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 . A method for synthesizing an isoprenoid or an isoprenoid precursor via a mevalonate pathway in a host cell, wherein the method comprises:
 i) culturing a transformed host cell in a suitable medium, wherein the transformed host cell is a prokaryote that does not normally synthesize isopentenyl pyrophosphate (IPP) through the mevalonate pathway, and wherein the host cell comprises one or more nucleic acids heterologous to the host cell, wherein the one or more heterologous nucleic acids comprises nucleotide sequences that encode two or more mevalonate pathway enzymes, wherein said two or more mevalonate pathway enzymes comprises 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 and one or more additional mevalonate pathway enzymes selected from:
 (a) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA; 
 (b) an enzyme that converts HMG-CoA to mevalonate; 
 (c) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate; 
 (d) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate; and 
 (e) an enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate, said culturing providing for production of the two or more enzymes, resulting in synthesis of said isoprenoid or isoprenoid precursor in a recoverable amount of at least about 1 mg/L; and 
   ii) recovering the produced isoprenoid or isoprenoid precursor.   
     
     
         2 . The method of  claim 1 , wherein the one or more heterologous nucleic acids is integrated into the chromosome of the host cell. 
     
     
         3 . The method of  claim 1 , wherein the one or more heterologous nucleic acids is contained in at least one extrachromosomal expression vector. 
     
     
         4 . The method of  claim 3 , wherein the one or more heterologous nucleic acids is present in a single expression vector. 
     
     
         5 . The method of  claim 3 , wherein each of the one or more heterologous nucleic acids is contained within a separate expression vector. 
     
     
         6 . The method of  claim 3 , wherein at least two of the one or more heterologous nucleic acids are contained in a single expression vector. 
     
     
         7 . The method of  claim 3 , wherein the one or more heterologous nucleic acids is contained in two expression vectors. 
     
     
         8 . A method for synthesizing isopentenyl pyrophosphate (IPP) via a mevalonate pathway in a host microorganism, the method comprising: culturing a transformed host microorganism in a suitable medium, the transformed host microorganism comprising one or more nucleic acids heterologous to the host microorganism, wherein the host microorganism is a prokaryote that does not normally synthesize IPP through the mevalonate pathway, wherein the one or more nucleic acids comprises nucleotide sequences encoding two or more enzymes selected from:
 a) an enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA, wherein said enzyme is from Ralstonia,  Saccharomyces , or  Escherichia coli , wherein said enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA is present as the first step in the synthesis of the IPP;   b) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA, wherein said enzyme is from  Blattella  or  Saccharomyces;      c) an enzyme that converts HMG-CoA to mevalonate, wherein said enzyme is from  Sulfolobus, Haloferax , or  Saccharomyces;      d) a  Saccharomyces  enzyme that phosphorylates mevalonate to mevalonate 5-phosphate;   e) a  Saccharomyces  enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate; and   f) a  Saccharomyces  enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate, said culturing providing for production of the enzymes, wherein said production of said two or more enzymes results in production of IPP.   
     
     
         9 . The method of  claim 1 , wherein the method further comprises reacting isopentenyl pyrophosphate with dimethylallyl pyrophosphate or a polyprenyl pyrophosphate in the presence of at least one enzyme to provide a polyprenyl pyrophosphate isoprenoid precursor. 
     
     
         10 . The method of  claim 9 , wherein the one or more heterologous nucleic acids further comprises:
 g) a nucleic acid comprising a nucleotide sequence coding for an enzyme that converts isopentenyl pyrophosphate to dimethylallyl pyrophosphate.   
     
     
         11 . The method of  claim 1  wherein the isoprenoid precursor is IPP, and wherein the IPP is further modified enzymatically by the action of isopentenyl pyrophosphate isomerase and one or more polyprenyl pyrophosphate synthases to provide an isoprenoid selected from the group consisting of a monoterpene, sesquiterpene, diterpene, sesterterpene, triterpene, tetraterpene, and a steroid. 
     
     
         12 . The method of  claim 11 , wherein the isoprenoid is a monoterpene. 
     
     
         13 . The method of  claim 12 , wherein the monoterpene is selected from the group consisting of limonene, citranellol, and geraniol. 
     
     
         14 . The method of  claim 11 , wherein the isoprenoid is a sesquiterpene. 
     
     
         15 . The method of  claim 14 , wherein the sesquiterpene is selected from the group consisting of periplanone B, arteinisinin, ginkgolide B, forskolin, and farnesol. 
     
     
         16 . The method of  claim 12 , wherein the isoprenoid is a diterpene. 
     
     
         17 . The method of  claim 1 , wherein the prokaryote is  Escherichia coli.    
     
     
         18 . The method of  claim 7 , wherein the prokaryote is  Escherichia coli.    
     
     
         19 . The method of  claim 8 , wherein the prokaryote is  Escherichia coli.    
     
     
         20 . The method of  claim 11 , wherein the prokaryote is  Escherichia coli.    
     
     
         21 . The method of  claim 19 , wherein the enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA is from  E. coli.    
     
     
         22 . The method of  claim 19 , wherein the enzyme that condenses acetyl-CoA with acetoacetyl-CoA is from  Saccharomyces.    
     
     
         23 . A method for synthesizing isopentenyl pyrophosphate (IPP) via a mevalonate pathway in a host microorganism, wherein the method comprises: culturing a transformed host microorganism in a suitable medium, the transformed host microorganism comprising at least two operons heterologous to the host microorganism, wherein each of said two operons comprises nucleotide sequences encoding enzymes in the mevalonate pathway, and wherein the host microorganism is a prokaryote that does not normally synthesize IPP through the mevalonate pathway; wherein the mevalonate pathway comprises:
 (a) an enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA, wherein said enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA is present as the first step in the synthesis of IPP;   (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, said culturing providing for production of the enzymes, wherein said production of said two or more enzymes results in production of IPP.   
     
     
         24 . The method of  claim 23 , wherein said at least two operons are contained in a single extrachromosomal expression vector. 
     
     
         25 . The method of  claim 23 , wherein at least one of said at least two operons is contained in a different extrachromosomal expression vector from another of said at least two operons. 
     
     
         26 . The method of  claim 23 , wherein at least one of said at least two operons is integrated into a chromosome of said transformed host microorganism. 
     
     
         27 . The method of  claim 23 , wherein said transformed host microorganism also comprises a heterologous nucleic acid comprising a nucleotide sequence encoding an enzyme that converts IPP to dimethylallyl pyrophosphate, and the method further comprises reacting the IPP with dimethylallyl pyrophosphate and a polyprenyl pyrophosphate synthase to provide a polyprenyl pyrophosphate isoprenoid precursor. 
     
     
         28 . The method of  claim 23 , wherein said transformed host microorganism is  E. coli.    
     
     
         29 . The method of  claim 24 , wherein said transformed host microorganism is  E. coli.    
     
     
         30 . The method of  claim 25 , wherein said transformed host microorganism is  E. coli.    
     
     
         31 . The method of  claim 26 , wherein said transformed host microorganism is  E. coli.    
     
     
         32 . The method of  claim 28 , wherein said  E. coli  also produces IPP by a DXP pathway. 
     
     
         33 . The method of  claim 28 , wherein
 a) said enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA is from  Ralsionia, Saccharomyces , or  Escherichia coli;      b) said enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA is from  Blattella  or  Saccharomyces;      c) said enzyme that converts HMG-CoA to mevalonate is from  Sulfolobus, Haloferax , or  Saccharomyces ; and   d) said enzymes that phosphorylate mevalonate to mevalonate 5-phosphate, that convert mevalonate 5-phosphate to mevalonate 5-pyrophosphate, and that convert mevalonate 5-pyrophosphate to isopentenyl pyrophosphate, are from  Saccharomyces.      
     
     
         34 . The method of  claim 33 , wherein the enzyme that condenses two molecules of acetyl-CoA to form acetoacetyl-CoA is from  E. coli.    
     
     
         35 . The method of  claim 33 , wherein the enzyme that condenses acetyl-CoA to acetoacetyl-CoA to form HMG-CoA is  Saccharomyces.    
     
     
         36 . The method of  claim 1 , wherein the two or more enzymes are from at least two distinct organisms. 
     
     
         37 . The method of  claim 1 , wherein at least one of the two or more enzymes is from an organism other than  Saccharomyces cerevisiae.    
     
     
         38 . The method of  claim 1 , wherein the one or more nucleic acids comprises nucleotide sequences encoding three enzymes in the mevalonate pathway. 
     
     
         39 . The method of  claim 1 , wherein the one or more nucleic acids comprises nucleotide sequences encoding four enzymes in the mevalonate pathway. 
     
     
         40 . The method of  claim 1 , wherein the one or more nucleic acids comprises nucleotide sequences encoding six enzymes in the mevalonate pathway. 
     
     
         41 . The method of  claim 23 , wherein the IPP is further modified enzymatically by the action of isopentenyl pyrophosphate isomerase to provide dimethylallyl pyrophosphate (DMAPP). 
     
     
         42 . The method of  claim 41 , wherein the DMAPP is further modified enzymatically with one or more polyprenyl pyrophosphate synthases to provide an isoprenoid. 
     
     
         43 . The method of  claim 41 , wherein the isoprenoid is a monoterpene. 
     
     
         44 . The method of  claim 43 , wherein the monoterpene is selected from limonene, citranellol, and geraniol. 
     
     
         45 . The method of  claim 42 , wherein the isoprenoid is a sesquiterpene. 
     
     
         46 . The method of  claim 45 , wherein the sesquiterpene is selected from periplanone B, artemisinin, ginkgolide B, forskolin, and farnesol. 
     
     
         47 . The method of  claim 42 , wherein the isoprenoid is a diterpene. 
     
     
         48 . The method of  claim 42 , wherein the isoprenoid is a triterpene. 
     
     
         49 . The method of  claim 42 , wherein the isoprenoid is a tetraterpene. 
     
     
         50 . The method of  claim 42 , wherein the isoprenoid is a steroid.