Method for producing ascorbic acid intermediates

Abstract

The present invention relates to non-fermentative methods for the production of ASA intermediates, KDG, DKG and KLG and methods for the regeneration of co-factor. The invention provides genetically engineered host cells comprising heterologous nucleic acid encoding enzymes useful in the process.

1 - 17 . (canceled)  
     
     
         18 . A process for the non-fermentative production of 2-keto-gulonic acid (2-KLG) from a carbon source, comprising the following steps in any order: 
 a. enzymatically oxidizing the carbon source by a first dehydrogenase enzyme having oxidative activity to a first oxidation product;    b. enzymatically oxidizing the first oxidation product by a second dehydrogenase enzyme having oxidative activity to a second oxidation product;    c. enzymatically oxidizing the second oxidation product by a third dehydrogenase enzyme having oxidative activity to a third oxidation product; and    d. enzymatically reducing the third oxidation product by a reductase enzyme to 2-KLG,    wherein at least one of said first dehydrogenase, said second dehydrogenase and said third dehydrogenase requires an oxidized form of an enzymatic co-factor and the reductase enzyme requires a reduced form of said enzymatic co-factor and the oxidized form of the co-factor and the reduced form of the co-factor are recycled between and coupled to at least one of the oxidizing steps and the reducing step, and    wherein the oxidized form of said co-factor is NADP +  or NAD + .    
     
     
         19 . (canceled)  
     
     
         20 . The process of  claim 18 , wherein sad first dehydrogenase requires the oxidized form of said enzymatic co-factor.  
     
     
         21 . The process of  claim 18 , wherein said carbon source is glucose and said first dehydrogenase is a glucose dehydrogenase.  
     
     
         22 . The process of  claim 21 , wherein said glucose dehydrogenase is obtained from a bacterial, yeast or fungal source.  
     
     
         23 . The process of  claim 22 , wherein said glucose dehydrogenase is obtained from a  Thermophlasma acidophilum , a  Cryptococcus unlguttalafus  or a  Bacililus  species.  
     
     
         24 . (canceled)  
     
     
         25 . The process of  claim 18 , wherein the first dehydrogenase, the second dehydrogenase, the third dehydrogenase or the reductase is immobilized.  
     
     
         26 . The process of  claim 18 , wherein the first dehydrogenase, the second dehydrogenase, the third dehydrogenase or the reductase is in solution.  
     
     
         27 . The process of  claim 25 , wherein said second dehydrogenase enzyme is a gluconate dehydrogenase.  
     
     
         28 . The process of  claim 25 , wherein said third dehydrogenase enzyme is a 2-keto-D-gluconate dehydrogenase.  
     
     
         29 . (canceled)  
     
     
         30 . The process of  claim 18 , wherein said reductase enzyme is obtained from a bacterial, yeast or fungal source.  
     
     
         31 . (canceled)  
     
     
         32 . The process of  claim 18 , wherein said reductase enzyme is a 2,5-diketo-D-gluconate reductase.  
     
     
         33 . The process of  claim 18 , wherein said first oxidation product is gluconate, said second oxidation product is 2-keto-D-gluconate, and said third oxidation product is 2-diketo-D-gluconate.  
     
     
         34 . The process of  claim 18  that proceeds in an environment comprising recombinant host cells.  
     
     
         35 . The process of  claim 34  wherein said host cell is viable.  
     
     
         36 . The process of  claim 34  wherein said host cell is non-viable.  
     
     
         37 . The process of  claim 34 , wherein said recombinant host cells comprise members of Enterobacteriaceae.  
     
     
         38 . The process of  claim 34 , wherein the process proceeds in an environment comprising recombinant host cell membranes and wherein at least one of said first dehydrogenase, said second dehydrogenase and said third dehydrogenase enzymes are bound to said host cell membranes.  
     
     
         39 . The process of  claim 37 , wherein said recombinant host cell is a  Pantoea  species.  
     
     
         40 . The process of  claim 39 , wherein said recombinant host cell is  Pantoea citrea.    
     
     
         41 . The process of  claim 40 , wherein said recombinant host cell has a in a naturally occurring glucose dehydrogenase.  
     
     
         42 . The process of  claim 41 , wherein said mutation is in a membrane bound glucose dehydrogenase.  
     
     
         43 . The process of  claim 41 , wherein said host cell further comprises nucleic acid encoding a heterologous glucose dehydrogenase.  
     
     
         44 . The process of  claim 43 , wherein said heterologous glucose dehydrogenase is obtained from  Thermophlasma acidophilum, Cryptococcus uniquttalatus , or a  Bacillus  species.  
     
     
         45 . The process of  claim 18  wherein said oxidized form of said enzymatic cofactor is NADP+ and said reduced form of said enzymatic cofactor is NADPH.  
     
     
         46 . The process of  claim 18  wherein said oxidized form of said enzymatic cofactor is NAD and said reduced form is NADH.  
     
     
         47 . The process of  claim 18  that is continuous.  
     
     
         48 . The process of  claim 18  that is batch.  
     
     
         49 . The process of  claim 18 , wherein the process further proceeds in an environment comprising organic solvents.  
     
     
         50 . The process of  claim 18 , wherein the process further proceeds in an environment comprising long polymers.  
     
     
         51 . The process of  claim 18  further comprising the step of obtaining ascorbic acid (ASA) from said 2-KLG.  
     
     
         52 - 54 . (canceled)  
     
     
         55 . The host cell of claim  52  further comprising a nucleic acid encoding a heterologous glucose dehydrogenase.  
     
     
         56 . The host cell of  claim 55  further comprising a nucleic acid encoding a heterologous reductase enzyme.  
     
     
         57 . (canceled)  
     
     
         58 . The process of  claim 18 , wherein said 2-KLG is further purified via electrodialysis.  
     
     
         59 . (canceled)  
     
     
         60 . The process of  claim 18 , wherein said process further proceeds in an environment comprising salt.  
     
     
         61 . The process of  claim 60  wherein the salt includes ammonium sulfate, sodium acetate, ammonium acetate, ammonium chloride, sodium sulfate, potassium phosphate, sodium phosphate, sodium chloride, KCl, NH 4 Cl, K 2 SO 4  and NaI.  
     
     
         62 . The process of  claim 60 , wherein the salt concentration is less than or equal to 500 mM.  
     
     
         63 . The process of  claim 30 , wherein said reductase enzyme is obtained from a  Corynebacterium  or  Erwinia  strain.  
     
     
         64 . A process for the non-fermentative production of 2-keto-L-gulonic acid (2-KLG) comprising, 
 a) obtaining  Pantoea  cells which comprise a mutation in a naturally occurring membrane bound glucose dehydrogenase (GDH) gene, wherein said mutation results in the inactivation of the naturally occurring GDH gene product;    b) providing a bioreactor with said  Pantoea  cells, glucose, GDH from a source other than the naturally occurring GDH, and 2,5-diketo-D-gluconate (2,5-DKG) reduce; and    c) allowing enzymatic oxidation by the GDH of step b) and enzymatic reduction by the 2,5-DKG reductase in the bioreactor to yield 2-KLG    wherein the GDH of step b) requires an oxidized form of an enzyme co-factor and the 2,5-DKG reductase requires a reduced form of said enzymatic co-factor and the oxidized co-factor and the reduced-cofactor are recycled between the GDH and the 2,5-DKG reductase, wherein the oxidized form of said cofactor is NADP +  or NAD + .    
     
     
         65 . The process of  claim 64 , wherein the 2,5-DKG reductase is provided in solution.  
     
     
         66 . The process of  claim 64 , wherein the GDH is provided in solution.  
     
     
         67 . The process of  claim 64  wherein the  Pantoea  cells are recombinant cells.  
     
     
         68 . The process of  claim 67 , wherein the  Pantoea  cells comprise a nucleic acid encoding a heterologous 2,5-DKG reductase.  
     
     
         69 . The process of  claim 67 , wherein the  Pantoea  cells comprise a nucleic acid encoding a heterologous glucose dehydrogenase.  
     
     
         70 . The process of  claim 64 , wherein the oxidized co-factor is NAD +  and the reduced co-factor is NADH.  
     
     
         71 . The process of  claim 64 , wherein the oxidized cofactor is NADP +  and the reduced co-factor is NADPH.  
     
     
         72 . The process of  claim 64 , wherein said glucose dehydrogenase is obtained from a  Thermophlasma acidophilum , a  Cryptococcus uniguttalatus  or a  Bacillus  species.  
     
     
         73 . The process of  claim 64 , wherein the  Pantoea  cells are  P. citrea.    
     
     
         74 . The process of  claim 64  that is continuous.  
     
     
         75 . The process of  claim 64  that is batch.  
     
     
         76 . The process of  claim 64  further comprising providing the bioreactor with organic solvents.  
     
     
         77 . The process of  claim 64  further comprising providing the bioreactor with salts.  
     
     
         78 . The process of  claim 77 , wherein the salts are selected from the group consisting of ammonium sulfate, sodium acetate, ammonium acetate, ammonium chloride, sodium sulfate, potassium phosphate, sodium phosphate, sodium chloride, potassium chloride, ammonium chloride and NaI.  
     
     
         79 . The process of  claim 64  further comprising purifying the 2-KLG.