Transgenic plants used as a bioreactor system

Abstract

The present invention relates generally to the use of plants as bioreactors for the production of molecules having useful properties such as inter alia polymers, metabolites, proteins, pharmaceuticals and nutraceuticals. More particularly, the present invention contemplates the use of grasses, and even more particularly C4 grasses, such as sugarcane, for the production of a range of compounds such as, for example, polyhydroxyalkanoates, pHBA, vanillin, indigo, adipic acid, 2-phenylethanol, 1,3-propanediol, sorbitol, fructan polymers and lactic acid as well as other products including, inter alia, other plastics, silks, carbohydrates, therapeutic and nutraceutic proteins and antibodies. The present invention further extends to transgenic plants and, in particular, transgenic C4 grass plants, capable of producing the compounds noted above and other products, and to methods for generating such plants. The ability to utilize the high growth rate and efficient carbon fixation of C4 grasses is advantageous, in that it obviates the significant growth penalties observed in other plants, and results in high yields of desired product without necessarily causing concomitant deleterious effects on individual plants. In addition, the C4 grass, sugarcane, is particularly advantageous, as in addition to the features common to all C4 grasses, this plant accumulates sucrose. This sucrose store provides a ready supply of carbon based compounds and energy which may further obviate any deleterious effects on the growth of the plant associated with the production of the product. The present invention provides, therefore, a bioreactor system comprising a genetically modified plant designed to produce particular metabolic or biosynthetic products of interest.

1 - 54 . (canceled) 
     
     
         55 . A method of producing a bioproduct in a genetically modified C4 grass which C4 grass substantially does not produce the bioproduct prior to genetic modification, said method comprising introducing a genetic sequence encoding an enzyme required for synthesis of the bioproduct or a precursor of said bioproduct into a cell or group of cells of said C4 grass, regenerating a C4 grass from said cell or group of cells and growing the C4 grass or genetically modified progeny therefrom under conditions sufficient to produce the bioproduct. 
     
     
         56 . The method of  claim 55  wherein the bioproduct is selected from the list comprising a polyhydroxyalkanoate (PHA), p-hydroxybenzoic acid (pHBA), vanillin, sorbitol and fructan. 
     
     
         57 . The method of  claim 56  wherein the biproduct is polyhydroxyalkanoate (PHA) and the genetic sequence comprises one or more genetic sequences selected from the list comprising:
 (i) a nucleotide sequence encoding a phaA; 
 (ii) a nucleotide sequence encoding phaB; 
 (iii) a nucleotide sequence encoding phaC; 
 (iv) a nucleotide sequence encoding phaC1; 
 (v) a nucleotide sequence encoding phaG; 
 (vi) a nucleotide sequence encoding phaJ; 
 (vii) SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:10 or SEQ ID NO:12 or a nucleotide sequence having at least 60% identity thereto after optimal alignment, or capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:10 or SEQ ID NO:12 or a complementary form thereof under low stringency conditions; 
 (viii) SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:13 or SEQ ID NO:15 or a nucleotide sequence having at least 60% identity thereto after optimal alignment, or capable of hybridizing to SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:13 or SEQ ID NO:15 or a complementary form thereof under low stringency conditions; 
 (ix) SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:16 or SEQ ID NO:18 or a nucleotide sequence having at least 60% identity thereto after optimal alignment, or capable of hybridizing to SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:16 or SEQ ID NO:18 or a complementary form thereof under low stringency conditions; 
 (x) SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:22 or SEQ ID NO:24 or SEQ ID NO:25 or SEQ ID NO:27 or a nucleotide sequence having at least 60% identity thereto after optimal alignment, or capable of hybridizing to SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:22 or SEQ ID NO:24 or SEQ ID NO:25 or SEQ ID NO:27 or a complementary form thereof under low stringency conditions; 
 (xi) SEQ ID NO:28 or SEQ ID NO:30 or a nucleotide sequence having at least 60% identity thereto after optimal alignment, or capable of hybridizing to SEQ ID NO:28 or SEQ ID NO:30 or a complementary form thereof under low stringency conditions; 
 (xii) SEQ ID NO:31 or SEQ ID NO:33 or a nucleotide sequence having at least 60% identity thereto after optimal alignment, or capable of hybridizing to SEQ ID NO:31 or SEQ ID NO:33 or a complementary form thereof under low stringency conditions; 
 
     
     
         58 . The method of  claim 56  wherein the bioproduct is p-hydroxybenzoic acid (pHBA) and the genetic sequence comprises one or more genetic sequences selected from the list comprising a nucleotide sequence encoding hydroxycinnamoyl-CoA hydratase/lysase, a nucleotide sequence encoding chorismate pyruvate lyase, a nucleotide sequence encoding the ubiC gene from  E. coli , a nucleotide sequence encoding the HCHL gene from  Pseudomonas fluorescens    
     
     
         59 . The method of  claim 56  wherein the bioproduct is vanillin and the enzyme encoded by the genetic sequence is selected from the list comprising 3-dehydroshikimate dehyhratase, catechol-O-methyltransferase, aryl aldehyde dehybrogenase, feruloyl-CoA synthetase, enoyl-CoA hydratase and enoyl-CoA aldolase. 
     
     
         60 . The method of  claim 56  wherein the bioproduct is sorbitol and the enzyme encoded by the genetic sequence is glucose-fructose oxidoreductase. 
     
     
         61 . The method of  claim 60  wherein the glucose-fructose oxidoreductases is encoded by the polynucleotide sequence set forth in GenBank Accession number Z80356, or a homolog thereof having at least 60% identity thereto after optimal alignment, or capable of hybridizing to GenBank Accession number Z80356 or a complementary form thereof under low stringency conditions. 
     
     
         62 . The method of  claim 60  wherein the glucose-fructose oxidoreductase is encoded by the polynucleotide sequence set forth in GenBank Accession number M97379, or a homolog thereof having at least 60% identity thereto after optimal alignment, or capable of hybridizing to GenBank Accession number M97379 or a complementary form thereof under low stringency conditions. 
     
     
         63 . The method of  claim 56  wherein the bioproduct is fructan and the enzyme encoded by the genetic sequence is selected from the list comprising fructosyltransferase and levan sucrase. 
     
     
         64 . The method of  claim 63  wherein the fructosyltransferase is encoded by the polynucleotide sequence set forth in GenBank Accession number AY150365, or a homolog thereof having at least 60% identity thereto after optimal alignment, or capable of hybridizing to GenBank Accession number AY150365 or a complementary form thereof under low stringency conditions. 
     
     
         65 . A vector comprising a genetic sequence encoding an enzyme of  claim 55 . 
     
     
         66 . The vector of  claim 65  wherein the vector is an expression vector. 
     
     
         67 . A genetically modified C4 grass cell or group of cells comprising an introduced genetic sequence encoding an enzyme of  claim 55 . 
     
     
         68 . A genetically modified C4 grass cell or group of cells comprising an introduced genetic sequence encoding a vector of  claim 65 . 
     
     
         69 . A genetically modified C4 grass plant comprising a cell or group of cells of  claim 67  or genetically modified progeny thereof. 
     
     
         70 . Seeds or other reproductive material from the plant of  claim 68 . 
     
     
         71 . A product produced in a genetically modified plant or genetically modified cells or parts of a plant by the method of  claim 55 .