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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

GC/MS total ion chromatograms of hydrocarbon extracts (left panels) and photomicrographs (right panels) of R. setigera.A) 1st culture cycle upon the onset of auxosporulation, B) 10th culture cycle after auxosporulation and C) 20th culture cycle after auxosporulation. Ion peaks appearing between 23 to 25 minutes were identified as C25 HBIs and peaks appearing between 33 to 36 minutes were identified as C30 HBIs based on comparisons of their mass spectra to R. setigera HBIs identified in previous studies.
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f8: GC/MS total ion chromatograms of hydrocarbon extracts (left panels) and photomicrographs (right panels) of R. setigera.A) 1st culture cycle upon the onset of auxosporulation, B) 10th culture cycle after auxosporulation and C) 20th culture cycle after auxosporulation. Ion peaks appearing between 23 to 25 minutes were identified as C25 HBIs and peaks appearing between 33 to 36 minutes were identified as C30 HBIs based on comparisons of their mass spectra to R. setigera HBIs identified in previous studies.

Mentions: To monitor changes in the HBI composition of R. setigera, samples were collected for hydrocarbon analysis by GC/MS at the end of each 15 day culture period from the 1st (Cy1), 10th (Cy10) and 20th (Cy20) culture cycles after auxosporulation as representative samples of R. setigera at different stages of its life cycle. Significant changes in HBI composition were notably observed between cycles, wherein a shift from C30 HBIs to C25 HBIs as the major hydrocarbon component in cells was evident as culture cycles progressed after the auxosporulation event (Fig. 8A-C). Decreasing cell size and increasing cell densities were also noted in the succeeding culture cycles after auxosporulation (Table 1).


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)

GC/MS total ion chromatograms of hydrocarbon extracts (left panels) and photomicrographs (right panels) of R. setigera.A) 1st culture cycle upon the onset of auxosporulation, B) 10th culture cycle after auxosporulation and C) 20th culture cycle after auxosporulation. Ion peaks appearing between 23 to 25 minutes were identified as C25 HBIs and peaks appearing between 33 to 36 minutes were identified as C30 HBIs based on comparisons of their mass spectra to R. setigera HBIs identified in previous studies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: GC/MS total ion chromatograms of hydrocarbon extracts (left panels) and photomicrographs (right panels) of R. setigera.A) 1st culture cycle upon the onset of auxosporulation, B) 10th culture cycle after auxosporulation and C) 20th culture cycle after auxosporulation. Ion peaks appearing between 23 to 25 minutes were identified as C25 HBIs and peaks appearing between 33 to 36 minutes were identified as C30 HBIs based on comparisons of their mass spectra to R. setigera HBIs identified in previous studies.
Mentions: To monitor changes in the HBI composition of R. setigera, samples were collected for hydrocarbon analysis by GC/MS at the end of each 15 day culture period from the 1st (Cy1), 10th (Cy10) and 20th (Cy20) culture cycles after auxosporulation as representative samples of R. setigera at different stages of its life cycle. Significant changes in HBI composition were notably observed between cycles, wherein a shift from C30 HBIs to C25 HBIs as the major hydrocarbon component in cells was evident as culture cycles progressed after the auxosporulation event (Fig. 8A-C). Decreasing cell size and increasing cell densities were also noted in the succeeding culture cycles after auxosporulation (Table 1).

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