2001 — 2002 |
Han, Kyung-Hwan Kamdem, Donatien |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Sger: Biochemical Genomics of Rubber Biosynthesis @ Michigan State University
ABSTRACT: MCB-0091088 Han and Kamdem Rubber (cis-1,4-polyisoprene) is produced in varying quantities and qualities by about 2,000 plant species. This isoprenoid polymer has no identified physiological function in plants, but it has many important industrial uses due to its elasticity, flexibility, and resilience. Although natural rubber is present in many species, only that from the tree Hevea brasiliensis is used commercially. This is due to its combination of high quality, high yield, and ease of harvest. Rubber biosynthesis in plants presents three interesting questions: 1) Why do plants make this metabolically expensive polymer? 2) How is rubber synthesized? 3) What determines the chain length of rubber molecules? The answers to these questions are of keen commercial and scientific interest, but our ability to answer them hinges on the cloning of the rubber polymerase gene(s). Several groups have tried unsuccessfully to clone the gene using conventionally available methods. The recent advances in genomics, however, offer a new opportunity. This project uses a simple but innovative concept to clone the gene(s) encoding rubber polymerase. The investigators will first generate about 1,000 EST's from a subtracted cDNA library that is enriched for latex genes. These will represent a catalog of the genes abundantly expressed in the rubber-producing tissue of H. brasiliensis. The EST's will then be compared with annotated public databases and will be assigned putative functional identifications if possible. The EST's with homology to any genes that are known to function in processes unrelated to the rubber biosynthetic pathway (i.e., the isoprenoid pathway) will be eliminated from further experiments. The remaining cDNA clones will be introduced into an array of yeast strains, each of which will then bear a plasmid expressing a latex cDNA. Yeast already have all the components of the isoprenoid pathway leading to rubber biosynthesis except for the gene(s) encoding the rubber polymerase itself. Ectopic expression of the rubber polymerase gene(s) from Hevea trees may enable the yeast cells to produce natural rubber. The investigators will use two approaches to determine this. In one they will directly test each transformed yeast strain for the ability to synthesize rubber. If, however, yeast require more than one H.brasiliensis gene product for rubber biosynthesis, this approach will not work. Because of that possibility, they will also prepare pools of extracts of the yeast strains, with each pool containing extracts from numerous strains. These pools will then be assayed for in vitro rubber biosynthesis. The investigators will progressively narrow down any pool(s) showing positive results until they identify the individual strain(s) expressing the polymerase gene(s). This pooling elevates yeast complementation to a genomic scale.
Natural rubber is an extremely important product with a vast range of uses. It is superior to synthetic, petroleum-based versions for many applications. Unfortunately, its ready availability could be endangered by numerous events such as crop failure or political turmoil. If successful, this novel project will provide society with an efficient and simple alternative: natural rubber harvested from yeast cultures. It will also enable scientists to learn more about natural polymerization and to engineer rubber compounds with special chemical features.
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