Accumulation of Squalene in a Microalga <i>Chlamydomonas reinhardtii</i> by Genetic Modification of Squalene Synthase and Squalene Epoxidase Genes

<div><p>Several microalgae accumulate high levels of squalene, and as such provide a potentially valuable source of this useful compound. However, the molecular mechanism of squalene biosynthesis in microalgae is still largely unknown. We obtained the sequences of two enzymes involved in squalene synthesis and metabolism, squalene synthase (CrSQS) and squalene epoxidase (CrSQE), from the model green alga <i>Chlamydomonas reinhardtii</i>. CrSQS was functionally characterized by expression in <i>Escherichia coli</i> and CrSQE by complementation of a budding yeast <i>erg1</i> mutant. Transient expression of CrSQS and CrSQE fused with fluorescent proteins in onion epidermal tissue suggested that both proteins were co-localized in the endoplasmic reticulum. <i>CrSQS</i>-overexpression increased the rate of conversion of ¹⁴C-labeled farnesylpyrophosphate into squalene but did not lead to over-accumulation of squalene. Addition of terbinafine caused the accumulation of squalene and suppression of cell survival. On the other hand, in <i>CrSQE</i>-knockdown lines, the expression level of <i>CrSQE</i> was reduced by 59–76% of that in wild-type cells, and significant levels of squalene (0.9–1.1 μg mg^–1 cell dry weight) accumulated without any growth inhibition. In co-transformation lines with <i>CrSQS</i>-overexpression and <i>CrSQE</i>-knockdown, the level of squalene was not increased significantly compared with that in solitary <i>CrSQE</i>-knockdown lines. These results indicated that partial knockdown of <i>CrSQE</i> is an effective strategy to increase squalene production in <i>C</i>. <i>reinhardtii</i> cells.</p></div

Knockdown of <i>CrSQE</i> gene in parental line (PL) UVM4.

<p>PL UVM4 and five knockdown lines (SQEKD-1 to SQEKD-5) were analyzed. A) qRT-PCR analysis of the <i>CrSQE</i> gene expression of each gene was normalized to that of the <i>CRY1</i> gene. Asterisks above the bars indicate significant differences (*<i>p</i> < 0.05, **<i>p</i> < 0.01). B) Squalene content was measured in each line. DW; cell dry weight. ND; not detected. C) Contents of ergosterol (grey bars), putative 7-dehydroporiferasterol (open bars) and squalene (hatched bars) were measured in each line. D) Doubling time in each line was calculated by measuring absorbance at 730 nm. Data in all experiments indicate mean value ± SD from three biological replicates.</p

Functional characterization of CrSQS in <i>E</i>. <i>coli</i>.

<p>A) SDS-polyacrylamide gel electrophoresis of crude extracts from cells harboring the empty vector pET-21b (lane 1), cells expressing <i>CrSQS</i> (lane 2) and purified CrSQS using a TALON Metal affinity chromatography. B) Normal-phase thin-layer chromatogram of the reaction products derived from (1–¹⁴C) farnesyl diphosphate (FPP). Crude extracts from cells harboring the empty vector (lanes 1–3), from cells expressing CrSQS (lanes 4–6) and purified CrSQS protein (lanes 7–9) were assayed for SQS activity using (1–¹⁴C) FPP and Mg²⁺ with NADPH (lanes 1–2, 4–5, and 7–8 are experimental replicates) or without NADPH (lanes 3, 6, 9). Authentic (1–¹⁴C) FPP was loaded in lane 10 as a negative control. The positions of origin, solvent front and authentic squalene are indicated on the left. Open triangles on the right indicate position of signals from putative dehydrosqualene and 12-hydroxysqualene in lane 9.</p

Overexpression of <i>CrSQS</i> gene and knockdown of <i>CrSQE</i> gene in parental line (PL) UVM4.

<p>A) Immunoblotting analysis with antibodies against the HA epitope tag and α-tubulin in the <i>CrSQE</i>-KD-5 lines harboring a <i>CrSQS</i>-expression plasmid. qRT-PCR analysis of the <i>CrSQS</i> (B) and <i>CrSQE</i> (C) genes in each line. Expression of each gene was normalized to that of the <i>CRY1</i> gene. D) Squalene content of the each line. DW; cell dry weight. Data in all experiments indicate mean value ± SD from three biological replicates.</p

Effects of terbinafine (TBF) on squalene and sterol biosynthesis and cell viability.

<p>A) Contents of ergosterol (grey bars), putative 7-dehydroporiferasterol (open bars) and squalene (hatched bars) in the cells treated with different concentrations of TBF. DW; cell dry weight. B) Images of cultures treated with TBF. C) Chlorophyll content in cells treated with TBF. Data in all experiments are mean value ± SD from three biological replicates.</p

Complementation of a yeast <i>erg1</i> mutant KLN1 by <i>CrSQE</i> expression.

<p>Three independent lines harboring the pAUR123-CrSQE (CrSQE-1 to CrSQE-3), two yeast lines harboring the pAUR123-ScERG1 (ScERG1-1 and ScERG1-2), and a control line harboring an empty pAUR123 vector (VC) were streaked onto YPD medium without ergosterol under the aerobic conditions (A) or with ergosterol under anaerobic conditions (B).</p

Overexpression of <i>CrSQS</i> gene in <i>C</i>. <i>reinhardtii</i> line UVM4.

<p>A) Immunoblotting analysis with antibodies against the HA epitope tag and α-tubulin in transgenic UVM4 lines harboring a <i>CrSQS</i>-expression plasmid. B) qRT-PCR analysis of <i>CrSQS</i> gene expression in a parental line (PL) UVM4 and two overexpression lines, <i>CrSQS</i>-ox-2 and <i>CrSQS</i>-ox-5. Expression of each gene was normalized to <i>CRY1</i>, which encodes ribosomal protein S14 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120446#pone.0120446.ref043" target="_blank">43</a>]. C) Incorporation of (1–¹⁴C) FPP into squalene in each line. D) Content of ergosterol (grey bars) and putative 7-dehydroporiferasterol (open bars) in each line. Asterisks above the bars indicate significant differences (**<i>p</i> < 0.01). DW; cell dry weight. ND; not detected. Data in all experiments indicate mean value ± SD from three biological replicates.</p

Subcellular localization of CrSQS and CrSQE in onion epidermal cells.

<p>A) Schematic diagrams of expression plasmids for GFP- and mCherry-fusion proteins. pGEP-CrSQS: GFP was fused at the C terminus to full-length CrSQS. pGFP-CrSQSTMD: GFP was fused at the C terminus to the C-terminal 118 residues of CrSQS including two predicted transmembrane domains (TMDs). pTMD-mCherry-CrSQE: mCherry was fused at its N terminus to the N-terminal 100 residues of CrSQE including a predicted TMD and at its C terminus to the C-terminal 423 residues of CrSQE including two predicted TMDs. pmCherry-AtSQS1TMD: mCherry was fused at the C-terminus to the C-terminal 67 residues of <i>A</i>. <i>thaliana</i> SQS1 including a predicted TMD, which had been shown to be localized in the endoplasmic reticulum [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120446#pone.0120446.ref022" target="_blank">22</a>]. 35S, cauliflower mosaic virus 35S promoter; NOS, terminator of nopaline synthase. B) The fusion proteins were expressed transiently in onion epidermal cells by particle bombardment. Fluorescence was observed using a laser scanning confocal microscope. Bars = 50 μM</p