Multigene expression vectors for the biosynthesis of products via multienzyme biological pathways

Abstract

The use of multigene vectors for the preparation of transformed host cells and plants is disclosed. Multigene vectors reduce the number of transformations required, and leads to increased production of polyhydroxyalkanoate polymer in the resulting transformed host cells and plants.

What is claimed is:  
     
         1 . An isolated nucleic acid segment comprising: 
 a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein;    a second nucleic acid sequence encoding a β-ketoacyl reductase protein; and    a third nucleic acid sequence encoding a β-ketothiolase protein.    
     
     
         2 . The isolated nucleic acid segment of  claim 1 , further comprising a fourth nucleic acid sequence encoding a threonine deaminase protein.  
     
     
         3 . The isolated nucleic acid segment of  claim 1 , further comprising a fourth nucleic acid sequence encoding a deregulated threonine deaminase protein.  
     
     
         4 . The isolated nucleic acid segment of  claim 1 , wherein: 
 the first nucleic acid sequence further encodes a chloroplast transit peptide;    the second nucleic acid sequence further encodes a chloroplast transit peptide; and    the third nucleic acid sequence further encodes a chloroplast transit peptide.    
     
     
         5 . A recombinant vector comprising operatively linked in the 5′ to 3′ direction: 
 a promoter that directs transcription of a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence;  
 a first nucleic acid sequence;  
 a second nucleic acid sequence;  
 a third nucleic acid sequence;  
 a 3′ transcription terminator; and  
 a 3′ polyadenylation signal sequence; wherein: 
 the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence encode different proteins; and  
 the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence are independently selected from the group consisting of a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein, a nucleic acid sequence encoding a β-ketoacyl reductase protein, and a nucleic acid sequence encoding a β-ketothiolase protein.  
 
 
     
     
         6 . The recombinant vector of  claim 5 , wherein the promoter directs transcription of the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence in plants.  
     
     
         7 . The recombinant vector of  claim 5 , wherein the promoter is a viral promoter.  
     
     
         8 . The recombinant vector of  claim 5 , wherein the promoter is a CMV 35S promoter, an enhanced CMV 35S promoter, or an FMV 35S promoter.  
     
     
         9 . The recombinant vector of  claim 5 , wherein the promoter is an enhanced CMV 35S promoter.  
     
     
         10 . The recombinant vector of  claim 5 , wherein the promoter is a tissue specific promoter.  
     
     
         11 . The recombinant vector of  claim 5 , wherein the promoter is a Lesquerella hydroxylase promoter or a 7S conglycinin promoter.  
     
     
         12 . The recombinant vector of  claim 5 , wherein the promoter is a Lesquerella hydroxylase promoter.  
     
     
         13 . The recombinant vector of  claim 5 , wherein: 
 the first nucleic acid sequence further encodes a chloroplast transit peptide;    the second nucleic acid sequence further encodes a chloroplast transit peptide; and    the third nucleic acid sequence further encodes a chloroplast transit peptide.    
     
     
         14 . A recombinant vector comprising: 
 a first element comprising operatively linked in the 5′ to 3′ direction: 
 a first promoter that directs transcription of a first nucleic acid sequence;  
 a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein;  
 a first 3′ transcription terminator; and  
 a first 3′ polyadenylation signal sequence;  
   a second element comprising operatively linked in the 5′ to 3′ direction: 
 a second promoter that directs transcription of a second nucleic acid sequence;  
 a second nucleic acid sequence encoding a β-ketoacyl reductase protein;  
 a second 3′ transcription terminator; and  
 a second 3′ polyadenylation signal sequence; and  
   a third element comprising operatively linked in the 5′ to 3′ direction: 
 a third promoter that directs transcription of a third nucleic acid sequence;  
 a third nucleic acid sequence encoding a β-ketothiolase protein;  
 a third 3′ transcription terminator; and  
 a third 3′ polyadenylation signal sequence.  
   
     
     
         15 . The recombinant vector of  claim 14 , wherein the β-ketothiolase protein: 
 catalyzes the condensation of two molecules of acetyl-CoA to produce acetoacetyl-CoA; and  
 catalyzes the condensation of acetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA.  
 
     
     
         16 . The recombinant vector of  claim 14 , wherein the β-ketoacyl reductase protein: 
 catalyzes the reduction of acetoacetyl-CoA to β-hydroxybutyryl-CoA; and  
 catalyzes the reduction of β-ketovaleryl-CoA to β-hydroxyvaleryl-CoA.  
 
     
     
         17 . The recombinant vector of  claim 14 , wherein the polyhydroxyalkanoate synthase protein is selected from the group consisting of: 
 a polyhydroxyalkanoate synthase protein that catalyzes the incorporation of β-hydroxybutyryl-CoA into P(3HB) polymer; and    a polyhydroxyalkanoate synthase protein that catalyzes the incorporation of β-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV) copolymer.    
     
     
         18 . The recombinant vector of  claim 14 , wherein: 
 the β-ketothiolase protein comprises a transit peptide sequence that directs transport of the β-ketothiolase protein to the plastid;    the β-ketoacyl reductase protein comprises a transit peptide sequence that directs transport of the β-ketoacyl reductase protein to the plastid; and    the polyhydroxyalkanoate synthase protein comprises a transit peptide sequence that directs transport of the polyhydroxyalkanoate synthase protein to the plastid.    
     
     
         19 . The recombinant vector of  claim 14 , further comprising a nucleic acid sequence encoding a threonine deaminase protein.  
     
     
         20 . The recombinant vector of  claim 14 , further comprising a nucleic acid sequence encoding a deregulated threonine deaminase protein.  
     
     
         21 . The recombinant vector of  claim 14 , wherein: 
 the first promoter directs transcription of the first nucleic acid sequence in plants;    the second promoter directs transcription of the second nucleic acid sequence in plants; and    the third promoter directs transcription of the third nucleic acid sequence in plants.    
     
     
         22 . The recombinant vector of  claim 14 , wherein the first promoter, second promoter, and third promoter are viral promoters.  
     
     
         23 . The recombinant vector of  claim 14 , wherein: 
 the first promoter is a CMV 35S promoter, an enhanced CMV 35S promoter, or an FMV 35S promoter;    the second promoter is a CMV 35S promoter, an enhanced CMV 35S promoter, or an FMV 35S promoter; and    the third promoter is a CMV 35S promoter, an enhanced CMV 35S promoter, or an FMV 35S promoter.    
     
     
         24 . The recombinant vector of  claim 14 , wherein: 
 the first promoter is an enhanced CMV 35S promoter;    the second promoter is an enhanced CMV 35S promoter; and    the third promoter is an enhanced CMV 35S promoter.    
     
     
         25 . The recombinant vector of  claim 14 , wherein: 
 the first promoter is a tissue specific promoter;    the second promoter is a tissue specific promoter; and    the third promoter is a tissue specific promoter.    
     
     
         26 . The recombinant vector of  claim 14 , wherein: 
 the first promoter is a Lesquerella hydroxylase promoter or a 7S conglycinin promoter;    the second promoter is a Lesquerella hydroxylase promoter or a 7S conglycinin promoter; and    the third promoter is a Lesquerella hydroxylase promoter or a 7S conglycinin promoter.    
     
     
         27 . The recombinant vector of  claim 14 , wherein: 
 the first promoter is a Lesquerella hydroxylase promoter;    the second promoter is a Lesquerella hydroxylase promoter; and    the third promoter is a Lesquerella hydroxylase promoter.    
     
     
         28 . The recombinant vector of  claim 14 , wherein: 
 the first nucleic acid sequence further encodes a chloroplast transit peptide;    the second nucleic acid sequence further encodes a chloroplast transit peptide; and    the third nucleic acid sequence further encodes a chloroplast transit peptide.    
     
     
         29 . A transformed host cell comprising a recombinant vector, wherein the recombinant vector comprises: 
 a first element comprising operatively linked in the 5′ to 3′ direction: 
 a first promoter that directs transcription of a first nucleic acid sequence;  
 a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein;  
 a first 3′ transcription terminator; and  
 a first 3′ polyadenylation signal sequence;  
   a second element comprising operatively linked in the 5′ to 3′ direction: 
 a second promoter that directs transcription of a second nucleic acid sequence;  
 a second nucleic acid sequence encoding a β-ketoacyl reductase protein;  
 a second 3′ transcription terminator; and  
 a second 3′ polyadenylation signal sequence; and  
   a third element comprising operatively linked in the 5′ to 3′ direction: 
 a third promoter that directs transcription of a third nucleic acid sequence;  
 a third nucleic acid sequence encoding a β-ketothiolase protein;  
 a third 3′ transcription terminator; and  
 a third 3′ polyadenylation signal sequence.  
   
     
     
         30 . The transformed host cell of  claim 29 , wherein the transformed host cell is a bacterial cell.  
     
     
         31 . The transformed host cell of  claim 29 , wherein the transformed host cell is a fungal cell.  
     
     
         32 . The transformed host cell of  claim 29 , wherein the transformed host cell is a plant cell.  
     
     
         33 . The transformed host cell of  claim 29 , wherein: 
 the first nucleic acid sequence further encodes a chloroplast transit peptide;    the second nucleic acid sequence further encodes a chloroplast transit peptide; and    the third nucleic acid sequence further encodes a chloroplast transit peptide.    
     
     
         34 . A transformed host cell comprising: 
 a first element comprising operatively linked in the 5′ to 3′ direction: 
 a first promoter that directs transcription of a first nucleic acid sequence;  
 a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein;  
 a first 3′ transcription terminator; and  
 a first 3′ polyadenylation signal sequence;  
   a second element comprising operatively linked in the 5′ to 3′ direction: 
 a second promoter that directs transcription of a second nucleic acid sequence;  
 a second nucleic acid sequence encoding a β-ketoacyl reductase protein;  
 a second 3′ transcription terminator; and  
 a second 3′ polyadenylation signal sequence; and  
   a third element comprising operatively linked in the 5′ to 3′ direction: 
 a third promoter that directs transcription of a third nucleic acid sequence;  
 a third nucleic acid sequence encoding a β-ketothiolase protein;  
 a third 3′ transcription terminator; and  
 a third 3′ polyadenylation signal sequence;  
   wherein the first element, second element, and third element are cointegrated between a single left Ti border sequence and a single right Ti border sequence.    
     
     
         35 . The transformed host cell of  claim 34 , wherein the transformed host cell is a fungal cell.  
     
     
         36 . The transformed host cell of  claim 34 , wherein the transformed host cell is a plant cell.  
     
     
         37 . The transformed host cell of  claim 34 , wherein the transformed host cell is a tobacco, wheat, potato, Arabidopsis, corn, soybean, canola, oil seed rape, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.  
     
     
         38 . The transformed host cell of  claim 34 , wherein: 
 the first nucleic acid sequence further encodes a chloroplast transit peptide;    the second nucleic acid sequence further encodes a chloroplast transit peptide; and    the third nucleic acid sequence further encodes a chloroplast transit peptide.    
     
     
         39 . A transformed plant comprising: 
 a first element comprising operatively linked in the 5′ to 3′ direction: 
 a first promoter that directs transcription of a first nucleic acid sequence;  
 a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein;  
 a first 3′ transcription terminator; and  
 a first 3′ polyadenylation signal sequence;  
   a second element comprising operatively linked in the 5′ to 3′ direction: 
 a second promoter that directs transcription of a second nucleic acid sequence;  
 a second nucleic acid sequence encoding a β-ketoacyl reductase protein;  
 a second 3′ transcription terminator; and  
 a second 3′ polyadenylation signal sequence; and  
   a third element comprising operatively linked in the 5′ to 3′ direction: 
 a third promoter that directs transcription of a third nucleic acid sequence;  
 a third nucleic acid sequence encoding a β-ketothiolase protein;  
 a third 3′ transcription terminator; and  
 a third 3′ polyadenylation signal sequence;  
   wherein the first element, second element, and third element are cointegrated between a single left Ti border sequence and a single right Ti border sequence.    
     
     
         40 . The transformed plant of  claim 39 , wherein the transformed plant is a tobacco, wheat, potato, Arabidopsis, corn, soybean, canola, oil seed rape, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.  
     
     
         41 . The transformed plant of  claim 39 , wherein: 
 the first nucleic acid sequence further encodes a chloroplast transit peptide;    the second nucleic acid sequence further encodes a chloroplast transit peptide; and    the third nucleic acid sequence further encodes a chloroplast transit peptide.    
     
     
         42 . A method of preparing transformed host cells, the method comprising: 
 selecting a host cell;    transforming the selected host cell with a recombinant vector comprising: 
 a first element comprising operatively linked in the 5′ to 3′ direction: 
 a first promoter that directs transcription of the first nucleic acid sequence;  
 a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein;  
 a first 3′ transcription terminator; and  
 a first 3′ polyadenylation signal sequence;  
 
 a second element comprising operatively linked in the 5′ to 3′ direction: 
 a second promoter that directs transcription of the second nucleic acid sequence;  
 a second nucleic acid sequence encoding a β-ketoacyl reductase protein;  
 a second 3′ transcription terminator; and  
 a second 3′ polyadenylation signal sequence; and  
 
 a third element comprising operatively linked in the 5′ to 3′ direction: 
 a third promoter that directs transcription of the third nucleic acid sequence;  
 a third nucleic acid sequence encoding a β-ketothiolase protein;  
 a third 3′ transcription terminator; and  
 a third 3′ polyadenylation signal sequence; and  
 
   obtaining transformed host cells; wherein the transformed host cells produce polyhydroxyalkanoate polymer.    
     
     
         43 . A method of preparing transformed host cells, the method comprising: 
 selecting a host cell;    transforming the selected host cell with a recombinant vector comprising 
 operatively linked in the 5′ to 3′ direction:  
 a promoter that directs transcription of a first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence;  
 a first nucleic acid sequence;  
 a second nucleic acid sequence;  
 a third nucleic acid sequence;  
 a 3′ transcription terminator; and  
 a 3′ polyadenylation signal sequence; and  
   obtaining transformed host cells; wherein: 
 the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence encode different proteins;  
 the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence are independently selected from the group consisting of a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein, a nucleic acid sequence  
   regenerating the transformed host plant cells to produce transformed plants; wherein: 
 the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence encode different proteins;  
 the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence are independently selected from the group consisting of a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein, a nucleic acid sequence encoding a β-ketoacyl reductase protein, and a nucleic acid sequence encoding a β-ketothiolase protein; and  
 the transformed plants produce polyhydroxyalkanoate polymer.  
   
     
     
         46 . A method of producing polyhydroxyalkanoate comprising: 
 obtaining the transformed host cell of  claim 29  or  claim 34;     culturing the transformed host cell under conditions suitable for the production of polyhydroxyalkanoate; and    recovering polyhydroxyalkanoate from the transformed host cell.    
     
     
         47 . The method of  claim 46 , wherein the polyhydroxyalkanoate is poly(3-hydroxybutyrate), poly(4-hydroxybutyrate), or poly(3-hydroxybutyrate-co-4-hydroxybutyrate).  
     
     
         48 . A method of producing polyhydroxyalkanoate comprising: 
 obtaining the transformed plant of  claim 39;     growing the transformed plant under conditions suitable for the production of polyhydroxyalkanoate; and    recovering polyhydroxyalkanoate from the transformed plant.    
     
     
         49 . The method of  claim 48 , wherein the polyhydroxyalkanoate is poly(3-hydroxybutyrate), poly(3-hydroxyvalerate), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate).