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

Victor Marco Emmanuel N Ferriols,Kentaro Takada,Ryoko Yaginuma,Shigeru Okada,Masao Adachi,Shigeki Matsunaga

doi:10.1038/srep10246

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.

Highlights

For isoprenoid biosynthesis through a series of experiments using isotopic labeling techniques
Using the information obtained from the expressed sequence tag (EST) databases to design gene specific primers, we were able to isolate an RsFPPS cDNA sequence spanning 1465 bp with an open reading frame (ORF) of 1299 bp encoding a protein with 432 amino acids and an estimated molecular weight of 48.94 kDa
The deduced amino acid sequence of RsFPPS cDNA had a high degree of similarity with other known Farnesyl pyrophosphate (FPP) synthases[20,21,22,27,29,30]

Summary

Introduction

For isoprenoid biosynthesis through a series of experiments using isotopic labeling techniques. In the context of R. setigera, FPPS presents an interesting research target assuming that a C15 isoprenoid unit is used as one of the precursors for the production of C25 and C30 HBIs. the aim of this study was to clone and characterize a potential FPPS cDNA from R. setigera, to seek a possible role of the enzyme encoded by this cDNA as the supplier of precursors for HBI biosynthesis, and to determine changes in its gene expression in relation to the position of R. setigera in its life cycle. The aim of this study was to clone and characterize a potential FPPS cDNA from R. setigera, to seek a possible role of the enzyme encoded by this cDNA as the supplier of precursors for HBI biosynthesis, and to determine changes in its gene expression in relation to the position of R. setigera in its life cycle It is to our knowledge the first instance that an FPP synthase was cloned and characterized from the extensively diverse diatoms

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Conclusion