Dual expression of plastidial GPAT1 and LPAT1 regulates triacylglycerol production and the fatty acid profile in Phaeodactylum tricornutum
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Metabolic engineering has emerged as a potential strategy for improving microalgal lipid content through targeted changes to lipid metabolic networks. However, the intricate nature of lipogenesis has impeded metabolic engineering. Therefore, it is very important to identify the crucial metabolic nodes and develop strategies to exploit multiple genes for transgenesis. In an attempt to unravel the microalgal triacylglycerol (TAG) pathway, we overexpressed two key lipogenic genes, glycerol-3-phosphate acyltransferase (GPAT1) and lysophosphatidic acid acyltransferase (LPAT1), in oleaginous Phaeodactylum tricornutum and determined their roles in microalgal lipogenesis.
Engineered P. tricornutum strains showed enhanced growth and photosynthetic efficiency compared with that of the wild-type during the growth phase of the cultivation period. However, both the cell types reached stationary phase on day 7. Overexpression of GPAT1 and LPAT1 increased the TAG content by 2.3-fold under nitrogen-replete conditions without compromising cell growth, and they also orchestrated the expression of other key genes involved in TAG synthesis. The transgenic expression of GPAT1 and LPAT1 influenced the expression of malic enzyme and glucose 6-phosphate dehydrogenase, which enhanced the levels of lipogenic NADPH in the transgenic lines. In addition, GPAT1 and LPAT1 preferred C16 over C18 at the sn-2 position of the glycerol backbone.
Overexpression of GPAT1 together with LPAT1 significantly enhanced lipid content without affecting growth and photosynthetic efficiency, and they orchestrated the expression of other key photosynthetic and lipogenic genes. The lipid profile for elevated fatty acid content (C16-CoA) demonstrated the involvement of the prokaryotic TAG pathway in marine diatoms. The results suggested that engineering dual metabolic nodes should be possible in microalgal lipid metabolism. This study also provides the first demonstration of the role of the prokaryotic TAG biosynthetic pathway in lipid overproduction and indicates that the fatty acid profile can be tailored to improve lipid production.
KeywordsDiatoms GPAT1 LPAT1 Biofuel Triacylglycerol biosynthesis Prokaryotic TAG pathway
lysophosphatidic acid acyltransferase
ATPase gamma subunit
oxygen-evolving enhancer protein
photosystem II subunit
phosphatidic acid phosphatase
Phaeodactylum tricornutum is a unicellular model pennate diatom that can accumulate lipids particularly when it is subjected to environmental stresses [1, 2, 3, 4, 5]. Recently, P. tricornutum has emerged as a model candidate for lipid enhancement by metabolic engineering, owing to its high lipid content and the availability of a sequenced genome and genetic tool kit. However, engineered strains accumulate lipids to less than their theoretical maximum [6, 7] and contain a wide range of fatty acid moieties that hinder their commercial exploitation [8, 9]. Furthermore, the triacylglycerol (TAG) biosynthetic pathway is complex and is regulated by various metabolic nodes. Therefore, there is a pressing need to identify the key metabolic nodes and unravel their regulatory mechanisms so that the full potential of microalgal fuel production potential can be realized.
Triacylglycerols in microalgae are primarily stored in lipid droplets (LDs) and serve as energy reservoirs . In microalgae, TAG can be synthesized via the de novo synthetic pathway, the fatty acyl-CoA-dependent Kennedy pathway, and/or the acyl-CoA independent PDAT pathway. The Kennedy pathway comprises three sequential acylations of a glycerol backbone that are catalyzed by glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAT), and diacylglycerol acyltransferase (DGAT) . The GPAT, LPAT, and DGAT transfer the acyl moiety to the sn-1, sn-2, and sn-3 positions of a glycerol backbone for TAG assembly, respectively. This leads to the generation of lysophosphatidic acid (LPA), phosphatidic acid (PA), diacylglycerol (DAG), and the end product TAG. In higher plants, TAG is assembled in the endoplasmic reticulum (ER) from its precursor DAG and stored in cytosolic LDs. However, in Chlamydomonas reinhardtii, Fan et al.  found that TAG is produced from DAG in the chloroplast and accumulated in both the chloroplasts and cytosol, thus implying the existence of prokaryotic plastid and eukaryotic ER-localized TAG biosynthetic pathways.
GPAT and LPAT catalyze the initial committed steps. This leads to the formation of key substrates for TAG biosynthesis, and hence, they are considered to be the rate-limiting enzymes [13, 14]. In higher plants, ten GPAT isoforms have been identified, of which two have been reported to be involved in the Kennedy pathway [15, 16, 17]. In the model diatom P. tricornutum, GPAT overexpression significantly enhances TAG content and alters the fatty acid profile . Our previous study showed that overexpression of LPAT significantly increases TAG content and polyunsaturated fatty acid levels in P. tricornutum . Plastidial LPAT has prokaryotic catalytic activity and prefers C16 fatty acids, whereas ER-localized LPAT prefers C18 fatty acids [20, 21, 22]. Interestingly, Chlamydomonas and related green algae have been shown to possess ER-LPAT, but they exhibit greater activity on C16:0 as plastidial LPATs . Therefore, it is important to understand the acyl-specific catalytic mechanism underlying algal LPAT as well as its pivotal role in producing TAG.
We introduced both GPAT1 and LPAT1 genes into P. tricornutum cells to produce microalgal strains with high lipid yields. Molecular analyses revealed the expression of the two genes in the transgenic lines. We obtained transgenic strains that showed significantly elevated TAG contents without affecting algal biomass. This study also investigated the potential mechanisms underlying lipid accumulation triggered by GPAT1 and LPAT1 overexpression and elucidated the prokaryotic pathway for TAG assembly in diatoms. A presumptive pathway that transports TAG precursors from the chloroplast to the LDs is proposed for diatoms.
Materials and methods
Algal strain and culture conditions
Phaeodactylum tricornutum (Strain CCMP-2561) was procured from the Provasoli-Guillard National Center for Marine Algae and Microbiota (East Boothbay, USA). It was maintained in f/2 medium at 20 ± 0.5 °C under a 12 h:12 h light/dark cycle with 200 μmol photons m−2 s−1 irradiance. Before preparation of the transgenic cells, the algal cells were acclimated in modified f/2 medium without Si. In the nitrogen (N) or phosphorus (P)-depleted experiments, the cells grown under N-replete conditions were harvested by centrifugation (4400×g for 10 min), washed with an N or P-free medium, and resuspended in N or P-free medium. In the NADPH inhibition experiment, different concentrations of sesamol (0–2 mM) were added to the medium, and the cells were harvested after 48 h treatment for further analysis. The analyses of the samples taken from the media containing different concentrations of sesamol were performed in triplicate. In addition, cells harvested on day 4 were used for biomass analysis while cells harvested on day 7 were used for the determination of lipid productivity.
Construction and transformation of the double overexpression system
Total RNA was isolated from algal cells harvested on day 7 using a Plant RNA Kit (Omega, USA) and transcribed into cDNA with a HiScript II Reverse Transcriptase Kit (Vazyme, China) according to the manufacturer’s instructions. The full-length coding regions for GPAT1 (Accession No.: XP_002177014.1) and LPAT1 (Accession No.: XP_002176893.1) were amplified using the primers shown in Additional file 1: Table S1. The GPAT1 and LPAT1 gene fragments were purified using a gel extraction kit (Omega, USA) and cloned into the expression vectors pHY18 and pHY21, respectively, through the homologous recombination method, using a CloneExpress II One Step Kit (Vazyme, China). The recombinant plasmids (pHY18-GPAT1 and pHY21-LPAT1) were first linearized and then electroporated into algal cells using Gene Pulser Xcell equipment (Bio-Rad, USA) at a 1:1 ratio (w/w) according to Wang et al. . The transformed cells were grown in f/2 liquid medium without antibiotics for at least 2 days, then selected on f/2 solid medium with chloramphenicol (250 mg L−1) and zeocin (100 mg L−1). Genomic PCR was performed to verify the integration of the expressions containing the GPAT1 and LPAT1 cassettes in the diatom. The PCR method has been previously described by Kang et al. . Briefly, both WT and transgenic cells were harvested, and their genomic DNA was extracted. Then the genomic DNA was used as the template for the PCR. Taq PCR StarMix (GenStar, China) was also used to run the PCR. The antibiotic gene Shble and CAT in the expression cassette were amplified using primers Shble-f, Shble-r, CAT-f, and CAT-r, whereas the 18S rDNA gene was detected using primers 18s-f and 18s-r (Additional file 1: Table S1). The expected PCR product lengths of Shble, CAT, and the 18s rDNA were 349 bp, 500 bp, and 498 bp, respectively.
Quantitative real-time PCR, western blotting, and enzymatic activity assays
Total RNA was extracted for qRT-PCR using a Plant RNA Kit (Omega, USA) and its concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The cDNA was synthesized using HiScript II Q RT SuperMix for qPCR (Vazyme, China). The qRT-PCR reactions were performed in eight-strip real-time PCR tubes containing 20 μL AceQ qPCR SYBR Green Master Mix (Vazyme, China). The relative transcript abundance was calculated by the 2−ΔΔCt method after the expression had been normalized to that of the endogenous housekeeping gene β-actin. Three biological replicates were analyzed. The primers for the qRT-PCR are listed in Additional file 1: Table S1.
Western blot analysis was used to examine the expression of the target proteins. Total protein was extracted from algal cells with RIPA lysis buffer (Beyotime, China) containing phenylmethanesulfonyl fluoride (PMSF, Beyotime, China). The total protein concentration was determined with a BCA protein quantification kit (Beyotime, China). After quantification, the proteins were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electro-transferred to a polyvinylidene difluoride membrane pre-activated by methanol. The membrane was incubated with primary anti c-Myc antibody (1:3000, Abcam, UK) or anti-flag antibody (1:2000, Sigma, USA) overnight at 4 °C after it had been blocked with skimmed milk for 1 h at 4 °C. This was followed by incubation with HRP-conjugated goat anti-rabbit secondary antibody (1:5000, CST, USA) for 2 h at 4 °C. Then, the membrane was washed three times in pre-cooled PBST (Sangon, China) and developed with a chemiluminescence system (Millipore, USA). Endogenous β-actin was used as an internal control.
The activity levels of GPAT1, LPAT1 and malic enzyme (ME) were measured using a plant GPAT activity spectrophotometry assay kit (Bangyi, China), a plant LPAT activity spectrophotometry assay kit (Bangyi, China), and a ME colorimetric quantitative detection kit (Ke Ming Co., China), respectively, according to the manufacturer’s instructions.
Measurement of photosynthetic parameters
The effective photochemical efficiency of photosystem II (Fv/Fm) and the electron transport rate (ETR) were measured using a PhytoPAM Phytoplankton Analyzer (Walz, Germany). Non-photochemical quenching (NPQ) was calculated using the formula: NPQ = (Fm − Fm′)/Fm′ . Chlorophyll a and c were quantified using an Agilent 1200 HPLC system (Agilent Technologies, USA) with a Symmetry C8 column as described previously . Cell density was calculated daily through the direct count method, with a microscope and a bright-line Neubauer hemocytometer. Specific growth rate (μ) of culture in log phase was calculated following the equation reported by Nur et al. .
Lipid, protein, and carbohydrate analysis
The relative neutral lipid (NL) content was determined with the Nile-red staining method as described previously with minor modification . Briefly, 50 μL Nile red solution (0.1 mg mL−1 in acetone) was added to 5 mL algal culture and incubated for 20 min in the dark at 37 °C. Then the mixture was transferred to a 96-well plate, and the relative fluorescence intensity was measured with a microplate reader (Bio-Tek, USA) at an excitation wavelength of 480 nm and an emission wavelength of 592 nm. Total lipids (TLs) were determined gravimetrically according to a previously described method . Approximately, 50 mg lyophilized algal cells were ground to a powder in liquid nitrogen and transferred to 10 mL tubes. Then 3.8 mL mixed solvent (methanol/chloroform/water, 2:1:0.8, v/v/v) was added, and the mixture was sonicated for 15 min at 200 W. Then 2 mL of chloroform/water (1:1, v/v) was added, and the solution was mildly vortexed. The suspension was separated into two layers by centrifugation at 2000 rpm for 5 min. The upper phase was discarded, and the lower phase was collected in a pre-weighed tube. The extract was dried under a stream of N2 and weighed.
The TAG was determined by extracting the total lipids from the algal cells as described as above and dissolving them in chloroform. The lipids were separated by thin layer chromatography on a silica plate, using the developing solvent hexane/diethyl ether/acetic acid (85:15:1, v/v/v). The separated TAG was visualized by iodine vapor, scraped off the plate, and dissolved in chloroform. The solution was centrifuged at 12,000×g for 10 min, and the supernatant was collected, dried by N2 steam flow, and TAG was gravimetrically determined. Fatty acid composition was determined as fatty acid methyl esters with a gas chromatography–mass spectrophotometer equipped with an NIST 147 spectrum library, according to the method described by Balamurugan et al. . The peak areas were normalized to the internal standard methyl nonadecylate (Aladdin, China).
The total carbohydrate content was obtained following the phenol–sulfuric acid method . Briefly, cell pellets were resuspended in 1 mL ddH2O. Then 1 mL 5% phenol solution was added to the mix, followed by 5 mL sulfuric acid (95–98%, v/v). The solution was incubated in a water bath at 25 °C for 10 min, and the absorbance of the solution was read at a wavelength of 483 nm. Based on the curve of glucose standard, carbohydrate content per liter of culture was obtained. This value was divided by cell density to get carbohydrate content per cell. Total soluble protein was extracted with the method described above and determined using a BCA protein quantification kit (Beyotime, China). According to the standard curve of bovine serum albumin, total soluble protein per liter of culture was calculated.
Confocal microscopy and subcellular localization analysis
One milliliter of culture was stained with 10 μL Nile red solution (0.1 mg mL−1 in acetone) and incubated at 37 °C for 30 min in the dark. The stained cells were observed under an LSM880 laser-scanning confocal microscope (Zeiss, Germany) with an excitation wavelength of 514 nm, an emission wavelength of 596 nm, and a detection wavelength range of 539–652 nm.
The subcellular localization of GPAT1 and LPAT1 was determined by fusing EGFP with the 3′-terminus of the target gene in pPhAP1-EGFP following the previous method . The recombinant plasmids containing the target genes were electroporated into algal cells. The cells were observed under an LSM880 confocal laser scanning microscope (Zeiss, Germany), and the EGFP fluorescence was observed at an excitation wavelength of 488 nm and an emission wavelength range of 510–555 nm. Chlorophyll auto-fluorescence was detected at an excitation wavelength of 488 nm and an emission wavelength range of 625–720 nm.
All experiments were carried out in triplicate, and the data are expressed as mean ± SD. All statistical tests were performed using the SPSS statistical package 19.0. Paired t-tests were used to compare two groups. The results were considered to be significantly different at p < 0.05 (*) or p < 0.01 (**).
Results and discussion
Sequence analysis and construction of the expression system
The sequence structure of GPAT1 and LPAT1 predicted using SMART (http://smart.embl-heidelberg.de/)  and ChloroP (http://www.cbs.dtu.dk/services/ChloroP/)  showed that GPAT1 contained a chloroplastic signal peptide, a LPLAT superfamily and two transmembrane regions, whereas LPAT1 had a plastidial signal peptide, a transmembrane region, and a PlsC domain (Additional file 1: Figure S1), thus suggesting that they are both membrane proteins localized to the chloroplast. However, the subcellular localization of GPAT1 and LPAT1 predicted using HECTAR  revealed that LPAT1 localized to the chloroplasts, whereas GPAT1 localized to the mitochondria (Additional file 1: Table S2). The results indicated that bioinformatic prediction is inaccurate. To further validate the localization of GPAT1 and LPAT1, we used pPhAP1 with EGFP to express the fusion protein for GPAT1 or LPAT1 (Additional file 1: Figure S2B and D). The confocal microscopy images showed that both GPAT1 and LPAT1 localized to the chloroplasts (Additional file 1: Figure S3), thus verifying the previous immunoelectron microscopy results [18, 19].
The full-length coding sequences for GPAT1 and LPAT1 were PCR amplified and inserted into the vectors pHY-18 and pHY-21, respectively, for co-overexpression (Additional file 1: Figure S2A and C).
Verification of transgenic strains using molecular approaches
Effect of GPAT1 and LPAT1 overexpression on photosynthesis
Specific growth rate, biomass and lipid productivity of overexpression lines (OE-1 and OE-2) and wild-type (WT)
Specific growth rate (μ)a
0.542 ± 0.069
0.772 ± 0.093**
0.785 ± 0.078**
Biomass (DCW, g L−1)
1.284 ± 0.132
1.671 ± 0.151**
1.664 ± 0.177**
Total lipid productivity (mg day−1 109 cells−1)
1.023 ± 0.071
2.737 ± 0.195**
2.779 ± 0.209**
TAG productivity (mg day−1 109 cells−1)
0.689 ± 0.053
1.953 ± 0.142**
2.037 ± 0.139**
Co-overexpression altered the contents of cellular components
Effect of collective GPAT1 and LPAT1 overexpression on key genes involved in photosynthesis and TAG synthesis
TAG synthesis mediated by GPAT1 and LPAT1
In this study, we report the functional importance of the TAG biosynthetic machinery in the overproduction of engineered fatty acids after the double overexpression of GPAT1 and LPAT1 in oleaginous P. tricornutum. The TAG content and maximum lipid content was considerably elevated in the OE lines, but cellular biomass did not decrease. GPAT1 and LPAT1 localized to plastids. Their increased expression elevated photosynthetic efficiency during the early growth phase and triggered the expression of lipogenic genes. LPAT1 shows prokaryotic activity, which prefers the transfer of the 16-carbon acyl group to the sn-2 position on TAG. These results suggest that the plastidial TAG pathway is involved in enhancing cellular lipid content. Collectively, the results provide valuable insights that may be used to genetically improve lipid production by manipulating suitable metabolic targets.
XW, HPD, WDY and HYL designed the study. XW and WW carried out experiments and analyzed the data. XW, SB, and HPD wrote the manuscript. JSL and HYL organized and supported the project. All authors read and approved the final manuscript.
This study was funded by the National Natural Science Foundation of China (41725002, 41671463, 41576123, 41576132), the Guangdong Natural Science Foundation (2018A030313164, 2016A030312004, 2014A030308010), and the innovation and strengthening project of Guangdong Ocean University (GDOU2014050201, GDOU2013010203, GDOU2013050201). We thank Prof. Han-Jia Lin from National Taiwan Ocean University for kindly providing the vector pPhAP1 for protein subcellular localization.
The authors declare that they have no competing interests.
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