Biotin biosynthesis in bacillus subtilis
AssigneeDsm IP Assets B.V.
InventorsStanley Grant Bower, John B. Perkins, R. Rogers Yocum, Janice G. Pero
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Abstract
The invention generally provides the genes of the biotin biosynthetic operon of Bacillus subtilis, and closely related species thereof, and well as constructions useful for high level production of biotin.
Claims17
An isolated polypeptide encoded by a fragment of SEQ ID NO:
- 1, said fragment comprising a polynucleotide selected from the group consisting of nucleotides 1208-2554 of SEQ ID NO:
- 1 (bioA), nucleotides 4408-5415 of SEQ ID NO:
- 1 (bioB), nucleotides 3710-4405 of SEQ ID NO:
- 1 (bioD), nucleotides 2544-3713 of SEQ ID NO:
- 1 (bioF), nucleotides 439-1218 of SEQ ID NO:
- 1 (bioW), nucleotides 5484-6671 of SEQ ID NO:
- 1 (bioI), and nucleotides 6748-7509 of SEQ ID NO:
- 1 (orf2).
An isolated polypeptide comprising an amino acid sequence encoded by a polynucleotide selected from the group consisting of nucleotides 1208-2554 of SEQ ID NO:
- 1 (bioA), nucleotides 4408-5415 of SEQ ID NO:
- 1 (bioB), nucleotides 3710-4405 of SEQ ID NO:
- 1 (bioD), nucleotides 2544-3713 of SEQ ID NO:
- 1 (bioF), nucleotides 439-1218 of SEQ ID NO:
- 1 (bioW), nucleotides 5484-6671 of SEQ ID NO:
- 1 (bioI), and nucleotides 6748-7509 of SEQ ID NO:
- 1 (orf2), wherein the polynucleotide is isolated from a microorganism selected from the group consisting of Bacillus subtilis, B. pumilus, B. licheniformis, B. amyloliquefaciens, B. megaterium, B. cereus, and B. thuringiensis.
An isolated polypeptide encoded by bioI (nucleotides 5484-6671 of SEQ ID NO:1) or a polynucleotide which hybridizes under the following conditions to bioI or the complement of bioI:
- hybridization in 5×SSC (0.75M NaCl and 0.075M Na citrate, pH 7.0), 10-50% formamide, 1×Denhardt's solution (0.02% bovine serum albumin, 0.02% Ficoll, 0.02% pyrollidone), and 100 microgram/ml denatured salmon sperm DNA at 37-42° C., wherein the hybridizing polynucleotide hybrid encodes a polypeptide having cytochrome P-450 activity.
An isolated polypeptide encoded by bioA (nucleotides 1208-2554 of SEQ ID NO:
- 1) or a polynucleotide which hybridizes under the following conditions to bioA or the complement of bioA:
- hybridization in 5×SSC (0.75M NaCl and 0.075M Na citrate, pH 7.0), 10-50% formamide, 1×Denhardt's solution (0.02% bovine serum albumin, 0.02% Ficoll, 0.02% pyrollidone), and 100 microgram/ml denatured salmon sperm DNA at 37-42° C., wherein the hybridizing polynucleotide encodes a polypeptide having DAPA aminotransferase activity.
An isolated polypeptide encoded by bioB (nucleotides 4408-5415 of SEQ ID NO:
- 1) or a polynucleotide which hybridizes under the following conditions to bioB or the complement of bioB:
- hybridization in 5×SSC (0.75M NaCl and 0.075M Na citrate, pH 7.0), 10-50% formamide, 1×Denhardt's solution (0.02% bovine serum albumin, 0.02% Ficoll, 0.02% pyrollidone), and 100 microgram/ml denatured salmon sperm DNA at 37-42° C., wherein the hybridizing polynucleotide encodes a polypeptide having biotin synthetase activity.
An isolated polypeptide encoded by bioD (nucleotides 3710-4405 of SEQ ID NO:
- 1) or a polynucleotide which hybridizes under the following conditions to bioD or the complement of bioD:
- hybridization in 5×SSC (0.75M NaCl and 0.075M Na citrate, pH 7.0), 10-50% formamide, 1×Denhardt's solution (6.02% bovine serum albumin, 0.02% Ficoll, 0.02% pyrollidone), and 100 microgram/ml denatured salmon sperm DNA at 37-42° C., wherein the hybridizing polynucleotide encodes a polypeptide having DTB synthetase activity.
An isolated polypeptide encoded by bioF (nucleotides 2544-3713 of SEQ ID NO:
- 1) or a polynucleotide which hybridizes under the following conditions to bioF or the complement of bioF:
- hybridization in 5×SSC (0.75M NaCl and 0.075M Na citrate, pH 7.0), 10-50% formamide, 1×Denhardt's solution (0.02% bovine serum albumin, 0.02% Ficoll, 0.02% pyrollidone), and 100 microgram/ml denatured salmon sperm DNA at 37-42° C., wherein the hybridizing polynucleotide encodes a polypeptide having 7-KAP synthetase activity.
An isolated polypeptide encoded by bioW (nucleotides 439-1218 of SEQ ID NO:
- 1) or a polynucleotide which hybridizes under the following conditions to bioW or the complement of bioW:
- hybridization in 5×SSC (0.75M NaCl and 0.075M Na citrate, pH 7.0), 10-50% formamide, 1×Denhardt's solution (0.02% bovine serum albumin, 0.02% Ficoll, 0.02% pyrollidone), and 100 microgram/ml denatured salmon sperm DNA at 37-42° C., wherein the hybridizing polynucleotide encodes a polypeptide having pimelyl-CoA synthetase activity.
An isolated polypeptide encoded by orf2 (nucleotides 6748-7509 of SEQ ID NO:
- 1) or a polynucleotide which hybridizes under the following conditions to orf2 or the complement of orf2:
- hybridization in 5×SSC (0.75M NaCl and 0.075M Na citrate, pH 7.0), 10-50% formamide, 1×Denhardt's solution (0.02% bovine serum albumin, 0.02% Ficoll, 0.02% pyrollidone), and 100 microgram/ml denatured salmon sperm DNA at 37-42° C., wherein the hybridizing polynucleotide encodes a polypeptide having β-ketoreductase activity.
Dependencies are inferred from the claim text.