US2005080248A1PendingUtilityA1

Multimeric oxidoreductases

62
Priority: Jul 30, 2003Filed: Jul 27, 2004Published: Apr 14, 2005
Est. expiryJul 30, 2023(expired)· nominal 20-yr term from priority
Inventors:Robert M. CaldwellM. Harunur RashidFernando Valle
C12P 7/58C12N 15/74C12N 9/002C12P 7/60C12N 9/0004C12N 15/78
62
PatentIndex Score
0
Cited by
0
References
0
Claims

Abstract

The present invention concerns multimeric oxidoreductase complexes which function in the enzymatic conversion of a carbon substrate, said complexes having a dehydrogenase subunit and a cytochrome C subunit. The invention further relates to polynucleotides coding for the multimeric complexes and methods of use thereof.

Claims

exact text as granted — not AI-modified
1 . An isolated polynucleotide coding for a multimeric oxidoreductase complex, said complex having oxidoreductase activity and comprising a cytochrome C subunit and a dehydrogenase subunit, wherein the complex is selected from the subgroups consisting of 
 a) SEQ ID NO: 1 and SEQ ID NO: 20;    b) SEQ ID NO: 8 and SEQ ID NO: 27;    c) SEQ ID NO: 5 and SEQ ID NO: 23;    d) SEQ ID NO: 7 and SEQ ID NO: 28;    e) SEQ ID NO: 4 and SEQ ID NO: 22;    f) SEQ ID NO: 9 and SEQ ID NO: 26;    g) SEQ ID NO: 2 and SEQ ID NO: 21;    h) SEQ ID NO: 10 and SEQ ID NO: 25;    i) SEQ ID NO: 6 and SEQ ID NO: 24;    j) SEQ ID NO: 3 and SEQ ID NO: 29;    k) SEQ ID NO: 18 and SEQ ID NO: 34;    l) SEQ ID NO: 14 and SEQ ID NO: 35;    m) SEQ ID NO: 16 and SEQ ID NO: 31;    n) SEQ ID NO: 19 and SEQ ID NO: 38;    o) SEQ ID NO: 12 and SEQ ID NO: 32;    p) SEQ ID NO: 13 and SEQ ID NO: 37;    q) SEQ ID NO: 15 and SEQ ID NO: 36;    r) SEQ ID NO: 17 and SEQ ID NO: 33;    s) SEQ ID NO: 11 and SEQ ID NO: 30; and    t) a sequence having at least 95% sequence identity to any one of the sequences represented in subgroups a)-t):    
     
     
         2 . The isolated polynucleotide of  claim 1 , wherein said polynucleotide codes for the multimeric complex selected from the subgroup of 
 c) which includes SEQ ID NO. 5 and SEQ ID NO: 23;    e) which includes SEQ ID NO: 4 and SEQ ID NO: 22;    i) which includes SEQ ID NO: 6 and SEQ ID NO: 24;    j) which includes SEQ ID NO: 3 and SEQ ID NO: 29; or    n) which includes SEQ ID NO: 19 and SEQ ID NO: 38 and    a sequence having at least 95% sequence identity to any one of the subgroups c), e), i), j) and n).    
     
     
         3 . The isolated polynucleotide of  claim 2 , wherein the polynucleotide includes a third coding region for the multimeric complex comprising, 
 SEQ ID NO: 41 for subgroup c);    SEQ ID NO: 42 for subgroup e);    SEQ ID NO: 39 for subgroup i);    SEQ ID NO: 40 for subgroup j);    SEQ ID NO: 57 for subgroup n); and    a sequence having at least 95% sequence identity thereto.    
     
     
         4 . A multimeric oxidoreductase complex having oxidoreductase activity and an amino acid sequence comprising a sequence selected from the subgroups consisting of, 
 a) SEQ ID NO: 1 and SEQ ID NO: 20;    b) SEQ ID NO: 8 and SEQ ID NO: 27;    c) SEQ ID NO: 5 and SEQ ID NO: 23;    d) SEQ ID NO: 7 and SEQ ID NO: 28;    e) SEQ ID NO: 4 and SEQ ID NO: 22;    f) SEQ ID NO: 9 and SEQ ID NO: 26;    g) SEQ ID NO: 2 and SEQ ID NO: 21;    h) SEQ ID NO: 10 and SEQ ID NO: 25;    i) SEQ ID NO: 6 and SEQ ID NO: 24;    j) SEQ ID NO: 3 and SEQ ID NO: 29;    k) SEQ ID NO: 18 and SEQ ID NO: 34;    l) SEQ ID NO: 14 and SEQ ID NO: 35;    m) SEQ ID NO: 16 and SEQ ID NO: 31;    n) SEQ ID NO: 19 and SEQ ID NO: 38;    o) SEQ ID NO: 12 and SEQ ID NO: 32;    p) SEQ ID NO: 13 and SEQ ID NO: 37;    q) SEQ ID NO: 15 and SEQ ID NO: 36;    r) SEQ ID NO: 17 and SEQ ID NO: 33;    s) SEQ ID NO: 11 and SEQ ID NO: 30; and    t) a sequence having at least 95% sequence identity to any one of the sequences represented in the subgroups of a)-t).    
     
     
         5 . The multimeric oxidoreductase complex of  claim 4 , wherein said complex is selected from the subgroups of 
 c) which includes SEQ ID NO. 5 and SEQ ID NO: 23;    e) which includes SEQ ID NO: 4 and SEQ ID NO: 22;    i) which includes SEQ ID NO: 6 and SEQ ID NO: 24;    j) which includes SEQ ID NO: 3 and SEQ ID NO: 29; or    n) which includes SEQ ID NO: 19 and SEQ ID NO: 38; and    a sequence having at least 95% identity to any one of the sequences of subgroups c), e), i), j) or n).    
     
     
         6 . The multimeric oxidoreductase complex of  claim 4 , wherein said complex has gluconate dehydrogenase activity or 2-keto-D-gluconate dehydrogenase activity.  
     
     
         7 . An isolated polynucleotide coding for a multimeric oxidoreductase complex which functions in the enzymatic conversion of a carbon substrate to a desired product, said complex having an amino acid sequence comprising an alpha subunit, a gamma subunit and a cytochrome C subunit, wherein 
 the alpha subunit has dehydrogenase activity and is selected from the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO:  16 , SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 and sequences having at least 95% sequence identity thereto;    the gamma subunit is selected from the sequences of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 and sequences having at least 95% sequence identity thereto; and    the cytochrome C subunit is selected from the group of sequences of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and sequences having at least 95% sequence identity thereto.    
     
     
         8 . An isolated polynucleotide coding for a multimeric oxidoreductase complex having oxidoreductase activity and which functions in the enzymatic conversion of glucose to a desired product, said complex comprising an alpha subunit, a gamma subunit and a cytochrome C subunit, wherein 
 the alpha subunit is selected from the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and sequences having at least 95% sequence identity thereto;    the gamma subunit is selected from the sequences of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, and sequences having at least 95% sequence identity thereto; and    the cytochrome C subunit is selected from the group of sequences of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and sequences having at least 95% sequence identity thereto.    
     
     
         9 . The isolated polynucleotide of  claim 8 , wherein the polynucleotide coding for the alpha subunit has the sequence selected from the group of SEQ ID NOs. 59-68.  
     
     
         10 . The isolated polynucleotide of  claim 8 , wherein the polynucleotide coding for the cytochrome C subunit has the sequence selected from the group of SEQ ID NOs. 78-87.  
     
     
         11 . The isolated polynucleotide of  claim 8 , wherein the desired product is an ascorbic acid intermediate.  
     
     
         12 . An isolated polynucleotide encoding a dehydrogenase protein comprising an amino acid sequence selected from the group of SEQ ID NO: 1 through SEQ ID NO: 19 and amino acid sequences having at least 90% sequence identity thereto.  
     
     
         13 . The isolated polynucleotide of  claim 12 , wherein the dehydrogenase has the amino acid sequence selected from the group of SEQ ID NO: 1 through SEQ ID NO: 10.  
     
     
         14 . The isolated polynucleotide of  claim 12 , wherein the dehydrogenase has the amino acid sequence selected from the group of SEQ ID NO: 11 through SEQ ID NO: 19.  
     
     
         15 . An isolated polynucleotide encoding a cytochrome C protein comprising an amino acid sequence selected from the group of SEQ ID NO: 20 through SEQ ID NO: 38 and amino acid sequences having at least 90% sequence identity thereto.  
     
     
         16 . The isolated polynucleotide of  claim 15 , wherein the cytochrome C protein has the amino acid sequence selected from the group of SEQ ID NO: 20 through SEQ ID NO: 29.  
     
     
         17 . The isolated polynucleotide of  claim 15 , wherein the cytochrome C protein has the amino acid sequence selected from the group of SEQ ID NO: 30 through SEQ ID NO: 38.  
     
     
         18 . An isolated polynucleotide encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 39-SEQ ID NO: 57, wherein said protein comprises a subunit in a multimeric oxidoreductase complex.  
     
     
         19 . An isolated multimeric oxidoreductase complex encoded by an operon comprising three coding regions, said operon selected from subgroups, 
 a) SEQ ID NO: 104, SEQ ID NO: 59, and SEQ ID NO: 78;    b) SEQ ID NO: 106, SEQ ID NO: 66 and SEQ ID NO: 85;    c) SEQ ID NO: 99, SEQ ID NO: 63 and SEQ ID NO: 81;    d) SEQ ID NO: 102, SEQ ID NO: 65 and SEQ ID NO: 86;    e) SEQ ID NO: 100, SEQ ID NO: 62 and SEQ ID NO: 80;    f) SEQ ID NO: 103, SEQ ID NO: 67 and SEQ ID NO: 84;    g) SEQ ID NO: 101, SEQ ID NO: 60 and SEQ ID NO: 79;    h) SEQ ID NO: 105, SEQ ID NO: 68 and SEQ ID NO: 83;    i) SEQ ID NO: 97, SEQ ID NO: 64 and SEQ ID NO: 82;    j) SEQ ID NO: 98, SEQ ID NO: 61 and SEQ ID NO: 87;    k) SEQ ID NO: 110, SEQ ID NO: 76 and SEQ ID NO: 92;    l) SEQ ID NO: 113, SEQ ID NO: 72 and SEQ ID NO: 93;    m) SEQ ID NO: 111,SEQ ID NO: 89 and SEQ ID NO: 74;    n) SEQ ID NO: 115, SEQ ID NO: 77 and SEQ ID NO: 96;    o) SEQ ID NO: 70, SEQ ID NO: 108 and SEQ ID NO: 90;    p) SEQ ID NO: 112, SEQ ID NO: 71 and SEQ ID NO: 95;    q) SEQ ID NO: 114, SEQ ID NO: 73 and SEQ ID NO: 94;    r) SEQ ID NO: 109, SEQ ID NO: 75 and SEQ ID NO: 91;    s) SEQ ID NO: 88, SEQ ID NO: 107 and SEQ ID NO: 69; and    t) a sequence having at least 97% sequence identity to any one of the sequences represented in subgroups a)-t).    
     
     
         20 . The isolated multimeric oxidoreductase complex of  claim 19 , wherein the operon comprising three coding regions is selected from subgroups 
 c) which includes SEQ ID NO: 99, SEQ ID NO: 63 and SEQ ID NO: 81;    e) which includes SEQ ID NO: 100, SEQ ID NO: 62 and SEQ ID NO: 80;    i) which includes SEQ ID NO: 97, SEQ ID NO: 64 and SEQ ID NO: 82;    j) which includes SEQ ID NO: 98, SEQ ID NO: 61 and SEQ ID NO: 87;    n) which includes SEQ ID NO: 115, SEQ ID NO: 77 and SEQ ID NO: 96;    and sequences having at least 97% sequence identity to any one of the sequences represented in each subgroup.    
     
     
         21 . A vector comprising a polynucleotide as claimed in claims  1 ,  7  or  8 .  
     
     
         22 . A vector comprising a polynucleotide as claimed in claims  12 ,  15  and  18 .  
     
     
         23 . A vector comprising an operon encoding the multimeric oxidoreductase complex of  claim 19 .  
     
     
         24 . A bacterial host cell transformed with a vector of  claim 21 ,  22  or  23 .  
     
     
         25 . The bacterial host cell of  claim 24 , wherein the bacterial host cell is a Enterobacteriaceae strain.  
     
     
         26 . The bacterial host cell of  claim 25 , wherein the strain is a  Pantoea, Klebsiella, Erwinia  or  E. coli  strain.  
     
     
         27 . The bacterial host cell of  claim 24 , wherein the bacterial host cell is a Pseudomonadacea strain.  
     
     
         28 . A method of enhancing the enzymatic conversion of a polyol substrate in a bacterial host cell comprising, 
 contacting an altered bacterial host cell with a polyol substrate under suitable culture conditions, wherein the altered bacterial host cell is obtained by the transformation of a bacterial host cell with a vector according to  claim 21 , wherein said polynucleotide is expressed in the altered bacterial host cell and    allowing the enzymatic conversion of the polyol substrate to a desired product wherein, the enzymatic conversion of said polyol substrate is enhanced compared to the enzymatic conversion of the polyol substrate from a corresponding unaltered bacterial host cell.    
     
     
         29 . The method according to  claim 28 , wherein the enzymatic conversion of the polyol substrate is an oxidative conversion.  
     
     
         30 . The method according to  claim 28 , wherein the enzymatic conversion of the polyol substrate is a reductive conversion.  
     
     
         31 . A method for producing a desired product comprising, 
 contacting an altered bacterial host cell with a polyol substrate under suitable culture conditions, wherein the altered bacterial host cell is obtained by the transformation of a bacterial host cell with a vector according to  claim 21  and wherein the polynucleotide encoding the multimeric oxidoreductase complex is expressed in the altered bacterial host cell and    producing a desired product from an enzymatic conversion of the polyol substrate by the multimeric oxidoreductase complex to said desired product.    
     
     
         32 . The method according to  claim 31 , wherein the polyol substrate is glucose, sorbitol or glycerol.  
     
     
         33 . The method according to  claim 31 , wherein the desired product is an ascorbic acid (AsA) intermediate.  
     
     
         34 . The method according to  claim 33 , wherein the AsA intermediate is 2-keto-D-gluconate or 2,5-diketo-D-gluconate.  
     
     
         35 . The method according to  claim 31 , wherein the desired product is an organic acid.  
     
     
         36 . The method according to  claim 31 , wherein the host cell is selected from strains of  E. coli, Pantoea, Klebsiella,  and  Erwinia.    
     
     
         37 . The method according to  claim 31  further comprising recovering the desired product.

Cited by (0)

No later patents cite this yet.

References (0)

No backward citations on record.