Abstract:

Objective: Natural rubber biosynthesis is the typical plant isoprenoid metabolic pathway. The MVA is one of the major metabolic pathways for providing the 5-carbon component units for the subsequent natural rubber or terpenoids biosynthesis. The farnesyl pyrophosphate, forming by the polymerization of two IPP molecules with one DMAPP molecule, is identified as the primer of rubber synthesis in nature. To characterize the farnesyl pyrophosphate synthases (FPS) in rubber-producing plants is of great assistance not only in illustrating the rubber biosynthesis mechanism, but also in providing a solid enzymatic foundation for natural rubber genetic breeding. Method: Homologues analysis was applied to investigate the primary amino acids and the secondary structure between these rubber-producing plants including rubber trees (Hevea brasiliensis). The two highly homologous FPS-encoding genes with high expression were selected to construct prokaryotic expressed vectors. The two rubber tree FPS genes expressed their recombinant products were purified and further confirmed by MALDI TOF mass spectrometry detection. After the in vitro enzymatic reaction assay, these two FPS proteins were supplemented to the in vitro rubber synthesis efficacy analysis. Result: Rubber tree harbored three predicted homologous FPS proteins with two conserved motifs 'DDIMD' and 'DDYXD' and similar tertiary structures. The amino acids similarity of the homologous proteins encoded by the two highly-expressed FPS genes was reached 95%, indicating the later gene copy events during its genome evolution. HbFPS1 and HbFPS2 genes can be correctly expressed in Escherichia coli, while the HbFPS 3 existed mostly in the inclusion body, reflecting the probable affection by the obvious difference of hydrophilicity or hydrophobicity in their N-terminal amino acids. The HbFPS1 and HbFPS2 proteins exhibited direct FPP synthesis capacity by using the precursor IPP and DMAPP despite the theoretical precursor GPP in the in vitro enzymatic reaction. Meanwhile, the addition of HbFPS1 or HbFPS2 also respectively increased the in vitro rubber synthesis efficacy in a dose-related manner by using the native rubber particles in the reaction system, and peak improvement was observed at 100 μg level. Conclusion: The amino acids differentiation may affect the catalytic activity between the two highly homologues redundant HbFPS1 and HbFPS2 proteins with the HbFPS3. HbFPS1 and HbFPS2 are indeed the exact functional enzymes that participate in the natural rubber biosynthesis process, especially harboring the ability to generate FPP by direct polyreaction of IPP and DMAPP per se. We establish an in vitro protein functional analysis platform via the natural rubber particles, and confirm the significant enhancement in the natural rubber synthesis efficacy by HbFPS1 and HbFPS2 enzymes when supplemented at an appropriate level. Fine-tuning the rubber tree HbFPS1 and HbFPS2 enzymatic activities will be conducive to the genetic improvement and breeding of novel rubber tree varieties with high quality and high yield.

Key words: Hevea brasiliensis, farnesyl pyrophosphate synthase, natural rubber biosynthesis, latex