Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1999-03-02
2000-12-05
Achutamurthy, Ponnathapu
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C12P 1942
Patent
active
061565458
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a biosynthetic process for preparing cobalamins. More precisely, it relates to a process for amplifying the production of cobalamins and, more specifically, of coenzyme B.sub.12 by means of recombinant DNA techniques and/or by means of adding a novel cobalamin precursor. Finally, the present invention relates to a process for preparing recombinant strains which are of use in the process for preparing cobalamins according to the present invention.
Vitamin B.sub.12 is a member of a class of molecules which are termed cobalamins and whose structure is presented, in particular, in WO91/11518.
Cobalamins are synthesized almost exclusively by bacteria in accordance with a complex process which is also described in WO91/11518. Because of the high degree of complexity of the biosynthetic mechanisms, cobalamins, and in particular vitamin B.sub.12, are principally produced at the industrial level using large-volume cultures of the bacteria Pseudomonas denitrificans, Propionobacterium shermanii and Propionobacterium freudenreichii.
It is widely known that cobalamins are synthesized by certain microorganisms from the following substrates: aminolaevulinic acid, S-adenosyl-L-methionine, cobalt, glutamine, (R)-1-amino-2-propanol and 5,6-dimethylbenzimidazole.
Of the abovementioned precursors, 5,6-dimethylbenzimidazole is synthesized by the microorganisms which produce cobalamins. There appear to exist two biosynthetic pathways for 5,6-dimethyl-benzimidazole: one is characteristic of aerobic microorganisms and involves molecular oxygen while the other is employed by anaerobic microorganisms. Only one gene involved in the anaerobic pathway has been isolated; this is the cobT gene of Salmonella typhimurium (Trzebiatowski et al., 1994). No gene for synthesizing 5,6-dimethylbenzimidazole has to date been identified in the aerobic microorganisms. The quantity of 5,6-dimethylbenzimidazole synthesized by microorganisms is often limiting.
As a consequence, 5,6-dimethylbenzimidazole is prepared chemically and added to the production media. Elimination of this addition to the media would therefore offer a definite advantage.
So far, no process for the industrial preparation of cobalamins has mentioned the addition of precursors other than cobalt and 5,6-dimethyl-benzimidazole. Some strains which only produce cobalamins on media containing (R)-1-amino-2-propanol have recently been described (Crouzet et al., 1990, Grabau et al., 1992). (R)-1-Amino-2-propanol could, therefore, be used for improving the production of cobalamins. However, its use for a possible industrial fermentation is elaborate and expensive since, on the one hand, (R)-1-amino-2-propanol is an irritant and volatile product and, on the other hand, it can inhibit growth of the microorganism. It would be particularly advantageous, therefore, to find another precursor of the (R)-1-amino-2-propanol residue of cobalamins which does not suffer from these drawbacks. In this regard, a pathway for biosynthesizing (R)-1-amino-2-propanol from L-threonine via aminoacetone has been described in certain microorganisms. However, L-threonine does not complement the abovementioned strains.
More generally, it can be advantageous, for improving the production of cobalamins, to increase the quantity of their precursors in the medium, in particular if these precursors are limiting. This approach can be effected either by adding the limiting precursor, or one of its derivatives or analogues, directly to the medium, or by amplifying the in-situ synthesis of this precursor in the producer strain using genetic techniques, in particular recombinant DNA technology.
To that end, knowledge of the biosynthetic pathways of the cobalamins, and of their precursors, is a key step for improving the production of cobalamins.
Thus, most of the steps in the pathway for biosynthesizing vitamin B12 have recently been characterized in Pseudomonas denitrificans (Blanche et al., 1995). No less than 22 cob genes involved in cobalamin biosynthesis have been isolated and the funct
REFERENCES:
Pollich et al. Identification and Sequence Analysis of Genes Involved in Late Steps of Cobalamin (Vitamin B12) Synthesis in Rhodobacter capsulatus. Journal of Bacteriology (Aug. 1995) 177(15):4481 -4487.
Stamford NPJ. Genetics and enzymology of the B12 pathway. Ciba Foundation Symposium (1994) 180:247-266.
Brey et al. Cloning and Multiple Genes Involved with Cobalamin (Vitamin B12) Biosynthesis in Bacillus megaterium. Journal of Bacteriology (Aug. 1986) 167(2): 623-630.
Escalante-Semerena et al. The Cobll and Coblll Regions of the Cobalamin (Vitamin B12) Biosynthetic Operon of Salmonella typhimurium (Jan. 1992) 174(1): 24-29.
Blanche Francis
Cameron Beatrice
Crouzet Joel
Debussche Laurent
Remy Elisabeth
Achutamurthy Ponnathapu
Kerr Kathleen
Rhone-Poulenc Rorer S.A.
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