# Biosynthesis of Natural Rubber: Current State and Perspectives
## 2 Biosynthesis of Natural Rubber and the Involved Genes/Proteins  
### Biosynthesis and Physiological Roles of Natural Rubber  
Natural rubber consists mainly of _cis_-1,4-polyisoprene, of which the constituent monomer isopentenyl pyrophosphate (IPP) is synthesized from the mevalonate (MVA) pathways and possibly also from the 2-C-methyl-d-erythitol-4-phosphate (MEP) pathway in plants (Figure 1). Its biosynthesis is catalyzed by rubber transferase [EC.2.5.1.20] and involves three steps: initiation, polymerization, and termination. Rubber chain initiation needs an oligomeric allyic pyrophosphates initiator which could be dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP) or _cis_-allyic pyrophosphate in vitro [1; 12]. However, the low binding constant of FPP compared with other initiators, the _trans-trans-cis_ sequence detected by NMR at the initiating terminal of rubber molecules, and the cytosolic location of FPP suggested that FPP is the main initiator in vivo [1; 13; 14; 15]. The rubber transferase needs divalent cations, such as Mg\({}^{2+}\) or Mn\({}^{2+}\), as \"activity cofactors\" and the elementary IPP incorporation process is very similar to those of cationic polymerization [13; 16; 17; 18]. Initiation starts by an enzyme-divalent cationic cofactor-assisted ionization of the carbon-oxygen bond of the initiator and yields an allylic cation plus pyrophosphate counteranion [19] (Figure 2). Subsequently, the vinylidene group of IPP adds to the allylic carbocation yielding a tertiary carbocation, which, via proton elimination, regenerates an allylic pyrophosphate end [19]. With the continuously incorporation of IPP monomers, the rubber molecules grow as a \"living carbocation polymerization\" and the _cis-trans_ stereoregulation depends on specific rubber transferases which generate certain type of double band after IPP addition and proton elimination [19]. The termination of the polymerization, or in other words the molecular weight determinant, remains unclear. Natural rubber exhibits broad molecular weight distribution and species-specific molecular weight difference, indicating that multiple factors may be involved besides certain regulatory proteins. In vitro rubber biosynthesis assay revealed that the concentration of FPP and IPP and the ratio of them affect the rubber biosynthetic rate and rubber molecular weight. Increasing IPP concentrations lead to increased rubber molecular weight, whereas increasing FPP concentrations have the opposite effect [20]. Magnesium concentrations were also demonstrated to regulate substrate affinity and rubber molecular weight in vitro [13; 16]. In addition, through recent latex proteome analyses, the termination process was proposed to involve an ubiquitin-proteasome-mediated proteolysis system for protein degradation [21]. However, the detailed mechanism needs to be further investigated. Figure 1: Biosynthesis of natural rubber and _trans_-polyisoprene and the related isoprenoid pathway. (**A**) Biosynthetic pathway of natural rubber and _trans_-polyisoprene, and the related 2-C-methyl-D-erythnitol-4-phosphate (MEP), mevalonate (MVA) and the oligomeric allylic pyrophosphates synthetic pathway, “n” depends on species [2]; (**B**) the exclusively commercial _cis_-polyisoprene producing plant—Para rubber tree _Hevea brasiliensis_, with the lower left picture showing rubber tapping; (**C**) the _trans_-polyisoprene producing plant—hardy rubber tree _Euconomia ulmoides_, with the lower left picture showing _trans_-polyisoprene containing in its fruit [5]. Intermediates in MEP pathway: G3P, glyceraldehyde-3-phosphate; DXP, 1-deoxy-d-xylulose-5-phosphate; MEP, 2-C-methyl-d-erythnitol-4-phosphate; CDP-ME, 4-(cytidine-5’-diphospho)-2-Cmethyl-D-erythnitol; CDP-MEP, 2-phospho-4-(cytidine-5’-diphospho)-2-C-methyl-d-erythnitol; ME-cPP, 2-C-methyl-d-erythnitol-2,4-cyclodiphosphate; HBMPP, 4-hydroxy-3-methylbut-2-enyl diphosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate.