Genes involved in the biosynthesis of




Дата канвертавання21.04.2016
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TITLE

GENES INVOLVED IN THE BIOSYNTHESIS OF

ISOPENTENYL DIPHOSPHATE in Hevea brasiliensis latex

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology and botany. More specifically, this invention pertains to nucleic acid fragments encoding enzymes useful for the bioproduction of isopentenyl diphosphate.



BACKGROUND OF THE INVENTION

Plants synthesize a variety of hydrocarbons built up of isoprene units (C5H8), termed polyisoprenoids (Tanaka, Y. In Rubber and Related Polyprenols. Methods in Plant Biochemistry; Dey, P.M. and Harborne, J.B., Eds., Academic Press: San Diego, 1991; Vol. 7, pp 519 536). Those with from 45 to 115 carbon atoms, and varying numbers of cis- and trans- (Z- and E-) double bonds, are termed polyprenols, while those of longer chain length are termed rubbers (Tanaka, Y. In Minor Classes of Terpenoids. Methods in Plant Biochemistry; Dey, P.M. and Harborne, J.B., Eds., Academic Press: San Diego, 1991; Vol. 7, pp 537 542). The synthesis of these compounds is carried out by a family of enzymes termed prenyltransferases, which catalyze the sequential addition of C5 isopentenyl diphosphate units to an initiator molecule (Figure 1). In Hevea brasiliensis rubber, the C5 units are added in the cis-configuration, and thus the prenyltransferas(s) involved are termed cis- or Z-prenyltransferases.

Two distinct pathways for the synthesis of isopentenyl diphosphate (IPP) are now known to be present in living organisms (Lichtenthaler et al., Physiol. Plantarum 101:643 652 (1997)). In one pathway, which is confined in plants to plastids, glyceraldehyde 3 phosphate and pyruvate are precursors of IPP (Lichtenthaler et al., FEBS Letts. 400:271-274 (1997)). In the second (cytoplasmic) pathway, acetate is converted to IPP via the intermediate mevalonic acid (Newman, J.D., Chappell, J. Isoprenoid biosynthesis in plants: carbon partitioning within the cytoplasmic pathway. Crit Rev Biochem Mol Biol. 1999;34(2):95 106; Bach TJ, Boronat A, Campos N, Ferrer A, Vollack KU, Mevalonate biosynthesis in plants. Crit Rev Biochem Mol Biol. 1999;34(2):107 22). The latter pathway, the acetate/mevalonate pathway, has long been assumed to be the sole pathway operating in the rubber-synthesizing latex of Hevea brasiliensis. In this pathway, acetate is converted to IPP by the sequential action of the following six enzymes: acetyl-coA acetyltransferase, HMG-coA synthase, HMG-coA reductase, mevalonate kinase, phosphomevalonate kinase and mevalonate diphosphate decarboxylase (Figure 2).

Of the minimum of six genes encoding the enzymes of this pathway in Hevea brasiliensis, only those for HMG-coA reductase have been cloned. Two cDNAs, encoding enzymes termed HMGR1 and HMGR2, were isolated using a heterologous hybridization probe, and genomic southern blotting confirmed the presence of at least two genes for HMG-coA reductase in the Hevea brasiliensis genome (Chye et al., Plant Mol. Biol. 16:567 577 (1991)). An EST homologous with HMGR1 was also identified in a Hevea brasiliensis latex library (Han et al., Tree Physiol. 20:503 510 (2000)). A gene encoding a third isoform of HMG-coA reductase in Hevea, termed HMGR3, has also been reported (Chye et al (1992) Plant Mol. Biol. 19: 473-484). Of the other five genes, although several have been identified in other plant species, no Hevea brasiliensis homologs have been identified or their genes isolated.

The initiator molecules used for the elaboration of polyprenols and rubbers are also derived from IPP, and are allylic terpenoid diphosphates such as dimethylallyldiphosphate (DMAPP), but more usually the C10 compound geranyl diphosphate (GPP), the C15 compound farnesyl diphosphate (FPP) or the C20 compound geranylgeranyl diphosphate (GGPP) (Figure 1). DMAPP is generated from IPP by the action of an isomerase enzyme termed IPP isomerase. Genes encoding this enzyme have been isolated from a number of species, including Hevea brasiliensis (Oh et al., J. Plant Physiol. 157:549 557 (2000)). The allylic diphosphates GPP, FPP and GGPP are synthesized by trans- or E prenyltransferases, using DMAPP and IPP. Genes encoding the enzymes which synthesize these allylic terpenoid diphosphates have been cloned from a number of organisms, including plants (McGarvey et al., Plant Cell 7:1015 1026 (1995); Chappell, J., Annu. Rev. Plant Physiol. Plant Mol. Biol. 46:521 547 (1995)). All of these gene products condense isoprene units in the trans-configuration.

There are several suggested functions for plant polyisoprenoids. Terpenoid quinones are most likely involved in photophosphorylation and respiratory chain phosphorylation. Rubbers have been implicated in plant defense against herbivory, possibly serving to repel and entrap insects and seal wounds in a manner analogous to plant resins. The roles of the C45 C115 polyprenols remain unidentified, although as with most secondary metabolites they too most likely function in plant defense. Short-chain polyprenols may also be involved in protein glycosylation in plants, by analogy with the role of dolichols in animal metabolism.


CLAIMS


What is claimed is:

1. An isolated nucleic acid molecule encoding an isopentenyl diphosphate pathway enzyme, selected from the group consisting of:

(a) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13;

(b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1X SSC, 0.1% SDS at 65 °C, and washed with 2X SSC, 0.1% SDS followed by 0.1X SSC, 0.1% SDS; and

(c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).

2. The isolated nucleic acid molecule of Claim 1 selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

3. A polypeptide encoded by the isolated nucleic acid molecule of Claim 1.

4. The polypeptide of Claim 3 selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

5. An isolated nucleic acid molecule comprising a first nucleotide sequence encoding an acetyl-coA acetyltransferase enzyme that has at least 65% identity over length of 411 amino acids based on the CLUSTALW method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:8 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.

6. An isolated nucleic acid molecule comprising a first nucleotide sequence encoding an HMG-coA synthase enzyme that has at least 82% identity over length of 464 amino acids based on the CLUSTALW method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:9 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.

7. An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a mevalonate kinase enzyme that has at least 68% identity over length of 386 amino acids based on the CLUSTALW method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:11 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.

8. An isolated nucleic acid molecule comprising a first nucleotide sequence encoding an phosphomevalonate kinase enzyme that has at least 73% identity over length of 503 amino acids based on the CLUSTALW method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:12 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.

9. An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a mevalonate diphosphate decarboxylase enzyme that has at least 77% identity over length of 415 amino acids based on the CLUSTALW method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:13 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.

10. A chimeric gene comprising the isolated nucleic acid molecule of any one of Claims 1 or 5 9 operably linked to suitable regulatory sequences.

11. A transformed host cell comprising the chimeric gene of Claim 10.

12. The transformed host cell of Claim 11 wherein the host cell is selected from the group consisting of bacteria, yeast, filamentous fungi, algae and green plants.



13. The transformed host cell of Claim 12 wherein the host cell is selected from the group consisting of Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Flavobacterium, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Escherichia, Erwinia, Pseudomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus, Methanobacterium and Klebsiella.


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