Strains and growth conditions
The marine protist T. roseum ATCC28210 was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The culture of T. roseum was maintained on 3% agar slants containing the basal medium supplemented with 2% glucose (w/v) and 0.2% yeast extract (w/v). The basal medium contained (g/L): NaCl, 25; MgSO4·7H2O, 5; KCl, 1; KH2PO4, 0.1; CaCO3, 0.2; (NH4)2SO4, 0.2; NaHCO3, 0.1; monosodium glutamate, 2.0. T. roseum cells were grown in the medium at 25 °C with orbital shaking at 180 rpm.
The Saccharomyces cerevisiae quadruple mutant H1246 MATα (dga1Δ lroΔ are1Δ are2Δ), gift from Antoni Banas, University of Gdansk and Medical University of Gdansk (Poland), was grown at 28 °C in YPD medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose. Its derivative strains for heterologous expression of the empty vector pESC-Ura, TrWSD4, or TrWSD5 were grown at 28 °C in synthetic complete medium lacking uracil and containing 0.67% (w/v) yeast nitrogen base with ammonium sulfate and 2% glucose.
Escherichia coli DH5α was used for cloning and cultured at 37 °C in Luria–Bertani broth with the 100 μg/mL ampicillin. E. coli Rosetta (DE3) was used as the expression host for the overproduction of recombinant TrWSD4 and TrWSD5. All strains used in this study are listed in Additional file 7: Table S2.
RNA isolation and reverse transcription PCR
Total RNA was isolated from the cultures of T. roseum using Trizol reagent (Invitrogen) according to the manufacturer’s instruction. Two micrograms of RNA was reverse transcribed and used for cDNA synthesis using the PrimeScript™ Double Strand cDNA Synthesis Kit (Takara) as described by the manufacture. The PCR products were cloned into pMD19-T Simple vector for DNA sequencing.
All protein sequences used for phylogenetic analysis were obtained from the public database at NCBI (http://www.ncbi.nlm.nih.gov/). The phylogenetic tree was constructed after protein sequence alignments using the neighbor-joining (NJ) method which was performed with MEGA 4.0 . The molecular distance of the aligned sequences was calculated according to the poisson correction model. All gap and missing data in the alignments were accounted for by pairwise deletion. Branch points were tested for significance by bootstrapping with 1000 replications. The organisms and GenBank accession numbers for proteins used for phylogenetic analyses are shown on the corresponding phylogenetic trees.
Construction of plasmids for enzyme overproduction in E. coli
Sequence information from annotation of the T. roseum genome was used to design cDNA primers. After obtaining the full-length cDNA sequences, the open reading frames of TrWSD4 were amplified from total cDNAs of the protist T. roseum with Phusion High-Fidelity DNA Polymerase (Thermo) using the primer pair TrWSD4ec-for/TrWSD4ec-rev containing restriction sites (BamHI and XhoI) appropriate for cloning into pMD19-T Simple vector (Takara). The fidelity of PCR was confirmed by DNA sequencing. The resulting plasmid was digested with BamHI and XhoI, and the ORF of TrWSD4 was subcloned into the BamHI and XhoI sites of pET23b (Novagen), generating a C-terminal His6-tagged TrWSD4 construct.
To construct the plasmid for overproduction of TrWSD5 in E. coli, the coding sequence of TrWSD5 was codon optimized, synthesized by GenWiz (Shanghai, China) with the deletion of a predicted C-terminal transmembrane region (residues 472–494), and amplified by PCR using the primer pair TrWSD5ec-for/TrWSD5ec-rev containing restriction sites (BamHI and EcoRI) appropriate for cloning into pMD19-T Simple vector. The resulting plasmid was digested with BamHI and EcoRI and the codon-optimized ΔTmTrWSD5 (missing residues 472–494) was subcloned into the BamHI and EcoRI sites of the E. coli expression vector pMBP-C (BioVector, Beijing) to generate a C-terminal His6-tagged ΔTmTrWSD5 construct. The vectors pET23b carrying TrWSD4 and pMBP-C carrying ΔTmTrWSD5 were transformed into E. coli Rosetta (DE3) for recombinant enzyme overproduction. All the primers used in this study are listed in Additional file 7: Table S2.
Construction of plasmids for heterologous expression in yeast
Full-length TrWSD4 open reading frame was amplified by PCR using the primer pair TrWSD4sc-for/TrWSD4sc-rev containing restriction sites at the 5′ ends (EcoRI to the sense primer and SpeI to the antisense primer). The PCR product was cloned into the pMD19-T Simple vector and sequenced to confirm the fidelity of the construct. Full-length cDNA of TrWSD4 in pMD19-T Simple vector was digested with EcoRI and SpeI and cloned into the EcoRI and SpeI restriction sites of the pESC-URA expression vector (Agilent Technologies).
To construct the plasmid for heterologous expression of TrWSD5 in yeast, the coding sequence of TrWSD5 was codon optimized, synthesized by GenWiz (Shanghai, China) and amplified by PCR using the primer pair TrWSD5sc-for/TrWSD5sc-rev containing restriction sites at the 5′ ends (EcoRI to the sense primer and SpeI to the antisense primer). The PCR product was cloned into the pMD19-T Simple vector and sequenced to confirm the fidelity of the construct. The codon-optimized form of TrWSD5 cDNA in pMD19-T Simple vector was digested with EcoRI and SpeI and cloned into the EcoRI and SpeI restriction sites of the pESC-URA vector.
Enzyme overproduction in E. coli
For production of the TrWSD4 and TrWSD5 recombinant enzymes in E. coli Rosetta (DE3), starter cultures for the constructs of TrWSD4-pET23b and TrWSD5-pMBP-C were grown overnight at 37 °C in LB media containing 100 μg/mL ampicillin. Aliquots from these cultures were used to inoculate 200 mL cultures that were grown at 37 °C until mid-log phase has been reached. The cultures were induced to overexpress the recombinant WS/DGAT enzymes by the addition of 0.8 mM isopropylthio-β-galactoside (IPTG) and growth was continued for additional 6 h. The bacterial cells were harvested by centrifugation at 3000 rpm for 5 min, and lysed by sonication on ice for 15 min. Cell debris was removed by centrifugation at 12,000 rpm at 4 °C for 15 min. The clarified lysate was used to purify soluble TrWSD4–6 × His and TrWSD5–6 × His by a HisTrap FF column (GE Healthcare) according to the manufacturer’s instructions. After that, recombinant proteins were transferred to a 10 kDa molecular weight cutoff Amicon Ultra-15 centrifugal filter unit (EMD Millipore) for concentration and buffer exchange into 50 mM sodium-phosphate buffer (pH 7.4). The purified proteins were supplemented with 5% (v/v) glycerol and stored at −20 °C.
Heterologous expression of TrWSD4 and TrWSD5 in yeast
The TrWSD4-pESC-URA and TrWSD5-pESC-URA constructs were transformed into the yeast mutant H1246 using the PEG/lithium acetate method . Yeast cells transformed with the empty pESC-URA plasmid were used as control. Yeast transformants were selected by growth on synthetic complete medium lacking uracil (SC-ura), supplemented with 2% (w/v) agar and 2% glucose. The positive colonies were transferred into liquid SC-ura media supplemented with 2% (w/v) glucose and grown at 28 °C overnight. The overnight cultures were diluted to OD600 = 0.4 in SC-ura medium supplemented with 2% (w/v) galactose and 1% (w/v) raffinose to induce protein expression. The cultures supplemented with 0.1% (w/v) palmitic acid together with 0.1% (w/v) hexadecanol were grown at 28 °C for 48 h and then harvested by centrifugation at 3500 rpm for 5 min. The resulting pellets were washed three times with distilled water before being used for further lipid analysis.
Assay for WS/DGAT activity
Protein concentration of the purified TrWSD4 and TrWSD5 was determined using a bicinchoninic acid protein assay kit (TIANGEN). WS/DGAT activity assays were performed using a previously described method . WS activity was indirectly measured using the coupled reaction of the Ellman’s reagent [5,5′-dithio-bis(2-nitrobenzoic acid), DTNB] and free CoA released during the reaction of esterification of fatty alcohol and fatty acyl-CoA. The reaction mixture (200 μL) consisted of 25 mM sodium phosphate buffer (pH 7.4), 1 mg/mL DTNB (dissolved in DMSO), appropriate amount of purified recombinant enzymes (about 5 μg, unless otherwise specified), and the substrates of fatty alcohol and fatty acyl-CoA. Assay reactions were preincubated at 37 °C for 5 min before the reactions were initiated by the addition of the purified recombinant enzyme or acyl-CoA. The release of free CoA was monitored for additional 60 min at 412 nm (ε = 14,150 M−1 cm−1) on a microplate reader SpectraMax M2 (Molecular Devices). DGAT activity was determined in the same manner as described for the WS assay except that diacylglycerol (DAG, 100 μM) was used as the substrate instead of fatty alcohol. All assays were run at 37 °C, unless otherwise stated. To determine kinetic parameters for WS-catalyzed reaction, 5 μg TrWSD4 or TrWSD5 was used for each enzyme assay with specific acyl-CoA as substrate. The concentration of hexadecanol was constant at 100 μM and the concentrations of acyl-CoAs with different chain length and saturation were varied from 15 to 90 μM (15, 30, 45, 60, 75, and 90 μM). To test the effect of different temperatures on WS activity, spectrophotometric assays were conducted in 25 mM sodium phosphate buffer containing 90 μM palmitoyl-CoA and 100 μM hexadecanol as the substrates at temperatures ranging from 7 to 57 °C (7, 17, 27, 37, 47, and 57 °C). To test the effect of salt concentration on WS activity, WS assays were carried out at 37 °C in the same reaction mixture containing the substrates of 90 μM decanoyl-CoA and 100 μM hexadecanol together with different concentrations of NaCl (400, 800, 1200, and 1600 mM). WS and DGAT activities of TrWSD4 and TrWSD5 are from a representative experiment performed in triplicate.
Determination of kinetic parameters of WS-catalyzed reaction
To obtain the kinetic constants for TrWSD4- or TrWSD5-catalyzed esterification between acyl-CoA and fatty alcohol, the standard assay was performed at 37 °C in 200 μL microplates. Initial rates (V
0) were determined as the maximal slope of A412 against time curves using SoftMax Pro 5.3 software (Molecular Devices). The Michaelis–Menten parameters were determined by non-linear regression analysis or linear regression analysis for which the parameters were calculated according to the equation. Upon varying [S] to create saturation curves, values of V
0 were fitted to the Michaelis–Menten equation V
0 = V
max[S]/([S] + K
m) (where V
max is the maximum velocity and K
m is the Michaelis–Menten constant) using GraphPad Prism 7.0 to yield V
max and K
m. For certain substrates of acyl-CoAs on which substrate inhibition was observed, the Michaelis–Menten equation was modified to V
0 = V
m + [S](1 + [S]/K
i)}, where K
i is the inhibition constant. Values of K
cat were calculated as V
Measurement of acyl-CoA and fatty alcohol specificity
Acyl-CoA and fatty alcohol specificity of TrWSD4 and TrWSD5 was determined by the same spectrophotometric assays as described for WS/DGAT activity assay. Acyl-CoA specificity was measured using hexadecanol as acyl acceptor, whereas alcohol specificity was determined with palmitoyl-CoA as acyl donor. In assays for acyl-CoA specificity of TrWSD4 and TrWSD5, different molecular species of acyl-CoAs were added to the above reaction mixture. Acyl-CoA substrates used in this study include: octanoyl-Coenzyme A, decanoyl-Coenzyme A and palmitoyl-Coenzyme A which were purchased from Sigma-Aldrich, and lauroyl Coenzyme A, myristoyl Coenzyme A, stearoyl Coenzyme A, (6Z,9Z,12Z-octadecatrienoyl) Coenzyme A, (5Z,8Z,11Z,14Z-arachidonoyl) Coenzyme A, and (5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl) Coenzyme A which were obtained from Avanti Polar Lipids (Alabama, USA).
Lipid analysis by TLC and GC–MS
Yeast cells (50 mL) were harvested after 48 h of induction and washed twice with distilled water. Neutral lipids were extracted using the method described by Blight and Dyer . Separation of TAG, WE, and polar lipids was performed by running silica gel 60 plates (Merck) in a developing solvent of hexane:diethylether:acetic acid (70:30:1[v/v/v]) and identified by co-migration with known standards of WE and TAG. Lipids on the TLC plate were visualized under UV light after spraying with primuline (Sigma). Spots corresponding to WEs were scraped off the TLC plates and resolved in chloroform. The wax composition was determined using gas chromatography (GC) and mass spectrometry (MS). GC (7890A; Agilent technologies) was equipped with a flame ionization detector (FID) and a HP-5 capillary column (30 m × 0.25 mm having a film thickness of 0.25 μm, Agilent technologies). The flow rate of the carrier gas (He) was constant at 1 mL min−1. The temperature of injector and detector was 280 °C. The column temperature was controlled at 40 °C for 2 min, raised by 20 °C min−1 to 300 °C, and held for 15 min at 300 °C. MS analysis was carried out with an Agilent 5795C mass spectrometer and the parameters for MS analysis were as follows: the temperature of electron ionization (EI) ion source was 230 °C, electron energy was 70 eV, and temperature of quadruples was 150 °C. The wax sample was diluted in 10 μL chloroform and 1 μL was injected into the system. Identification of wax constitutes was made on the basis of their retention times in comparison with an internal library of spectra.