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

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.

Figure 1. Simplified hypothetical biosynthetic pathway for the formation of representative C₂₅ and C₃₀ highly branched isoprenoids produced by R. setigera.

Red structures (E,E-FPP) represent the C₁₅ isoprenoid unit to which a C₁₀ (blue structures, GPP or NPP) or another C₁₅ (orange structures, E,E-FPP or Z,E-FPP) isoprenoid unit attaches at C-6 to produce the trans- or cis- forms of C₂₅ and C₃₀ HBIs respectively through 1’-6 coupling. Biosynthetic scheme is patterned after those proposed in previous studies (Masse et al., 2004b; Belt et al., 2006).

Figure 2. FPPS phylogenetic tree.

R. setigera FPPS was compared against those from animal, plant, yeast, algae, and bacteria sources using the Neighbor Joining method based on their amino acid sequences. Alphanumeric codes to the right of the binomial names correspond to accession numbers on the NCBI database. The scale bar on the bottom-left is representative of the degree of difference among sequences wherein a distance of 0.2 infers a 20% difference among sequences.

Figure 3. Multiple amino acid sequence alignment of R. setigera FPPS.

Comparisons were made against those from mouse, yeast, bacteria, and plants. Conserved domains typical of FPP synthases are marked by Roman numerals (I-VII) and a bar above the sequences. The first and second aspartate rich regions in domains II and VI respectively are indicated by boxes.

Figure 4. Representative LC/MS extracted ion chromatograms (EIC).

A) Enzyme reaction with IPP and GPP as substrates, B) enzyme reaction with IPP and DMAPP as substrates, C) GPP and FPP standards at 100 μM concentrations. GPP and FPP eluted at retention times of 4.0 and 4.9 minutes respectively.

Figure 5. RsFPPS enzyme kinetics.

Purified recombinant RsFPPS was subjected to enzyme assays to determine the effect of varying concentrations of the allylic substrates A) DMAPP and C) GPP on enzyme activity. Parallel reactions using IPP as the counter substrate against B) DMAPP (50 μM) and D) GPP (100 μM) were also conducted. Inset values (A-D) are the means (±S.D.) of the derived kinetic constants K_m (μM) and k_cat(min⁻¹). Values for k_cat were calculated using the dimeric form of the enzyme.

Figure 6. GC/MS total ion chromatogram of enzyme reaction products using IPP and DMAPP as substrates.

Upper panels show prominent peaks corresponding to A) geraniol (GOH) and B) E,E-farnesol (E,E-FOH) with minor peaks for nerol (NOH) and Z,E-farnesol (Z,E-FOH) marked with asterisks (*). Lower panels are magnified images of the boxed areas in the upper panels. Other minor peaks in the upper panels are either contaminants or artefacts of treatment with alkaline phosphatase as determined in control alkaline phosphatase reactions using GPP or E,E-FPP standards.

Figure 7. 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.

Figure 8. GC/MS total ion chromatograms of hydrocarbon extracts (left panels) and photomicrographs (right panels) of R. setigera.

A) 1^st culture cycle upon the onset of auxosporulation, B) 10^th culture cycle after auxosporulation and C) 20^th 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.