Methods of increasing the production of cobalamins using cob gene expression

Abstract

Novel polypeptides involved in the biosynthesis of cobalamines and/or cobamides, in particular coenzyme B 12 , genetic material responsible for expressing these polypeptides, and a method for preparing them, are described. A method for amplifying the production of cobalamines, and particularly coenzyme B 12 , using recombinant DNA techniques, are also described.

1 - 57 . (canceled)  
     
     
         58 . A method for increasing the production of cobalamins, cobamides, cobalamin precursors, or cobamide precursors, wherein said method comprises: 
 a) introducing a plasmid comprising a DNA sequence selected from the group consisting of the cobA, cobB, cobC, cobD, cobE, cobF, cobG, cobH, cobI, cobJ, cobK, cobL, cobM, cobN, cobO, cobP, cobQ, cobS, cobT, cobU, cobV, cobW, and cobX genes of  P. denitrificans  and homologs of said genes resulting from the degeneracy of the genetic code into a microorganism capable of producing cobalamins or cobamides;    b) culturing said microorganism under conditions suitable for the synthesis of cobalamins, cobamides, cobalamin precursors, or cobamide precursors, wherein said culture conditions are also suitable for expression of said DNA; and    c) recovering the cobalamins, cobamides, cobalamin precursors, or cobamide precursors produced.    
     
     
         59 . The method of  claim 58 , wherein said recovery step comprises: 
 a) solubilization;    b) conversion to a cyanoform; and    c) purification.    
     
     
         60 . The method of  claim 58 , wherein said cobalamin is coenzyme B 12 .  
     
     
         61 . The method of  claim 58 , wherein said cobalamin precursor or cobamide precursor is selected from the group consisting of decobaltocorrinoids and corrinoids.  
     
     
         62 . A method for increasing the production of cobalamins, cobamides, cobalamin precursors, or cobamide precursors, wherein said method comprises: 
 a) introducing a DNA selected from the group consisting of the cobA, cobB, cobC, cobD, cobE, cobF, cobG, cobH, cobI, cobJ, cobK, cobL, cobM, cobN, cobO, cobP, cobQ, cobS, cobT, cobU, cobV, cobW, and cobX genes of  P. denitrificans  and homologs of said genes resulting from the degeneracy of the genetic code into a microorganism capable of producing cobalamins or cobamides;    b) culturing said microorganism under conditions suitable for the synthesis of cobalamins, cobamides, cobalamin precursors, or cobamide precursors, wherein said culture conditions are also suitable for expression of said DNA; and    c) recovering the cobalamins, cobamides, cobalamin precursors, or cobamide precursors produced.    
     
     
         63 . The method of  claim 62 , wherein said recovery step comprises: 
 a) solubilization;    b) conversion to a cyanoform; and    c) purification.    
     
     
         64 . The method of  claim 62 , wherein said cobalamin is coenzyme B 12 .  
     
     
         65 . The method of  claim 62 , wherein said cobalamin precursor or cobamide precursor is selected from the group consisting of decobaltocorrinoids and corrinoids.  
     
     
         66 . The method of  claim 58  or  62  wherein said microorganism is  P. denitrificans  strain SC510 RifR.  
     
     
         67 . A method for increasing the industrial production of cobalamins, cobamides, cobalamin precursors, or cobamide precursors, wherein said method comprises: 
 a) introducing at least one plasmid comprising a DNA sequence selected from the group consisting of cobA, cobB, cobC, cobD, cobE, cobF, cobG, cobH, cobI, cobJ, cobK, cobL, cobM, cobN, cobO, cobP, cobQ, cobS, cobT, cobU, cobV, cobW, and cobX genes of  P. denitrificans  and homologs of said genes resulting from the degeneracy of the genetic code into a microorganism producing cobalamins or cobamides;    b) culturing said microorganism under conditions suitable for the synthesis of cobalamins, cobamides, cobalamin precursors, or cobamide precursors, wherein industrial production comprises culture conditions suitable for expression of said DNA and suitable for production of at least 100 grams of cells; and    c) recovering the cobalamins, cobamides, cobalamin precursors, or cobamide precursors produced,    wherein the industrial production of cobalamins, cobamides, cobalamin precursors, or cobamide precursors by said microorganism is increased by the introduction of said plasmid.    
     
     
         68 . The method of  claim 67 , wherein said host cell is selected from  Pseudomonas denitrificans, Rhizobium meliloti,  and  Agrobacterium tumefaciens.    
     
     
         69 . The method of  claim 68 , wherein said microorganism is  P. denitrificans  strain SC510 RifR.  
     
     
         70 . The method of any one of claims  67 - 69 , wherein said cobalamin is coenzyme B 12 .  
     
     
         71 . The method of  claim 67 , wherein said cobalamin precursor or cobamide precursor is selected from the group consisting of decobaltocorrinoids and corrinoids.  
     
     
         72 . The method of  claim 67 , wherein said at least one plasmid comprises the cobF, cobG, cobH, cobI, cobJ, cobK, cobL, and cobM genes of  P. denitrificans  or homologs of said genes resulting from the degeneracy of the genetic code.  
     
     
         73 . The method of  claim 72 , wherein said host cell is selected from  Pseudomonas denitrificans, Rhizobium meliloti,  and  Agrobacterium tumefaciens.    
     
     
         74 . The method of  claim 73 , wherein said microorganism is  P. denitrificans  strain SC510 RifR.  
     
     
         75 . The method of any one of claims  72 - 74 , wherein said cobalamin is coenzyme B 12 .  
     
     
         76 . The method of  claim 72 , wherein said cobalamin precursor or cobamide precursor is selected from the group consisting of decobaltocorrinoids and corrinoids.  
     
     
         77 . The method of  claim 72 , wherein said at least one plasmid further comprises the cobA and cobE genes of  P. denitrificans  or homologs of said genes resulting from the degeneracy of the genetic code.  
     
     
         78 . The method of  claim 77 , wherein said host cell is selected from  Pseudomonas denitrificans, Rhizobium meliloti,  and  Agrobacterium tumefaciens.    
     
     
         79 . The method of  claim 78 , wherein said microorganism is  P. denitrificans  strain SC510 RifR.  
     
     
         80 . The method of any one of claims  77 - 79 , wherein said cobalamin is coenzyme B 12 .  
     
     
         81 . The method of  claim 77 , wherein said cobalamin precursor or cobamide precursor is selected from the group consisting of decobaltocorrinoids and corrinoids.  
     
     
         82 . The method of  claim 77 , wherein said at least one plasmid further comprises the cobA and cobE genes of  P. denitrificans  or homologs of said genes resulting from the degeneracy of the genetic code.  
     
     
         83 . The method of  claim 82 , wherein said host cell is selected from  Pseudomonas denitrificans, Rhizobium meliloti,  and  Agrobacterium tumefaciens.    
     
     
         84 . The method of  claim 83 , wherein said microorganism is  P. denitrificans  strain SC510 RifR.  
     
     
         85 . The method of any one of claims  82 - 84 , wherein said cobalamin is coenzyme B 12 .  
     
     
         86 . The method of  claim 82 , wherein said cobalamin precursor or cobamide precursor is selected from the group consisting of decobaltocorrinoids and corrinoids.  
     
     
         87 . A method for increasing the industrial production of cobalamins, cobamides, cobalamin precursors, or cobamide precursors, wherein said method comprises: 
 a) introducing at least one plasmid comprising a DNA sequence selected from the group consisting of cobA, cobB, cobC, cobD, cobE, cobF, cobG, cobH, cobI, cobJ, cobK, cobL, cobM, cobN, cobO, cobP, cobQ, cobS, cobT, cobU, cobV, cobW, and cobX genes of  P. denitrificans  and homologs of said genes resulting from the degeneracy of the genetic code into a microorganism capable of producing cobalamins or cobamides;    b) culturing said microorganism under conditions suitable for the synthesis of cobalamins, cobamides, cobalamin precursors, or cobamide precursors, wherein industrial production comprises culture conditions suitable for expression of said DNA and suitable for production of at least 100 grams of cells; and    c) recovering the cobalamins, cobamides, cobalamin precursors, or cobamide precursors produced,    wherein the industrial production of cobalamins, cobamides, cobalamin precursors, or cobamide precursors by said microorganism is increased by the introduction of said plasmid.    
     
     
         88 . The method of  claim 87 , wherein said host cell is selected from  Pseudomonas denitrificans, Rhizobium meliloti,  and  Agrobacterium tumefaciens.    
     
     
         89 . The method of  claim 88 , wherein said microorganism is  P. denitrificans  strain SC510 RifR.  
     
     
         90 . The method of any one of claims  87 - 89 , wherein said cobalamin is coenzyme B 12 .  
     
     
         91 . The method of  claim 87 , wherein said cobalamin precursor or cobamide precursor is selected from the group consisting of decobaltocorrinoids and corrinoids.  
     
     
         92 . The method of  claim 87 , wherein said at least one plasmid comprises the cobF, cobG, cobH, cobI, cobJ, cobK, cobL, and cobM genes of  P. denitrificans  or homologs of said genes resulting from the degeneracy of the genetic code.  
     
     
         93 . The method of  claim 92 , wherein said host cell is selected from  Pseudomonas denitrificans, Rhizobium meliloti,  and  Agrobacterium tumefaciens.    
     
     
         94 . The method of  claim 93 , wherein said microorganism is  P. denitrificans  strain SC510 RifR.  
     
     
         95 . The method of any one of claims  92 - 94 , wherein said cobalamin is coenzyme B 12 .  
     
     
         96 . The method of  claim 92 , wherein said cobalamin precursor or cobamide precursor is selected from the group consisting of decobaltocornrinoids and corrinoids.  
     
     
         97 . The method of  claim 92 , wherein said at least one plasmid further comprises the cobA and cobE genes of  P. denitrificans  or homologs of said genes resulting from the degeneracy of the genetic code.  
     
     
         98 . The method of  claim 97 , wherein said host cell is selected from  Pseudomonas denitrificans, Rhizobium meliloti,  and  Agrobacterium tumefaciens.    
     
     
         99 . The method of  claim 98 , wherein said microorganism is  P. denitrificans  strain SC510 RifR.  
     
     
         100 . The method of any one of claims  97 - 99 , wherein said cobalamin is coenzyme B 12 .  
     
     
         101 . The method of  claim 97 , wherein said cobalamin precursor or cobamide precursor is selected from the group consisting of decobaltocorrinoids and corrinoids.  
     
     
         102 . The method of  claim 87 , wherein said at least one plasmid further comprises the cobA and cobE genes of  P. denitrificans  or homologs of said genes resulting from the degeneracy of the genetic code.  
     
     
         103 . The method of  claim 102 , wherein said host cell is selected from  Pseudomonas denitrificans, Rhizobium meliloti,  and  Agrobacterium tumefaciens.    
     
     
         104 . The method of  claim 103 , wherein said microorganism is  P. denitrificans  strain SC510 RifR.  
     
     
         105 . The method of any one of claims  102 - 104 , wherein said cobalamin is coenzyme B 12 .  
     
     
         106 . The method of  claim 102 , wherein said cobalamin precursor or cobamide precursor is selected from the group consisting of decobaltocorrinoids and corrinoids.  
     
     
         107 . The method of any one of claims  67 - 69 ,  71 - 74 ,  76 - 79 ,  81 - 84 ,  87 - 89 ,  91 - 94 ,  96 - 99 ,  101 - 104 , and  106 , wherein said recovery step comprises: 
 a) solubilization;    b) conversion to a cyanoform; and    c) purification.    
     
     
         108 . The method of  claim 70 , wherein said recovery step comprises: 
 a) solubilization;    b) conversion to a cyanoform; and    c) purification.    
     
     
         109 . The method of  claim 75 , wherein said recovery step comprises: 
 a) solubilization;    b) conversion to a cyanoform; and    c) purification.    
     
     
         110 . The method of  claim 80 , wherein said recovery step comprises: 
 a) solubilization;    b) conversion to a cyanoform; and    c) purification.    
     
     
         111 . The method of  claim 85 , wherein said recovery step comprises: 
 a) solubilization;    b) conversion to a cyanoform; and    c) purification.    
     
     
         112 . The method of  claim 90 , wherein said recovery step comprises: 
 a) solubilization;    b) conversion to a cyanoform; and    c) purification.    
     
     
         113 . The method of  claim 95 , wherein said recovery step comprises: 
 a) solubilization;    b) conversion to a cyanoform; and    c) purification.    
     
     
         114 . The method of  claim 100 , wherein said recovery step comprises: 
 a) solubilization;    b) conversion to a cyanoform; and    c) purification.    
     
     
         115 . The method of  claim 105 , wherein said recovery step comprises: 
 a) solubilization;    b) conversion to a cyanoform; and    c) purification.