Table of Contents
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.
New Domestic Sources of Natural Rubber
Cornish, Zhiqang Pan, and Ralph A. Backhaus
- NATURAL RUBBER BIOSYNTHESIS
- LOCALIZATION OF RUBBER TRANSFERASE
- PARTICLE-BOUND RUBBER TRANSFERASE
- A PARTICLE-BOUND 48.5 kD PROTEIN FROM GUAYULE
- MOLECULAR CLONING OF THE RPP GENE
- Fig. 1
- 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
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 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
22.214.171.124) (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.
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
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).
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.
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).
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).
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.
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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
Last update September 10, 1997