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

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).

1 - 114 . (canceled) 
     
     
         115 . A transformed prokaryotic cell that synthesizes an isoprenoid or an isoprenoid precursor via a mevalonate pathway,
 wherein the transformed prokaryotic cell comprises one or more nucleic acids heterologous to the prokaryotic cell, wherein the one or more heterologous nucleic acids comprise nucleotide sequences that encode:   (a) an enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA as the first step in the synthesis of the isoprenoid or isoprenoid precursor;   (b) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA;   (c) an enzyme that converts HMG-CoA to mevalonate;   (d) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate;   (e) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate; and   (f) an enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate, wherein culturing of said transformed prokaryotic cell in a suitable medium provides for production of the enzymes and synthesis of the isoprenoid or isoprenoid precursor.   
     
     
         116 . The transformed prokaryotic cell of  claim 115 , wherein the one or more heterologous nucleic acids is contained in at least one extrachromosomal expression vector. 
     
     
         117 . The transformed prokaryotic cell of  claim 115 , wherein the one or more heterologous nucleic acids is present in a single expression vector. 
     
     
         118 . The transformed prokaryotic cell of  claim 115 , wherein the isoprenoid precursor is mevalonate. 
     
     
         119 . The transformed prokaryotic cell of  claim 115 , wherein the transformed prokaryotic cell further comprises a heterologous nucleic acid comprising a nucleotide sequence encoding isopentenyl pyrophosphate isomerase. 
     
     
         120 . The transformed prokaryotic cell of  claim 119 , wherein the isoprenoid precursor is IPP, and wherein the IPP is further modified enzymatically by the action of the isopentenyl pyrophosphate isomerase to generate dimethylallyl pyrophosphate (DMAPP). 
     
     
         121 . The transformed prokaryotic cell of  claim 120 , wherein the transformed prokaryotic cell further comprises a heterologous nucleic acid comprising a nucleotide sequence encoding one or more polyprenyl pyrophosphate synthases. 
     
     
         122 . The transformed prokaryotic cell of  claim 121 , wherein the DMAPP is further modified enzymatically with the one or more polyprenyl pyrophosphate synthases to provide an isoprenoid. 
     
     
         123 . The transformed prokaryotic cell of  claim 122 , wherein the isoprenoid is a tetraterpene. 
     
     
         124 . The transformed prokaryotic cell of  claim 122 , wherein the isoprenoid is lycopene. 
     
     
         125 . The transformed prokaryotic cell of  claim 115 , wherein the transformed prokaryotic cell is of the genus  Escherichia.    
     
     
         126 . The transformed prokaryotic cell of  claim 115 , wherein the transformed prokaryotic cell is an  Escherichia coli.    
     
     
         127 . A transformed prokaryotic cell that synthesizes an isoprenoid or an isoprenoid precursor via a mevalonate pathway,
 wherein the transformed prokaryotic cell comprises one or more nucleic acids heterologous to the prokaryotic cell, wherein the one or more heterologous nucleic acids comprise nucleotide sequences that encode:   (a) an enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA as the first step in the synthesis of the isoprenoid or isoprenoid precursor;   (b) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA;   (c) an enzyme that converts HMG-CoA to mevalonate;   (d) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate;   (e) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate;   (f) an enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate (IPP); and   (g) an enzyme that isomerizes IPP to dimethylallyl pyrophosphate (DMAPP); wherein culturing of said transformed prokaryotic cell in a suitable medium provides for production of the enzymes and synthesis of the isoprenoid or isoprenoid precursor.   
     
     
         128 . The transformed prokaryotic cell of  claim 127 , wherein the one or more heterologous nucleic acids is contained in at least one extrachromosomal expression vector. 
     
     
         129 . The transformed prokaryotic cell of  claim 127 , wherein the one or more heterologous nucleic acids is present in a single expression vector. 
     
     
         130 . The transformed prokaryotic cell of  claim 127 , wherein the isoprenoid precursor is mevalonate. 
     
     
         131 . The transformed prokaryotic cell of  claim 127 , wherein the transformed prokaryotic cell further comprises a heterologous nucleic acid comprising a nucleotide sequence encoding isopentenyl pyrophosphate isomerase. 
     
     
         132 . The transformed prokaryotic cell of  claim 131 , wherein the isoprenoid precursor is IPP, and wherein the IPP is further modified enzymatically by the action of the isopentenyl pyrophosphate isomerase to generate dimethylallyl pyrophosphate (DMAPP). 
     
     
         133 . The transformed prokaryotic cell of  claim 132 , wherein the transformed prokaryotic cell further comprises a heterologous nucleic acid comprising a nucleotide sequence encoding one or more polyprenyl pyrophosphate synthases. 
     
     
         134 . The transformed prokaryotic cell of  claim 133 , wherein the DMAPP is further modified enzymatically with the one or more polyprenyl pyrophosphate synthases to provide an isoprenoid. 
     
     
         135 . The transformed prokaryotic cell of  claim 134 , wherein the isoprenoid is a tetraterpene. 
     
     
         136 . The transformed prokaryotic cell of  claim 134 , wherein the isoprenoid is lycopene. 
     
     
         137 . The transformed prokaryotic cell of  claim 127 , wherein the transformed prokaryotic cell is of the genus  Escherichia.    
     
     
         138 . The transformed prokaryotic cell of  claim 127 , wherein the transformed prokaryotic cell is an  Escherichia coli.    
     
     
         139 . A transformed  Escherichia coli  host cell that synthesizes an isoprenoid or an isoprenoid precursor via a mevalonate pathway,
 wherein the transformed  Escherichia coli  host cell comprises one or more nucleic acids heterologous to the  Escherichia coli  host cell, wherein the one or more heterologous nucleic acids comprise nucleotide sequences that encode:   (a) an enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA as the first step in the synthesis of the isoprenoid or isoprenoid precursor;   (b) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA;   (c) an enzyme that converts HMG-CoA to mevalonate;   (d) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate;   (e) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate; and   (f) an enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate, wherein culturing of said transformed  Escherichia coli  host cell in a suitable medium provides for production of the enzymes and synthesis of the isoprenoid or isoprenoid precursor.   
     
     
         140 . A transformed  Escherichia coli  host cell that synthesizes an isoprenoid or an isoprenoid precursor via a mevalonate pathway,
 wherein the transformed  Escherichia coli  host cell comprises one or more nucleic acids heterologous to the  Escherichia coli  host cell, wherein the one or more heterologous nucleic acids comprise nucleotide sequences that encode:   (a) an enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA as the first step in the synthesis of the isoprenoid or isoprenoid precursor;   (b) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA;   (c) an enzyme that converts HMG-CoA to mevalonate;   (d) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate;   (e) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate;   (f) an enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate (IPP); and   (g) an enzyme that isomerizes IPP to dimethylallyl pyrophosphate (DMAPP); wherein culturing of said transformed  Escherichia coli  host cell in a suitable medium provides for production of the enzymes and synthesis of the isoprenoid or isoprenoid precursor.   
     
     
         141 . A transformed cell having increased isoprenoid production as compared to a non-transformed cell, said cell comprising one or more isolated polynucleotides which together encode the enzymes of the complete mevalonate pathway. 
     
     
         142 . The cell of  claim 141 , wherein the cell is a bacterial cell, a plant cell or a microalgae cell. 
     
     
         143 . The cell of  claim 142 , wherein the bacterial cell is an  E. coli  cell. 
     
     
         144 . The cell according to  claim 141 , wherein one of the one or more isolated polynucleotides comprises a polynucleotide sequence encoding isopentenyl pyrophosphate (IPP) isomerase. 
     
     
         145 . A descendant transformed cell of the transformed cell of  claim 141 , wherein said descendant cell exhibits increased isoprenoid production as compared to a non-transformed cell of the same type. 
     
     
         146 . A transformed cell having increased isoprenoid production as compared to a non-transformed cell, wherein said cell is produced by a method of:
 providing one or more isolated polynucleotides which together encode the enzymes of the complete mevalonate pathway;
 providing a plurality of target cells; 
 introducing said isolated polynucleotide(s) into said target cells; 
 selecting target cells which have been transformed with said polynucleotide(s); and 
 growing said transformed cells under conditions whereby additional generations of descendant transformed cells are produced, said transformed cells exhibiting increased isoprenoid production as compared to non-transformed cells of the same type. 
   
     
     
         147 . A cell culture comprising the transformed cell of  claim 146 . 
     
     
         148 . A method of making isopentenyl pyrophosphate (IPP) using the cell of any of  claim 146 . 
     
     
         149 . A method of making isopentenyl pyrophosphate (IPP) via a mevalonate pathway in cells comprising:
 providing a plurality of target cells that do not naturally produce IPP through the mevalonate pathway or that do not naturally possess the mevalonate pathway;   providing one or more isolated polynucleotide(s) which together encode the enzymes of the complete mevalonate pathway;   introducing said isolated polynucleotide(s) into said target cells encoding the enzymes of the complete mevalonate pathway;   selecting target cells which have been transformed with said polynucleotide(s); and   culturing the transformed target cells under conditions whereby additional generations of descendant transformed cells are produced, said transformed cells exhibiting increased IPP production as compared to non-transformed cells of the same type.   
     
     
         150 . The method of  claim 149 , wherein one of the one or more isolated polynucleotides comprises a polynucleotide sequence encoding isopentenyl pyrophosphate (IPP) isomerase. 
     
     
         151 . The method of  claim 150 , wherein the method further comprises converting IPP to dimethylallyl diphosphate (DMAPP). 
     
     
         152 . The method of  claim 149 , wherein the target cells are bacterial cells. 
     
     
         153 . The method of  claim 152 , wherein the bacterial cells are  E. coli  cells. 
     
     
         154 . 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 bacterial 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 the bacterial cells;   selecting the transformed bacterial cells; and   culturing the transformed bacterial cells under conditions whereby additional generations of descendant transformed bacterial cells are produced and wherein the complete mevalonate pathway is active in those transformed bacterial cells.   
     
     
         155 . The method of  claim 154 , wherein the at least one polynucleotide comprises an open reading frame encoding at least one of the group comprising:
 (a) an enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA as the first step in the synthesis of the isoprenoid or isoprenoid precursor;   (b) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA;   (c) an enzyme that converts HMG-CoA to mevalonate;   (d) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate; and   (e) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate.   
     
     
         156 . The method of  claim 155 , wherein the at least one polynucleotide further comprises an open reading frame encoding IPP isomerase. 
     
     
         157 . The method of  claim 156 , wherein the at least one polynucleotide is a polycistronic polynucleotide encoding all of the enzymes of the complete mevalonate pathway. 
     
     
         158 . The method of  claim 154 , 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. 
     
     
         159 . The method of  claim 155 , wherein the bacterial cells are  E. coli  cells.