# Biosynthesis of Natural Rubber: Current State and Perspectives
## 4 Possibility of Natural Rubber Biosynthesis In Vitro or in Genetically Engineered Microorganisms  
Identification of the synthetic or regulatory genes involved in natural rubber provides useful information for improving rubber yield in rubber-producing plants. For example, overexpression of an _EuPI_ showed 3- to 4-fold increase of _trans_-polyisoprene content in transgenic lines of _E. ulmoides_[119]. However, for perennial woody plants such as _H. brasiliensis_, it would take years of growth to see the phenotype. Although more plants species have been investigated and domesticated for industrial rubber production purposes, the plant-derived rubber production and harvest is a time-consuming and labor-intensive process. The rubber-producing plants need land to be planted and usually have a relatively long mature period. Their growth also depends on climate and might be disrupted by diseases and pests. Is it possible to synthesize rubber in vitro or in genetically engineered microorganisms? So far, almost all IPP incorporation assays and CPT/REF/SRPP activity assays have used rubber particles or latex washed bottom fraction as an essential component for investigating rubber synthesis in vitro, which indicated the indispensability of rubber particles in rubber synthesis. [PERSON] et al. introduced the cell-free wheat germ system-expressed HbHRT1 or LsCPT3 to the detergent-washed rubber particles separately [38]. Both recombinant HbHRT1 and LsCPT3 showed rubber transferase activity and synthesized rubber was obtained in vitro. They also revealed the formation of the protein complex consisting of HRT1, REF and HRBP which functions as the natural rubber biosynthetic machinery on rubber particles [38]. However, the study could not eliminate the possibility that some unknown proteins that persist on the detergent-washed rubber particles have rubber transferase activity or work as an indispensable component in the rubber transferase complex. Oil bodies share similar properties with rubber particles [4]. It is possible to prepare artificial oil bodies with triacylglycerol, phospholipids, and oleosins which can be used to purify recombinant proteins fused with oleosins [120; 121]. Reconstituting rubber particles using natural rubber, phospholipids, and recombinant rubber transferase candidates such as CPT/REF/SRPP/CPTL isoforms would reveal the minimum structure unit of the rubber synthetic machinery. However, the cost price of substrates like FPP and IPP might be a stumbling block to the synthesis of natural rubber in vitro. Compared with the in vitro system, synthesis of natural rubber by fermentation of engineered microorganisms from renewable sugar exhibits more advantages (Figure 5). The biosynthesis of a mass of basic compounds with relatively low molecule weight has been achieved in engineered microorganisms including monomers for synthetic rubber, such as isoprene and styrene [122; 123]. Synthesis of some biopolymers such as polyhydroxyalkanoic acids (PHAs) which consist of a wide range of different HAs was also found in many bacteria [124]. Therefore, is it possible to synthesize rubber in engineered microorganisms? As the biogenesis of rubber particles was supposed to occur in ER or Golgi and the activity of rubber transferase might be modified by the eukaryotic post-translation modification, the endomembrane-containing eukaryotic organism would be a suitable host for ectopic rubber biosynthesis. Oleaginous yeasts, such as _Yarrowia lipolytica_ or _Cryptococcus curvatus_, may be ideal candidates, because they are able to accumulate up to about 50% (dry weight) of storage carbohydrates in oil bodies which resemble the rubber particles [4; 125]. Researchers suggested that disruption of the isoprenoid branch pathway _squalene synthase_ (\(SQS/ERG\)) gene might lead to a considerable increase in the flow of substrates to rubber biosynthesis [124].