US2006263863A1PendingUtilityA1

Manipulation of genes of the mevalonate and isoprenoid pathways to create novel traits in transgenic organisms

59
Individually held — no corporate assignee on recordPriority: Jul 31, 2000Filed: Jul 18, 2006Published: Nov 23, 2006
Est. expiryJul 31, 2020(expired)· nominal 20-yr term from priority
Inventors:Frederick M. HahnAdelheid R. Kuehnle
C12N 9/90C12P 9/00C12N 15/8209C12N 15/52C12N 15/8243C12N 15/8214
59
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Claims

Abstract

Disclosed are the uses of specific genes of the mevalonate and isoprenoid biosynthetic pathways, and of inactive gene sites (the pseudogene) to (1) enhance biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate and isoprenoid pathway derived products in the plastids of transgenic plants and microalgae, (2) create novel antibiotic resistant transgenic plants and microalgae, and (3) create a novel selection system and/or targeting sites for mediating the insertion of genetic material into plant and microalgae plastids. The specific polynucleotides to be used, solely or in any combination thereof, are publicly available from GeneBank and contain open reading frames having sequences that upon expression will produce active proteins with the following enzyme activities: (a) acetoacetyl CoA thiolase (EC 2.3.1.9), (b) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (EC 4.1.3.5), (c) HMG-CoA reductase (EC 1.1.1.34), (d) mevalonate kinase (EC 2.7.1.36), (e) phosphomevalonate kinase (EC 2.7.4.2), (f) mevalonate diphosphate decarboxylase (EC 4.1.1.33), (g) isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2), and (h) phytoene synthase (EC 2.5.1.32).

Claims

exact text as granted — not AI-modified
1 . A method of causing activity of the complete mevalonate pathway in plastids that naturally possess only the non-mevalonate pathway, comprising: 
 providing target cells comprising plastids that naturally possess only the non-mevalonate pathway;    transforming the cells with at least one polynucleotide encoding at least one enzyme of the mevalonate pathway such that expression of the at least one polynucleotide results in the enzymes of the complete mevalonate pathway being present in plastids;    selecting transformed target cells; and    culturing the transformed target cells under conditions whereby additional generations of descendant transformed cells are produced and wherein the complete mevalonate pathway is active in plastids of those transformed cells.    
     
     
         2 . The method of  claim 1 , wherein the at least one polynucleotide comprises an open reading frame encoding IPP isomerase.  
     
     
         3 . The method of  claim 1 , wherein the at least one polynucleotide is a polycistronic polynucleotide encoding all of the enzymes of the complete mevalonate pathway.  
     
     
         4 . The method of  claim 1 , wherein more than one kind of isolated polynucleotide is used to transform the target cell, with no single polynucleotide encoding all enzymes of the complete mevalonate pathway.  
     
     
         5 . The method of  claim 1 , wherein the at least one polynucleotide encodes at least one enzyme attached to a plastid targeting sequence.  
     
     
         6 . The method of  claim 2 , wherein the at least one polynucleotide encodes at least one enzyme attached to a plastid targeting sequence.  
     
     
         7 . The method of  claim 3 , wherein the at least one polynucleotide encodes at least one enzyme attached to a plastid targeting sequence.  
     
     
         8 . The method of  claim 4 , wherein at least one polynucleotide encodes at least one enzyme attached to a plastid targeting sequence.  
     
     
         9 . The method according to  claim 1 , wherein the target cells are microalgae.  
     
     
         10 . The method according to  claim 2 , wherein the target cells are microalgae.  
     
     
         11 . The method according to  claim 3 , wherein the target cells are microalgae.  
     
     
         12 . The method according to  claim 4 , wherein the target cells are microalgae.  
     
     
         13 . The method according to  claim 5 , wherein the target cells are microalgae.  
     
     
         14 . The method according to  claim 6 , wherein the target cells are microalgae.  
     
     
         15 . The method according to  claim 7 , wherein the target cells are microalgae.  
     
     
         16 . The method according to  claim 8 , wherein the target cells are microalgae.  
     
     
         17 . The method according to  claim 1 , wherein the target cells are plant cells.  
     
     
         18 . The method according to  claim 2 , wherein the target cells are plant cells.  
     
     
         19 . The method according to  claim 3 , wherein the target cells are plant cells.  
     
     
         20 . The method according to  claim 4 , wherein the target cells are plant cells.  
     
     
         21 . The method according to  claim 5 , wherein the target cells are plant cells.  
     
     
         22 . The method according to  claim 6 , wherein the target cells are plant cells.  
     
     
         23 . The method according to  claim 7 , wherein the target cells are plant cells.  
     
     
         24 . The method according to  claim 8 , wherein the target cells are plant cells.  
     
     
         25 . The method according to  claim 17 , further comprising regenerating said transformed cells into a transformed plant.  
     
     
         26 . The method according to  claim 18 , further comprising regenerating said transformed cells into a transformed plant.  
     
     
         27 . The method according to  claim 19 , further comprising regenerating said transformed cells into a transformed plant.  
     
     
         28 . The method according to  claim 20 , further comprising regenerating said transformed cells into a transformed plant.  
     
     
         29 . The method according to  claim 21 , further comprising regenerating said transformed cells into a transformed plant.  
     
     
         30 . The method according to  claim 22 , further comprising regenerating said transformed cells into a transformed plant.  
     
     
         31 . The method according to  claim 23 , further comprising regenerating said transformed cells into a transformed plant.  
     
     
         32 . The method according to  claim 24 , further comprising regenerating said transformed cells into a transformed plant.  
     
     
         33 . A method of causing activity of the complete mevalonate pathway in bacteria that naturally possess only the non-mevalonate pathway, comprising: 
 providing target bacterial cells that naturally possess only the non-mevalonate pathway;    transforming the bacteria with at least one polynucleotide encoding at least one enzyme of the mevalonate pathway such that expression of the at least one polynucleotide results in the enzymes of the complete mevalonate pathway being present in the bacteria;    selecting transformed bacteria; and    culturing the transformed bacteria under conditions whereby additional generations of descendant transformed bacteria are produced and wherein the complete mevalonate pathway is active in those transformed bacteria.    
     
     
         34 . The method of  claim 33 , wherein the at least one polynucleotide comprises an open reading frame encoding IPP isomerase.  
     
     
         35 . The method of  claim 33 , wherein the at least one polynucleotide is a polycistronic polynucleotide encoding at least most of the enzymes of the complete mevalonate pathway.  
     
     
         36 . The method of  claim 33 , wherein more than one kind of isolated polynucleotide is used to transform the target cell, with no single polynucleotide encoding all enzymes of the complete mevalonate pathway.  
     
     
         37 . The method of  claim 33 , wherein the bacteria are  E. coli.    
     
     
         38 . The method of  claim 34 , wherein the bacteria are  E. coli.    
     
     
         39 . The method of  claim 35 , wherein the bacteria are  E. coli.    
     
     
         40 . The method of  claim 36 , wherein the bacteria are  E. coli.

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