Pichia pastoris formate dehydrogenase and uses therefor

Abstract

This invention relates to a recombinant Pichia pastoris formate dehydrogenase (FDH) enzyme that catalyzes the oxidation of formate to carbon dioxide and the simultaneous reduction of nicotinamide adenine dinucleotide (NAD+) to its reduced form (NADH). Also related are isolated nucleic acids encoding P. pastoris FDH polypeptides, and fragments and variants thereof, as well as vectors and host cells comprising these nucleic acids. Further related are isolated, recombinant P. pastoris FDH polypeptides, and fragments and variants thereof, and antibodies that specifically bind to P. pastoris FDH polypeptides, fragments, or variants. The invention also relates to methods of obtaining isolated P. pastoris FDH nucleic acids, polypeptides, and antibodies, and methods of using P. pastoris FDH in various reactions for industrial or pharmaceutical applications.

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         15 . A recombinant polypeptide comprising amino acid sequence SEQ ID NO:5.  
     
     
         16 . A recombinant polypeptide comprising the NAD-binding domain of amino acid sequence SEQ ID NO:5, wherein said recombinant polypeptide has formate dehydrogenase activity, and wherein the NAD-binding domain of amino acid sequence SEQ ID NO:5 is amino acids 117-309 of SEQ ID NO:5.  
     
     
         17 . A recombinant polypeptide comprising the catalytic domain of amino acid sequence SEQ ID NO:5, wherein said recombinant polypeptide has formate dehydrogenase activity, and wherein the catalytic domain of amino acid sequence SEQ ID NO:5 is amino acids 16-115 of SEQ ID NO:5.  
     
     
         18 . A recombinant polypeptide comprising an amino acid sequence that shares at least 95% sequence identity to the recombinant polypeptide of  claim 15 , wherein said recombinant polypeptide has formate dehyrogenase activity.  
     
     
         19 . A recombinant polypeptide that is a formate dehydrogenase encoded by the nucleotide sequence contained in the plasmid in the ATCC deposit designated PTA-3691.  
     
     
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         22 . A method of producing nicotinamide adenine dinucleotide (NAD+) in a reduced form (NADH) comprising: incubating a recombinant polypeptide of  claim 15  with formate and NAD+ under conditions to allow oxidation of the formate and reduction of the NAD+, thereby producing NADH.  
     
     
         23 . A method of reducing a substrate comprising: incubating a recombinant polypeptide of  claim 15  with formate, nicotinamide adenine dinucleotide (NAD+), substrate, and a reducing enzyme under conditions to allow oxidation of the formate, reduction of the NAD+, and thereby allow reduction of the substrate.  
     
     
         24 . A method of reducing a ketone to produce an alcohol comprising: incubating a recombinant polypeptide of  claim 15  with formate, nicotinamide adenine dinucleotide (NAD+), the ketone and ketone reductase under conditions to allow oxidation of the formate, reduction of the NAD+, and reduction of the ketone, thereby producing the alcohol.  
     
     
         25 . The method of  claim 24  wherein said ketone is a 2-pentanone ketone and said ketone reductase is an isolated  Gluconobacter oxydans  2-keto reductase reducing enzyme having the amino acid sequence as set forth in SEQ ID NO:24.  
     
     
         26 . A recombinant polypeptide of  claim 16 , wherein said recombinant polypeptide further comprises the catalytic domain of amino acid sequence SEQ ID NO:5, and wherein the catalytic domain of amino acid sequence SEQ ID NO:5 is amino acids 16-115 of SEQ ID NO:5.  
     
     
         27 . The recombinant polypeptide of  claim 26 , wherein said recombinant polypeptide shares at least 95% sequence identity with amino acid sequence SEQ ID NO:5.  
     
     
         28 . The recombinant polypeptide of  claim 15 , wherein said recombinant polypeptide consists of amino acid sequence SEQ ID NO:5.  
     
     
         29 . A method of producing nicotinamide adenine dinucleotide (NAD+) in a reduced form (NADH) comprising: incubating a recombinant polypeptide of  claim 16  with formate and NAD+ under conditions to allow oxidation of the formate and reduction of the NAD+, thereby producing NADH.  
     
     
         30 . A method of producing nicotinamide adenine dinucleotide (NAD+) in a reduced form (NADH) comprising: incubating a recombinant polypeptide of  claim 17  with formate and NAD+ under conditions to allow oxidation of the formate and reduction of the NAD+, thereby producing NADH.  
     
     
         31 . A method of producing nicotinamide adenine dinucleotide (NAD+) in a reduced form (NADH) comprising: incubating a recombinant polypeptide of  claim 18  with formate and NAD+ under conditions to allow oxidation of the formate and reduction of the NAD+, thereby producing NADH.  
     
     
         32 . A method of reducing a substrate comprising: incubating a recombinant polypeptide of  claim 16  with formate, nicotinamide adenine dinucleotide (NAD+), substrate, and a reducing enzyme under conditions to allow oxidation of the formate, reduction of the NAD+, and thereby allow reduction of the substrate.  
     
     
         33 . A method of reducing a substrate comprising: incubating a recombinant polypeptide of  claim 17  with formate, nicotinamide adenine dinucleotide (NAD+), substrate, and a reducing enzyme under conditions to allow oxidation of the formate, reduction of the NAD+, and thereby allow reduction of the substrate.  
     
     
         34 . A method of reducing a substrate comprising: incubating a recombinant polypeptide of  claim 18  with formate, nicotinamide adenine dinucleotide (NAD+), substrate, and a reducing enzyme under conditions to allow oxidation of the formate, reduction of the NAD+, and thereby allow reduction of the substrate.  
     
     
         35 . A method of reducing a ketone to produce an alcohol comprising: incubating a recombinant polypeptide of  claim 16  with formate, nicotinamide adenine dinucleotide (NAD+), the ketone and ketone reductase under conditions to allow oxidation of the formate, reduction of the NAD+, and reduction of the ketone, thereby producing the alcohol.  
     
     
         36 . A method of reducing a ketone to produce an alcohol comprising: incubating a recombinant polypeptide of  claim 17  with formate, nicotinamide adenine dinucleotide (NAD+), the ketone and ketone reductase under conditions to allow oxidation of the formate, reduction of the NAD+, and reduction of the ketone, thereby producing the alcohol.  
     
     
         37 . A method of reducing a ketone to produce an alcohol comprising: incubating a recombinant polypeptide of  claim 18  with formate, nicotinamide adenine dinucleotide (NAD+), the ketone and ketone reductase under conditions to allow oxidation of the formate, reduction of the NAD+, and reduction of the ketone, thereby producing the alcohol.