Geranyl diphosphate synthase large subunit, and methods of use

Details Geranyl diphosphate synthase large subunit, and methods of use Geranyl diphosphate synthase large subunit, and methods of use

1999-10-18

2001-10-16

Brusca, John S. (Department: 1631)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S325000, C435S419000, C435S252300, C435S320100, C536S023600

Reexamination Certificate

active

06303330

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to nucleic acid sequences which code for geranyl diphosphate synthase large subunit, such as geranyl diphosphate synthase large subunit from
Mentha piperita
, and to vectors containing the sequences, host cells containing the sequences and methods of producing recombinant geranyl diphosphate synthase large subunit and its mutants.
BACKGROUND OF THE INVENTION
Geranyl diphosphate synthase (GPP synthase) is one of a family of enzymes called prenyltransferases that catalyze C
5
elongation reactions to form the linear (acyclic) precursors of the various terpenoid families. GPP synthase catalyzes the condensation of dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) to form geranyl diphosphate (GPP) which is the immediate, C
10
acyclic precursor of the monoterpenes (Wise, M. L. and Croteau, R., in Cane, D. E., ed., “Comprehensive Natural Products Chemistry: Isoprenoids, Vol. 2”, Elsevier Science, Oxford, 1999. ps. 97-154) (FIG.
1
). Farnesyl diphosphate synthase (FPP synthase), a related prenyltransferase, utilizes GPP and IPP as substrates to form farnesyl diphosphate (FPP), which is the immediate, C
15
precursor of the sesquiterpenes (FIG.
1
). Another prenyltransferase, geranylgeranyl diphosphate synthase (GGPP synthase), catalyzes the condensation of farnesyl diphosphate (or DMAPP) and IPP to form geranylgeranyl diphosphate (GGPP) which is the immediate C
20
precursor of the diterpene family (FIG.
1
). Other types of prenyltransferases can utilize FPP, GGPP and IPP as substrates to form very long chain molecules, such as natural rubber. Poulter C. D. and Rilling, H. C.,
Accts. Chem. Res
. 11: 307-313 (1978); Scolnik, P. A. and Bartley, G.,
Plant Mol Biol. Rep
. 14: 305, 307 (1996); Ogura, K. and Koyama, T.,
Chem. Rev
. 98: 1263-1276 (1998).
The basic reaction mechanism for all of these prenyltransferases is the same, and consists of three steps (see
FIG. 2
in which the reaction catalyzed by geranyl diphosphate synthase is presented as illustrative of the general reaction mechanism). With reference to
FIG. 2
, in the first step an allylic diphosphate ester (2a) is ionized to the stable carbonium ion (2b). The carbonium ion then attacks the double bond of isopentenyl diphosphate (2c) to yield another carbonium ion (2d). In the final step of the cycle, a proton is eliminated from the newly formed carbonium ion (2d) to form a terpenoid containing a new allylic double bond (2e). In the reaction catalyzed by GPP synthase, the allylic diphosphate ester is dimethyl allyl diphosphate (FIG.
1
and FIG.
2
). In the reactions catalyzed by FPP synthase and GGPP synthase the allylic diphosphate ester is geranyl diphosphate and farnesyl diphosphate, respectively (FIG.
1
).
Unlike FPP synthase and GGPP synthase, which produce GPP as an intermediate and which are nearly ubiquitous (Ogura, K. and Koyama, T., in Ogura, K. and Sankawa, U., eds., “Dynamic Aspects of Natural Products Chemistry” Kodansha/Harwood Academic Publishers, Tokyo, pp. 1-23, 1997), geranyl diphosphate synthase is largely restricted to plant species that produce abundant quantities of monoterpenes. Because both FPP synthase and GGPP synthase produce only negligible levels of GPP as a free intermediate on route to FPP and GGPP ((Ogura, K. and Koyama, T., in Ogura, K. and Sankawa, U., eds., “Dynamic Aspects of Natural Products Chemistry” Kodansha/Harwood Academic Publishers, Tokyo, pp. 1-23, 1997)), it is geranyl diphosphate synthase that provides the crucial link between primary metabolism and monoterpene biosynthesis and that serves as the essential driver of monoterpene biosynthesis (Wise, M. L. and Croteau, R., supra).
Any attempt, therefore, to exploit recombinant methods to increase the yield of monoterpene-producing (essential oil) species, or to genetically engineer the monoterpene biosynthetic pathway into any non-producing species (e.g., field crops, fruit-bearing plant species and animals) requires access to a geranyl diphosphate synthase gene or cDNA clone. Co-expression of geranyl diphosphate synthase along with a selected monoterpene synthase, such as (−)-limonene synthase (Colby et al.,
J Biol. Chem
. 268:23016-23024, 1993), and any subsequent pathway enzymes, should allow production of the corresponding monoterpene product(s) from simple carbon substrates, such as glucose, in any living organism.
Monoterpenes are utilized as flavoring agents in food products, and as scents in perfumes (Arctander, S., in
Perfume and Flavor Materials of Natural Origin
, Arctander Publications, Elizabeth, N.J.; Bedoukian, P. Z. in
Perfumery and Flavoring
Materials, 4th edition, Allured Publications, Wheaton, Ill., 1995; Allured, S., in
Flavor and Fragrance Materials
, Allured Publications, Wheaton, Ill., 1997. Monoterpenes are also used as intermediates in various industrial processes. Dawson, F. A., in
The Amazing Terpenes
, Naval Stores Rev., Mar./Apr., 6-12, 1994. Monoterpenes are also implicated in the natural defense systems of plants against pests and pathogens. Francke, W. in Muller, P. M. and Lamparsky, D., eds.,
Perfumes: Art, Science and Technology
, Elsevier Applied Science, NY, N.Y., 61-99, 1991; Harborne, J. B., in Harborne, J. B. and Tomas-Barberan, F. A., eds., Ecological Chemistry and Biochemistry of Plant Terpenoids, Clarendon Press, Oxford, 399-426, 1991; Gershenzon, J and Croteau, R in Rosenthal, G. A. and Berenbaum, M. R., eds., Herbivores: Their Interactions with Secondary Plant Metabolites, Academic Press, San Diego, 168-220, 1991.
There is also substantial evidence that monoterpenes are effective in the prevention and treatment of cancer (Elson, C. E. and Yu, S. G.,
J Nutr
. 124: 607-614, 1994.). Thus, for example, limonene, perrilyl alcohol and geraniol have each been shown to have chemotherapeutic activity against a very broad range of mammalian cancers (see, for example, (1) limonene, Elegbede et al.,
Carcinogenesis
5:661-665, 1984; Elson et al.,
Carcinogenesis
9:331-332, 1988; Maltzman et al.,
Carcinogenesis
10:781-785, 1989; Wattenberg, L. W. and Coccia, J. B.,
Carcinogenesis
12:115-117, 1991; Wattenberg, L. W. and Coccia, J. B.,
Carcinogenesis
12:115-117, 1991; Haag et al.,
Cancer Res
. 52:4021-4026, 1992; Crowell, P. L. and Gould, M. N.,
CRC Crit. Rev. Oncogenesis
5:1-22, 1994; (2) perillyl alcohol, Mills et al.,
Cancer Res
. 55:979-983, 1995; Haag, J. D. and Gould, M. N.,
Cancer Chemother. Pharmacol
. 34:477-483, 1994; Stark et al.,
Cancer Lett
. 96:15-21, 1995 and (3) geraniol, Shoff et al.,
Cancer Res
. 51:37-42, 1991; Yu et al.,
J. Nutr
. 125:2763-2767, 1995; Burke et al.,
Lipids
32:151-156, 1997.).
Cancer cells can be modified to produce therapeutic amounts of a monoterpene having anti-cancer properties by targeting the cognate monoterpene synthase protein to cancer cells, or by introducing a monoterpene synthase gene into cancer cells. This approach to cancer therapy is complicated, however, by the fact that the natural distribution of geranyl diphosphate synthase is largely restricted to plant species that produce abundant quantities of monoterpenes. Thus, animal cells do not naturally produce the monoterpene precursor geranyl diphosphate. Consequently, the genetic manipulation of cancer cells to produce endogenous monoterpenes having anti-cancer properties requires the introduction of a gene encoding geranyl diphosphate synthase, together with a gene encoding a monoterpene synthase that produces a monoterpene having anti-cancer properties. Similarly, if the protein targeting approach is utilized, both geranyl diphosphate synthase protein and monoterpene synthase protein must be targeted to cancer cells.
Standard protein targeting techniques can be used to introduce geranyl diphosphate synthase along with a monoterpene synthase, such as limonene synthase (Colby et al.,
J. Biol. Chem
. 268:23016-23024, 1993), into animal cells with specific targeting to tumors. See, e.g., Wearley, L. L.,
Critical Reviews in Therapeutic Drug Carrier Systems
, 8(4): 331-394, 1991; Sheldon, K et

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