Limits...
Molecular Cloning and Characterisation of Farnesyl Pyrophosphate Synthase from Tripterygium wilfordii.

Zhao YJ, Chen X, Zhang M, Su P, Liu YJ, Tong YR, Wang XJ, Huang LQ, Gao W - PLoS ONE (2015)

Bottom Line: TwFPSs maintained their capability to synthesise FPP in vitro when purified as recombinant proteins from E. coli.Consistent with the endogenous role of FPS in FPP biosynthesis, TwFPSs were highly expressed in T. wilfordii roots, and were up-regulated upon methyl jasmonate (MeJA) treatment.The global gene expression profiles suggested that the TwFPSs might play an important regulatory role interpenoid biosynthesis in T. wilfordii, laying the groundwork for the future study of the synthetic biology of natural terpene products.

View Article: PubMed Central - PubMed

Affiliation: Capital Medical University School of Traditional Chinese Medicine, Capital Medical University, Beijing, People's Republic of China.

ABSTRACT
Farnesylpyrophosphate synthase (FPS) catalyzes the biosynthesis of farnesyl pyrophosphate (FPP), which is an important precursor of sesquiterpenoids such as artemisinin and wilfordine. In the present study, we report the molecular cloning and characterization of two full-length cDNAs encoding FPSs from Tripterygium wilfordii (TwFPSs). TwFPSs maintained their capability to synthesise FPP in vitro when purified as recombinant proteins from E. coli. Consistent with the endogenous role of FPS in FPP biosynthesis, TwFPSs were highly expressed in T. wilfordii roots, and were up-regulated upon methyl jasmonate (MeJA) treatment. The global gene expression profiles suggested that the TwFPSs might play an important regulatory role interpenoid biosynthesis in T. wilfordii, laying the groundwork for the future study of the synthetic biology of natural terpene products.

No MeSH data available.


GC—MS analysis of reaction products catalyzed by purified recombinant TwFPS incubated with IPP and DMAPP.A Control (the empty vector). B The reaction products catalyzed by purified recombinant TwFPS1 (IPP and DMAPP were added to the reaction mixture). C The reaction products catalyzed by purified recombinant TwFPS2 (IPP and DMAPP were added to the reaction mixture). D GC—MS analysis of dephosphorylated FPP (farnesol) as standards. E The mass spectrogram of the reaction products catalyzed by purified recombinant TwFPS1.F The mass spectrogram of the reaction products catalyzed by purified recombinant TwFPS2. G The mass spectrogram of the dephosphorylated FPP(farnesol).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4418688&req=5

pone.0125415.g005: GC—MS analysis of reaction products catalyzed by purified recombinant TwFPS incubated with IPP and DMAPP.A Control (the empty vector). B The reaction products catalyzed by purified recombinant TwFPS1 (IPP and DMAPP were added to the reaction mixture). C The reaction products catalyzed by purified recombinant TwFPS2 (IPP and DMAPP were added to the reaction mixture). D GC—MS analysis of dephosphorylated FPP (farnesol) as standards. E The mass spectrogram of the reaction products catalyzed by purified recombinant TwFPS1.F The mass spectrogram of the reaction products catalyzed by purified recombinant TwFPS2. G The mass spectrogram of the dephosphorylated FPP(farnesol).

Mentions: The purified proteins were assayed for farnesylpyrophosphate synthase catalytic activity. When the purified enzyme was incubated with DMAPP and IPP, the products had the same retention time as the farnesol standards (Fig 5A–5D). The GC retention time (RT) of farnesol was 29.588 min; TwFPS1 samples of the product, 29.590 min; and TwFPS2 samples of the product, 29.584 min. The blank control sample was not detected in the corresponding characteristic peak. Under GC-MS analysis (Fig 5E–5G), the FPS sample product qualified as farnesol, which had the characteristic peaks, including m/z = 222.0 (Molecular ion: M+) and m/z = 69.10 (CH3(CH3) = CHCH2-). Thus, these results indicated that the coding regions of TwFPSs encode functional FPP synthase.


Molecular Cloning and Characterisation of Farnesyl Pyrophosphate Synthase from Tripterygium wilfordii.

Zhao YJ, Chen X, Zhang M, Su P, Liu YJ, Tong YR, Wang XJ, Huang LQ, Gao W - PLoS ONE (2015)

GC—MS analysis of reaction products catalyzed by purified recombinant TwFPS incubated with IPP and DMAPP.A Control (the empty vector). B The reaction products catalyzed by purified recombinant TwFPS1 (IPP and DMAPP were added to the reaction mixture). C The reaction products catalyzed by purified recombinant TwFPS2 (IPP and DMAPP were added to the reaction mixture). D GC—MS analysis of dephosphorylated FPP (farnesol) as standards. E The mass spectrogram of the reaction products catalyzed by purified recombinant TwFPS1.F The mass spectrogram of the reaction products catalyzed by purified recombinant TwFPS2. G The mass spectrogram of the dephosphorylated FPP(farnesol).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418688&req=5

pone.0125415.g005: GC—MS analysis of reaction products catalyzed by purified recombinant TwFPS incubated with IPP and DMAPP.A Control (the empty vector). B The reaction products catalyzed by purified recombinant TwFPS1 (IPP and DMAPP were added to the reaction mixture). C The reaction products catalyzed by purified recombinant TwFPS2 (IPP and DMAPP were added to the reaction mixture). D GC—MS analysis of dephosphorylated FPP (farnesol) as standards. E The mass spectrogram of the reaction products catalyzed by purified recombinant TwFPS1.F The mass spectrogram of the reaction products catalyzed by purified recombinant TwFPS2. G The mass spectrogram of the dephosphorylated FPP(farnesol).
Mentions: The purified proteins were assayed for farnesylpyrophosphate synthase catalytic activity. When the purified enzyme was incubated with DMAPP and IPP, the products had the same retention time as the farnesol standards (Fig 5A–5D). The GC retention time (RT) of farnesol was 29.588 min; TwFPS1 samples of the product, 29.590 min; and TwFPS2 samples of the product, 29.584 min. The blank control sample was not detected in the corresponding characteristic peak. Under GC-MS analysis (Fig 5E–5G), the FPS sample product qualified as farnesol, which had the characteristic peaks, including m/z = 222.0 (Molecular ion: M+) and m/z = 69.10 (CH3(CH3) = CHCH2-). Thus, these results indicated that the coding regions of TwFPSs encode functional FPP synthase.

Bottom Line: TwFPSs maintained their capability to synthesise FPP in vitro when purified as recombinant proteins from E. coli.Consistent with the endogenous role of FPS in FPP biosynthesis, TwFPSs were highly expressed in T. wilfordii roots, and were up-regulated upon methyl jasmonate (MeJA) treatment.The global gene expression profiles suggested that the TwFPSs might play an important regulatory role interpenoid biosynthesis in T. wilfordii, laying the groundwork for the future study of the synthetic biology of natural terpene products.

View Article: PubMed Central - PubMed

Affiliation: Capital Medical University School of Traditional Chinese Medicine, Capital Medical University, Beijing, People's Republic of China.

ABSTRACT
Farnesylpyrophosphate synthase (FPS) catalyzes the biosynthesis of farnesyl pyrophosphate (FPP), which is an important precursor of sesquiterpenoids such as artemisinin and wilfordine. In the present study, we report the molecular cloning and characterization of two full-length cDNAs encoding FPSs from Tripterygium wilfordii (TwFPSs). TwFPSs maintained their capability to synthesise FPP in vitro when purified as recombinant proteins from E. coli. Consistent with the endogenous role of FPS in FPP biosynthesis, TwFPSs were highly expressed in T. wilfordii roots, and were up-regulated upon methyl jasmonate (MeJA) treatment. The global gene expression profiles suggested that the TwFPSs might play an important regulatory role interpenoid biosynthesis in T. wilfordii, laying the groundwork for the future study of the synthetic biology of natural terpene products.

No MeSH data available.