Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide encodes an inhibitory rna molecule
Reexamination Certificate
1998-10-02
2002-02-19
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide encodes an inhibitory rna molecule
C800S278000, C800S286000, C435S193000, C435S320100, C435S419000, C435S468000, C536S023600
Reexamination Certificate
active
06348641
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to purified proteins, recombinant nucleic acid sequences, hosts transformed with such sequences, and processes for producing caffeine-free or caffeine-reduced beverages. More particularly, the invention provides recombinant nucleic sequences that suppress the expression of caffeine in coffee plants and in coffee beans harvested from them.
Coffee is prepared from the roasted ground beans of the plants of the genus Coffea, generally from the species
Coffea arabica
(arabica coffee),
Coffea canephora
(robusta coffee), and the like. Coffee plants produce the alkaloid caffeine, which is present in their dried fruit, coffee beans. Because many coffee drinkers prefer coffee without caffeine, a number of processes have been developed to remove caffeine from coffee beans. However, all of these processes also result in the removal of substances other than caffeine from the beans, thereby adversely affecting the taste of coffee brewed from the treated beans. Although a few naturally occurring caffeine-free coffees and related genera are known (Mascarocoffea spp. and
Coffea bengalensis
), they have no commercial value. (Charrier and Berthaud (1975), “Variation Of Caffeine Content In The Coffea Genus”, Cafe' Cacao The', 14:251-264). Accordingly, there is a need for a method for producing decaffeinated coffee beans that does not result in the removal of substances from the beans other than caffeine.
Caffeine is a naturally occurring purine alkaloid produced by coffee and tea plants, among others. It is believed that caffeine synthesis protects the plants from insects. Coffee plants synthesize caffeine from the nucleoside xanthosine in four sequential reactions as shown in FIG.
1
. (For a review, see Suzuki, T., Ashihara, H. and Waller, G. R. (1992),
Phytochemistry
31: 2575). The first step in the pathway is the methylation of the nucleoside xanthosine by S-adenosylmethionine, which is catalyzed by the enzyme xanthosine-N
7
-methyltransferase (XMT). The product, 7-methylxanthosine, is hydrolyzed (a ribose is removed) to 7-methylxanthine, and undergoes further methylations to theobromine and caffeine. It is to be expected that interruption of this sequence of synthetic reactions would block caffeine synthesis. Accordingly, a strategy for selectively reducing or eliminating caffeine from coffee plants is to inhibit or eliminate synthesis of specific enzymes in the pathway for caffeine biosynthesis.
In one embodiment, this invention relates to genetic alteration of coffee plants to inhibit or eliminate synthesis of XMT. In the preferred embodiments, synthesis of XMT is suppressed by transforming coffee plant cells with a nucleic acid sequence that codes on transcription for an RNA that is either sense or antisense to the messenger RNA (mRNA) that codes on expression for XMT.
It is expected that the invention can be used to suppress caffeine synthesis in tea (genus Camellia, e.g.,
Camellia sinensis
) and cola (genus Cola, e.g.,
Cola acuminata
), as well as related alkaloids in chocolate (genus Theobroma, e.g.,
Theobroma cacao
). Thus, the invention may be generalized to produce other caffeine-free beverages and food products, including tea, cocoa, and other chocolate-based beverages or foods.
SUMMARY OF THE INVENTION
The invention provides purified proteins, DNA sequences that code on expression therefore, and recombinant DNA sequences, including hosts transformed with such sequences, for transforming coffee plants to reduce or eliminate the synthesis of caffeine. The DNA sequences are characterized in that they code on expression for an enzyme, xanthosine-N
7
-methyltransferase (XMT), that is the first step in the pathway for caffeine synthesis in coffee. The base sequence of that DNA and the predicted amino acid sequence of XMT is provided.
In one embodiment of the invention, coffee plant cells are transformed by means of transforming vectors that comprise a transcriptional initiation region (promoter) operably linked to a nucleic acid sequence that codes on transcription for an RNA that is complementary (antisense) to a mRNA that codes for at least one enzyme in the pathway for caffeine biosynthesis, the transcribed RNA having a length sufficient to interfere with the expression of the enzyme. In another embodiment of the invention, coffee plant cells are transformed by means of transforming vectors that comprise a nucleic acid sequence that codes on transcription for an RNA that shows substantial homology (sense) to a mRNA that codes for the enzyme, and that has a length sufficient to interfere with the expression of the enzyme.
Thus, for example, coffee plant cells may be transformed with a nucleic acid sequence that codes for an RNA that is sense or antsense to the mRNA for XMT, the enzyme active in the first step of the pathway for caffeine synthesis. Expression of either sense or antisense nucleic acid sequences in the transformed cells suppresses or eliminates the production of caffeine, although other aspects of cellular metabolism are not affected.
By the method of the invention, transformed coffee plant cells have been obtained in which caffeine production has been reduced by up to 98% compared with untransformed coffee plant cells. The invention also provides coffee plants regenerated from such transformed coffee plant cells, and coffee beans from these coffee plants.
A feature of the invention is that individual transformed coffee plants can be selected that produce, for example, no caffeine, or 1%, 2%, 10%, 25%, 50%, 75%, 90% of the normal amount of caffeine. Thus, “decaffeinated” coffee could include “100% decaf”, “99% decaf”, “98% decaf”, 90% decaf, “75% decaf”, “50% decaf”, “25% decaf”, “10% decaf”, or the like. The generally accepted level of caffeine for a coffee to be called “decaffeinated” is less than 2% to 3% of the normal amount of caffeine. Thus, the transformed coffee plant cell preferably exhibits greater than 98% reduction in caffeine.
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Napoli et al. Plant Cell 2:279-289, Apr. 1990.*
Ecker, J.R. and R.W. Davis, Inhibition of Gene Expression in Plant Cells By Expression of Antisense RNA,PNAS(USA) 83: 5372-5376, Aug. 1986.
Smith, C.J.S. et al. Antisense RNA Inhibition of Polygalacturonase Gene Expression in Transgenic Tomatoes,Nature334: 724-726, 1988.
Schulthess B.H. & T.W. Baumann. Are Xanthosine and 7-Methylxanthosine Caffeine Precursors?Phytochemistry39: 1363-1370, 1995.
Hofgen, R. et al. A Visible Marker for Antisense mRNA Experssion in Plants: Inhibition of Chlorophyll Synthesis With a Glutamate-1-Semialdehyde Aminotransferase Antisense Gene.PNAS(USA) 91: 1726-1730, 1994.
Ashihara, T.S.H. & G.R. Waller. Review Article Number 68: Purine and Purine Alkaloid Metabolism inCamelliaandCoffeaPlants.Phytochemistry31: 2575-2584, 1992.
Spiral, J. & V. Petiard, Development of a transformation method for coffee and regeneration of transgenic coffee plants (Abstract). Colloq. Sci. Int. Cafe [C.R.] 1993, 15th (vol. 1), pp. 115-122.
Schulthess, B.H., Morath, P. and T.W. Baumann. Caffeine biosynthesis starts with the metabolically channelled formation of 7-methyl-XMP—A new hypothesis. Phytochemistry 41 (1):169-175 (1996).
Kato, M. et al. Caffeine biosynthesis in young leaves ofCamellia sinensis:in vitro studies on N-methyltransferase activity involved in the conversion of xanthosine to caffeine. Physiologia Planatarum 98 (32): 629-636 (1996).
Moisyadi Istefo
Neupane Kabi Raj
Stiles John I.
Fox David T.
Jones Day Reavis & Pogue
University of Hawai'i
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