4

Biosynthesis of NaturalRubber and Other NaturalPolyisoprenoids

Dr. Norimasa Ohya1, Prof. Dr. Tanetoshi Koyama2

1 Department of Material and Biological Chemistry, Faculty of Science, YamagataUniversity, Koshirakawa-cho 1 ± 4-12, Yamagata 990 ± 8560, Japan;Tel.:�81-236-28-4583; Fax:�81-236-28-4510; E-mail: ohya@sci.kj.yamagata-u.ac.jp

2 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University,Katahira 2 ± 1-1, Aoba-ku, Sendai 980 ± 8577, Japan; Tel.:�81-22-217-5621;Fax:�81-22-217-5620; E-mail: koyama@icrs.tohoku.ac.jp

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751.1 Occurrence of Polyisoprene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751.2 Chemical Properties of Rubber . . . . . . . . . . . . . . . . . . . . . . . . . . . 761.3 The Latex Vessel System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 761.4 Composition of Latex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771.5 Molecular Weight of Rubber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

2 Historical Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792.1 Rubber Precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792.2 Rubber Formation on Rubber Particles . . . . . . . . . . . . . . . . . . . . . . 812.3 Stereochemistry of Rubber Biosynthesis . . . . . . . . . . . . . . . . . . . . . . 822.4 Initiator of Rubber Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

3 Biosynthesis Pathway in Hevea brasiliensis . . . . . . . . . . . . . . . . . . . . . 833.1 Formation of IPP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833.2 Initiation of Rubber Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . 833.3 Chain-Elongation of Rubber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 853.4 13C-NMR Study of Rubber Molecules . . . . . . . . . . . . . . . . . . . . . . . . 86

4 Enzymes in Hevea brasiliensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 874.1 Enzymes for the Formation of Mevalonic Acid . . . . . . . . . . . . . . . . . . 874.2 Enzymes for the Formation of IPP . . . . . . . . . . . . . . . . . . . . . . . . . 884.3 Rubber Transferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

5 Genetic Basis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

73

5.1 Rubber Biosynthesis-Related Proteins . . . . . . . . . . . . . . . . . . . . . . . 905.1.1 Rubber Elongation Factor (REF) . . . . . . . . . . . . . . . . . . . . . . . . . . 905.1.2 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGR) . . . . . . . . . 915.1.3 Isopentenyl Diphosphate Isomerase (IDPI) . . . . . . . . . . . . . . . . . . . . 925.1.4 Farnesyl Diphosphate Synthase (FPS) . . . . . . . . . . . . . . . . . . . . . . . 925.1.5 Small Rubber Particle Protein (SRPP) . . . . . . . . . . . . . . . . . . . . . . . 935.2 Defense-Related Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 935.2.1 Hevein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 935.2.2 Chitinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 935.2.3 b-1,3-Glucanase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945.2.4 Hever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945.3 Latex Allergens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945.4 Other Enzymes in Hevea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

6 Rubber Biosynthesis Pathways and Genes in Guayule and other Organisms . 956.1 Guayule Rubber Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 956.2 Rubber Synthesis in Indian Rubber Tree . . . . . . . . . . . . . . . . . . . . . . 966.3 Rubber Synthesis in Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

7 Production in Transgenic Plants and Recombinant Microorganisms . . . . . . 97

8 Outlook and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

9 Relevant Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 999.1 Stimulation of Rubber Yield and Rubber-Producing Plants . . . . . . . . . . . 999.2 Rubber Biosynthetic Genes and their Transformation . . . . . . . . . . . . . . 999.3 Synthetic Use of Enzymes from H. brasiliensis . . . . . . . . . . . . . . . . . . 1019.4 Enzymes from Hevea and their DNA Sequences . . . . . . . . . . . . . . . . . 101

10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

ACAT acetyl-CoA acetyltransferaseDMAPP dimethylallyl diphosphateFPP farnesyl diphosphateGGPP geranylgeranyl diphosphateGPP geranyl diphosphateGUS b-glucuronidaseHMG-CoA 3-hydroxy-3-methylglutaryl-CoAHMGR 3-hydroxy-3-methylglutaryl coenzyme A reductaseHnl (S )-hydroxynitrile lyaseIDPI isopentenyl diphosphate isomeraseIPP isopentenyl diphosphateMnSOD manganese-containing superoxide dismutaseMVA mevalonic acidMVA-P 5-phosphomevalonic acid

4 Biosynthesis of Natural Rubber and Other Natural Polyisoprenoids74

MVA-PP 5-diphosphomevalonic acidNPP neryl diphosphatePAGE polyacrylamide gel electrophoresisPCR polymerase chain reactionREF rubber elongation factorSDS sodium dodecyl sulfateUPS undecaprenyl diphosphate synthaseWRP washed rubber particle

1

Introduction

1.1

Occurrence of Polyisoprene

Over 2000 species of higher plants producepolyisoprene (Backhaus, 1985). The rubbermolecule is a high-molecular weight polymerconsisting of isoprene units in the cis-config-uration. Rubber, having elasticity, has beenwidely applied to many products, such astires, gloves,balloonsand balls for sports.Therubber for practical use was obtained frommany species until the twentieth century.However, Hevea brasiliensis has been estab-lished as only one commercial rubber sourcedue to its good yield of rubber and excellentphysical properties of the rubber product(Archer and Audley, 1973). The latter, afteruse, has been recycled as reclaimed rubberbecause of the ease of recycling. Naturalrubber also contributes to the global environ-ment preservation by reducing the amount ofcarbon dioxide in the atmosphere (Kodama,1999). The amount absorbed is estimated at90,000,000tonsperyearbasedonthequantityof rubber produced and the growth of rubbertrees (Rahaman, 1994). When rubber hasbeen collected from each tree for about 20years, the rubber tree is used for furniture andfloormaterials,etc.as thewoodisexcellent forsuch purposes. While the tropical rainforestis being decreased in size year by year byburning of the soil and the trimming of trees,the rubber tree ± which produces useful

compounds and in itself also eventuallybecomes a wood resource ± has attractedattention as a substitute for the tropicalrainforest. During World War II it was notpossible for the USA and European countriesto import rubber from plantations in Asia,and consequently the main supply source ofthe rubber at that time was from Pertheniumargentatum (guayule) and Taraxacum koksa-ghyz (Russian dandelion). Both of these weretypical rubber-bearing plants growing intemperate zones, and they became establish-ed as emergency resources for rubber duringthis period of crisis. After World War II,guayule (as a North American desert shrub)was investigated as being an alternativesource of natural rubber, which containsresin, rubber-soluble triglycerides and higherterpenes (Backhaus, 1985). Ficus elastica(Indian rubber tree) was one of the firstplants of the tropical regions of Asiato be grown for rubber production (Polha-mus, 1962). Dyera costulata (Jelutong), Comamacrocarpa (sorva) and Coma utilis (sorvinha)also produce rubber that contains largeamounts of resins. Among lower organisms,two major classes of fungi ± Ascomycetes andBasidiomycetes ± produce low-molecularweight rubber (Stewart et al. , 1955; Kuriharaet al. , 1962).

Relatively few plant species, for exampleMimusops balata (Balata) and Plaquium gutta(Gutta percha), produce high-molecularweight trans-polyisoprene. Gutta was onceused principally for cable insulation, beltingand golf-ball covers, although synthetic poly-

1 Introduction 75

mers have replaced most of these natural rawmaterials in many applications. Achras sapota(Chicle) in Mexico, Guatemala and Venezue-la exceptionally produces the mixture of cis-and trans-polyisoprene, which is an econom-ically important resource for using as a rawmaterial inchewinggum(ArcherandAudley,1973).

1.2

Chemical Properties of Rubber

The fact that the fundamental structure ofnatural rubber consists of isoprene units,C5H8, was established in the early twentiethcentury using chemical techniques such asthermal degradation, ozonolysis and hydro-genation. X-ray diffraction studies on naturalrubber have demonstrated that the doublebond in isoprene units is in the cis-config-uration.

Both 3,4- and 1,2-units are formed togetherwith cis-1,4- and trans-1,4-units in syntheticcis-1,4-polyisoprene. Normally, synthetic pol-yisoprene is considered as a copolymer con-sisting of isoprene units linked together withthese four types of bonding.

In 1956, polyisoprene consisting of essen-tially all cis-1,4-units was synthesized with aZiegler-type initiator system prepared fromtitanium tetrachloride and alkyl aluminum.1H-NMR analysis of the polyisoprene synthe-sized by Ti-Al catalysis showed it to becomposed of 99% cis-1,4, 0.3 ± 1.0% cis-3,4,and 0.0 ± 0.7% trans-1,4-units (Campos-lopes, 1978). Although synthetic cis-1,4-poly-isoprene has a marked structural resem-blance to natural rubber, it shows consider-able difference in physical and mechanicalproperties. Furthermore, significant differ-ences between Hevea and guayule rubbershave been observed for processibility andvulcanizing properties. It may be reasonableto interpret that the differences betweenHevea and guayule rubber are derived from

some non-rubber components because theformer occurs as latex, while the latter isobtained by solvent extraction of crushedshrubs. These facts suggest the presence ofsome structural characteristics in Hevearubber other than isomeric units. In Hevearubber a small number of non-isoprenegroups attached to the main hydrocarbonchain is observed by chemical and spectro-scopic analyses.

Hevea rubber is obtained as a latex whichcontains about 35% by weight of rubberparticles. It also includes about 0.5% pro-teins, 0.6% phospholipids and 0.09% toco-trienols as non-rubber components. Solidrubber,which is obtained from latex, containsabout 2.8% acetone-soluble fraction (toco-trienols, fatty acids, sterols, etc.), 2.5% pro-tein fraction and 0.2% ash fraction (mainlymagnesium and potassium phosphates) aswell as the 94 ± 95% of rubber hydrocarbon(Backhaus, 1985). In addition to the isopreneunits, the Hevea rubber molecule has alsobeen reported to contain abnormal groupssuch as esters (Eng et al. , 1994), aldehydes(Subramaniam, 1976, 1977) and epoxides(Burfield, 1976, 1986). The Mooney viscosityofHevea rubber increases onstorage, which iscalled storage hardening; this is presumed tooccur by crosslinking of rubber chains byabnormal group. However, the detailed struc-ture and function of these groups have not yetbeen identified.

1.3

The Latex Vessel System

Rubber is packaged in subcellular rubberparticles located in latex vessels in thoseplantsthatcontainlatex,suchasH.brasiliensisand F. elastica. The white-colored latex isobtained from the trunk of Hevea tree bytapping. Some Lactarius mushrooms exudelatex of various colors, including white,yellow, brown, blue, etc. according to species.

4 Biosynthesis of Natural Rubber and Other Natural Polyisoprenoids76

Lactarius volemus produces a high quantity oflatex that contains about 1 ± 7% rubber, basedon the dry weight of the fruiting body (Tanakaet al. , 1990; Ohya and Tanaka, 1998). How-ever, not all plants that contain latex producerubber (Fahn, 1979). In laticiferous plantshaving no rubber, the latex is constituted byother isoprenoid compounds or resins (Boo-ner and Galston, 1947; Esau, 1965). However,in guayule ± a non-laticiferous plant ± therubber particles accumulate in the parenchy-ma cells of the stem and root (Backhaus andWalsh, 1983).Therubbercontentperdry totalplant of guayule is much higher than that ofH. brasiliensis, because in guayule all theparenchymalatentlyhasrubber-synthesizingability, while in H. brasiliensis only a relativelysmall number of latex vessels can producerubber (Anonymous, 1977; Leong et al.,1982). At the time of the first commercialproduction of rubber, research had alreadycommenced into the secretory tissue inH. brasiliensis. During the 1960s, researchinto the organization of the Hevea tree and itscomponent cell was carried out using cyto-logical methods for the purpose of improvinglatex productivity. The structure of the latexvessel in Hevea was described in detail byDickenson (1969) and Gomez and Moir(1979). The latex vessel is located mainly inthe secondary phloem of the trunk, and isintermingled with a sieve tube, although latexisalsopresent intheflowers, fruitsandleaves.Adjoined latex tubes form a series of concen-tric, circular rings by fusing to each other atcontact points. The contacted wall is pene-trated at several places to yield a continuousnetwork. The wood is connected with thephloem, through which the latex exits via thevascular layer, simultaneously transportingmaterials such as water, minerals and rubberprecursors that are stored in the wood. Sincerapid regeneration of rubber occurs in thelatex vessel after tapping, the Hevea tree canbe tappedonalternatedays to yieldaconstant,

or even increasing, amount of rubber over along period of time (Booner, 1967). Detailedinformation on the structure of latex will beprovided in Chapter 6 of this volume (Witit-suwannakul, 2001).

1.4

Composition of Latex

It has long been known that Hevea latexcontains a large number of non-rubber con-stituents present in relatively small amountsin natural rubber, as well as rubber particles.Many of these are dissolved in the aqueousserum of latex, while others are adsorbed onthe surface of the rubber particles or sus-pended in the latex. Non-rubber compoundsare not only of biological significance withregard to the structure and origin of rubber,but also affect the physical and chemicalproperties of the latex. The major compo-nents of the non-rubber compounds areidentified as inorganic salts, amino acids,proteins, inositols and carbohydrates.

Detailed microscopic observation of thelaticifers in H. brasiliensis shows that latex iscontained in the cytoplasm, with three mainparticulate components ± rubber particles,lutoid particles and Frey-Wyssling complex ±are present in major amounts, in addition totypical components such as nuclei, mito-chondria and ribosomes (Dickenson, 1969).As shown in Figure 1, Hevea latex whenultracentrifuged, is separated into threefractions: (i) the top layer, which containsthe cream of rubber particles; (ii) the yellowlayer, which contains the Frey-Wyssling par-ticlesandtheclearserum;and(iii) thebottomlayer containing predominantly the lutoid.

The rubber particles, which constitute 25 ±45% of the volume of the fresh Hevea latexrange in diameter from 50 � to 3 mm (Gomezand Moir, 1979). The particles are usuallyspherical, but the larger ones in latex frommature trees are often pear-shaped (Schoon

1 Introduction 77

and van der Bie, 1955; Dickenson, 1963). Thehydrophobic rubber molecules are protectedfrom the hydrophilic medium by a complexfilm of proteins and lipids (Ho et al., 1975).As illustrated in Figure 2, the rubber particlescomprise a sphere consisting of rubber, andare surrounded by spherical shells insidewhich are contained phospholipids and pro-teins (Gomez and Moir, 1979). Triglycerides,sterols, sterol esters, tocotrienols and otherlipidsalso combinewithrubber particles.Thefresh Hevea rubber particles have isoelectricpoints ranging from pH 4.0 to 4.6, depending

on the clone, indicating that the relativeproportions of the adsorbed proteins areclonal characteristic (Bowler, 1953). Thebimodal distribution of particle size has beenreported for H. brasiliensis, where Hevea latexhad peaks in the regions of 0.30 and 0.70 mm(Pendle and Swinyard, 1991). In the case ofwashed rubber particles isolated fromH. brasiliensis, F. elastica and P. argentatum,F. elastica rubber particles are substantiallylarger (three-fold mean diameter) than thoseof the other two species (Cornish, 1993).

Lutoids are most abundant non-rubberparticles in Hevea latex. These are vacuoleshaving spherical membrane-bounded bodieswith a diameter of 0.5 ± 3 mm (Dickenson,1969). Within the lutoids there is an aqueousenvironment containing dissolved substan-ces such as acids, minerals, proteins andsugars. Acid phosphatase and lysozyme havebeen detected in these particles, and inaddition the lutoids contain mainly character-istic acid hydrolases ± suggesting that theparticles are analogous to lysosomes (Pujar-niscle, 1968; Jacob and Sontag, 1974; Tataet al. , 1976). Lutoids possess one of thefactors that lead to cessation of latex flow,because the dilution reaction that occursinside the latex vessel on tapping causesswelling of the osmotically sensitive lutoidsremaining in the tube (Boatman, 1966;Southorn, 1969; Subramaniam, 1972).

The Frey-Wyssling complexes are spheri-cal, 4 ± 6 mm in diameter, and bound with adouble membrane (Dickenson, 1969). Theparticle is a composite organelle containingsmall particles of lipids and carotenoids; theyellow color is due to the presence ofcarotenoidpigments.Thehighlycomplicatedstructure of a Frey-Wyssling complex sug-gests that it has an important function in themetabolism of Hevea latex. Since the particlescontain plastoquinone and plastochromanol(in which b-carotene is synthesized) they areassumed to be modified plastids.

4 Biosynthesis of Natural Rubber and Other Natural Polyisoprenoids78

Fig. 1 Ultracentrifugation of Hevea brasiliensis latex.

Fig. 2 Schematic drawing of the rubber moleculesurface.

1.5

Molecular Weight of Rubber

Rubber from H. brasiliensis is a high-molec-ular weight polymer with broad molecularweight distribution (Subramaniam, 1972,1977, 1980). The molecular weight of Hevearubber isolated from freshly tapped latex ofdifferent clonal origins, has been shown tohave either a distinctly bimodal distribution,or a unimodal distribution with a shoulder inthe low molecular weight region. The high-and low-molecular weight peaks are usuallycentered around 106 and 105. In contrast, themolecular weight distribution of guayulerubber has a single peak, irrespective of theoriginandthe averagemolecularweight is2 ±8 � 105 (Hager et al. , 1979). Most rubber-bearing plants produce inferior quality rub-ber with a low molecular weight of �5 � 104.The molecular weight distributions of rub-bers from several species represent thedegree of polymerization carried out by therubber transferase of the particular species(Backhaus, 1985). A molecular weight dis-tribution of this sort may well be related insome ways to the biosynthesis mechanism or

the structure of the molecular chains of Hevearubber. It can be presumed that there are twotypes of enzyme which contribute to thebiosynthesis of rubber of different molecularweights as a result of difference in reactivity(Hager et al. , 1979). However, so far only oneenzyme (rubber transferase) ± which extendsthe length of the polyisoprene chain ± hasbeen discovered (Archer and Cockbain,1969), and there is no evidence for theexistence of two types of enzyme with differ-ent activities. The molecular weight distribu-tion can vary for different clone, and appearsto be genetically controlled in H. brasiliensis(Subramaniam, 1977).

2

Historical Outline

2.1

Rubber Precursors

The general pathway to rubber biosynthesiswas established during 1950s and 1960s.Biochemical studies presumed that rubberformation took place in the latex and started

2 Historical Outline 79

Fig. 3 Types of molecular weight distribution curves of natural rubber (Subramaniam, 1980). Type 1: Skewedunimodal distribution, with a `shoulder` or a plateau in the low-molecular weight region. Type 2: Distinctlybimodal distribution, where the height of the low-molecular weight peak is only half (or less) than that of thehigh-molecularweightpeak.Type3:Distinctlybimodaldistribution,where thepeakheight in the low-molecularweight region is nearly equal or slightly less than that of the high-molecular weight region.

from carbohydrate via isopentenyl diphos-phate (IPP), as shown in Figure 4 (Lynen,1969). This pathway is thought to involve atleast 17 steps from simple sugar. Each step isnaturally dependent on a particular enzymeor enzyme system. Radioactive tracer techni-ques have been applied to elucidate theincorporation of individual precursors (Ly-nen and Henning, 1960).

It is generally assumed that sugars areutilized as the main source of carbon forrubber formation. A small amount of 14C wasincorporated into rubber when Hevea latexwas incubated with radioactive sucrose, glu-cose and fructose (Teas and Bandursky,1956). Although, it has been shown thatsugars were converted to pyruvate in Hevealatex, there is little direct proof of incorpo-ration of 14C into rubber (d'Auzac, 1964;Bealing, 1969). However, in the case ofguayule, the rubber content in a callus cultureincreased with increasing amount of sucrose

in the medium. The conversion from pyru-vate or acetate to rubber was demonstrated byincubation of Hevea latex with the radio-labeled compounds (Harris and Kekwick,1961; d'Auzac, 1964). Arreguin et al. (1951)showed that the [14C]acetate was incorporatedinto rubber by using guayule plants, whileRabinowotz and Teas (1961) showed that [1-14C]acetate and [2-14C]acetate were incorpo-rated into Hevea rubber in the ratio of 2:3,which was expected on the basis of the knownpathway of polyisoprenoid biosynthesis (Ra-binowotz and Teas, 1961). Teas and Bandur-sky (1956) established that Hevea latex con-tained all the enzymes and cofactors neededfor rubber formation from acetate. However,the conversion efficiency from pyruvate oracetate to rubber is very low. This poorconversion is considered to be partially dueto the low activity of enzyme required for theconsumption of the compounds, and also tothe rapid conversion of pyruvate to ethanol in

4 Biosynthesis of Natural Rubber and Other Natural Polyisoprenoids80

Fig. 4 Biosynthetic pathway of natural rubber (proposed by Lynen, 1969).

Hevea latex (Archer and Audley, 1967). It is tobe expected that acetyl-CoA and acetoacetyl-CoA are incorporated into rubber, but there isno direct proof that these compounds areconverted into the rubber in Hevea latex.Hepper and Audley (1969) found a high-levelincorporation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) into rubber on incubationwith Hevea latex. Ozonolysis of rubber ob-tained from 14C-labeled HMG-CoA showedthat it was not decomposed to the low-molecular precursors but was utilized fordirect rubber molecule synthesis. On theother hand, the free acid (3-hydroxy-3-meth-ylglutaric acid) was not incorporated intorubber, but inhibited the formation of rubberfrom HMG-CoA. It was demonstrated that[14C]mevalonic acid (MVA) was incorporatedinto high- molecular weight rubber in Hevealatex (Park and Bonner, 1958; Kekwick et al.,1959). By the degradation of rubber from[14C]MVA, Park and Bonner (1958) showedthat the positions of the individual carbonatoms in the repeating isoprene unit werederived from MVA. The incorporation ofHMG-CoA to rubber changes by the season,but the incorporation of MVA to rubber isconstant (Hepper and Audley, 1969). Theformation of MVA from HMG-CoA wasestimated to be an important rate-limitingstep in the regulation of rubber formation, aswell in cholesterol biosynthesis (Lynen, 1967;Hepper and Audley, 1969). MVA is convertedinto IPP via 5-phoshomevalonate and 5-diphosphomevalonate in Hevea latex, andthese enzymes which catalyzed the reactionswere isolated (Williamson and Kekwick,1965; Skilleter et al., 1966). It was demon-strated that [14C]IPP was incorporated intorubber on incubation with Hevea latex first byLynen (1969), and also by Archer et al. (1967).The degree of incorporation of IPP was foundto be as high as 97%, and the incorporation ofIPP to rubber was faster than that of MVA(Lynen and Henning, 1960; Archer et al.,

1967). The enzymatic incorporation of[14C]IPP was also observed for the washedrubberparticles fromthe latexofF. elasicaandfor the case of incubation with the extractsfrom guayule leaves (Archer et al., 1967).These findings indicate that IPP was thedirect precursor of rubber molecule. Theconversion from acetate to IPP has beenfound in various sources, including Hevealatex serum and guayule extracts (Lynen,1969; Archer et al., 1967). A supply of ATP isrequired for these reactions, and glycolysis isgenerally assumed to account for this (Lynen,1969). All enzymes required for IPP forma-tion exist in the latex serum (Archer, 1980).

2.2

Rubber Formation on Rubber Particles

The site of rubber biosynthesis within the treehas been extensively studied. Using electronmicroscopy, it was established that rubbersynthesis was carried out only in the latexvessel of the rubber tree, though rubber wasdetected in other tissues. Studies performedduring the 1960s suggested that rubber syn-thesis tookplaceonthe surfaceofpre-existingrubber particles. This hypothesis was con-firmed by many experiments using washedrubber particle (WRP), which incorporatedrepeated dilution and centrifugation. Hence,the polymerizing enzyme was thought tobond to the surface of rubber particles.

The enzyme that catalyzes rubber forma-tion has been identified as rubber transferase[EC.2.5.1.20] (Archer and Audley, 1987; Lightand Dennis, 1989; Light et al., 1989). It hasbeen shown that IPP is incorporated intorubber at the surface of the rubber particles inthe latex by reaction with the terminal allylicdiphosphate group of rubber molecules(Archer, 1965; McMullen and McSweeney,1966). Lynen (1969) indicated the hypothet-ical scheme outlining the event at the inter-

2 Historical Outline 81