Patent EA-002588-B1 (EA)
Title: Method for biosynthesis of cobalamins, strains of bacteria producing cobalamin and process for producing the same
Inventor: BLANCHE FRANCIS
IPC: C12P19/42
Filed: 1997-05-05
Abstract: 1. A process for amplifying the biosynthesis of cobalamins in a prokaryotic microorganism, which produces cobalamins, wherein a recombinant strain of the microorganism is used as said microorganism, transformed with a DNA fragment which comprises the bluE and bluF genes of Rhodobacter capsulatus, encoding an enzyme involved in the biosynthesis O-phospho-L-threonine or a homologous fragment and/or a fragment which is hybridized with said bluE and bluF genes and encodes the enzyme involved in the biosynthesis O-phospho-L-threonine and culture said strain under conditions appropriate expression of said enzyme and production of cobalamins. 2. The process for amplifying the biosynthesis of cobalamins by fermentation of the prokaryotic microorganism, producing cobalamins, wherein a cobalamin precursor O-phospho-L-threonine is added into the fermentation medium. 3. The process for amplifying the biosynthesis of cobalamins by fermentation of the prokaryotic microorganism, producing cobalamins, wherein a recombinant strain of an aerobic microorganism is used as said microorganism, transformed with a DNA fragment which comprises the bluE and bluF genes of Rhodobacter capsulatus, encoding an enzyme involved in the biosynthesis 5,6-dimethylbenzimidazole or a homologous fragment and/or a fragment which is hybridized with said bluE and bluF genes and encodes the enzyme involved in the biosynthesis 5,6-dimethylbenzimidazole and culture said strain under aerobic conditions appropriate expression of said enzyme and production of cobalamins. 4. The process according to claims 1, 3, wherein said DNA fragment comprises the 6.8 kb BamH1 fragment of plasmid pER1. 5. The process according to claim 1, wherein said DNA fragment comprises the 2.1 kb EcoR1/Clal fragment of plasmid pER2. 6. The process according to claim 1, wherein said DNA fragment comprises the 1.6 kb EcoRI/EcoRV fragment of plasmid pAHW25 or of plasmid pER2. 7. The process according to any ofclams 1 to 6 wherein recovering vitamin B12 after the biosynthesis. 8. The process according to any of clams 1 to 7 wherein said strain of the recombinant microorganism is a strain of Psuedornonas denitrificans or strain of Agrobacterium radiobacter. 9. A process for preparing recombinant strain of bacteria producing cobalamin, wherein said bacteria are transformed with a DNA fragment which comprises the bluE and bluF genes of Rhodobacter capsulatus, encoding an enzyme involved in the biosynthesis O-phospho-L-threonine or a homologous fragment and/or a fragment which is hybridized with said bluE and bluF genes and encodes the enzyme involved in the biosynthesis O-phospho-L-threonine. 10. A process for preparing recombinant strain of bacteria producing cobalamin, wherein said bacteria are transformed with a DNA fragment which comprises the bluE and bluF genes of Rhodobacter capsulatus, encoding an enzyme involved in the biosynthesis biosynthesis 5,6-dimethylbenzimidazole or a homologous fragment and/or a fragment which is hybridized with said bluE and bluF genes and encodes the enzyme involved in the biosynthesis 5,6-dimethylbenzimidazole. 11. The process according to claim 9 or 10, wherein the strain of Psuedornonas denitrificans or strain of Agrobacterium radiobacter is produced. 12. The strain of bacteria, producing cobalamin prepared according to the process of any of 9 or 11 claims. 13. The strain of bacteria, producing cobalamin prepared according to the process of any of 10 or 11 claims.