Magnesium regulation of rubber biosynthesis

Natural rubber is an important compound with unique properties that are related to its molecular weight. There are over 2500 plant species that produce rubber, however only a few species produce the high molecular weight rubber and at a sufficiently high yield to be commercially viable. Hevea brasiliensis is the main source of commercial natural rubber, and the commercialization of Parthenium argentatum natural rubber only started in 2005. It is desirable to find new sources of natural rubber. This can be achieved by improving the quality and yield of a plant species that already produces natural rubber. Alternatively, one could genetically engineer a microorganism to produce rubber. The development of both options requires understanding the mechanisms regulating rubber biosynthesis and rubber molecular weight. This knowledge can be obtained with in vitro studies using purified rubber particles. Even though the rubber transferase gene has not yet been identified, the in vitro studies of rubber biosynthesis allows the engineering of a microorganism for rubber production, thus a platform host would be available once potential rubber transferase genes are cloned.; In the work presented here, it was shown that in vitro magnesium affects biosynthesis and the molecular weight of the rubber produced by the transferases from H. brasiliensis and P. argentatum. It was also shown that IPP-Mg is the substrate for the rubber transferase from H. brasiliensis and P. argentatum, and the affinity of the enzyme for IPP·Mg is dependent on the amount of magnesium supplemented to the medium. Thus, if plant cells can regulate their magnesium content, they could control rubber biosynthesis and rubber molecular weight by altering their amounts of magnesium.; As magnesium is essential for a number of functions in the cell, it was not known if it was possible to have cells with different amounts of magnesium. This hypothesis was tested in Saccharomyces cerevisiae. The S. cerevisiae wild type strain (BY4741) has two known magnesium transporters (ALR1 and ALR2) to the plasma membrane. Strains with deletion in one or both magnesium transporters were created. The total intracellular level of magnesium of the alr1 and alr1 alr2 strains was lower than the wild type or alr2 strain. The total intracellular level of magnesium of the alr1 and alr1 alr2 strains could be varied by changing the amount of magnesium supplemented to the medium. The change in total intracellular level of magnesium was accompanied by changes in the total intracellular level of other elements. In general strains with lower levels of magnesium had a higher level of manganese.; The total intracellular level of magnesium could also be altered by constitutively expressing magnesium transporters from S. cerevisiae ( ALR1 and ALR2) or Arabidopsis thaliana (AtMRS2-1, AtMRS2-2, AtMRS2-3, AtMRS2-4, AtMRS2-5 and AtMGT10) under the control of the PGK1 promoter. The total intracellular level of magnesium varied from 0.4 to 2.5 times the wild type level.