Methods of increasing the production of cobalamins using cob...

Details Methods of increasing the production of cobalamins using cob... Methods of increasing the production of cobalamins using cob...

1995-04-21

2003-12-02

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

Reexamination Certificate

active

06656709

ABSTRACT:

The present invention relates to new polypeptides involved in the biosynthesis of cobalamins and/or cobamides, and especially of coenzyme B
12
. It also relates to the genetic material responsible for the expression of these polypeptides, as well as to a method by means of which they may be prepared. It relates, lastly, to a method for amplification of the production of cobalamins, and more especially of coenzyme B
12
, by recombinant DNA techniques.
Vitamin B
12
belongs to the B group of vitamins. It is a water-soluble vitamin which has been identified as the factor enabling patients suffering from pernicious anaemia to be treated. It is generally prescribed to stimulate haematopoiesis in fatigue subjects, but it is also used in many other cases comprising liver disorders and nervous deficiencies or as an appetite stimulant or an active principle with tonic activity, as well as in dermatology (Berck, 1982, Fraser et al., 1983). In the industrial rearing of non-ruminant animals, the feed being essentially based on proteins of vegetable origin, it is necessary to incorporate vitamin B
12
in the feed rations in amounts of 10 to 15 mg per tonne of feed (Barrère et al., 1981).
Vitamin B
12
belongs to a class of molecules known as cobalamins, the structure of which is presented in FIG.
1
. Cobamides differ from cobalamins in the base of the lower nucleotide, which is no longer 5,6-dimethylbenzimidazole but another base, e.g. 5-hydroxybenzimidazole for vitamin B
12
-factor III synthesised, inter alia, by
Clostridium thermoaceticum
and
Methanosarcina barkeri
(Iron et al., 1984). These structural similarities explain the fact that the metabolic pathways of biosynthesis of cobalamins and cobamides are, for the most part, shared.
Cobalamins are synthesised almost exclusively by bacteria, according to a complex and still poorly understood process which may be divided into four steps (FIG.
2
):
i) synthesis of uroporphyrinogen III (or uro'gen III), then
ii) conversion of uro'gen III to cobyrinic acid, followed by
iii) conversion of the latter to cobinamide, and
iv) construction of the lower nucleotide loop with incorporation of the particular base (5,6-dimethylbenzimidazole in the case of cobalamins).
For coenzyme B
12
, it is probable that the addition of the 5′-deoxyadenosyl group occurs shortly after the corrin ring-system is synthesised (Huennekens et al., 1982).
In the case of cobamides, only the step of synthesis and incorporation of the lower base is different.
The first part of the biosynthesis of cobalamins is very well known, since it is common to that of haemes as well as to that of chlorophylls (Battersby et al., 1980). It involves, successively, &dgr;-aminolevulinate synthase (EC 2.3.137), &dgr;-aminolevulinate dehydrase (EC 4.2.1.24), porphobilinogen deaminase (EC 4.3.1.8) and uro'gen III cosynthase (EC 4.2.1.75), which convert succinyl-CoA and glycine to uro'gen III. However, the first step takes place in some organisms [e.g.
E. coli
(Avissar et al., 1989) and in methanogenic bacteria (Kannangara et al., 1989), for example] by the conversion by means of a multi-enzyme complex of glutamic acid to &dgr;-aminolevulinic acid.
Between uro'gen III and cobyrinic acid, only three intermediate derivatives have been purified to date; they are the factors FI, FII and FIII, which are oxidation products, respectively, of the three intermediates precorrin-1, precorrin-2 and precorrin-3, which correspond to the mono-, di- and trimethylated derivatives of uro'gen III (FIG.
3
); these intermediates are obtained by successive transfers of methyl groups from SAM (S-adenosyl-L-methionine) to uro'gen III at positions C-2, C-7 and C-20, respectively. The other reactions which take place to give cobyrinic acid are, apart from five further transfers of methyl groups from SAM at C-17, C-12, C-1, C-15 and C-5, elimination of the carbon at C-20, decarboxylation at C-12 and insertion of a cobalt atom (FIG.
4
). These biosynthetic steps have been deduced from experiments performed in vitro on acellular extracts of
Propionibacterium shermanii
or of
Clostridium tetanomorphum
. In these extracts, cobyrinic acid is obtained by conversion of uro'gen III after incubation under suitable anaerobic conditions (Batterby et al., 1982). No intermediate between precorrin-3 and cobyrinic acid capable of being converted to corrinoids by subsequent incubation with extracts of cobalamin-producing bacteria has been isolated to date. The difficulty of isolating and identifying these intermediates is linked to
i) their great instability,
ii) their sensitivity to oxygen, and
iii) their low level of accumulation in vivo.
In this part of the pathway, only one enzyme of
Pseudomonas denitrificans
has been purified and studied; it is SAM:uro'gen III methyltransferase (Blanche et al., 1989), referred to as SUMT.
Between cobyrinic acid and cobinamide, the following reactions are performed:
i) addition of the 5′-deoxyadenosyl group (if coenzyme B
12
is the compound to be synthesised),
ii) amidation of six of the seven carboxyl functions by addition of amine groups, and
iii) amidation of the last carboxyl function (propionic acid chain of pyrrole ring D) by addition of (R)-1-amino-2-propanol (FIG.
2
).
Whether there was really an order in the amidations was not elucidated (Herbert et al., 1970). Lastly, no assay of activity in this part of the pathway has been described, except as regards the addition of the 5′-deoxyadenosyl group (Huennekens et al., 1982).
The final step of the biosynthesis of a cobalamin, e.g. coenzyme B
12
, comprises four successive phases described in
FIG. 5
(Huennekens et al., 1982), namely:
i) phosphorylation of the hydroxyl group of the aminopropanol residue of cobinamide to cobinamide phosphate, then
ii) addition of a guanosine diphosphate by reaction with guanosine 5′-triphosphate; the compound obtained is GDP-cobinamide (Friedmann, 1975), which
iii) reacts with 5,6-dimethylbenzimidazole, itself synthesised from riboflavin, to give adenosylcobalamin 5′-phosphate (Friedmann et al., 1968), which
iv) on dephosphorylation leads to coenzyme B
12
(Schneider and Friedmann, 1972).
Among bacteria capable of producing cobalamins, the following may be mentioned in particular:
Agrobacterium tumefaciens
Agrobacterium radiobacter
Bacillus megaterium
Clostridium sticklandii
Clostridium tetanomorphum
Clostridium thermoaceticum
Corynebacterium XG
Eubacterium limosum
Methanobacterium arbophilicum
Methanobacterium ivanovii
Methanobacterium ruminantium
Methanobacterium thermoautotrophicum
Methanosarcina barkeri
Propionobacterium shermanii
Protaminobacter ruber
Pseudomonas denitrificans
Pseudomonas putida
Rhizobium meliloti
Rhodopseudomonas sphaeroides
Salmonella typhimurium
Spirulina platensis
Streptomyces antibioticus
Streptomyces aureofaciens
Streptomyces griseus
Streptomyces olivaceus
At the industrial level, as a result of the great complexity of the biosynthetic mechanisms, the production of cobalamins, and especially of vitamin B
12
, is exclusively microbiological. It is carried out by large-volume cultures of the bacteria
Pseudomonas denitrificans, Propionibacterium shermanii
and
Propionibacterium freudenreichii
(Florent, 1986). The strains used for the industrial production are derived from wild-type strains; they may have undergone a large number of cycles of random mutation and then of selection of improved clones for the production of cobalamins (Florent, 1986). The mutations are obtained by mutagenesis with mutagenic agents or by physical treatments such as treatments with ultraviolet rays (Barrère et al., 1981). By this empirical method, random mutations are obtained and improve the production of cobalamins. For example, it is described that, from the original strain of
Pseudomonas denitrificans
initially isolated by Miller and Rosenblum (1960, U.S. Pat. No. 2,938,822), the production of this microorganism was gradually increased in the space of ten years, by the techniques

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