Assessment on the oil accumulation by knockdown of triacylglycerol lipase in the oleaginous diatom Fistulifera solaris

Strain and culture conditions

The marine diatom Fistulifera solaris JPCC DA0580 was routinely maintained in half-strength Guillard’s f medium (f/2) dissolved in artificial seawater (ASW) (Marine art SF-1, Tomita Pharmaceutical Co., Ltd., Tokushima, Japan)¹⁴. The cells were cultivated at 25 ℃ under continuous light at 130 µmol photons/m²/s using fluorescent lamps for plant cultivation Plantlux (Toshiba Co., Tokyo, Japan). The photosynthetic photon flux density was measured using a luminometer HD2302.01 at the wavelength ranging from 400 to 700 nm with a probe LP471PAR (Delta OHM S.r.l, Caselle di Selvazzano, Italy). To check the cell concentrations and oil body volumes of wild type and 4 transformant clones, the diatom cells were cultivated in conical flasks containing 50 mL of f/2 medium (the initial cell concentrations of 1 × 10⁶ cells/mL) at 25 °C under continuous illumination at 50 µmol photons/m²/s on the shaker (~ 120 rpm). G-418 (500 μg/mL, an aminoglycoside antibiotic, Merck KGaA, Darmstadt, Germany) was added to the culture of the transformant clones.

The 10f medium which contain tenfold more nutrition components than f medium¹⁵ was used as nutrient-replete condition, and ASW was used as the nutrient-depleted condition. F. solaris wild type and transformants were cultured in the nutrient-replete condition with the initial cell concentration of 1.0 × 10⁶ cells/mL for 72 h in flat-shaped flasks containing 500 mL of 10f medium (the initial cell concentrations of 1 × 10⁶ cells/mL) at 25 °C under continuous illumination at 130 µmol photons/m²/s with aeration using sterile air containing 2% CO₂ at the flow rate of 0.8 L/L/min (vvm) in flat-shaped flasks under the light, temperature, and aeration conditions mentioned above. The cultured cells were collected by centrifugation (8500×g for 10 min at 25 °C), and the collected cells were washed twice using ASW, and suspended in ASW (the nutrient-depleted condition), followed by cultivation for 120 h. Cell counts and oil body volume were evaluated at every 24 h.

Plasmid construction and transformation

The KD vectors targeting two Tgl1 homoeologous genes, namely Tgl1-a (Gene ID: fso:g13598, DDBJ/EMBL/GenBank accession: GAX22768.1) and Tgl1-b (fso:g10609. GAX19798.1) were constructed by utilization of pSP-NPT/H4⁹. The antisense fragments, with restriction sites (XbaI), were positioned upstream of the terminator region (227 bp) from a fucoxanthin chlorophyll a/c-binding protein A (fcpA). The two antisense fragments, Tgl1 antisense 1 fragment (236 bp) and Tgl1 antisense 2 fragment (248 bp) (Supplementary Fig. S1), containing 23 bp of effective siRNA fragments (5′-ACA TTG TGA TAG GTT TCT ACC AA-3′ and 5′-CAA TGT CAA TGG CAT GAA TCC AA-3′, respectively) were designed by siDirect v2.0¹⁶, and amplified by PCR from F. solaris genomic DNA. These fragments were then inserted into the XbaI site within the expression vector pSP-NPT/H4 to construct pSP-ANT1 and pSP-ANT2, respectively (Supplementary Fig. S2). Supplementary Table S1 shows the list of primers used for construction of these vectors.

The constructed KD vector (pSP-ANT1 or pSP-ANT2) were introduced into F. solaris by microparticle bombardment using the Biolistic PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, Inc., Hercules, CA, USA), as described previously⁹. After particle bombardment, the cells (5 × 10⁷ cells per plate) were spread on the f/2 agar medium containing 500 µg/mL of G-418, and incubated at 25 ℃ under the continuous light for 2–3 weeks.

Quantitative real time PCR

F. solaris wild type and the transformants were cultured in flat flasks containing f/2 medium for 72 h and were collected by centrifugation. Total RNA was extracted from the collected cells (1 × 10⁸ cells) using NucleoSpin RNA kit (TaKaRa Bio Inc., Shiga, Japan). cDNA was synthesized with 1 μg of total RNA by Prime Script II 1st strand cDNA Synthesis Kit (TaKaRa Bio Inc., Shiga, Japan). The prepared cDNA (2 ng) was used as the template of quantitative real time PCR (qRT-PCR) with Fast SYBR Green Master Mix (Applied Biosystem, Foster City, CA) and ViiA™ 7 real time PCR system (Life Technologies, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Primers used for qRT-PCR were summarized in Supplementary Table S1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a housekeeping gene for normalization of the expression levels with the following equation,

where E_{tgl1,transformant} and E_{gapdh,transformant} are expression levels of Tgl1 and GAPDH genes in each transformant, respectively, and E_tgl1,WT and E_gapdh,WT are expression those in wild type, respectively, analyzed by qRT-PCR.

Confocal fluorescence microscopy

The microalgal cells stained with BODIPY 505/515 (at least 20 cells of wild type and transformants) were observed using a confocal microscope FLUOVIEW FV1000 (Olympus Corp., Tokyo, Japan). The oil body volumes were calculated by Volocity using confocal microscopic images, as described in previous studies^10,11.

Lipid extraction and thin layer chromatography

The cells were incubated in the 10f medium for 72 h to induce lipid degradation, and subsequently lyophilized. The lyophilized cells (15 mg) were suspended in 6 mL of chloroform:methanol (2:1, v/v), and viciously stirred. The mixture was collected by centrifugation at 1000×g for 10 min. After the supernatant was collected, the cell pellet was suspended to 3 mL of chloroform:methanol (2:1, v/v), and repeated the above treatment 2 more times. All the supernatant was collected and mixed in the new tube. Next, 1.25 mL of 0.1 M KCl solution was added, and the mixture was collected by centrifugation at 1000×g for 10 min. The lower layer of organic solvent was collected, and 10 mg of anhydrous sodium sulfate was added. Finally, the solvent was filtered using the polytetrafluoroethylene (PTFE) syringe filter (0.2 µm, Advantec, Tokyo, Japan) and dried under argon gas.

Extracted total lipids (200 µg) from wild type and transformants cells were spotted onto Glass HPTLC Silica gel 60 plates (Merck Millipore, Massachusetts、U.S.A.), and were separated using petroleum ether: diethyl ether: methanol: acetic acid (90:7:2:0.5, v/v). The detection was performed by exposing the plates to iodine vapor. After spot identification, TAG content was determined by ImageJ¹⁷. Triolein, diolein, and monoolein (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan) were mixed in the chloroform at the weight ratio of 1:1:1:497 as standards of a triacylglycerol (TAG), diacylglycerol (DAG) and monoacylglycerol (MAG) on the TLC analysis.

For extraction of neutral lipids to estimate the oil productivity, the neutral lipids were extracted using n-hexane as described previously¹⁸.

Gas chromatography–mass spectrometry

Total lipid extracts of F. solaris wild and transformants were transesterified by heating with 1.5 M HCl-methanol at 100 °C for 1 h. After methanolysis, the fatty acid methyl esters (FAMEs) were extracted three times with hexane. The crude extracts were filtered using the polytetrafluoroethylene (PTFE) syringe filter and dried under argon gas. GC–MS-QP2010 Plus (Shimadzu Corporation, Kyoto, Japan) was used to determine the generated FAME compositions. Oven temperatures were programmed at 140 °C for 1 min, then to 240 °C at 4 °C/min and holding at 240 °C for 10 min. FAMEWAX column (30 m, 0.25 mm ID, 0.25 μm, Restek Corporation, USA) was used for separation of FAMEs. A standard fatty acid mixture, FIM-FAME-7 mixture (Matreya, State College, PA, USA) was used. FAMEs were identified by comparing the peak retention times and mass spectra of samples with a standard fatty acid mixture.