Production of polyhydroxyalkanoate in plants

Abstract

The invention relates to the genetic manipulation of plants to produce polyhydroxyalkanoate, particularly in the peroxisomes. Methods for producing such polymers in plants and host cells are provided. Such methods find use in producing biodegradable thermoplastics in plants and other organisms. Nucleotide molecules, expression cassettes, and genetically manipulated host cell, plants, plant tissues, and seeds are also provided.

1 . A plant genetically manipulated to produce polyhydroxyalkanoate in its peroxisomes, said plant comprising in its genome: 
 a stably integrated first DNA construct comprising a promoter that drives expression in a plant cell operably linked to a first coding sequence, wherein said first coding sequence encodes a polyhydroxyalkanoate synthase and is operably linked to a nucleotide sequence encoding a peroxisome-targeting signal; and    a stably integrated second DNA construct comprising a promoter that drives expression in a plant cell operably linked to a second coding sequence, wherein said second coding sequence is operably linked to a nucleotide sequence encoding a peroxisome-targeting signal and said second coding sequence is selected from the group consisting of: 
 (a) a nucleotide sequence encoding a 2-enoyl-CoA hydratase that is capable of catalyzing the synthesis of R-(−)-3-hydroxyacyl-CoA;  
 (b) a nucleotide sequence set forth in SEQ ID NO: 21;  
 (c) a nucleotide sequence comprising the 2-enoyl-CoA hydratase domain of a multifunctional protein-2.  
 (d) a nucleotide sequence set forth in SEQ ID NO: 4;  
 (e) a nucleotide sequence set forth in SEQ ID NO: 1; and  
 (f) a nucleotide sequence encoding a multifunctional protein-2, wherein the dehydrogenase activity of said multifunctional protein has been eliminated.  
   
     
     
         2 . The plant of  claim 1 , wherein said promoters are selected from the group consisting of seed-preferred promoters, chemical-regulatable promoters, germination-preferred promoters, and leaf-preferred promoters.  
     
     
         3 . The plant of  claim 1 , wherein said polyhydroxyalkanoate synthase is capable of catalyzing the synthesis of polyhydroxyalkanoate copolymers.  
     
     
         4 . The plant of  claim 3 , wherein said polyhydroxyalkanoate synthase is encoded by a nucleotide sequence selected from the group consisting SEQ ID NOs: 8-12.  
     
     
         5 . The plant of  claim 1  further comprising in its genome a stably integrated third DNA construct comprising a promoter that drives expression in a plant cell operably linked to a third coding sequence, wherein said third coding sequence encodes a 3-ketoacyl-CoA reductase and is operably linked to a nucleotide sequence encoding a peroxisome-targeting signal.  
     
     
         6 . The plant of  claim 1 , wherein said third coding sequence comprises at least a portion of a nucleotide sequence selected from the group consisting of: 
 (a) a nucleotide sequence encoding a 3-ketoacyl-CoA reductase that is capable of utilizing NADH;    (b) a nucleotide sequence set forth in SEQ ID NO: 3.    (c) a nucleotide sequence set forth in SEQ ID NO: 22;    (d) the nucleotide sequence set forth in SEQ ID NO: 1;    (e) a nucleotide sequence encoding a multifunctional protein-2, wherein the hydratase activity of said multifunctional protein has been eliminated;    (f) the nucleotide sequence set forth in SEQ ID NO: 6; and    (g) a nucleotide sequence set forth in SEQ ID NO: 23.    
     
     
         7 . The plant of  claim 5  further comprising in its genome a stably integrated fourth DNA construct comprising a promoter that drives expression in a plant cell operably linked to fourth coding sequence, wherein said fourth coding sequence encodes an acetyl-CoA:acetyl transferase and is operably linked to a nucleotide sequence encoding a peroxisome-targeting signal.  
     
     
         8 . The plant of  claim 7 , wherein said fourth coding sequence comprises at least a portion of the nucleotide sequence set forth in SEQ ID NO: 24.  
     
     
         9 . The plant of  claim 7  further comprising in its genome a fifth DNA construct comprising a promoter that drives expression in a plant cell operably linked to a fifth coding sequence, wherein said fifth coding sequence encodes an NADH kinase or an NAD +  kinase and said fifth coding sequence is operably linked to a nucleotide sequence encoding a peroxisome-targeting signal  
     
     
         10 . The plant of  claim 9 , wherein said fifth coding sequence comprises at least a portion of a nucleotide sequence selected from the group consisting SEQ ID NOs: 25-27.  
     
     
         11 . The plant of  claim 1 , wherein said plant is an oilseed plant.  
     
     
         12 . The plant of  claim 11 , wherein said oilseed plant has been genetically manipulated for the enhanced production of short-chain or modified fatty acids.  
     
     
         13 . The plant of  claim 11 , wherein said oilseed plant comprises in its genome a DNA construct comprising a coding sequence for an acyl-CoA oxidase operably linked to a promoter that drives expression in a plant.