Method of vitamin production

Abstract

The invention provides a method of producing α-tocopherol and α-tocopheryl esters. The method comprises using a biological system to produce farnesol or geranylgeraniol. Then, the farnesol or geranylgernaiol is chemically converted into α-tocopherol or an α-tocopheryl ester.

What is claimed is:  
     
         1 . A method for producing α-tocopherol or α-tocopheryl esters comprising: 
 (a) biologically producing a compound selected from the group consisting of farnesol and geranylgeraniol; and  
 (b) chemically converting said compound into α-tocopherol or an α-tocopheryl ester.  
 
     
     
         2 . The method of  claim 1 , wherein said compound is geranylgeraniol having a first, second, third and fourth olefin moieties and wherein said step of chemically converting comprises: 
 (a) reducing at least one of said second, third and fourth olefin moieties of said geranylgeraniol to form an allylic alcohol; and    (b) forming α-tocopherol or an α-tocopheryl ester from said allylic alcohol and a hydroquinone.    
     
     
         3 . The method of  claim 2 , wherein said reducing step (a) comprises: 
 adding a protecting group to said geranylgeraniol to protect the first olefin moiety of said geranylgeraniol from reduction; and    reducing at least one of the second, third and fourth olefin moieties of said protected geranylgeraniol; and    removing said protecting group from said reduced protected geranylgeraniol to provide said allylic alcohol.    
     
     
         4 . The method of  claim 3 , wherein said protecting group is a hydroxy protecting group.  
     
     
         5 . The method of  claim 4 , wherein said hydroxy protecting group is an ester.  
     
     
         6 . The method of  claim 5 , wherein said ester is selected from the group consisting of isobutyrate and pivaloate.  
     
     
         7 . The method of  claim 2 , wherein said reducing step comprises hydrogenation.  
     
     
         8 . The method of  claim 2 , wherein said allylic alcohol comprises phytol.  
     
     
         9 . The method of  claim 2 , wherein formation of said α-tocopherol or an α-tocopheryl ester comprises contacting said allylic alcohol with an acid in the presence of said hydroquinone.  
     
     
         10 . The method of  claim 2 , wherein said hydroquinone is a trimethyl hydroquinone.  
     
     
         11 . The method of  claim 10 , wherein said trimethyl hydroquinone is 2,3,5-trimethyl hydroquinone.  
     
     
         12 . The method of  claim 1 , wherein said compound is farnesol and wherein said step of chemically converting comprises: 
 (a) converting farnesol to a first intermediate comprising sufficient carbon atoms to form a trimethyltridecyl substituent group of vitamin E when said intermediate is reacted with a hydroquinone to form vitamin E; and    (b) reacting said intermediate compound with a hydroquinone to form vitamin E.    
     
     
         13 . The method of  claim 12 , wherein said converting step comprises: 
 oxidizing said farnesol to farnesal; and    forming a methyl ketone from said farnesal.    
     
     
         14 . The method of  claim 13 , further comprising: 
 reducing olefin moieties in said methyl ketone to form an alkyl methyl ketone; and    forming an allylic alcohol from said alkyl methyl ketone.    
     
     
         15 . The method of  claim 12 , wherein said intermediate comprises isophytol.  
     
     
         16 . The method of  claim 12 , wherein said hydroquinone is a trimethyl hydroquinone.  
     
     
         17 . The method of  claim 16 , wherein said trimethyl hydroquinone is 2,3,5-trimethyl hydroquinone.  
     
     
         18 . The method of  claim 1 , wherein said compound is farnesol and wherein said step of chemically converting comprises: 
 (a) oxidizing said farnesol to farnesal;    (b) forming a methyl ketone from said farnesal;    (c) reducing olefin moieties in said methyl ketone to form an alkyl methyl ketone;    (d) forming an allylic alcohol from said alkyl methyl ketone, wherein said allylic alcohol comprises a sufficient number of carbon atoms to form at least a trimethyltridecyl substituent group of vitamin E when vitamin E is formed from said allylic alcohol and a corresponding hydroquinone; and    (e) forming vitamin E from said compound and a hydroquinone.    
     
     
         19 . The method of  claim 18 , wherein said allylic alcohol comprises isophytol.  
     
     
         20 . The method of  claim 18 , wherein said hydroquinone is a trimethyl hydroquinone.  
     
     
         21 . The method of  claim 20 , wherein said trimethyl hydroquinone is 2,3,5-trimethyl hydroquinone.  
     
     
         22 . The method of  claim 1 , wherein said compound is farnesol.  
     
     
         23 . The method of  claim 1 , wherein said compound is geranylgeraniol.  
     
     
         24 . A method for producing α-tocopherol or α-tocopheryl esters comprising: 
 (a) culturing a microorganism in a fermentation medium to produce a product selected from the group consisting of farnesyl phosphate and farnesol, wherein the action of squalene synthase of said microorganism is reduced;  
 (b) recovering said product; and  
 (c) chemically converting said product into α-tocopherol or an α-tocopheryl ester.  
 
     
     
         25 . The method of  claim 24 , wherein said fermentation medium comprises a squalene synthase inhibitor.  
     
     
         26 . The method of  claim 24 , wherein said microorganism is genetically modified to decrease the action of squalene synthase.  
     
     
         27 . The method of  claim 26 , wherein said microorganism is further genetically modified to increase the action of HMG-CoA reductase.  
     
     
         28 . The method of  claim 27 , wherein the action of HMG-CoA reductase is increased by overexpression of HMG-CoA reductase or the catalytic domain thereof in the microorganism.  
     
     
         29 . The method of  claim 28 , wherein said microorganism is further genetically modified to increase the action of a protein selected from the group consisting of acetoacetyl Co-A thiolose, HMG-CoA synthase, mevalonate kinase, phosphomevalonate kinase, phosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthase, D-1-deoxyxylulose 5-phosphate synthase, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase.  
     
     
         30 . The method of  claim 29 , wherein the microorganism has been genetically modified to increase the action of farnesyl pyrophosphate synthase.  
     
     
         31 . The method of  claim 30 , wherein the microorganism has been genetically modified to overexpress farnesyl pyrophosphate synthase.  
     
     
         32 . The method of  claim 24 , wherein said microorganism is an erg9 mutant.  
     
     
         33 . The method of  claim 32 , wherein said microorganism comprises a erg9Δ::HIS3 deletion/insertion allele.  
     
     
         34 . The method of  claim 24 , wherein said recovering step comprises recovering said product from said microorganism.  
     
     
         35 . The method of  claim 24 , wherein said product is secreted into said fermentation medium by said microorganism and wherein said step of recovering comprises purification of said product from said fermentation medium.  
     
     
         36 . The method of  claim 24 , wherein said product is intracellular farnesyl phosphate and farnesol and said step of recovering comprises isolating said farnesyl phosphate and farnesol from said microorganism.  
     
     
         37 . The method of  claim 24 , wherein said product is intracellular farnesyl phosphate and said step of recovering further comprises dephosphorylating said farnesyl phosphate to produce farnesol.  
     
     
         38 . The method of  claim 24 , wherein said microorganism is a fungi.  
     
     
         39 . The method of  claim 38 , wherein said fungi has been genetically modified to express at least a portion of the enzymes in the mevalonate independent pathway.  
     
     
         40 . The method of  claim 39 , wherein said fungi has been genetically modified to express an enzyme selected from the group consisting of D-1-deoxyxylulose 5-phosphate synthase, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase.  
     
     
         41 . The method of  claim 38 , wherein said fungi is a yeast and said yeast is blocked in the ergosterol pathway and is genetically modified to take up exogenous sterols under aerobic conditions.  
     
     
         42 . The method of  claim 24 , wherein said microorganism is a bacteria.  
     
     
         43 . The method of  claim 42 , wherein said bacteria has been genetically modified to express at least a portion of the enzymes in the mevalonate pathway.  
     
     
         44 . The method of  claim 43 , wherein said bacteria has been genetically modified to express an enzyme selected from the group consisting of acetoacetyl Co-A thiolose, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and phosphomevalonate decarboxylase.  
     
     
         45 . The method of  claim 24 , wherein said microorganism is a microalgae.  
     
     
         46 . The method of  claim 45 , wherein said microalgae is selected from the group consisting of Chlorella and Prototheca.  
     
     
         47 . The method of  claim 24 , wherein said microorganism has been genetically modified to increase phosphatase action.  
     
     
         48 . A method for producing α-tocopherol or α-tocopheryl esters comprising: 
 (a) culturing a microorganism in a fermentation medium to produce a product selected from the group consisting of geranylgeranyl phosphate and geranylgeraniol, wherein the action of squalene synthase of said microorganism is reduced;  
 (b) recovering said product; and  
 (c) chemically converting said geranylgeraniol into α-tocopherol or an α-tocopheryl ester.  
 
     
     
         49 . The method of  claim 48 , wherein said fermentation medium comprises a squalene synthase inhibitor.  
     
     
         50 . The method of  claim 48 , wherein said microorganism is genetically modified to decrease the action of squalene synthase.  
     
     
         51 . The method of  claim 50 , wherein said microorganism is further genetically modified to increase the action of HMG-CoA reductase.  
     
     
         52 . The method of  claim 48 , wherein the action of HMG-CoA reductase is increased by overexpression of HMG-CoA reductase or the catalytic domain thereof in the microorganism.  
     
     
         53 . The method of  claim 52 , wherein said microorganism is further genetically modified to increase the action of a protein selected from the group consisting of acetoacetyl Co-A thiolose, HMG-CoA synthase, mevalonate kinase, phosphomevalonate kinase, phosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthase, geranylgeranyl pyrophosphate synthase, D-1-deoxyxylulose 5-phosphate synthase, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase.  
     
     
         54 . The method of  claim 53 , wherein the microorganism has been genetically modified to increase the action of farnesyl pyrophosphate synthase.  
     
     
         55 . The method of  claim 54 , wherein the microorganism has been genetically modified to overexpress farnesyl pyrophosphate synthase.  
     
     
         56 . The method of  claim 53 , wherein the microorganism has been genetically modified to increase the action of geranylgeranyl pyrophosphate synthase.  
     
     
         57 . The method of  claim 54 , wherein the microorganism has been genetically modified to overexpress geranylgeranyl pyrophosphate synthase.  
     
     
         58 . The method of  claim 48 , wherein said microorganism is an erg9 mutant.  
     
     
         59 . The method of  claim 58 , wherein said microorganism comprises a erg9Δ::HIS3 deletion/insertion allele.  
     
     
         60 . The method of  claim 48 , wherein said recovering step comprises recovering said product from said microorganism.  
     
     
         61 . The method of  claim 48 , wherein said product is secreted into said fermentation medium by said microorganism and wherein said step of recovering comprises purification of said product from said fermentation medium.  
     
     
         62 . The method of  claim 48 , wherein said product is intracellular geranylgeranyl phosphate and geranylgeraniol and said step of recovering comprises isolating said geranylgeranyl phosphate and geranylgeraniol from said microorganism.  
     
     
         63 . The method of  claim 48 , wherein said product is intracellular geranylgeranyl phosphate and said step of recovering further comprises dephosphorylating said geranylgeranyl phosphate to produce geranylgeraniol.  
     
     
         64 . The method of  claim 48 , wherein said microorganism is a fungi.  
     
     
         65 . The method of  claim 64 , wherein said fungi has been genetically modified to express at least a portion of the enzymes in the mevalonate independent pathway.  
     
     
         66 . The method of  claim 65 , wherein said fungi has been genetically modified to express an enzyme selected from the group consisting of D-1-deoxyxylulose 5-phosphate synthase, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase.  
     
     
         67 . The method of  claim 64 , wherein said fungi is a yeast and said yeast is blocked in the ergosterol pathway and is genetically modified to take up exogenous sterols under aerobic conditions.  
     
     
         68 . The method of  claim 48 , wherein said microorganism is a bacteria.  
     
     
         69 . The method of  claim 68 , wherein said bacteria has been genetically modified to express at least a portion of the enzymes in the mevalonate pathway.  
     
     
         70 . The method of  claim 69 , wherein said bacteria has been genetically modified to express an enzyme selected from the group consisting of acetoacetyl Co-A thiolose, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and phosphomevalonate decarboxylase.  
     
     
         71 . The method of  claim 48 , wherein said microorganism is a microalgae.  
     
     
         72 . The method of  claim 71 , wherein said microalgae is selected from the group consisting of Chlorella and Prototheca.  
     
     
         73 . The method of  claim 48 , wherein said microorganism has been genetically modified to increase phosphatase action.  
     
     
         74 . A method for producing α-tocopherol or α-tocopheryl esters comprising: 
 (a) biologically producing a first compound selected from the group consisting of geranylgeraniol and geranylgeranyl pyrophosphate;  
 (b) contacting said first compound with a geranylgeranyl reductase to form a second compound selected from the group consisting of phytol and phytyl diphosphate; and  
 (c) chemically converting said second compound into α-tocopherol or an α-tocopheryl ester.  
 
     
     
         75 . A method, as claimed in  claim 74 , wherein said step of contacting is conducted in vivo using a microorganism having geranylgeranyl reductase activity.  
     
     
         76 . A method, as claimed in  claim 74 , wherein said step of contacting is conducted in a biotranformation process.  
     
     
         77 . A method, as claimed in  claim 74 , wherein said first compound is geranylgeraniol and said second compound is phytol.  
     
     
         78 . A method, as claimed in  claim 74 , wherein said first compound is geranylgeranyl pyrophosphate and said second compound is phytyl diphosphate.  
     
     
         79 . A method, as claimed in  claim 74 , wherein said step of chemically converting comprises reacting said second compound or a derivative thereof with a hydroquinone to form α-tocopherol or an α-tocopheryl ester.  
     
     
         80 . A method, as claimed in  claim 74 , wherein said step of contacting comprises purifying said first compound and transforming said first compound with isolated geranylgeranyl reductase or microorganisms having geranylgeranyl reductase activity.  
     
     
         81 . The method of  claim 80 , wherein said microorganism is further genetically modified to increase the action of geranylgeranyl reductase.  
     
     
         82 . The method of  claim 81 , wherein the action of geranylgeranyl reductase is increased by overexpression of geranylgeranyl reductase.  
     
     
         83 . A method for producing a compound selected from the group consisting of farnesol and geranylgeraniol comprising: 
 (a) culturing a microorganism in a fermentation medium comprising a compound selected from the group consisting of isoprenol and prenol to produce a product selected from the group consisting of farnesyl phosphate, farnesol, geranylgeranyl phosphate and geranylgeraniol; and    (b) recovering said product.    
     
     
         84 . The method of  claim 83 , wherein said microorganism is further genetically modified to increase the action of dimethylallyl transferase.  
     
     
         85 . The method of  claim 84 , wherein the action of dimethylallyl transferase is increased by overexpression of dimethylallyl transferase in the microorganism.  
     
     
         86 . The method of  claim 85 , wherein the microorganism has been genetically modified to increase the action of farnesyl pyrophosphate synthase.  
     
     
         87 . The method of  claim 86 , wherein the microorganism has been genetically modified to overexpress farnesyl pyrophosphate synthase.  
     
     
         88 . The method of  claim 85 , wherein the microorganism has been genetically modified to increase the action of geranylgeranyl pyrophosphate synthase.  
     
     
         89 . The method of  claim 86 , wherein the microorganism has been genetically modified to overexpress geranylgeranyl pyrophosphate synthase.  
     
     
         90 . The method of  claim 83 , wherein the microorganism has been genetically modified to increase the action of an enzyme selected from the group consisting of isoprenol kinase and prenol kinase.  
     
     
         91 . The method of  claim 83 , wherein the microorganism has been genetically modified to overexpress an enzyme selected from the group consisting of isoprenol kinase and prenol kinase.  
     
     
         92 . The method of  claim 83 , wherein said microorganism is an erg9 mutant.