Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Mutation employing a chemical mutagenic agent
Reexamination Certificate
1999-09-28
2001-10-16
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Mutation employing a chemical mutagenic agent
C435S252310
Reexamination Certificate
active
06303377
ABSTRACT:
BACKGROUND OF THE INVENTION
Biotin (vitamin B
8
or vitamin H), a coenzyme for carboxylation and decarboxylation reactions, is an essential metabolite for living cells. Exogenous biotin is required for most higher organisms; however many bacteria synthesize their own biotin.
The enzymatic steps involved in the biotin synthetic pathway from pimelyl-CoA (PmCoA) to biotin have been elucidated in
Escherichia coli
and
Bacillus sphaericus
(
FIG. 1
; reviewed in Perkins and Pero,
Bacillus subtilis and other Gram-Positive Bacteria
, ed. Sonenshein, Hoch, and Losick, Amer. Soc. of Microbiology, pp. 325-329, 1993). The steps include the conversions of 1) pimelyl-CoA to 7-keto-8-amino pelargonic acid; (7-KAP or KAPA) by 7-KAP synthetase (bioF); 2) 7-KAP to 7,8-diamino-pelargonic acid (DAPA) by DAPA aminotransferase (bioA); 3) DAPA to dethiobiotin (DTB) by DTB synthetase (bioD); and 4) DTB to biotin by biotin synthetase (bioB). Synthesis of PmCoA reportedly involves different enzymatic steps in different microorganisms. The
E. coli
genes involved in steps preceding pimelyl-CoA synthesis include bioC (Otsuka et al.,
J. Biol. Chem.
263:19577-19585 (1988)) and bioH (O'Regan et al.,
Nucleic Acids Res.
17:8004 (1989)). In
B. sphaericus
, two different genes, bioX and bioW, are thought to be involved in PmCoA synthesis. BioX is thought to be involved in pimelate biosynthesis (Gloeckler et al.,
Gene
87:63-70, 1990), and bioW has been shown to encode pimelyl-CoA synthetase which converts pimelic acid (PmA) to PmCoA (Ploux et al.,
Biochem. J.
287:685-690, 1992). Neither
B. sphaericus
gene, bioW or bioX, has significant sequence similarity with the
E. coli
bioC and bioH genes either at the nucleotide or protein level (Gloeckler et al., 1990, supra).
In
E. coli
, the biotin biosynthetic genes are located in three or more operons in the chromosome. The bioA gene is located in one operon and the bioBFCD genes are located in a second closely linked operon. The bioH gene is unlinked to the other bio genes (
FIG. 2
; Eisenberg, M. A. 1987 in
Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology
, vol. 1, Amer. Soc. Micro. Wash. D.C.).
In
B. sphaericus
, the organization of the bio genes is clearly different from that in
E. coli
. Gloeckler et al. (1990, supra) have isolated and characterized two unlinked DNA fragments from
B. sphaericus
that encode bio genes. One fragment contains an operon encoding the bioD, bioA, bioy, and bioB genes, and the other fragment contains an operon encoding the bioX, bioW and bioF genes (FIG.
2
). The order and clustering of bio genes is different in
E. coli
and
B. sphaericus
(FIG.
2
).
Fisher U.S. Pat. No. 5,110,731 provides a system for producing biotin wherein the genes of the biotin operon of
E. coli
are transformed into, and expressed in, a retention-deficient strain of
E. coli.
Gloeckler et al. U.S. Pat. No. 5,096,823 describes genes involved in the biosynthesis of biotin in
B. sphaericus
: bioA, bioD, bioF, bioC, and bioH.
B. sphaericus
genes for bioA and bioD were cloned into both
E. coli
and
B. subtilis
. The bioA and bioD genes were stably integrated into
B. subtilis Bio
−
auxotrophs, and prototrophic strains were selected.
GB 2,216,530-B2 (Jul. 8, 1992; Minister of Agr & Fisheries) provides plasmids containing gene(s) for
E. coli
bioA, bioB, bioC, bioD, and bioF isolated from other
E. coli
genetic material, e.g., control sequences. The plasmids are capable of replicating and being expressed in non-
E. coli
strains, preferably in yeast.
Three biotin synthesis deficient mutants of
B. subtilis
(bioA, bioB, and a gene termed bio112 which may be analogous to
E. coli
bioF) have been reported (Pai,
Jour. Bact.
121:1-8, 1975; and Gloeckler et al., 1990, supra).
Nippon Zeon Co. Ltd. U.S. Pat. No. 4,563,426 discloses biotin fermentation that includes adding pimelic acid after culturing for about 24 hours. Transgene SA and Nippon Zeon Co. Ltd. E.P. 0 379 428 discloses adding pimelic acid to a biotin fermentation medium.
SUMMARY OF THE INVENTION
The invention generally provides the genes of the biotin synthetic operon of
B. subtilis
and closely related species to be used for high level production of biotin. Specific aspects of the invention are described in greater detail below. We have specifically identified, cloned, and engineered a previously unknown gene (bioI), which encodes a cytochrome P-450-like enzyme. We have also developed a strategy to overexpress the entire
B. subtilis
bio operon (which, when engineered with a strong promoter, is unexpectedly toxic to
E. coli
) by cloning two bio operon fragments separately, combining them in vitro, and transforming the host organism with the resulting ligated construction. Cloning the two fragments was further complicated by difficulty obtaining the 5′ end of the operon, due to toxicity in
E. coli
. The invention particularly features the full-length operon obtained by the above strategy. These and other features of the invention are described in greater detail below.
In one aspect, therefore, the invention features vector-derived DNA comprising: (a) a gene that encodes a biotin biosynthetic enzyme of
Bacillus subtilis
, or of a species closely related to
Bacillus subtilis
; (b) a biologically active fragment of (a); or (c) a DNA sequence that is substantially homologous to (a) or (b). We use the term “vector-derived” to include DNA that can be used to transform a cell and the DNA included in such a cell after transformation. Such vector-derived DNA differs from naturally occurring DNA, either by mutation or by its inclusion in a molecule that is different from the DNA molecule in which it naturally occurs. Also, as used herein, a species which is “closely related” to
B. subtilis
includes a member of a 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
(
FIG. 3
; Priest, in
Bacillus subtilis and other Gram-Positive Bacteria
, supra pp. 3-16, hereby incorporated by reference; Stackebrandt, et al.
J. Gen. Micro.
133:2523-2529, 1987, hereby incorporated by reference).
As noted above, we have discovered a novel gene (bioi) present in
B. subtilis
and closely related species thereof, which is particularly important to deregulated production of biotin, and that gene is included in the DNA of preferred embodiments of the first aspect of the invention. Also preferably, at least bioA and bioB are included in the DNA of the first aspect. BioD, bioF, bioW, and ORF2 (encoding a &bgr;-keto reductase-like enzyme) may also be advantageously included in the DNA of the invention. At least two of the above-defined genes may be included in the DNA. The gene(s) may be operably linked to a transcriptional promoter, e.g., a constitutive promoter such as a promoter derived from the SP01 bacteriophage. The entire biotin operon of
Bacillus subtilis
, or a closely related species thereof, may be linked to a single transcriptional promoter. Moreover, we have learned that it is particularly useful to include a second promoter—i.e., one or more of the genes is operably linked to a first transcriptional promoter, and at least a second one of the genes is operably linked to a second transcriptional promoter. The first promoter may be operably linked to one or more of bioA, bioB, bioD, bioF, and bioW of
B. subtilis
, or a closely related species thereof. The other promoter may be operably linked to one or more of bioI, bioA, bioB, or a combination thereof. In a particularly preferred embodiment, the first promoter controlls transcription of the entire operon, and transcription of bioI, optionally with bioA and or bioB, is also controlled by the second promoter. The DNA may include a mutated regulatory site of
Bower Stanley Grant
Perkins John B.
Pero Janice G.
Yocum R. Rogers
Bryan Cave LLP
Fronda Christian L.
Haracz Stephen M.
Nashed Nashaat T.
Roche Vitamins Inc.
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