# Biosynthesis of Natural Rubber: Current State and Perspectives
## 2 Biosynthesis of Natural Rubber and the Involved Genes/Proteins
### Genes/Proteins Involved in Natural Rubber Biosynthesis  
#### cis-Prenyltransferase  
Genes/proteins involved in rubber synthesis were investigated for nearly 50 years. In 1969, [PERSON] and [PERSON] first suggested that rubber transferase from rubber particles was responsible for natural rubber biosynthesis [52]. In the following studies, researchers revealed that the rubber transferase should be a protein complex consisting of several subunits. The _cis_-prenyltransferases (CPTs), which belong to the prenyltransferase family, were demonstrated to have the activity of incorporating IPP into the polyprenyl chains and were thought to be the most likely candidate or at least one component of the rubber transferase. CPTs were cloned and characterized from both prokaryotes and eukaryotes, and were proved to catalyze the synthesis of dolichols which is important for protein glycosylation [53; 54]. They can be divided into two major classes: i) prokaryotic type including bacterial CPT and plastidial CPT; ii) eukaryotic type including cytosolic CPT from yeast, plants, and animals [55; 56] (Figure 3). The crystal structure of prokaryotic CPTs was characterized and they were proved to form homodimers, while the eukaryotic CPTs were demonstrated to be heteromeric enzymes composed of at least two heterogeneous subunits [55; 57; 58]. [PERSON] et al. cloned two CPTs from _H. brasiliensis_ latex and designated them as HRT1 and HRT2 (_Hovea_ rubber transferase) [59]. HRT1 and HRT2 share 87.3% identity of amino acid sequences, and both of them have all the five highly conserved regions which are important for catalytic function and substrate binding for _cis_-phenyl chain elongating enzymes [59; 60]. HRT1 and HRT2 are expressed predominantly in the latex and may be unstable cytosolic proteins when expressed independently [44; 59]. Enzyme activity assay of recombinant protein produced from _E. coli_ showed that only HRT2 could catalyze the formation of long-chain polyphenyl products with approximate sizes of  
Figure 3: Three dimensional (3D) structure and phylogenetic analysis of CPTs and CPT-Like proteins (CPTLs). (**A**) Crystal structure of _Microoccus luteus_ undecaprenyl diphosphate synthase (MIUPS) homodimer (UniProtKB-O82827). (**B**) 3D structure of HbHRT1 (gray)-HbHRBP (blue) heterodimer predicted by PyMOL. (**C**) Phylogenetic analysis of CPTs and CPTLs. A total of 60 CPTs/CPTLs sequences were collected from human, plants and microbes, and the Neighbor-Joining (NJ) tree was constructed by MEGA6.0. Three clades are presented in the NJ tree: 1) the homomeric CPTs which catalyze the biosynthesis of short-chain and medium-chain preols, including bacterial CPTs and plastidial CPTs; 2) the heteromeric CPTs which catalyze the biosynthesis of dolichol or natural rubber, including cytosolic CPTs from yeast, plants, and human; 3) the CPTLs which interact and form heterodimer with CPTs. The light orange part indicates CPTs involved in natural rubber biosynthesis. HbCPTs and TkCPTs/TbCPTs are separated into two subclades, indicating the polyphyletic origins of CPTs involved in natural rubber biosynthesis. Species abbreviations are: _At_, _Arabidopsis thaliana_; _Ec_, _Escherichia coli_; _Hb_, _Hovea brasiliensis_; _Hs_, _Homo sapiens_; _Ls_, _Lactuca sativa_; _Ml_, _Microoccus luteus_; _Ml_, _Mycobacterium tuberculosis_; _Sc_, _Saccharomyces cerevisiae_; _Sl_, _Solanum lycopersicum_; _Sp_, _Streptococcus pneumoniae_; _Os_, _Oryza sativa_; _Tb_, _Taraxucum bevacizumab_; _Tk_, _Taraxucum kok-saghyz_. \(2\times 10^{3}\)-1 \(\times\) 10\({}^{4}\) Da in the absence of washed bottom fraction particles, while both recombinant HRT1 and HRT2 expressed from eukaryotic cell systems showed polyisoprenoids-producing activity with chain lengths of C\({}_{80-100}\) without centrifuged latex fractions, indicating that post-translational modifications or latex-specific co-factors may be required to enable rubber transferase activity of HRT1/HRT2 [59; 61]. In addition, three CPTs were cloned and characterized from _T. brevicornicultatum_[62; 63].