# Advances in Genome Sequencing and Natural Rubber Biosynthesis in Rubber-Producing Plants
## 3 Natural Rubber Biosynthesis
### The Core Enzyme for Rubber Synthesis: Rubber Transferase Complex  
The rubber transferase complex, which can either be bound to the surface or embedded within the single layer of rubber particles, is the most vital enzyme complex responsible for the catalytic synthesis of natural rubber. Therefore, identifying the components of the rubber transferase complex is the basis for studying the mechanism of rubber biosynthesis. _cis_-prenyltransferase (CPT) is responsible for catalyzing the condensation of an allylic phenyl diphosphate to IPP and plays a crucial role in the biosynthetic activity of NR (Figure 1). Furthermore, CPT has the ability to terminate IPP condensation by effectively detecting the carbon chain length of the resulting product. This termination process depends on the structures of the catalytic site and the hydrophobic space, which facilitate the accommodation of the hydrophobic premyl products [6]. Two CPT genes, namely _Hexa brasiliensis rubber transferase 1_ (_HRT1_) and _HRT2_, were isolated from the latex of \(H\). _brasiliensis_[51, 52]. When expressed as recombinant proteins in the _E. coli_ system, HRT1 and HRT2 did not exhibit any enzymatic activity [51]. However, the addition of buffer-washed rubber particles significantly enhanced their activity. The observation suggests that HRTs play a pivotal role as key enzymes in the biosynthesis of NR, working in coordination with other unknown essential cofactors. Nogo-B receptor (NgBR) proteins serve as partners of _cis_-prenyltransferases, commonly referred to as _cis_-prenyltransferase-like (CPTL) proteins. Homologs of NgBR have been isolated from NR-producing plants, including _H. brasiliensis_ (_HbCPTL_), _L. sativa_ (_LsCPTL_), and _T. brevicorniculatum_ (_TbRTA_) [42, 53, 54]. Furthermore, NgBR interacts with the HRTs and the rubber elongation factor (REF) in the rubber tree. Therefore, it is also referred to as the HRT-REF bridging protein (HRBP) [42, 55]. REFs are highly abundant expressed proteins that are closely bound to rubber particles. In _H. brasiliensis_, the transcript levels of _REFs_ show a positive correlation with latex yields [56]. The REF interacts with the small rubber particle protein (SRPP) and itself, indicating its capability to form homodimer and heterodimer structures for functional purposes [57]. A cell-free translation-coupled system assay on washed rubber particles (WRPs) indicates that the REF protein may be involved in the stabilization or maintenance of RPs, thereby reducing the coagulation of latex [6]. Homologs of the SRPP have been identified in both rubber-producing plants and non-rubber-producing plants, and they are designated as the SRPP family or stress-related protein family. The classification of REF proteins as members of the SRPP family is based on their possession of a conserved REF domain [58]. The SRPPs are located on the surface of rubber particles due to their interactions with the REF, while their binding to the rubber particles is not as tight as that of the REF. Additionally, the identification of three proteins as constituents of a membrane-bound complex was accomplished by employing labeled \({}^{3}\)H-Bz-GPP(\(p\)). However, the specific protein sequence remained unidentified [59]. The identification of the components comprising the rubber transferase complex will facilitate further exploration into the in vitro synthesis of natural rubber and provide valuable insights for the synthesis of natural rubber using bioreactors.