Adaptive laboratory evolution empowers lipids and biomass overproduction in Chlorella vulgaris for environmental applications.

Microalgal strain improvement with commercial features is needed to generate green biological feedstock to produce lipids for bioenergy. Hence, improving algal strain with enhanced lipid content without hindering cellular physiological parameters is pivotal for commercial applications of microalgae. In this report, we demonstrated the adaptive laboratory evolution (ALE) by hypersaline conditions to improve the algal strains for increasing the lipid overproduction capacity of Chlorella vulgaris for environmental applications. The evolved strains (namely E2 and E2.5) without notable impairment in general physiological parameters were scrutinized after 35 cycles. Conventional gravimetric lipid analysis showed that total lipid accumulation was hiked by 2.2-fold in the ALE strains compared to the parental strains. Confocal observation of algal cells stained with Nile-red showed that the abundance of lipid droplets was higher in the evolved strains without any apparent morphological aberrations. Furthermore, evolved strains displayed notable antioxidant potential than the control cells. Interestingly, carbohydrates and protein content were significantly decreased in the evolved cells, indicating that carbon flux was redirected into lipogenesis in the evolved cells. Altogether, our findings demonstrated a potential and feasible strategy for microalgal strain improvement for simultaneous lipids and biomass hyperaccumulation.

Hydrolysis of organophosphorus by diatom purple acid phosphatase and sequential regulation of cell metabolism.

Phosphorus (P) limitation affects phytoplankton growth and population size in aquatic systems, and consequently limits aquatic primary productivity. Plants have evolved a range of metabolic responses to cope with P limitation, such as accumulation of purple acid phosphatases (PAPs) to enhance acquisition of phosphates. However, it remains unknown whether algae have evolved a similar mechanism. In this study, we examined the role of PAPs in the model microalga Phaeodactylum tricornutum. Expression of PAP1 was enhanced in P. tricornutum cells grown on organophosphorus compared to inorganic phosphate. PAP1 overexpression improved cellular growth and biochemical composition in a growth-phase dependent manner. PAP1 promoted growth and photosynthesis during growth phases and reallocated carbon flux towards lipogenesis during the stationary phase. PAP1 was found to be localized in the endoplasmic reticulum and it orchestrated the expression of genes involved in key metabolic pathways and translocation of inorganic P (Pi), thereby improving energy use, reducing equivalents and antioxidant potential. RNAi of PAP1 induced expression of its homolog PAP2, thereby compensating for the Pi scavenging activity of PAP1. Our results demonstrate that PAP1 brings about sequential regulation of metabolism, and provide novel insights into algal phosphorus metabolism and aquatic primary productivity.

Hyperaccumulation of fucoxanthin by enhancing methylerythritol phosphate pathway in Phaeodactylum tricornutum.

The established human health benefits of carotenoids along with the contemporary consumption of natural carotenoids bring the necessity to sustainable production of carotenoids. Among, marine diatoms have emerged as the potential biological resources for carotenoid production; however, their relatively lower yield in native strains provides the impetus to genetically improve the diatoms to cope with the burgeoning demand. In this study, we genetically improved the diatom Phaeodactylum tricornutum by overexpressing key carotenogenic genes involved in methylerythritol phosphate (MEP) pathway. The genes with lower relative transcript level under optimum conditions such as CMK and CMS were selected and overexpressed in P. tricornutum individually. Both CMK and CMS overexpressing lines exhibited elevated growth and photosynthesis. The expression of key carotenogenic genes such as PSY, PDS, ZDS, CRT, and LCYB was significantly upregulated. Furthermore, total carotenoid content was significantly increased; particularly, fucoxanthin content was increased by 1.83- and 1.82-fold in engineered lines CMK and CMS, respectively. Together, the results identify the potential metabolic targets and also uncover the crucial role of MEP pathway in redirecting metabolic precursors towards carotenogenesis. KEY POINTS: • Low abundant genes CMS and CMK of MEP pathway were overexpressed in the diatom • Total carotenoid content was increased, particularly fucoxanthin • Critical metabolic nodes were uncovered to accelerate fucoxanthin biosynthesis.

Potentiation of concurrent expression of lipogenic genes by novel strong promoters in the oleaginous microalga Phaeodactylum tricornutum.

There has been growing interest in using microalgae as production hosts for a wide range of value-added compounds. However, microalgal genetic improvement is impeded by lack of genetic tools to concurrently control multiple genes. Here, we identified two novel strong promoters, designated Pt202 and Pt667, and delineated their potential role on simultaneously driving the expression of key lipogenic genes in Phaeodactylum tricornutum. In silico analyses of the identified promoter sequences predicted the presence of essential core cis elements such as TATA and CAAT boxes. Regulatory role of the promoters was preliminarily assessed by using GUS reporter which demonstrated strong GUS expression. Thereafter, two key lipogenic genes including malic enzyme (PtME) and 5-desaturase (PtD5b), were overexpressed by the two promoters Pt202 and Pt667, respectively, in P. tricornutum. Combinatorial gene overexpression did not impair general physiological performance, meanwhile neutral lipid content was remarkably increased by 2.4-fold. GC-MS analysis of fatty acid methyl esters revealed that eicosapentaenoic acid (EPA; C20:5) was increased significantly. The findings augment a crucial kit to microalgal genetic tools that could facilitate the multiple-gene expression driven by various promoters, and promote microalgae for industrial bioproduction.

High-efficiency promoter-driven coordinated regulation of multiple metabolic nodes elevates lipid accumulation in the model microalga Phaeodactylum tricornutum.

BACKGROUND: Microalgal metabolic engineering holds great promise for the overproduction of a wide range of commercial bioproducts. It demands simultaneous manipulation of multiple metabolic nodes. However, high-efficiency promoters have been lacking. RESULTS: Here we report a strong constitutive promoter Pt211 in expressing multiple target genes in oleaginous microalga Phaeodactylum tricornutum. Pt211 was revealed to contain significant cis-acting elements. GUS reporter and principal genes glycerol-3-phosphate acyltransferase (GPAT) and diacylglycerol acyltransferase 2 (DGAT2) involved in triacylglycerol biosynthesis were tested under driven of Pt211 in P. tricornutum. GUS staining and qPCR analysis showed strong GUS expression. DGAT2 and GPAT linked with a designed 2A sequence exhibited higher transcript abundances than WT, while algal growth and photosynthesis were not impaired. CONCLUSION: The total lipid content increased notably by 2.6-fold compared to WT and reached up to 57.5% (dry cell weight). Overall, our findings report a strong promoter and a strategy for coordinated manipulation of complex metabolic pathways.

The role of diatom glucose-6-phosphate dehydrogenase on lipogenic NADPH supply in green microalgae through plastidial oxidative pentose phosphate pathway.

Commercial production of biofuel from oleaginous microalgae is often impeded by their slow growth rate than other fast-growing algal species. A promising strategy is to genetically engineer the fast-growing algae to accumulate lipids by expressing key lipogenic genes from oleaginous microalgae. However, lacking of strong expression cassette to transform most of the algal species and potential metabolic target to engineer lipid metabolism has hindered its biotechnological applications. In this study, we engineered the oxidative pentose phosphate pathway (PPP) of green microalga Chlorella pyrenoidosa for lipid enhancement by expressing a glucose-6-phosphate dehydrogenase (G6PD) from oleaginous diatom Phaeodactylum tricornutum. Molecular characterization of transformed lines revealed that heterologous PtG6PD was transcribed and expressed successfully. Interestingly, subcellular localization analyses revealed that PtG6PD was targeted to chloroplasts of C. pyrenoidosa. PtG6PD expression remarkably elevated NADPH content and consequently enhanced the lipid content without affecting growth rate. Collectively, this report represents a promising candidate to engineer lipid biosynthesis in heterologous hosts with notable commercial significance, and it highlights the potential role of plastidial PPP in supplying lipogenic NADPH in microalgae.

Glucose-6-phosphate dehydrogenase as a target for highly efficient fatty acid biosynthesis in microalgae by enhancing NADPH supply.

Oleaginous microalgae have great prospects in the fields of feed, nutrition, biofuel, etc. However, biomass and lipid productivity in microalgae remain a major economic and technological bottleneck. Here we present a novel regulatory target, glucose-6-phosphate dehydrogenase (G6PD) from the pentose phosphate pathway (PPP), in boosting microalgal lipid accumulation. G6PD, involved in the formation of NADPH demanded in fatty acid biosynthesis as reducing power, was characterized in oleaginous microalga Phaeodactylum tricornutum. In G6PD overexpressing microalgae, transcript abundance of G6PD increased by 4.4-fold, and G6PD enzyme activity increased by more than 3.1-fold with enhanced NADPH production. Consequently, the lipid content increased by 2.7-fold and reached up to 55.7% of dry weight, while cell growth was not apparently affected. The fatty acid composition exhibited significant changes, including a remarkable increase in monounsaturated fatty acids C16:1 and C18:1 concomitant with a decrease in polyunsaturated fatty acids C20:5 and C22:6. G6PD was localized to the chloroplast and its overexpression stimulated an increase in the number and size of oil bodies. Proteomic and metabolomic analyzes revealed that G6PD play a key role in regulating pentose phosphate pathway and subsequently upregulating NADPH consuming pathways such as fatty acid synthesis, thus eventually leading to lipid accumulation. Our findings show the critical role of G6PD in microalgal lipid accumulation by enhancing NADPH supply and demonstrate that G6PD is a promising target for metabolic engineering.

Identification of a malonyl CoA-acyl carrier protein transacylase and its regulatory role in fatty acid biosynthesis in oleaginous microalga Nannochloropsis oceanica.

Oleaginous microalgae hold great promises for biofuel production. However, commercialization of microalgal biofuels remains impracticable due to the lack of suitable industrial strains with high growth rate and lipid productivity. Engineering of metabolic pathways is a potential strategy for the improvement of microalgal strains for the production of lipids and also value-added products in microalgae. Malonyl CoA-acyl carrier protein transacylase (MCAT) has been reported to be involved in fatty acid biosynthesis. Here, we identified a putative MCAT in the oleaginous marine microalga Nannochloropsis oceanica. NoMCAT overexpressing N. oceanica showed a higher growth rate and photosynthetic efficiency. The neutral lipid content of engineered lines showed a significant increase by up to 31% compared to wild type. Gas chromatography-mass spectrometry analysis revealed that NoMCAT overexpression significantly altered the fatty acid composition. The composition of eicosapentaenoic acid (C20:5), which is a polyunsaturated fatty acid necessary for animal nutrition, increased by 8%. These results demonstrate the role of MCAT in enhancing fatty acid biosynthesis and growth in microalgae, and also provide an insight into metabolic engineering of microalgae with high industrial potential.

High light stress triggers distinct proteomic responses in the marine diatom Thalassiosira pseudonana.

BACKGROUND: Diatoms are able to acclimate to frequent and large light fluctuations in the surface ocean waters. However, the molecular mechanisms underlying these acclimation responses of diaotms remain elusive. RESULTS: In this study, we investigated the mechanism of high light protection in marine diatom Thalassiosira pseudonana using comparative proteomics in combination with biochemical analyses. Cells treated under high light (800 µmol photons m-2s-1) for 10 h were subjected to proteomic analysis. We observed that 143 proteins were differentially expressed under high light treatment. Light-harvesting complex proteins, ROS scavenging systems, photorespiration, lipid metabolism and some specific proteins might be involved in light protection and acclimation of diatoms. Non-photochemical quenching (NPQ) and relative electron transport rate could respond rapidly to varying light intensities. High-light treatment also resulted in increased diadinoxanthin + diatoxanthin content, decreased Fv/Fm, increased triacylglycerol and altered fatty acid composition. Under HL stress, levels of C14:0 and C16:0 increased while C20:5ω3 decreased. CONCLUSIONS: We demonstrate that T. pseudonana has efficient photoprotective mechanisms to deal with HL stress. De novo synthesis of Ddx/Dtx and lipid accumulation contribute to utilization of the excess energy. Our data will provide new clues for in-depth study of photoprotective mechanisms in diatoms.

The pivotal role of malic enzyme in enhancing oil accumulation in green microalga Chlorella pyrenoidosa.

BACKGROUND: The fast growing photosynthetic microalgae have been widely used in aquaculture, food, health, and biofuels. Recent findings in the diatom has proposed a pivotal role of NADP-malic enzyme in generation of NADPH as an important supply of reducing power for fatty acid biosynthesis. To test the lipogenic malic enzyme for fatty acid synthesis in green algae, here the malic enzyme gene PtME from the oleaginous diatom Phaeodactylum tricornutum was expressed in a representative green microalga Chlorella pyrenoidosa. RESULTS: The engineered C. pyrenoidosa strain showed higher enzymatic activity of malic enzyme which subsequently promoted fatty acid synthesis. The neutral lipid content was significantly increased by up to 3.2-fold than wild type determined by Nile red staining, and total lipid content reached 40.9 % (dry cell weight). The engineered strain exhibited further lipid accumulation subjected to nitrogen deprivation condition. Upon nitrogen deprivation, engineered microalgae accumulated total lipid up to 58.7 % (dry cell weight), a 4.6-fold increase over the wild type cells under normal culture condition. At cellular level, increased volume and number of oil bodies were observed in the engineered microalgal cells. CONCLUSIONS: These findings suggested that malic enzyme is a pivotal regulator in lipid accumulation in green microalga C. pyrenoidosa, and presenting a breakthrough of generating ideal algal strains for algal nutrition and biofuels.

Bioremediation of Catechol and Concurrent Accumulation of Biocompounds by the Microalga Crypthecodinium cohnii.

Burgeoning commercial applications of catechol have led to its excessive accumulation in the environment, thereby posing a severe ecological threat. Bioremediation has emerged as a promising solution. The potential of the microalga Crypthecodinium cohnii to degrade catechol and use the byproduct as a carbon source was investigated in this study. Catechol significantly increased C. cohnii growth and was rapidly catabolized within 60 h of cultivation. Transcriptomic analysis highlighted the key genes involved in catechol degradation. Real-time polymerase chain reaction (RT-PCR) analysis showed that transcription of key genes CatA, CatB, and SaID involved in the ortho-cleavage pathway was remarkably increased by 2.9-, 4.2-, and 2.4- fold, respectively. Key primary metabolite content was also markedly altered, with a specific increment in polyunsaturated fatty acids. Electron microscopy and antioxidant analysis showed that C. cohnii could tolerate catechol treatment without morphological aberrations or oxidative stress. The findings provide a strategy for C. cohnii in the bioremediation of catechol and concurrent polyunsaturated fatty acids (PUFA) accumulation.

Effective bioremediation of tobacco wastewater by microalgae at acidic pH for synergistic biomass and lipid accumulation.

Tobacco wastewater is too difficult to decontaminate which poses a significant environmental problem due to the harmful and toxic components. Chlorella pyrenoidosa is a typical microalgal species with potential in removal of organic/inorganic pollutants and proves to be an ideal algal-based system for wastewater treatment. However, the strategy of tobacco related wastewater treatment using microalgae is in urgent need of development. In this study, C. pyrenoidosa was used to evaluate the removal efficiency of artificial tobacco wastewater. Under various solid-to-liquid (g/L) ratios, 1:1 ratio and acidic pH 5.0 were optimal for C. pyrenoidosa to grow with high performance of removal capacity to toxic pollutants (such as COD, NH3-N, nicotine, nitrosamines and heavy metals) with the alleviation of oxidative damage. Algal biomass could reach up to 540.24 mg/L. Furthermore, carbon flux of C. pyrenoidosa was reallocated from carbohydrate and protein biosynthesis to lipogenesis with a high lipid content of 268.60 mg/L at pH 5.0. Overall, this study demonstrates an efficient and sustainable strategy for tobacco wastewater treatment at acidic pH with the production of valuable microalgal products, which provides a promising biorefinery strategy for microalgal-based wastewater bioremediation.

Supplementation with rac-GR24 Facilitates the Accumulation of Biomass and Astaxanthin in Two Successive Stages of Haematococcus pluvialis Cultivation.

The unicellular freshwater green alga Haematococcus pluvialis has attracted much research attention due to its biosynthetic ability for large amounts of astaxanthin, a blood-red ketocarotenoid that is used in cosmetics, nutraceuticals, and pharmaceuticals. Recently, numerous studies have investigated the functions of natural astaxanthin; however, the high cost of the production of astaxanthin from H. pluvialis cultures restricts its commercial viability. There is an urgent need to fulfill commercial demands by increasing astaxanthin accumulation from H. pluvialis cultures. In this study, we discovered that treatment of H. pluvialis cultures at the beginning of the macrozooid stage (day 0) with 1 µM rac-GR24, a synthetic analogue of strigolactones (a class of phytohormones), led to significant increases in biomass [up to a maximum dry cell weight (DCW) of 0.53 g/L] during the macrozooid stage and astaxanthin (from 0.63 to 5.32% of DCW) during the hematocyst stage. We elucidated that this enhancement of biomass accumulation during the macrozooid stage by rac-GR24 is due to its increasing CO2 utilization efficiency in photosynthesis and carbohydrate biosynthesis. We also found that rac-GR24 stimulated the overproduction of nicotinamide adenine dinucleotide phosphate (NADPH) and antioxidant enzymes in H. pluvialis cultures, which alleviated the oxidative damage caused by reactive oxygen species generated during the hematocyst stage due to the exhaustion of nitrogen supplies. Moreover, rac-GR24 treatment of H. pluvialis synergistically altered the activity of the pathways of fatty acid biosynthesis and astaxanthin esterification, which resulted in larger amounts of astaxanthin being generated by rac-GR24-treated cultures than by controls. In summary, we have developed a feasible and economic rac-GR24-assisted strategy that increases the amounts of biomass and astaxanthin generated by H. pluvialis cultures, and have provided novel insights into the mechanistic roles of rac-GR24 to achieve these effects.

Physiological and molecular responses in halotolerant Dunaliella salina exposed to molybdenum disulfide nanoparticles.

Molybdenum disulfide nanoparticles (MoS2 NPs) has emerged as the promising nanomaterial with a wide array of applications in the biomedical, industrial and environmental field. However, the potential effect of MoS2 NPs on marine organisms has yet to be reported. In this study, the effect of MoS2 NPs on the physiological index, subcellular morphology, transcriptomic profiles of the marine microalgae Dunaliella salina was investigated for the first time. exhibited "doping-like" effects on marine microalgae; Growth stimulation was 193.55%, and chlorophyll content increased 1.61-fold upon the addition of 50 µg/L MoS2 NPs. Additionally, exposure to MoS2 NPs significantly increased the protein and carbohydrate content by 2.03- and 1.56-fold, respectively. The antioxidant system was activated as well to eliminate the adverse influence of reactive oxygen species (ROS). Transcriptomic analysis revealed that genes involved in porphyrin synthesis, glycolysis/gluconeogenesis, tricarboxylic acid cycle and DNA replication were upregulated upon MoS2 NPs exposure, which supports the mechanistic role of MoS2 NPs in improving cellular growth and photosynthesis. The "doping-like" effects on marine algae suggest that the low concentration of MoS2 NPs might change the rudimentary ecological composition in the ocean.

Molybdenum disulfide nanoparticles concurrently stimulated biomass and ß-carotene accumulation in Dunaliella salina.

Molybdenum disulfide nanoparticles (MoS2 NPs) hold tremendous properties in wide domain of applications. In this study, the impact of MoS2 NPs was investigated on algal physiological and metabolic properties and a two-stage strategy was acquired to enhance the commercial potential of Dunaliella salina. With 50 µg/L of MoS2 NPs exposure, cellular growth and biomass production were promoted by 1.47- and 1.33-fold than that in control, respectively. MoS2 NPs treated cells were subject to high light intensity for 7 days after 30 days of normal light cultivation, which showed that high light intensity gradually increased ß-carotene content by 1.48-fold. Furthermore, analyses of primary metabolites showed that combinatorial approach significantly altered the biochemical composition of D. salina. Together, these findings demonstrated that MoS2 NPs at an optimum concentration combined with high light intensity could be a promising approach to concurrently enhance biomass and ß-carotene production in microalgae.

Rapid and Effective Electroporation Protocol for Nannochloropsis oceanica.

Electroporation refers to the application of high strength electric pulse to create transient pores in the membrane, thereby enabling the passage of hydrophilic molecules into the cells. Based on the properties of cell and cell wall, the electroporation parameters vary among the algal species. Here, we demonstrated the optimized protocol for successful introduction of recombinant DNA (~5000 bp) into Nannochloropsis oceanica. The linearized recombinant plasmid that harbors eGFP and Bh-sle as the reporter and marker gene, respectively, was electroporated into the electrocompetent N. oceanica cells at voltage of 2200 V, 50 µF, resistance at 600 Ω using electroporator, and the transformed cells were then screened by molecular analysis. The report exemplifies a straightforward and reliable electroporation strategy for generating transgenic N. oceanica cells.

Sustainable and stepwise waste-based utilisation strategy for the production of biomass and biofuels by engineered microalgae.

Waste streams have emerged as potential feedstocks for biofuel production via microbial bioconversion. Metabolic engineering of the microalga Phaeodactylum tricornutum in its lipid biosynthetic pathways has been conducted with an aim to improve lipid production. However, there has been only limited achievement in satisfying biofuel demands by utilising extracellular organic carbons from low-cost waste streams. Herein, we present a successive staged cultivation mode, based on a previously engineered strain that co-overexpresses two key triacylglycerol biosynthesis genes. We first optimised microalgal biomass and lipid production by using food waste hydrolysate and crude glycerol as the cultivation media. Food waste hydrolysate (5% v/v) is a low-cost organic carbon source for enhanced microalgal biomass production, and the resulting lipid concentration was 1.08-fold higher with food-waste hydrolysate than that of the defined medium. Additionally, the resultant lipid concentration after using crude glycerol (100 mM) was 1.24-fold higher than that using the defined medium. Two carbon feeding modes (hybrid and sequential) were also performed to investigate the potential of engineered P. tricornutum with preliminary mechanistic analyses. The biodiesel properties of lipids produced in the hybrid mode were evaluated for potential application prospects. Collectively, this study demonstrates a waste stream utilisation strategy for efficient and sustainable microalgal biofuel production.

Enhanced polyunsaturated fatty acid production using food wastes and biofuels byproducts by an evolved strain of Phaeodactylum tricornutum.

This study investigates the prospective of utilizing kitchen wastewater and food wastes, biofuels industry byproducts as alternative water and carbon sources. Kitchen wastewater did not impede cellular growth rate of the evolved Phaeodactylum strain E70, which indicates its potential as an alternative to freshwater resources. Among the organic wastes assessed, food waste hydrolysate significantly increased cell growth. Supplement of crude glycerol in cultivation medium enhances the total fatty acid content. Mixed food waste hydrolysate and crude glycerol remarkably increased both the cell density and total fatty acid content. Also, the supplement of butylated hydroxytoluene alleviated the oxidative stress induced by impurities in organic wastes and concomitantly increased microalgal total fatty acids and polyunsaturated fatty acids content. The experimental results reported in this study show that a waste-based biorefinery could lead to utilization of organic waste resources for the efficient production of value-added products.

Glucose-6-Phosphate Dehydrogenase from the Oleaginous Microalga Nannochloropsis Uncovers Its Potential Role in Promoting Lipogenesis.

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose-6-phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19-fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09-fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose-6-phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.

TAG pathway engineering via GPAT2 concurrently potentiates abiotic stress tolerance and oleaginicity in Phaeodactylum tricornutum.

BACKGROUND: Despite the great potential of marine diatoms in biofuel sector, commercially viable biofuel production from native diatom strain is impractical. Targeted engineering of TAG pathway represents a promising approach; however, recruitment of potential candidate has been regarded as critical. Here, we identified a glycerol-3-phosphate acyltransferase 2 (GPAT2) isoform and overexpressed in Phaeodactylum tricornutum. RESULTS: GPAT2 overexpression did not impair growth and photosynthesis. GPAT2 overexpression reduced carbohydrates and protein content, however, lipid content were significantly increased. Specifically, TAG content was notably increased by 2.9-fold than phospho- and glyco-lipids. GPAT2 overexpression elicited the push-and-pull strategy by increasing the abundance of substrates for the subsequent metabolic enzymes, thereby increased the expression of LPAAT and DGAT. Besides, GPAT2-mediated lipid overproduction coordinated the expression of NADPH biosynthetic genes. GPAT2 altered the fatty acid profile in TAGs with C16:0 as the predominant fatty acid moieties. We further investigated the impact of GPAT2 on conferring abiotic stress, which exhibited enhanced tolerance to hyposaline (70%) and chilling (10 ºC) conditions via altered fatty acid saturation level. CONCLUSIONS: Collectively, our results exemplified the critical role of GPAT2 in hyperaccumulating TAGs with altered fatty acid profile, which in turn uphold resistance to abiotic stress conditions.