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Cloning and characterization of farnesyl pyrophosphate synthase from the highly branched isoprenoid producing diatom Rhizosolenia setigera.

Ferriols VM, Yaginuma R, Adachi M, Takada K, Matsunaga S, Okada S - Sci Rep (2015)

Bottom Line: A reduction in HBI content from diatoms treated with an FPPS inhibitor, risedronate, suggested that RsFPPS supplies precursors for HBI biosynthesis.Furthermore, RsFPPS gene expression at various life stages of R. setigera in relation to auxosporulation were also analyzed.Herein, we present data on the possible role of RsFPPS in HBI biosynthesis, and it is to our knowledge the first instance that an FPPS was cloned and characterized from a diatom.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan [2] Institute of Aquaculture, University of the Philippines Visayas, Philippines.

ABSTRACT
The diatom Rhizosolenia setigera Brightwell produces highly branched isoprenoid (HBI) hydrocarbons that are ubiquitously present in marine environments. The hydrocarbon composition of R. setigera varies between C25 and C30 HBIs depending on the life cycle stage with regard to auxosporulation. To better understand how these hydrocarbons are biosynthesized, we characterized the farnesyl pyrophosphate (FPP) synthase (FPPS) enzyme of R. setigera. An isolated 1465-bp cDNA clone contained an open reading frame spanning 1299-bp encoding a protein with 432 amino acid residues. Expression of the RsFPPS cDNA coding region in Escherichia coli produced a protein that exhibited FPPS activity in vitro. A reduction in HBI content from diatoms treated with an FPPS inhibitor, risedronate, suggested that RsFPPS supplies precursors for HBI biosynthesis. Product analysis by gas chromatography-mass spectrometry also revealed that RsFPPS produced small amounts of the cis-isomers of geranyl pyrophosphate and FPP, candidate precursors for the cis-isomers of HBIs previously characterized. Furthermore, RsFPPS gene expression at various life stages of R. setigera in relation to auxosporulation were also analyzed. Herein, we present data on the possible role of RsFPPS in HBI biosynthesis, and it is to our knowledge the first instance that an FPPS was cloned and characterized from a diatom.

No MeSH data available.


Related in: MedlinePlus

Effect of risedronate on the growth and hydrocarbon content of R. setigera.Biomass of R. setigera after two-day incubation with risedronate is presented as cells ml−1. Total HBI content of R. setigera quatified by GC/MS is presented as pg cell−1. Values under different letters denote significant differences (p < 0.05) with capital and small letters corresponding to biomass and hydrocarbon content data respectively.
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f7: Effect of risedronate on the growth and hydrocarbon content of R. setigera.Biomass of R. setigera after two-day incubation with risedronate is presented as cells ml−1. Total HBI content of R. setigera quatified by GC/MS is presented as pg cell−1. Values under different letters denote significant differences (p < 0.05) with capital and small letters corresponding to biomass and hydrocarbon content data respectively.

Mentions: Upon observing that RsFPPS produces potential precursors for HBI biosynthesis, we conducted further experiments to assess how RsFPPS inhibition would affect overall hydrocarbon production. R. setigera cultures fifteen cycles after an auxosporulation event (which were mainly producing C25 HBIs) were subjected to doses of the FPPS-specific inhibitor risedronate in a range from 0 to 50 μM after a 4-day pre-incubation period (Fig. 7). In terms of overall growth, risedronate did not have a significant effect on the final biomass (cells•ml−1) of treated cells for concentrations up to 25 μM. But, overall biomass was reduced by around 25% in treatments with 50 μM risedronate (Fig. 7). Interestingly, total hydrocarbon content (pg•cell-1) was reduced in a dose dependent manner with total HBI content decreasing by 16% and 30% at risedronate treatments of 12.5 μM and 25 μM, respectively, compared to the control (Fig. 7). The HBI content of cells treated with 50 μM risedronate were not significantly different from those treated with 25 μM (Fig. 7). This amount can be attributed to HBIs produced during the pre-incubation period prior to the addition of risedronate. These results would suggest that FPPS does contribute C15 isoprenoids for HBI production in R. setigera.


Cloning and characterization of farnesyl pyrophosphate synthase from the highly branched isoprenoid producing diatom Rhizosolenia setigera.

Ferriols VM, Yaginuma R, Adachi M, Takada K, Matsunaga S, Okada S - Sci Rep (2015)

Effect of risedronate on the growth and hydrocarbon content of R. setigera.Biomass of R. setigera after two-day incubation with risedronate is presented as cells ml−1. Total HBI content of R. setigera quatified by GC/MS is presented as pg cell−1. Values under different letters denote significant differences (p < 0.05) with capital and small letters corresponding to biomass and hydrocarbon content data respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440519&req=5

f7: Effect of risedronate on the growth and hydrocarbon content of R. setigera.Biomass of R. setigera after two-day incubation with risedronate is presented as cells ml−1. Total HBI content of R. setigera quatified by GC/MS is presented as pg cell−1. Values under different letters denote significant differences (p < 0.05) with capital and small letters corresponding to biomass and hydrocarbon content data respectively.
Mentions: Upon observing that RsFPPS produces potential precursors for HBI biosynthesis, we conducted further experiments to assess how RsFPPS inhibition would affect overall hydrocarbon production. R. setigera cultures fifteen cycles after an auxosporulation event (which were mainly producing C25 HBIs) were subjected to doses of the FPPS-specific inhibitor risedronate in a range from 0 to 50 μM after a 4-day pre-incubation period (Fig. 7). In terms of overall growth, risedronate did not have a significant effect on the final biomass (cells•ml−1) of treated cells for concentrations up to 25 μM. But, overall biomass was reduced by around 25% in treatments with 50 μM risedronate (Fig. 7). Interestingly, total hydrocarbon content (pg•cell-1) was reduced in a dose dependent manner with total HBI content decreasing by 16% and 30% at risedronate treatments of 12.5 μM and 25 μM, respectively, compared to the control (Fig. 7). The HBI content of cells treated with 50 μM risedronate were not significantly different from those treated with 25 μM (Fig. 7). This amount can be attributed to HBIs produced during the pre-incubation period prior to the addition of risedronate. These results would suggest that FPPS does contribute C15 isoprenoids for HBI production in R. setigera.

Bottom Line: A reduction in HBI content from diatoms treated with an FPPS inhibitor, risedronate, suggested that RsFPPS supplies precursors for HBI biosynthesis.Furthermore, RsFPPS gene expression at various life stages of R. setigera in relation to auxosporulation were also analyzed.Herein, we present data on the possible role of RsFPPS in HBI biosynthesis, and it is to our knowledge the first instance that an FPPS was cloned and characterized from a diatom.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan [2] Institute of Aquaculture, University of the Philippines Visayas, Philippines.

ABSTRACT
The diatom Rhizosolenia setigera Brightwell produces highly branched isoprenoid (HBI) hydrocarbons that are ubiquitously present in marine environments. The hydrocarbon composition of R. setigera varies between C25 and C30 HBIs depending on the life cycle stage with regard to auxosporulation. To better understand how these hydrocarbons are biosynthesized, we characterized the farnesyl pyrophosphate (FPP) synthase (FPPS) enzyme of R. setigera. An isolated 1465-bp cDNA clone contained an open reading frame spanning 1299-bp encoding a protein with 432 amino acid residues. Expression of the RsFPPS cDNA coding region in Escherichia coli produced a protein that exhibited FPPS activity in vitro. A reduction in HBI content from diatoms treated with an FPPS inhibitor, risedronate, suggested that RsFPPS supplies precursors for HBI biosynthesis. Product analysis by gas chromatography-mass spectrometry also revealed that RsFPPS produced small amounts of the cis-isomers of geranyl pyrophosphate and FPP, candidate precursors for the cis-isomers of HBIs previously characterized. Furthermore, RsFPPS gene expression at various life stages of R. setigera in relation to auxosporulation were also analyzed. Herein, we present data on the possible role of RsFPPS in HBI biosynthesis, and it is to our knowledge the first instance that an FPPS was cloned and characterized from a diatom.

No MeSH data available.


Related in: MedlinePlus