Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1997-10-22
2001-11-06
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S471000, C435S183000, C435S193000, C435S252300, C435S252330, C435S320100, C530S350000, C530S371000, C536S023100, C536S023200, C536S023700, C536S023740, C536S024100
Reexamination Certificate
active
06312920
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to recombinant DNA technology. In particular the invention pertains to the cloning of the SAM operon genes from
Streptomyces fradiae
and the use of said genes and their encoded proteins to produce S-adenosylmethionine (SAM) in a recombinant host.
S-adenosylmethionine is a product of natural origin found in all living organisms. SAM is a product of considerable importance for its role in biological reactions such as transmethylations. While the enzymes that catalyze these reactions are varied in their substrate specificity they are practically universal in their requirement of S-adenosylmethionine as the ultimate methyl group donor. Some methyl transfer reactions are important in the synthesis of certain antibiotics, such as tylosin.
Tylosin is a macrolide antibiotic composed of a 16-membered branched lactone, tylactone, and residues of three attached sugars, mycaminose, mycarose, and mycinose. Tylosin is produced commercially by
Streptomyces fradiae
(ATCC 19609; NRRL 2702) and is used as an animal growth promotant and veterinary antibiotic. The multi-step biosynthesis of tylosin has been studied both physiologically and genetically (See generally, R. H. Baltz and E. T. Seno, “Genetics of
Streptomyces fradiae
and tylosin biosynthesis. Ann. Rev. Microbiol. 42, 547-74 (1988)). At least 13 biosynthetic genes and 2 regulatory genes are necessary for normal production of tylosin. Tylosin synthesis requires multiple methylation reactions, the last two of which are rate-limiting. In the last step a specific methyltransferase catalyzes the transfer of a methyl group from SAM to the tylosin precursor molecule, macrocin. Thus, the availability of SAM as the methyl group donor is essential in the synthesis of tylosin.
S-adenosylmethionine is produced when an adenosyl group is transferred from ATP to methionine. SAM is synthesized in the cell by the action of three enzymes encoded by the SAM operon—SAM synthetase, methyl transferase (MT), and methylene tetrahydrofolate reductase (MTHR).
SUMMARY OF THE INVENTION
The present invention provides, inter alia, isolated nucleic acid molecules comprising the SAM operon from
Streptomyces fradiae.
The invention also provides the protein products encoded by the SAM operon, in substantially purified form.
Having the cloned SAM operon of
Streptomyces fradiae
enables the production of S-adenosylmethionine in recombinant host cells.
In one embodiment the present invention relates to an isolated nucleic acid that encodes SAM synthetase from
Streptomyces fradiae,
said nucleic acid comprising nucleotide residues 986 through 2209 of the nucleotide sequence identified as SEQ ID NO. 1.
In another embodiment the present invention relates to an isolated nucleic acid that encodes MT from
Streptomyces fradiae,
said nucleic acid comprising nucleotide residues 2241 through 3341 of SEQ ID NO.1.
In another embodiment the present invention relates to an isolated nucleic acid that encodes MTHR from
Streptomyces fradiae,
said nucleic acid comprising nucleotide residues 3338 through 4255 of SEQ ID NO.1.
In another embodiment the present invention relates to a novel SAM synthetase from
Sreptomyces fradiae
in substantially purified form comprising the sequence identified as SEQ ID NO. 2.
In still another embodiment the present invention relates to a novel MT from
Streptomyces fradiae
in substantially purified form comprising the sequence identified as SEQ ID NO. 3.
In yet another embodiment the present invention relates to a novel MTHR from
Streptomyces fradiae
in substantially purified form comprising the sequence identified as SEQ ID NO.5.
In a further embodiment the present invention relates to a ribonucleic acid molecule encoding SAM synthetase, said ribonucleic acid molecule comprising residues 986 through 2209 of the sequence identified as SEQ ID NO. 6:
In yet another embodiment, the present invention relates to a recombinant DNA vector that incorporates the
Streptomyces fradiae
SAM operon genes in operable linkage to gene expression sequences enabling said genes to be transcribed and translated in a host cell.
In still another embodiment the present invention relates to homologous or heterologous host cells which have been transformed or transfected with one or more of the cloned SAM operon genes from
Streptomyces fradiae
such that said gene(s) is/are expressed in the host cell.
In a still further embodiment, the present invention relates to a method for producing S-adenosylmethionine in recombinant host cells transformed with the
S. fradiae
SAM synthetase gene.
REFERENCES:
patent: 5672497 (1997-09-01), Cox et al.
patent: 0 189 322 A2 (1986-01-01), None
patent: 0 647 712 A1 (1993-10-01), None
patent: WO 93/10223 (1993-05-01), None
patent: WO 94/08014 (1994-04-01), None
Fishman et al. Proceedings of the National Academy of Sciences, USA. vol. 84, pp. 8248-8252, Dec. 1997.*
M. L. Dickens, et al. “Cloning, Sequencing and Analysis of Aklaviketone Reductase fromStreptomyces sp. Strain C5.”Journal of Bacteriology178(11) :3384-3388 (Jun. 1996).
R. H. Baltz and E. T. Seno. “Genetics ofStreptomyces Fradiaeand Tylosin Biosynthesis.”Ann. Rev. Microbiol. 42:547-74 (1988).
N. J. Bauer, et al. “Purification, Characterization, and Kinetic Mechanism of S-Adenoxyl-L-methionine:Macrocin O-Methyltransferase fromStreptomyces fradiae.”The Journal of Biological Chemistry263(30) :15619-15625 (Oct. 25, 1988).
M. Bierman, et al. “Plasmid cloning vectors for the conjugal transfer of DNA fromEscherichia colitoStreptomycesspp.”Gene116:43-49 (1992).
DeHoff Bradley Stuart
Rosteck, Jr. Paul Robert
Cohen Charles E.
Eli Lilly and Company
Tucker Tina M.
Webster Thomas D.
Yucel Remy
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