentially between position 84 and position 85 of SEQ ID NO:1. 
     
     
       22. The mutant prenyl diphosphate synthase of claim 1, wherein the mutant synthesizes more farnesyl diphosphate than the wild-type geranylgeranyl diphosphate synthase of Sulfolobus acidocaldarius.