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Cornish, K., Z. Pan, and R.A. Backhaus. 1993. Engineering new domestic sources of natural rubber. p. 192-196. In: J. Janick and J.E. Simon (eds.), New crops. Wiley, New York.

Engineering New Domestic Sources of Natural Rubber

Katrina Cornish, Zhiqang Pan, and Ralph A. Backhaus

  1. NATURAL RUBBER BIOSYNTHESIS
  2. LOCALIZATION OF RUBBER TRANSFERASE
  3. PARTICLE-BOUND RUBBER TRANSFERASE
  4. A PARTICLE-BOUND 48.5 kD PROTEIN FROM GUAYULE
  5. MOLECULAR CLONING OF THE RPP GENE
  6. CONCLUSIONS
  7. REFERENCES
  8. Fig. 1
  9. Fig. 2
Natural rubber is considered a vital raw material by developed countries and is valued for its high performance characteristics. Synthetic rubber, derived from petroleum, is not as elastic or resilient and does not have the heat transfer properties of natural rubber. Although synthetic rubber is often blended with natural rubber, various products, such as airplane tires, cannot be made without the natural form. Also, synthetic rubber is a non-renewable resource whereas natural rubber should be available indefinitely from renewable plant sources. The only commercial source of natural rubber, at the moment, is the Brazilian rubber tree [Hevea brasiliensis (A. Juss.) Mill. Arg.]. The rubber is harvested by tapping into the pipe-like network of latex-containing laticifers that run beneath the bark, a labor-intensive procedure. The expense of tapping and the tree's tropical growth requirements make H. brasiliensis unsuitable for cultivation within the United States. However, because natural rubber is the second most costly raw material imported into the United States after petroleum, there is strong commercial incentive to develop a domestic rubber crop. Moreover, as plantation-grown H. brasiliensis is derived from clonal material grafted onto seedling root stocks all plants of a commercial line are genetically identical to each other. Thus, H. brasiliensis is vulnerable to crop failure should a particularly virulent disease arise. An alternative rubber crop capable of rapid scale up, using fast-growing annual plants or fermentation in a bioreactor, could furnish a protective buffer in the event of an import shortfall. Even if the crop was not profitable initially, the commercial competitiveness of domestic rubber should steadily improve if natural rubber prices increase as anticipated (Greek 1992).

We are attempting to develop a domestic source of natural rubber using a biotechnological approach. To this end, we intend to clarify the biochemistry of rubber formation and identify and isolate the enzymes and genes responsible for the cis-1,4-polymerization of isoprene unique to rubber producing plants. Once accomplished, it should be possible to isolate, then insert and express the appropriate genes into annual plants and/or microorganisms. These systems would then be optimized to produce large amounts of high quality rubber.

A considerable body of information exists on the biological mechanism of rubber biosynthesis and on the adjacent portions of the isoprenoid pathway. This includes the isolation and cloning of genes for enzymes involved in the production of allylic pyrophosphate initiators for new rubber molecules (Anderson et al. 1989a,b). However, before transformation experiments on potential domestic rubber-producing species can be realistically begun, a definitive isolation of the rubber transferase enzyme responsible for rubber molecule elongation, and then its gene, is required.

Parthenium argentatum Gray (guayule) is a promising candidate for a domestic commercial source as it produces high quality rubber in its bark. Unlike, H. brasiliensis, which has the complex laticiferous anatomy to support its rubber production (d'Auzac et al. 1989), P. argentatum simply produces rubber in generalized parenchyma cells in its bark tissue (Backhaus 1985). Furthermore, P. argentatum is native to the warm arid regions of the southwestern United States and is being cultivated there in various preliminary trials. Disadvantages exist in obtaining the rubber from this perennial species as the destructive harvest of mature plants is required. Woody shrubs, at least three years old, must be ground up before the rubber can be extracted. Also, high yields only result when the crop is irrigated and fertilized, and P. argentatum cannot tolerate the severe winters of the northern United States. These characteristics of the crop make it unsuitable for an emergency supply as it could not be rapidly scaled up. Nonetheless, this species is a good model system for studying rubber biosynthesis, and provides a source of genes that may be useful in increasing its own rubber yield and/or for transformation of other species.

NATURAL RUBBER BIOSYNTHESIS

Natural rubber biosynthesis is a side-branch of the ubiquitous isoprenoid pathway (Fig. 1). Natural rubber is made almost entirely of isoprene units derived from the precursor isopentenyl pyrophosphate (IPP). Also, trans-allylic pyrophosphates are essential for rubber formation as they are used to initiate all new rubber molecules. The elongation of the rubber molecule is catalyzed by the enzyme rubber transferase (RuT) (EC 2.5.1.20) (Backhaus 1985). We do not know where the rate-limiting steps in rubber biosynthesis are located. Simply adding more RuT to a rubber-producing plant may not enhance its yield. We may need to overexpress earlier portions of the isoprenoid pathway to supply adequate substrate levels to support an increased level of rubber biosynthesis. It will also be necessary to ensure that the vital downstream portions of the isoprenoid pathway are not made substrate deficient by increased activity of the rubber biosynthesis branch.

The first biochemical step essential for, though not unique to, rubber biosynthesis is the isomerization of the C5 IPP to dimethylallyl pyrophosphate (DMAPP) by the enzyme IPP-isomerase (Fig. 2). This is followed by prenyl transferase-catalyzed synthesis of the C10 (geranyl pyrophosphate, GPP), C15 (farnesyl pyrophosphate, FPP) and C20 (geranyl geranyl pyrophosphate, GGPP) allylic pyrophosphates by a series of additions of IPP (nonallylic pyrophosphate) in the trans configuration, to DMAPP. The prenyl transferases and the IPP-isomerase are soluble cytosolic or chloroplastic enzymes. In vivo, RuT appears to use FPP or GGPP to initiate rubber molecule formation (Tanaka 1989) although all the allylic pyrophosphates, from the C5 DMAPP to the C20 GGPP, can initiate rubber molecule formation in vitro (Archer and Audley 1987; Berndt 1963; Cornish 1992; Madhaven et al. 1989). Once the initiator is in place, RuT can then begin the cis-elongation of isoprene units from IPP. Simply put, the longer the rubber chain, the greater the quality of the finished product. The highest quality rubber has a molecular weight of around 1.5 million.

There are over 2,000 species of plants from about 300 genera as well as at least two fungal genera (Archer et al. 1963; Backhaus 1985) that are known to make natural rubber but most make a short-chain form. A termination step, probably independent of RuT itself, may well govern chain length. Thus, a biological system transformed with the RuT gene from a high molecular weight species, may still generate short chain (poor quality) rubber unless termination is regulated.

LOCALIZATION OF RUBBER TRANSFERASE

In order to isolate the RuT enzyme, it is first necessary to determine the location of the enzymatic reaction. Rubber is compartmentalized into cytosolic rubber particles both in laticiferous species such as H. brasiliensis (d'Auzac et al. 1989), and in species that produce rubber in parenchyma cells such as in the bark of P. argentatum (Backhaus 1985). These two species are unusual among rubber-producers as they both can make commercial-grade rubber. During rubber biosynthesis, isopentenyl pyrophosphate is obtained from the aqueous environment outside the rubber particles and is dephosphorylated and polymerized by the RuT enzyme. The developing isoprene polymers extend into the particle interior. This process can be assayed by following the incorporation of labeled isoprene from 14C-IPP into the newly-synthesized rubber chains (e.g. Archer and Audley 1987; Cornish and Backhaus 1990).

The nature of the reaction intimates that it takes place at the surface of the rubber particles. However, RuT may be cytosolic and associated only loosely with the particle or it may be particle-bound. This question was addressed experimentally using isolated rubber particles. Particles were prepared from both H. brasiliensis and from P. argentatum using a centrifugation/flotation procedure (Cornish and Backhaus 1990). Repeated washes, using this procedure, allowed the removal of soluble cytoplasmic components from the latex or bark homogenate. The isolated particles were then assayed for their RuT activity by incubating them in the presence of IPP and an allylic pyrophosphate initiator. When P. argentatum rubber particles were incubated with IPP and FPP no reduction of RuT activity was observed with washing, demonstrating the presence of a highly active bound RuT (Cornish and Backhaus 1990). Similar experiments demonstrated a bound RuT on H. brasiliensis rubber particles (K. Cornish unpubl. data), as has previously been reported (Archer et al. 1963; Berndt 1963). The bound RuT accounts for most, if not all, of the RuT activity in H. brasiliensis latex (K. Cornish unpubl. data). As no reduction of RuT activity with increasing purification of rubber particles was observed for either species, the RuT molecules are firmly associated with rubber particles in both H. brasiliensis (Archer et al. 1963; Berndt 1963) and P. argentatum (Benedict et al. 1990; Cornish and Backhaus 1990).

H. brasiliensis latex contains the prenyl transferases and IPP-isomerase necessary (Fig. 2) for initiator synthesis and some incorporation of label was observed when 14C-IPP was added to the latex. However, washing the particles to remove the cytoplasmic components of the latex completely eliminates this activity. Without the control treatment, where allylic pyrophosphate added back to the washed rubber particles more than restored the original whole latex level of rubber biosynthesis (K. Cornish unpubl. data), it would be easy to misinterpret the results as meaning that the washing procedure had removed RuT itself, instead of the initiator system. An overview of this aspect of rubber biochemistry and a discussion of possible misinterpretations in the published literature, has been presented in detail (Cornish 1992).

PARTICLE-BOUND RUBBER TRANSFERASE

Although these biochemical assays of rubber biosynthesis were obtained using intact rubber particles, instead of a purified RuT enzyme, the bound-RuT activity does behave as a single enzyme system. The dependence of RuT activity upon substrate concentration, in both P. argentatum and H. brasiliensis and for several different allylic pyrophosphate initiators, IPP and various cofactors, has simple enzyme kinetics, giving rise to linear Eadie-Hofstee plots (Cornish and Backhaus 1990; K. Cornish unpubl. data). If a multicomponent system was present, these plots of V against V/[S] would generate curved instead of straight lines. The experiments also showed that the substrate binding characteristics of RuT from both species are extremely similar, suggesting that the active site of the two RuT enzymes may also be closely related (Cornish 1992). The RuT catalytic site is probably contained within a single enzyme or enzyme complex because two spatially separate active sites would be unlikely to permit the two different initiation and elongation substrates to be attached to each other.

Protein analysis of isolated rubber particles has been attempted in efforts to distinguish the RuT enzyme from the other particle-bound proteins (Benedict et al. 1990; Backhaus et al. 1991). Despite the biochemical similarity of the P. argentatum and H. brasiliensis rubber biosynthetic systems, the particle-bound protein profiles proved quite distinct. Silver-stained one-dimensional SDS-PAGE analysis of washed rubber particles revealed at least 20 distinct proteins associated with H. brasiliensis particles (K. Cornish unpubl. data) but only 4 to 8 in P. argentatum (Backhaus et al. 1991). As it has proved difficult to obtain solubilized RuT activity from the particles it should prove much easier to isolate RuT from amongst the 4 to 8 P. argentatum proteins than from amongst the much larger number of H. brasiliensis particle-bound proteins.

A PARTICLE-BOUND 48.5 kD PROTEIN FROM GUAYULE

The P. argentatum particle-bound 48.5 kD glycoprotein (RPP) is of most interest at present for several reasons. This protein is located largely within the particle but at the surface with the glycosylated moiety protruding into the cytoplasm (Backhaus et al. 1991). This is an appropriate locale for RuT, which must polymerize a hydrophobic molecule to the particle interior while obtaining hydrophilic substrates from the cytosol. It is also the most abundant particle-bound protein and is present in all ages and lines of P. argentatum examined so far (Backhaus et al. 1991; Cornish and Backhaus 1990). Furthermore, other workers have reported solubilized RuT activity associated with this protein (Benedict et al. 1990).

MOLECULAR CLONING OF THE RPP GENE

The RPP protein was purified to homogeneity from washed rubber particles using preparative SDS-PAGE and electroelution. The purified RPP was then sent to the University of California, Davis, Sequencing Laboratory for analysis. The amino acid composition was consistent with RPP being a membrane protein, and the calculated pI of 6.17 matched the pI of 6.2 determined earlier with isoelectric focusing (Backhaus et al. 1991). As the N-terminus of intact RPP was blocked, the protein was cleaved with cyanogen bromide at its five methionine residues and the resulting six peptide fragments were sequenced. Oligonucleotides corresponding to sequences determined were synthesized and used to prime plus and minus strand amplification of P. argentatum bark cDNA, using the polymerase chain reaction (PCR). The longest clone, c18, so far obtained from a P. argentatum stembark lambda ZAP cDNA library, accounts for 70% of the RPP gene and includes 4 out of the six peptide fragments obtained from the CNBr digests (Z. Pan and R.A. Backhaus unpubl. data).

Once the full-length gene has been obtained transformation experiments using P. argentatum and other species will be performed in attempts to increase rubber yield and to determine, conclusively, the role of RPP. This should be possible because P. argentatum has been successfully transformed with GUS and kanamycin resistance using Agrobacteria (Backhaus et al. in press).

CONCLUSIONS

In conclusion, RuT is bound to the rubber particle in both species examined, and this may prove to be true for all rubber-producing species. Rubber biosynthesis is biochemically indistinguishable in P. argentatum and H. brasiliensis. This suggests that the RuT may be alike in these two species and that their RuT genes may be readily interchangeable. The most abundant protein bound to P. argentatum rubber particles is a 48.5 kD glycoprotein positioned just beneath the particle surface. Evidence suggests that this protein is RuT, and 70% of its gene has now been isolated.

REFERENCES


Fig. 1. A section of the isoprenoid pathway illustrating the position of natural rubber biosynthesis.

Fig. 2. The biosynthesis of natural rubber from iso-pentenyl pyrophosphate. Each new molecule of cis-1,4-polyisoprene requires an allylic pyrophosphate initiator before the isoprene units from IPP can be polymerized.
Last update September 10, 1997 aw