Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
2000-01-11
2002-08-20
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S041000, C435S183000, C435S252300, C435S254110, C435S254200, C435S320100, C435S440000, C536S023200
Reexamination Certificate
active
06436681
ABSTRACT:
This application is a 371 of International Application No. PCT/EP98/04097 published as WO 99/05285 which has an international filing date of Jul. 2, 1998.
FIELD OF THE INVENTION
The invention relates to a gene construct comprising a biotin gene having the sequence SEQ ID No. 1 or SEQ ID No. 3, to organisms which comprise this gene construct, to the use of these sequences or of the gene construct for preparing biotin, and to a process for preparing biotin.
BACKGROUND OF THE INVENTION
Biotin (vitamin H) plays an essential role as coenzyme in enzyme-catalyzed carboxylation and decarboxylation reactions. Biotin is thus an essential factor in living cells. Almost all animals and some microorganisms have to take biotin in from the outside because they are unable to synthesize biotin themselves. It is thus an essential vitamin for these organisms. Bacteria, yeasts and plants by contrast are themselves able to synthesize biotin from precursors (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326, DeMoll, E.,
Escherichia coli
and Salmonella, eds. Neidhardt, F. C. et al. ASM Press, Washington D.C., USA, 1996: 704-708, ISBN 1-55581-084-5).
Biotin synthesis has been investigated in bacterial organisms, specifically in the Gram-negative bacterium
Escherichia coli
and in the Gram-positive bacterium
Bacillus sphaericus
(Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326). The first intermediate known to date in
E. coli
is regarded as being pimelyl-CoA (Pm-CoA), which derives from fatty acid synthesis (DeMoll, E.,
Escherichia coli
and Salmonella, eds. Neidhardt, F. C. et al. ASM Press, Washington D.C., USA, 1996: 704-708, ISBN 1-55581-084-5 1996). The synthetic pathway for this biotin precursor in
E. coli
is substantially unknown at present (Ifuku 1993, Lemoine 1996). Two genes, bioC and bioH, whose corresponding proteins are responsible for the synthesis of Pm-CoA, have been identified. The enzymatic function of the gene products BioH and BioC is not known at present (Lemoine et al., Mol. Microbio. 19, 1996: 645-647, DeMoll, E.,
Escherichia coli
and Salmonella, eds. Neidhardt, F. C. et al. ASM Press, Washington D.C., USA, 1996: 704-708, ISBN 1-55581-084-5). Pm-CoA is converted into biotin in four further enzymatic steps. Starting from Pm-CoA there is initially condensation with alanine to give 7-keto-8-aminopelargonic acid (KAPA). The gene product for this conversion is BioF (KAPA synthetase). KAPA is transaminated by BioA (DAPA aminotransferase) with the cosubstrate S-adenosylmethionine to give 7,8-diaminopelargonic acid. The next step results, after an ATP-dependent carboxylation reaction, in dethiobiotin (DTB) and is catalyzed by BioD (dethiobiotin synthase). In the last step, DTB is converted into biotin. This step is catalyzed by BioB (biotin synthase). The genes bioF, bioA, bioD, and bioB coding for the proteins-which have been described are encoded in
E. coli
on a bidirectional operon. This operon is located between the &lgr; attachment site and the uvrB gene locus at about 17 minutes on the
E. coli
chromosome (Berlyn et al. 1996: 1715-1902). Two other genes are additionally encoded on this operon, one of which, bioc, has functions which have already been described in the synthesis of Pm-CoA, while it has not yet been possible to assign a function to an open reading frame behind bioA (WO94/8023, Otsuka et al., J. Biol. Chem. 263, 1988: 19577-85). Highly conserved homologs to the
E. coli
proteins BioF, A, D, B have been found in B. sphaericus,
B. subtilis
, Syneccocystis sp. (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326, Bower et al., J. Bacteriol. 175, 1996: 4122-4130, Kaneko et al., DNA Res. 3, 3, 1996: 109-136), archaebacteria such as Methanococcus janaschi, yeasts such as Saccharomyces cerevisiae (Zhang et al., Arch. Biochem. Biophys. 309, 1, 1994: 29-35) or in plants such as
Arabidopsis thaliana
(Baldet et al., C. R. Acad. Sci. III, Sci. Vie. 319, 2, 1996: 99-106)).
The synthesis of Pm-CoA appears to take place differently in the two Gram-positive microorganisms which have been investigated to date than in
E. coli
. It has not been possible to find any homologs of bioH and bioC (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326).
Biotin is an optically active substance with three centers of chirality. It is currently prepared commercially only in a multistage costly chemical synthesis.
As an alternative to this chemical synthesis, many attempts have been made to set up a fermentation process for preparing biotin using microorganisms. It has been possible, by cloning the biotin operon on multi-copy plasmids, to increase biotin synthesis in microorganisms transformed with these genes. A further increase in biotin synthesis has been achieved by deregulation of biotin gene expression via selection of birA mutants (Pai C. H., J. Bacteriol. 112, 1972: 1280-1287). Combining the two approaches, ie. expression of the plasmid-encoded biosynthesis genes in a regulation-deficient strain (EP-B-0 236 429), resulted in a further increase in productivity. In this case, either the biotin operon can remain under the control of its native bidirectional promoter (EP-B-0 236 429), or its genes can be placed under the control of a promoter which can be regulated externally (WO94/8023).
It has not been possible to achieve commercially adequate productivity by previous approaches to the preparation of biotin by fermentation in
E. coli
. It has emerged that the yield in the preparation of biotin by fermentation is caused by the incomplete conversion of DTB into biotin by the BioB gene product (biotin-synthase). Cells which harbor mutations in the bioB gene are unable to grow on DTB and thus to convert DTB into biotin. The chemical and enzymatic mechanism of the conversion of DTB into biotin is at present only incompletely understood and elucidated.
Intensive genetic investigations to date have been unable to identify further proteins involved in the reaction. Characterization of the conversion of DTB into biotin has hitherto been carried out only in bacterial and plant cell extracts (WO94/8023, EP-B-0 449 724, Sanyal et al. Arch. Biochem. Biophys., Vol. 326, No. 1, 1996: 48-56 and Biochemistry 33, 1994: 3625-3631, Baldet et al. Europ. J. Biochem. 217, 1, 1993: 479-485, Méjean et al. Biochem. Biophys. Res. Commun., Vol. 217, No. 3, 1995: 1231-1237, Ohshiro et al., Biosci. Biotechnol. Biochem., 58, 9, 1994: 1738-1741).
These investigations have shown that low molecular weight factors such as S-adenosylmethionine, NADPH, cysteine, thiamine, Fe
2+
, asparagine, serine, fructose 1,6-bisphosphate stimulate the synthesis of biotin (Ohshiro et al., Biosci. Biotechnol. Biochem., 58, 9, 1994: 1738-1741, Birch et al., J. Biol. Chem. 270, 32, 1995: 19158-19165, Ifuk et al., Biosci. Biotechnol. Biochem., 59, 2, 1995: 185-189). Besides these low molecular weight factors, other proteins which stimulate the synthesis of biotin from DTB in the presence of BioB have been identified. These are flavodoxin and flavodoxin-NADPH reductase (Birch et al., J. Biol. Chem. 270, 32, 1995: 19158-19165, Ifuku et al., Biosci. Biotechnol. Biochem., 59, 2, 1995: 185-189, Sanyal et al., Arch. Biochem. Biophys. 326, 1, 1996: 48-56).
The biotin synthesis and lipoic acid synthesis exhibit great homology. In both synthetic pathways there is insertion of a sulfur, or two sulfur atoms, between non-activated carbon atoms in the last stage of the synthesis. The synthesis of lipoic acid is at present only inadequately characterized (DeMoll, E.,
Escherichia coli
and Salmonella, eds. Neidhardt, F. C. et al. ASM Press, Washington D.C., USA, 1996: 704-708, ISBN 1-55581-084-5). To date, only two necessary genes have been identified in
E.coli
: lipA and lipB. Both genes are located in an operon, with an as yet uncharacterized open reading frame (=ORF) between the two genes. Another gene lplA is able to transfer lipoic acid via a lipoyl-AMP intermediate to lysine. This reaction is thus similar to the activity of birA. Homologous regions in the amino acid sequence have been iden
Hauer Bernhard
Schröder Hartwig
BASF - Aktiengesellschaft
Keil & Weinkauf
Slobodyansky Elizabeth
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