Ferriols Victor Marco Emmanuel N, Yaginuma Ryoko, Adachi Masao, Takada Kentaro, Matsunaga Shigeki, Okada Shigeru
1] Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan [2] Institute of Aquaculture, University of the Philippines Visayas, Philippines.
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan.
Sci Rep. 2015 May 21;5:10246. doi: 10.1038/srep10246.
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.
硅藻纤细根管藻（Rhizosolenia setigera Brightwell）产生高度分支的类异戊二烯（HBI）烃类，这些烃类广泛存在于海洋环境中。纤细根管藻的烃类组成在C25至C30的HBI之间变化，这取决于与复大孢子形成相关的生命周期阶段。为了更好地理解这些烃类是如何生物合成的，我们对纤细根管藻的法呢基焦磷酸合酶（FPP合酶，FPPS）进行了表征。一个分离得到的1465 bp cDNA克隆包含一个1299 bp的开放阅读框，编码一个含有432个氨基酸残基的蛋白质。RsFPPS cDNA编码区在大肠杆菌中的表达产生了一种在体外表现出FPPS活性的蛋白质。用FPPS抑制剂利塞膦酸盐处理的硅藻中HBI含量的降低表明，RsFPPS为HBI生物合成提供前体。气相色谱 - 质谱联用的产物分析还表明，RsFPPS产生少量香叶基焦磷酸和顺式 - FPP的顺式异构体，它们是先前已表征的HBI顺式异构体的候选前体。此外，还分析了纤细根管藻在与复大孢子形成相关的不同生命阶段的RsFPPS基因表达情况。在此，我们展示了关于RsFPPS在HBI生物合成中可能作用的数据，据我们所知，这是首次从硅藻中克隆并表征FPP合酶。
