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
1997-07-14
2004-05-18
Prouty, Rebecca E. (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,...
C435S252310, C435S128000, C435S130000, C435S193000, C435S440000, C435S320100, C536S023200
Reexamination Certificate
active
06737256
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention is in the general field of the biosynthesis of biotin vitamers.
Biotin biosynthesis in
Escherchia coli
and
Bacillus sphaericus
has been studied at both the biochemical and molecular biological levels (DeMoll, 1996. In F. C. Neidhardt et al., (eds.)
E. coli
and
Salmonella typhimurium
: Cellular and Molecular Biology, Second edition ed., vol 1., pp. 704-709, ASM Press, Washington, D.C.; Perkins et al., In A. L. Sonenshein et al. (eds.), In
Bacillus subtilis
and Other Gram Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics, pp. 319-334, American Society for Microbiology, Washington, D.C.; Eisenberg, 1987. In F. Neidhardt et al. (eds.),
E. coli
and
Salmonella typhimurium
, pp. 544-550. American Society for Microbiology, Washington, D.C.; Cronan, Cell 58:427-429, 1989, Izumi et al., Agric. Biol. Chem. 45:1983-1989, 1981; Gloeckler et al., Gene 87:63-70, 1990), although some steps and components in biotin synthesis remain to be elucidated (Ohshiro et al., Biosci. Biotech. Biochem. 58:1738-1741, 1994; Ifuku et al., Eur. J. Biochem. 224:173-178, 1994; Florentin et al., C. R. Acad. Sci. Paris 317:485-488, 1994; Birch et al., J. Biol. Chem. 270:19158-19165, 1995; Sanyal et al., Biochemistry 33:3625-3631, 1995). Several enzymes involved in the conversion of pimeloyl-CoA to biotin have been isolated and characterized from both of these bacterial species (Ploux et al., Biochem. J. 283:327-321, 1992; Izumi et al., Agric. Biol. Chem. 45:1983-1989, 1981; Eisenberg, supra, Huang et al., Biochemistry 34:10985-10995, 1995). KAPA synthase, the product of bioF, catalyzes the conversion of pimeloyl-CoA and alanine to 8-amino-7-ketopelargonic acid (KAPA). DAPA aminotransferase, the product of bioA, then transfers an amino group from a donor to KAPA yielding 7,8-diaminopelargonic acid (DAPA). Dethiobiotin synthetase (bioD) catalyzes the closure of the ureido-ring to produce dethiobiotin (DTB), and finally the product of bioB, biotin synthase, functions together with a number of other components including flavodoxin (Birch et al., supra; Ifuku et al., supra) S-adenosylmethionine (SAM) (Florentin, C. R. Acad. Sci. Paris 317:485-488, 1994; Ohshiro et al., supra; Sanyal et al., supra; Birch et al., supra) ferrodoxin NADP
+
reductase (Birch et al., supra; Sanyal et al., Arch. Biochem. Biophys. 326:48-56, 1996) and possibly cysteine (Florentin, C. R. Acad. Sci. Paris 317:485-488, 1994; Birch et al., supra; Sanyal et al., supra) to convert dethiobiotin to biotin. The compounds KAPA, DAPA, DTB, and biotin are collectively or singly referred to as vitamers or biotin vitamers.
In
E. coli
the genes that encode these enzymes are located in two divergently transcribed operons, controlled by a single operator that interacts with the BirA repressor (Cronan, Cell 58:427-429, 1989). In
B. sphaericus
, the genes are located in two separate operons (Gloeckler et al., supra. The early steps of the pathway, those involved in the synthesis of pimeloyl-CoA, are less well understood (Ifuku et al., Eur. J. Biochem. 224:173-178, 1994; Sanyal et al., J. Am. Chem. Soc. 116:2637-2638, 1994).
B. sphaericus
contains an enzyme, pimeloyl-CoA synthetase (bioW) that converts pimelic acid to pimeloyl CoA (Gloeckler et al., Gene 87:63-70, 1990), (Ploux et al., Biochem. J. 287:685-690, 1992).
E. coli
lacks this enzyme and cannot use pimelic acid as an intermediate in biotin synthesis (Gloeckler et al., supra; Ifuku et al., Eur. J. Biochem. 224:173-178, 1994; Sanyal et al., J. Am. Chem. Soc. 116:2637-2638, 1994).
E. coli
contains two genes, bioC which is located in the bio operon and bioH which is unlinked to the other bio genes, that both appear to be involved in the early steps of biotin biosynthesis leading up to pimeloyl-CoA, but their exact roles are unknown (Eisenberg, supra; Lemoine et al., Mol. Micro. 19:645-647, 1996).
B. subtilis
contains homologs of the
E. coli
and
B. sphaericus
bioA, bioB, bioD, and bioF genes. These four genes along with a homolog of the
B. sphaericus
bioW gene are arranged in a single operon in the order bioWAFDB, and are followed by two additional genes, bioI and orf2 (Bower et al., J. Bacteriol. 178:4122-4130, 1996). bioI and orf2 are generally dissimilar to other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P450s and is able to complement mutations in either
E. coli
bioC or bioH (Bower et al., supra. Mutations in bioI cause
B. subtilis
to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants can be overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis (Bower et al., supra.
The
B. subtilis
bio operon is preceded by a putative vegetative promoter sequence and contains, just downstream, a region of dyad symmetry with homology to the bio regulatory region of
B. sphaericus
(Bower et al., supra. Analysis of a bioW-lacZ translational fusion indicates that expression of the biotin operon is regulated by biotin and the
B. subtilis
birA gene. Strains deregulated for biotin synthesis can be engineered by replacing the promoter and regulatory region with a constitutive promoter as described in European Patent Application 0635572 A2, incorporated herein by reference. Production of biotin and biotin vitamers can be further improved by integration and amplification of the deregulated genes in the
B. subtilis
chromosome. Strain BI282, in European Patent Application 0635572 A2, herein incorporated by reference, is an example of such a strain.
SUMMARY OF THE INVENTION
We have found that the conversion of KAPA to DAPA is a serious bottleneck in the biosynthesis of biotin using engineered cells that are fed pimelic acid. As other controls on biotin biosynthesis are removed, the KAPA to DAPA conversion is unable to keep pace with KAPA production, resulting in a build-up of KAPA, without a concomitant increase in the final product. We have also discovered that an important component of the bottleneck is the availability and identity of the amino donor used in the KAPA to DAPA conversion. In general, providing adequate quantities of the amino donor is an important strategy for overcoming the bottleneck. Moreover, a DAPA aminotransferase able to use lysine and related compounds as a source of the amino group to be transfered in the reaction which produces DAPA from KAPA, can significantly improve biosynthetic yields of the downstream biotin vitamers, especially dethiobiotin (DTB).
Although we do not wish to be limited to one specific explanation for our finding to the exclusion of other factors, it appears that providing higher levels of an amino donor which can be used by the available aminotransferase substantially ameliorates the bottleneck discussed above. For example, bacterial production of the biotin vitamers by bacteria whose DAPA aminotransferase uses lysine as an amino donor can be dramatically improved by making sufficient lysine available, either by including it in the fermentation medium or by deregulating the lysine biosynthetic pathway. Such a strategy can also be applied to the use of DAPA aminotransferases of
B. subtilis
and close relatives, including members of the cluster of Bacillus spp. represented by
B. subtilis
. The cluster includes, e.g.,
B. subtilis, B. pumilus, B. licheniformis, B. amyloliquefaciens, B. megaterium, B. cereus
and
B. thuringiensis
. The members of the
B. subtilis
cluster are genetically and metabolically divergent from the more distantly related Bacillus spp. of clusters represented by
B. sphaericus
and
B. stearothermophilus
(Priest, In
Bacillus subtilis
and Other Gram-Positive Bacteria, supra pp. 3-16, hereby incorporated by reference; Stackebrant, et al.
J. Gen. Micro.
133:2523-2529, 1987, hereby incorporated by reference).
Accordingly, one aspect of the invention generally features a method of biosynthesizing (e.g., enzymatically or in fermentations using engineered cells) a biotin vitamer by culturing a bacterium that inclu
Perkins John B.
Pero Janice G.
Van Arsdell Scott W.
Yocum R. Rogers
Bryan Cave LLP
Prouty Rebecca E.
Raminez Delia
Roche Vitamins Inc.
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