Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-11-23
2001-05-15
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S193000, C435S233000, C435S072000, C435S006120, C435S069100
Reexamination Certificate
active
06232102
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to recombinant DNA technology. In particular the invention pertains to the cloning of a glycosyltransferase gene from
Amycolatopsis orientalis,
the use of the cloned gene to express and purify the encoded enzyme, and a method of using the cloned enzyme in the production of glycopeptide compounds.
The use of antibiotic compounds has had a profound impact on the practice of medicine in the United States and around the world. Two highly effective antibiotic compounds of the glycopeptide class, vancomycin and teichoplanin, have been approved for use in humans.
The glycopeptide antibiotics comprise natural and semi-synthetic compounds of highly functionalized linear heptapeptides having a core structure composed of either seven modified or unusual aromatic amino acids, or a mix of aromatic and aliphatic amino acids. Natural glycopeptide compounds have been found in a variety of bacterial genera including Streptomyces, Actinoplanes, Nocardia, Amycolatopsis, Kibdelosporangia, and Pseudonocardia. M. Zmijewski and J. Fayerman. “Glycopeptide Antibiotics,” In
Genetics and Biochemistry of Antibiotic Production,
Chap. 18. Ed. L.C. Vining and C. Studtard. Publ. Butterworth Heinemann, Boston (1995). Generally, glycopeptide compounds are differentiated by the placement of sugar substituents on the peptide core. In some instances differentiation arises from the positioning of fatty acid moieties on said sugar substituents. Research has shown that the sugar moieties attached to the core may have an effect on the biological activity of glycopeptide molecules.
At present, investigations into glycosylation of glycopeptides and glycopeptide cores are limited to preliminary observations on crude cellular extracts of bacterial strains that produce glycopeptide compounds. These experiments have demonstrated that the glycosylation reaction appears to involve one or more enzymatic activities which attach sugar residues onto a glycopeptide core. One study, for example, demonstrated a glycosylating activity in a crude cellular extract of a vancomycin-producing strain of
Amycolatopsis orientalis.
M. Zmijewski & B. Briggs. “Biosynthesis of vancomycin: identification of TDP-glucose:aglycosylvancomycin glucosyltransferase from
Amycolatopsis orientalis
” FEMS Microbiol. Lett. 59, 129-134 (1989).
The glycosylation of glycopeptide compounds, intrinsically interesting from a scientific point of view, presents a number of practical considerations that warrant continued study of this subject. Recently, a number of glycopeptide resistant strains of pathogenic organisms have been encountered within the clinical environment. This trend toward diminished efficacy of glycopeptide compounds is alarming because of a similar phenomenon in the case-of 9-lactam antibiotics. It is clear that the rise in antibiotic resistance has occured by a plurality of molecular mechanisms and that resistant organisms possess a diverse repertoire for counteracting the otherwise lethal effect of antibiotic compounds.
In light of the trend toward greater resistance, and in view of the absence of effective alternative treatments, there exists a pressing need to develop new antibiotic compounds. A useful strategy toward this end involves derivitizing presently available glycopeptide compounds by engineering in defined ways the placement and configuration of sugar moieties on the glycopeptide core structure. Achieving molecular rearrangements and substitutions of sugars on glycopeptide compounds by chemical means is difficult if not impossible in most cases. By contrast to chemical procedures, enzymatic methods, if available, would provide an effective means to engineer specific modifications onto the glycopeptide core.
The challenge to provide an enzymatic means for modifying glycopeptides or glycopeptide core molecules has been met by the present invention. Described herein are gtfE genes isolated from
Amycolatopsis orientalis
which encode glycosyltransferase enzyme GtfE, which adds D-glucose or D-xylose moieties onto the B ring of vancomycin and teichoplanin glyopeptides and core molecules.
BRIEF SUMMARY
The present invention is designed to meet the aforementioned need and provides, inter alia, the isolated gtfE gene and other nucleic acid molecules that encode the GtfE gene product from
Amycolatopsis orientalis
C329.4. The invention also provides the GtfE protein product of the
Amycolatopsis orientalis
gtfE gene, in substantially purified form. Both the native
Amycolatopsis orientalis
gene gtfE, which encodes the activity, as well as a PCR-derived variant thereof, are used to produce proteins which exhibit this activity in a recombinant host cell. The GtfE proteins produced by recombinant methods are useful in the production of novel glycopeptide compounds.
Having the cloned gtfE gene of Amycolatopsis orientalis enables the production of recombinant GtfE protein from which can be made novel derivatives of glycopeptide compounds.
In one embodiment the present invention relates to an isolated DNA molecule encoding GtfE protein, said DNA molecule comprising the nucleotide sequence identified as
ATGCGTGTGT TGTTGTCGAC CTGTGGGAGC CGCGGAGACG TCGAACCACT GGTGGCGTTG
60
SEQ ID NO. 1:
GCGGTGCGGT TGCGGGAGCG CGGCGCCGAG GTGCGGATGT GCGCGCCGCC GGACTGCGCG
120
GATCGGCTGG CCGAAGTCGA CGTGCCGCAT CTGCCCCTCG GTGCGTCGGC GCGCCCGTCG
180
GCCGGGCAGG CGAAACCCTT GACGGCCGAG GACATGCTCC GGTTCACGAC CGAGACGATC
240
GCCACGCAGT TCGAGCGGAT TCCGGCGGCC GCCGAAGGAT GCGCCGCGGT GGTGACGACC
300
GGCCTGCTGG CCGCCGCCAT CGGCGTGCGG TCGGTGGCCG AAAAGCTGGG CATCCCCTAC
360
TTCTATGGCT TCCACTGCCC GAGCTATGTG CCGTCGCCGT ACTATGCGCC TCCGCCGCCC
420
CTCGGCGAGC CGCCCGCACC GGACGGGACC GACATCCAGG CGCTGTGGGA GCGCAACAAC
480
CAGAGCGCCT ACCGGCGGTA CGGGGAGCCG CTCAACAGCA GGCGCGCCGC CATCGGCCTG
540
CCGCCGGTGG AGGACATCTT CGGCCACGGC TACACCGATC ACCCGTGGAT GGCGGCGGAC
600
CCGGTACTGG CCCCGCTGCA ACCCACGGAT CTCGACGCCG TGCAGACCGG GGCGTGGATC
660
CTGCCCGACG AACGACCGAT TTCCGCTGAG CTGGAGGCGT TCCTGGACGC CGGCGCACCA
720
CCGGTGTACC TGGGGTTCGG CAGCCTTCGC GCCCCCGCCG ACGCCGCGAA GGTGGCCATC
780
GAGGCGATCC GTGCCCACGG CCACCGGGTG ATCCTCTCCC GCGGCTGGGC CGATCTGGTC
840
CTGCCCGACG ACCGGGAGGA CTGTTTCGCC ATCGGCGAAG TGAATCAGCA GGTGCTGTTC
900
CGCCGGGTGG CCGCCGTCAT CCACCACGGC GGCGCGGGCA CGACCCACGT GGCCACGCGG
960
GCGGGCGTCC CCCAGATCCT GGTTCCCCAG ATCGCGGACC AGCCCTACTA CGCCGCCCGG
1020
GTGGCCGAAC TGGGGGTCGG TGTGGCGCAT GACGGCCCGA CCCCGACCTT CGACACGTTG
1080
TCGGCGGCGC TCACCAAGGC CCTCGCTCCG GAAACGCGCG TGCGAGCGGA AGCCGTGGCG
1140
GAAACGGTCC AGACGGACGG GGCCGCGGTG GCCGCGGACC TGTTGTTCGC CGCGGTGACC
1200
GGGAACCAGC CCGCCGTTCC CGCC
1224
In another embodiment the present invention relates to a Glycosyltransferase protein molecule, encoded by SEQ ID NO:1 wherein said Glycosyltransferase protein molecule comprises the sequence identified as SEQ ID NO. 2.
In a further embodiment the present invention relates to a ribonucleic acid molecule encoding GtfE protein, said ribonucleic acid molecule comprising the sequence identified as SEQ ID NO. 3:
In yet another embodiment, the present invention relates to a recombinant DNA vector which incorporates the
Amycolatopsis orientalis
gtfE gene in operable linkage to gene expression sequences enabling the gtfE gene 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 the cloned gtfE gene of
Amycolatopsis orientalis
such that the gtfE gene is expressed in the host cell.
In still another embodiment, the present invention relates to a method for producing glycopeptide compounds wherein GtfE protein produced by recombinant cells is utilized to add one or more sugar moieties onto a glycopeptide or glycopeptide core, in vitro or in vivo.
In yet another embodiment, the present invention relates to novel glycopeptide compounds.
REFERENCES:
S. K. Chung, et al. “Biosynthetic Studies f Aridicin Antibiotics: Microbial Transformations and Glycosylations by Protoplasts.”Journal of Antibiotics3
Baltz Richard H.
Solenberg Patricia J.
Treadway Patti J.
Cohen Charles E.
Eli Lilly and Company
Hutson Richard
Prouty Rebecca E.
Webster Thomas D.
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