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.

We claim:  
     
         1 . A process for the non-fermentative production of KDG or DKG from a carbon source comprising, enzymatically oxidizing the carbon source by at least one oxidative enzymatic activity to yield KDG or DKG.  
     
     
         2 . The process of  claim 1  wherein said KDG is further converted to erythorbate.  
     
     
         3 . The process of  claim 1  comprising oxidizing the carbon source by a first oxidative enzymatic activity to yield a first oxidative product and oxidizing said first oxidative product by a second oxidative enzymatic activity to yield KDG.  
     
     
         4 . The process of  claim 3  wherein said first oxidative enzymatic activity is a GDH activity and said second oxidative enzymatic activity is an GADH activity.  
     
     
         5 . The process of  claim 1  that proceeds in an environment comprising host cells.  
     
     
         6 . The process of  claim 5  wherein said host cell is non-viable.  
     
     
         7 . The process of  claim 5  wherein said host cell is viable.  
     
     
         8 . The process of  claim 5  wherein at least one oxidative enzyme is bound to said host cell membranes.  
     
     
         9 . The process of  claim 1  wherein at least one oxidative enzymatic activity is in solution.  
     
     
         10 . The process of  claim 8  wherein said host cell comprises a mutation in the naturally occurring nucleic acid encoding a KDGDH activity.  
     
     
         11 . The process of  claim 5  wherein said host cell is an member of the family Enterobacteriacea.  
     
     
         12 . The process of  claim 11  wherein said member is a Pantoea species.  
     
     
         13 . The process of  claim 1  wherein at least one oxidative enzymatic activity immobilized.  
     
     
         14 . The process of  claim 3 , further comprising the steps of enzymatically oxidizing the KDG by at least one oxidative enzyme to an oxidation product; and enzymatically reducing said oxidation product by at least one reducing enzyme to 2-KLG.  
     
     
         15 . A process for the non-fermentative production of 2-KLG from a carbon source, comprising the following steps in any order, enzymatically oxidizing the carbon source by at least one oxidative enzymatic activity to an oxidation product; and enzymatically reducing said oxidation product by at least one reducing enzymatic activity to 2-KLG.  
     
     
         16 . The process of  claim 15  wherein said carbon source is KDG.  
     
     
         17 . The process of  claim 15  wherein said oxidative enzymatic activity requires an oxidized form of an enzymatic co-factor and said reducing enzymatic activity requires a reduced form of said enzymatic co-factor and wherein said oxidized from of said co-factor and said reduced form of said co-factor are recycled between at least one oxidizing step and at least one reducing step.  
     
     
         18 . The process of  claim 15  comprising the following steps in any order: 
 a. enzymatically oxidizing the carbon source by a first oxidative enzymatic activity to a first oxidation product;  
 b. enzymatically oxidizing the first oxidation product by a second oxidative enzymatic activity to a second oxidation product;  
 c. enzymatically oxidizing the second oxidation product by a third oxidative enzymatic activity to a third oxidation product; and  
 d. enzymatically reducing the third oxidation product by a reducing enzymatic activity to 2-KLG.  
 
     
     
         19 . The process of  claim 18  wherein at least one of said first, second and third oxidative enzymatic activities requires an oxidized form of an enzymatic co-factor and said reducing enzymatic activity requires a reduced form of said enzymatic co-factor and wherein said oxidized form of said co-factor and said reduced form of said co-factor are recycled between at least one oxidizing step and the reducing step.  
     
     
         20 . The process of  claim 19  wherein said first oxidative enzymatic activity requires an oxidized form of said enzymatic co-factor.  
     
     
         21 . The process of  claim 18  wherein said carbon source is glucose and said first enzymatic activity is a glucose dehydrogenase activity.  
     
     
         22 . The process of  claim 21  wherein said glucose dehydrogenase activity is obtainable from a bacterial, yeast or fungal source.  
     
     
         23 . The process of  claim 22  wherein said glucose dehydrogenase activity is obtainable from a source including  T. acidophilum, Cryptococcus uniguttalatus  and Bacillus species.  
     
     
         24 . The process of  claim 19  wherein each of said first, said second enzyme and said third enzyme is a dehydrogenase activity.  
     
     
         25 . The process of  claim 19  wherein at least one of said first, said second, said third and said fourth enzymatic activities are immobilized.  
     
     
         26 . The process of  claim 19  wherein at least one of said first, said second, said third and said fourth enzymatic activities are in solution.  
     
     
         27 . The process of  claim 25  wherein said second enzyme is a GADH activity.  
     
     
         28 . The process of  claim 25  wherein said third enzyme is KDGDH activity.  
     
     
         29 . The process of  claim 25  wherein said fourth enzyme is a reductase activity.  
     
     
         30 . The process of  claim 29  wherein said reductase activity is obtainable from a bacterial, yeast or fungal source.  
     
     
         31 . The reductase activity of  claim 29  wherein said source includes Corynebacterium and Erwinia.  
     
     
         32 . The process of  claim 31  wherein said reductase activity is 2,5 DKG reductase.  
     
     
         33 . The process of  claim 18  wherein said first oxidation product is gluconate, said second oxidation product is 2-KDG, and said third oxidation product is 2,5-DKG.  
     
     
         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 Enterobacteriacea.  
     
     
         38 . The process of  claim 34  that proceeds in an environment comprising recombinant host cell membranes and wherein at least one of said first, said second and said third 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 mutation of at least one naturally occurring dehydrogenase activity.  
     
     
         42 . The process of  claim 41  wherein said mutation is in a membrane bound GDH activity.  
     
     
         43 . The process of  claim 41  wherein said host cell further comprises nucleic acid encoding a heterologous GDH activity.  
     
     
         44 . The process of  claim 43  wherein said heterologous GDH activity is obtainable from  T. acidophilum, Cryptococcus uniguttalatus,  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 1 ,  claim 15  or  claim 18  that is continuous.  
     
     
         48 . The process of  claim 1 ,  claim 15  or  claim 18  that is batch.  
     
     
         49 . The process of  claim 1 ,  claim 15  or  claim 18  that proceeds in an environment comprising organic solvents.  
     
     
         50 . The process of  claim 1 ,  claim 15  or  claim 18  that proceeds in an environment comprising long polymers.  
     
     
         51 . The process of  claim 14 ,  claim 15  or  claim 18  further comprising the step of obtaining ASA from said 2-KLG.  
     
     
         52 . A host cell comprising nucleic acid having a mutation in the gene encoding GHD activity.  
     
     
         53 . A host cell comprising nucleic acid having a mutation in the gene encoding KDGDH activity.  
     
     
         54 . The host cell of claims  52  or 53 that is a Pantoea species.  
     
     
         55 . The host cell of  claim 52  further comprising nucleic acid encoding a heterologous GDH activity.  
     
     
         56 . The host cell of  claim 55  further comprising nucleic acid encoding a heterologous reductase activity.  
     
     
         57 . The process of  claim 1  optionally comprising the step of recovering said KDG or DKG.  
     
     
         58 . The process of  claim 14 ,  claim 15  or  claim 18  wherein said 2-KLG is further purified via electrodialysis.  
     
     
         59 . The process of  claim 45  or  claim 46  wherein said co-factor is purified via nanofiltration.  
     
     
         60 . The process of  claim 18  that proceeds in an environment comprising salt.  
     
     
         61 . The process of claim  60  wherein the salt includes ammonium sulfate, sodium acetate, ammonium acetate, ammonium cloride, sodium sulfate, potassium phosphate, sodium phosphate, sodium cloride, , KCl, NH 4 Cl, K 2 SO 4  and Nal.  
     
     
         62 . The process of claim  60  that comprises a salt concentration between 0 mM and 500 mM.