Isopentenyl diphosphate isomerase from Hevea brasiliensis...

Details Isopentenyl diphosphate isomerase from Hevea brasiliensis... Isopentenyl diphosphate isomerase from Hevea brasiliensis...

1999-04-22

2001-11-13

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C800S298000, C800S295000, C435S166000, C435S183000, C435S468000, C435S471000, C435S483000, C435S484000, C435S488000, C435S814000, C435S320100, C435S254100, C435S252300, C435S252330, C536S023200, C536S024100, C536S023600

Reexamination Certificate

active

06316695

ABSTRACT:

BACKGROUND OF INVENTION
1. Field of the Invention
The present invention relates to a isopentenyl diphosphate isomerase from
Hevea Brasiliensis
and a rubber producing method using the same.
2. Description of the Prior Art
The isoprenoid biosynthetic pathway is ubiquitous to all living organisms and produces more than 23,000 compounds which play vital roles in the structure of cells, electron transport, photosynthesis, cell-to-cell signaling, and interactions between organisms (Ramos-Valdivia et al., 1997). Several important classes of compounds derived from this complex pathway include sterols, carotenoids, dolichols, ubiquinones, and prenylated proteins (Hahn and Poulter, 1995). All of these compounds are derived from the same building block, IPP. In most eukaryotes, IPP is synthesized from three molecules of acetyl-CoA via the mevalonic acid pathway(Chappell, 1995). Recently, a separate mevalonate-independent pathway was discovered in bacteria and plant(Lichtenthaler et al., 1997; Romer et al., 1993). This pathway utilizes three-carbon precursors such as glyceraldehyde phosphate, pyruvate, and dihydroxyacetone phosphate. The enzyme IPP isomerase (hereafter “IPI”) catalyzes the interconversion of IPP to its highly electrophilic isomer, dimethylallyl diphosphate (DMAPP). These two isomers serve as substrates for the synthesis of isoprenoid compounds.
Bioengineering of isoprenoid biosynthesis is of keen interest because of the commercial value of plant compounds such as essential oils, pharmaceuticals, rubber, and waxes. Although the interconversion of IPP and DMAPP is catalyzed by the same IPP isomerase, the equilibrium is in favor of DMAPP production(Sun et al., 1998). This isomerization reaction may be a rate-limiting step for isoprenoid biosynthesis. Expression of an exogenous IPI gene enhanced isoprenoid biosynthesis in
E. coli
(Kajiwara et al., 1997). Transgenic tobacco plants transformed with a
H. brasiliensis
HMGCoA reductase over-produced total sterols up to six-times than the control plants did (Schaller et al., 1995).
Natural rubber (cis-1,4-polyisoprene) is an important raw material for many industrial uses. Although rubber is produced in about 2,000 plant species(Backhaus, 1985),
H. brasiliensis
has been the only commercial source of natural rubber mainly due to its abundance in the tree, its quality and the ease of harvesting. Diminishing acreage of rubber plantation coupled with increasing demand has renewed research interests on the study of rubber biosynthesis and the development of an alternative rubber source. The first step in rubber biosynthesis is the isomerization of IPP to DMAPP by IPP isomerase. The successive head-to-tail condensation reactions of the five-carbon intermediates catalyzed by enzyme(s) referred to as rubber transferase (or polymerase) have been assumed to yield rubber. IPP isomerase activity has been found in bottom fraction and C-serum of rubber latex of
H. brasiliensis
(Koyama et al., 1996; Tangpakdee et al., 1997). The presence of a site-directed specific inhibitor of IPP isomerase, 3,4-oxido-3-methyl-1-butyl diphosphate (OMBPP), inhibited incorporation of IPP into rubber in in vitro rubber assay (Cornish, 1993).
IPP isomerase catalyzes the conversion of IPP to DMAPP, which is an essential step in the biosynthesis of all isoprenoids including carotenoids, growth regulators, and natural rubber. The enzyme from a wide variety of organisms has been studied for a review, see (Ramos-Valdivia et al., 1997). In higher plants, IPP isomerase has been purified and characterized from Capsicum chromoplasts(Dogbo and Camara, 1987),
Cinchona robusta
cell suspension (Ramos-Valdivia et al., 1997),
H. brasiliensis
(Koyama et al., 1996). To date relatively few IPP isomerase sequences from higher plants appear in the literature. Full-length genes for higher plant IPP isomerase are known only for Arabidopsis(Campbell et al., 1997) and two flowering plants Clarkia spp.(Blanc et al., 1996; Blanc and Pichersky, 1995). Partial sequence is also available for tobacco (Accession No. Y09634). However, none of the genes has been implicated in rubber biosynthesis.
As the isomerization of IPP to DMAPP is the first step in rubber biosynthesis, it is plausible for IPP isomerase to play a key role in rubber biosynthesis. Although IPP isomerase activity was observed in both bottom and C-serum fractions from Hevea latex (Tangpakdee et al., 1997), neither the protein nor cDNA encoding IPP isomerase has been isolated and studied from Hevea rubber tree.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide a isopentenyl diphosphate isomerase from
Hevea brasiliensis
and a rubber producing method using the same.
The present invention cloned the gene by screening a latex cDNA library using a PCR-generated probe. Despite the presumed-crucial role of the enzyme in rubber biosynthesis, the gene does not seem to be expressed as an abundant transcript in latex, which contains 30-50% (wt/wt) of cis-1,4-polyisoprene (rubber). In a separate study, the present invention have generated 245 expressed sequence tags (ESTs) to study gene expression profile in the latex of
H. brasiliensis
(Han et al., 1999). The gene encoding IPP isomerase was not identified among the ESTs. Furthermore, initial attempts to screen 1×10
6
or less plaques of the latex cDNA library failed to produce any hybridizing cDNA clones. After screening of more than 2×10
6
plaques only two hybridizing clones were obtained. Since DMAPP is the primer for the isoprenoid biosynthesis and must be present in any cell compartment where isoprenoids are biosynthesized, the low transcript level of IPIHb gene in the latex is unexpected.


REFERENCES:
patent: 4638028 (1991-01-01), Lui et al.
patent: 4983729 (1991-01-01), Sikora
Oh et al. Accession No. AF111842 Deposited, Dec. 1998.*
Jung et al. Accession No. AF031079, Deposited, Oct. 1997.*
Attanyaka et al. Plant Molecular Biology 16: 1079-1081, 1991.*
Blanc et al. Accession No. X82627, Deposited, Nov. 1994.*
Blanc V., et al., “Nucleotide Sequence of Clarkia breweri cDNA Cloone of Ipi1, a Gene Encoding Isopentenyl Pyrophosphate Isomerase”Plant Physiol, vol. 108, pp. 855-856, 1995.
Tangpakdee, J., et al. “Isopentenyl Diphosphate isomerase and Prenyl Transferase Activities in Bottom Fraction and C-Serum from Hevea Latex”Phytochemistry, vol. 45, No. 2, pp. 2661-267, 1997.
Cornish, K., “The separate roles of plant cis and trans prenyl transferases in cis-1,4-polyisoprene biosynthesis”Biochem. Journal, vol. 116, pp. 267-271, 1993.
Kajiwara, S., et at. “Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis inEscherichia coli” Biochem. Journal, vol. 324, pp. 421-426, 1997.
Valdivia, A., et al. “Purification and characterization of two isoforms of isopentenyl-diphosphate isomerase from elicitor-treated Cinchona robusta cells.”EJB1997 pp. 470-474, 1997.
Valdivia, A., et al. Elicitor-mediated induction of anthraquinone biosynthesis and regulation of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase activities in cell suspension cultures of Cinchona robusta How.Plantavol. 203, pp. 155-161, 1997.
Campbel, M., et al., “Analysis of the isopentenyl diphosphate isomerase gene family from Arabidopsis” Plant Molecular Biology, 36:323-328, 1997.

Affiliated with

Also associated with

Rating

Isopentenyl diphosphate isomerase from Hevea brasiliensis... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Isopentenyl diphosphate isomerase from Hevea brasiliensis..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Isopentenyl diphosphate isomerase from Hevea brasiliensis... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2607751