Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1998-09-01
2001-02-20
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S320100, C435S252300, C435S419000, C536S023200
Reexamination Certificate
active
06190895
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to nucleic acid sequences which code for a novel transketolase from peppermint (
Mentha x piperita
), and to vectors containing the sequences, host cells containing the sequences and methods of upregulating or downregulating the production or activity of the transketolases and their mutants.
BACKGROUND OF THE INVENTION
The isoprenoids comprise the largest family of natural products with over 20,000 individual compounds described to date (Connolly, J. D. & Hill, R. A.,
Dictionary of Terpenoids
(Chapman and Hall, London, 1991)). The isoprenoids play numerous functional roles in plants as hormones (gibberelins, abscisic acid), photosynthetic pigments (side chain of phytol carotenoids), electron carriers (side chain of plastoquinone), and structural components of membranes (phytosterols). Isoprenoids also serve in communication and defense, for example as attractants for pollinators and seed dispersers, and as competitive phytotoxins, antibiotics, and herbivore repellents and toxins (Harborne, J. B. in
Ecological Chemistry and Biochemistry of Plant Terpenoids
(Harborne, J. B., Tomas-Berbean, F. A, Eds.), pp. 399-426 (Clarendon Press, Oxford, 1991)).
Until recently, it was generally assumed that all isoprenoids were synthesized from acetyl-CoA via the classical mevalonate pathway (Spurgeon, S. L. & Porter, J. W., Eds., in
Biosynthesis of Isoprenoid Compounds,
Vol. 1, pp 1-46 (John Wiley, New York, 1983)). However, in 1993, Rohmer and co-workers (Rohmer, M. et al.,
Biochem. J.
295:517-524 (1993)) demonstrated that a non-mevalonate pathway, originating from pyruvate and glyceraldehyde-3-phosphate (GAP) (Rohmer, M. et al.,
J. Am. Chem. Soc.
118:2564-2566 (1996)), operated in several eubacteria, including
E. coli.
Evidence subsequently emerged that the plastid-derived isoprenoids of plants, including carotenoids and the prenyl side chains of chlorophyll and plastoquinone (Lichtenthaler, H. K. et al.,
FEBS Lett.
400:271-274 (1997)), as well as isoprene (Zeidler J. G. et al.,
Z. Naturforsch
52c:15-23 (1997)), monoterpenes (Eisenreich, W. et al.,
Tetrahedron Lett.
38:3889-3892 (1997)) and diterpenes (Eisenreich, W. et al.,
Proc. Natl. Acad. Sci. USA
93:6431-6436 (1996)); (Schwarz, M. K., PhD thesis, ETH, Zurich, Switzerland (1994)), are synthesized via the pyruvate/GAP route to isopentenyl diphosphate (IPP). This new pathway had been completely overlooked in the past.
The first dedicated reaction of this new enzymatic pathway to IPP is considered to involve a transketolase-type condensation involving pyruvate and GAP to form 1-deoxy-D-xylulose-5-phosphate (Rohmer, M. et al.,
J. Am. Chem. Soc.
118:2564-2566 (1996)); (Zeidler J. G. et al.,
Z. Naturforsch.
52c:15-23 (1997)); (Broers, S. T. J., PhD thesis, ETH, Zurich, Switzerland (1994)) (FIG.
1
). A recent abstract has described the cloning of a gene encoding 1-deoxyxylulose-5-phosphate synthase from
E. coli,
but no sequence information, or other descriptive information, was reported (Lois, L. M. et al., Third Terpnet Meeting of the European Network on Plant Isoprenoids, Poitiers, France, May 29-30 (1997)).
SUMMARY OF THE INVENTION
In accordance with the present invention, isolated nucleic acid sequences, such as the sequences set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7 which encode all or part of a 1-deoxyxylulose-5-phosphate synthase (abbreviated as DXPS) from peppermint (
Mentha x piperita
) have been isolated, identified and characterized. Thus, the present invention provides nucleic acid sequences encoding plant 1-deoxyxylulose-5-phosphate synthase proteins. In particular, the present invention provides nucleic acid sequences encoding 1-deoxyxylulose-5-phosphate synthase proteins from the essential oil plants. In another aspect the present invention provides for isolated, recombinant DXPS proteins, such as the proteins having the sequences set forth in SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. In other aspects, the present invention is directed to replicable recombinant cloning vehicles comprising a nucleic acid sequence, e.g., a DNA sequence, which codes for DXPS or for a base sequence sufficiently complementary to at least a portion of the DXPS DNA or RNA to enable hybridization therewith (e.g., antisense transketolase RNA or fragments of complementary transketolase DNA which are useful as polymerase chain reaction primers or as probes for transketolases from
Mentha x piperita
or related genes). In yet other aspects of the invention, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence of the invention. Thus, the present invention provides for the recombinant expression of the transketolase 1-deoxyxylulose-5-phosphate synthase (DXPS) from peppermint (
Mentha x piperita
) and related transketolases, and the inventive concepts may be used to facilitate the production, isolation and purification of significant quantities of recombinant transketolases (or of the primary enzyme products) for subsequent use, such as to obtain expression or enhanced expression of transketolases in plants to attain enhanced production of predator or pathogen defense compounds, or may be otherwise employed in an environment where the regulation or expression of transketolases are desired. In other aspects, the regulation of isoprenoid biosynthesis in plants by transforming, transfecting, infecting and/or injecting the plant with a recombinant cloning vehicle and/or DNA sequence of the invention to obtain expression of the transketolase DXPS or a related transketolse in the plant and thereby upregulate the pyruvate/glyceraldehyde-3-phosphate isoprenoid biosynthetic pathway. Thus, in addition to the new nucleic acid sequences and fragments thereof, the present invention includes new vectors containing the sequences, host cells containing the sequences, isolated recombinant transketolase (synthase) polypeptides and methods of producing recombinant transketolases ad their mutants.
REFERENCES:
Suggs et al, PNAS, 1981, vol. 78, pp. 6613-6617.
Database SwissProt, Accession Q38854, submitted by Mandal et al., publicly available Dec. 15, 1998.
Becker, Nucleic Acids Research, 1990, vol. 18, p. 203.
Rohmer, M. et al.,Biochem J., 295:517-524 (1993).
Zeidler, J.G. et al.,Z. Naturforsch, 52c:15-23 (1997).
Lois, L.M. et al., Third Terpnet Meeting of the European Network on Plant Isoprenoids, Poitiers, France, May 29-30 (1997).
Mandel, M.A. et al.,Plant J.9:649-658 (1996).
Youvan, D.C. et al.Cell37:949-957 (1984).
Kaneko, T. et al.,DNA Res.3:109-136 (1996).
Hawkins, C.F. et al.,FEBS Lett.255:77-82 (1989).
Kotani, H. et al.,Annu. Rev. Plant Physiol. Plant Mol. Biol.49:151-71 (1998).
Croteau Rodney Bruce
Lange Bernd Markus
McCaskill David G.
Wildung Mark Raymond
Achutamurthy Ponnathapu
Johnson Kindness PLLC
Mayhew Bradley S.
O'Connor Christensen
Washington State University Research Foundation
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