Microalgal Triacylglycerol Research Articles

Functional triacylglycerols from microalgae and their use in the formulation of functional foods—Review

Consumers increased demand for food products that promote health has compelled the food processing industry to develop food products that not only satiate hunger but also aid in the prevention of non-communicable diseases. Microalgae have emerged as a potential source of functional components like triacylglycerols containing essential fatty acids that could be used in the formulation of functional food products. These components help prevent non-communicable diseases due to their inherent characteristics like anti-oxidative, anti-cancer, anti-diabetic, anti-inflammatory, etc. The microalgae are concentrated sources of triacylglycerols containing essential fatty acids and are considered a potential replacer for fish oil in providing these functional ingredients also well accepted by the vegan. This review aims to cover the literature on the triacylglycerol content of microalgae with a focus on the functional triacylglycerol, the fatty acid composition of triacylglycerol extracted from different microalgal biomass, innovative extraction technologies for intensified extraction, the potential use of triacylglycerols from microalgae in the formulation of functional food products. Further, the present constraints and future prospectus in using microalgal triacylglycerol in functional food are also discussed.

Outdoor culture of Halamphora coffeaeformis in the semi-arid Pampa of Argentina: A comprehensive analysis of triacylglycerol production for biodiesel

The feasibility of microalgal triacylglycerol (TAG) production for biodiesel remains constrained by high production costs under outdoor conditions. The aim of this study was to evaluate the capacity of an Argentinian strain of the diatom Halamphora coffeaeformis grown in outdoor raceway ponds to produce TAG suitable for biodiesel. The strain was cultured in seawater enriched with a biofertilizer during summer and spring in the semi-arid Pampa of Argentina. The following variables were analysed: 1) growth rate; 2) TAG accumulation; 3) lipid content and quality; 4) dissolved nutrient and physicochemical properties; and 5) climatic conditions. Intracellular TAG accumulation was evaluated with fluorometry by Nile Red. The growth rate and biomass productivity of the species were similar in the two seasons, with approximate values of 1.35 div. day−1 and 61.25 kg.ha−1.day−1, respectively. Total lipid content increased about 4-fold with respect to inocula in the 2 cultures, reaching up to 44.7 % ash free dry weight (AFDW) in summer and 36.4 % AFDW in spring. However, lipid accumulation differed between seasons: neutral lipid accumulation began at 8 days of cultivation in summer and at 19 days in spring. Salinity was the main stressor that accelerated the TAG accumulation in the species, the nutrient levels did not reach limiting values. In spring, precipitation decreased salinity levels, reducing stress and delaying the onset of TAG accumulation. The biodiesel properties inferred from the fatty acid profile of the species meet international standards and their quality ensures good performance in cold climates. Furthermore, the reported lipid productivity of the species (~25.6 kg.ha−1.day−1) is higher than that of the soybean oil used commercially for biodiesel. Based on the results obtained, H. coffeaeformis has potential for exploitation as a renewable source of TAG for biodiesel production in arid and semi-arid environments with access to brackish or marine water.

Genetic compensation of triacylglycerol biosynthesis in the green microalga Chlamydomonas reinhardtii.

SUMMARYGenetic compensation has been proposed to explain phenotypic differences between gene knockouts and knockdowns in several metazoan and plant model systems. With the rapid development of reverse genetic tools such as CRISPR/Cas9 and RNAi in microalgae, it is increasingly important to assess whether genetic compensation affects the phenotype of engineered algal mutants. While exploring triacylglycerol (TAG) biosynthesis pathways in the model alga Chlamydomonas reinhardtii, it was discovered that knockout of certain genes catalyzing rate‐limiting steps of TAG biosynthesis, type‐2 diacylglycerol acyltransferase genes (DGTTs), triggered genetic compensation under abiotic stress conditions. Genetic compensation of a DGTT1 null mutation by a related PDAT gene was observed regardless of the strain background or mutagenesis approach, for example, CRISPR/Cas 9 or insertional mutagenesis. However, no compensation was found in the PDAT knockout mutant. The effect of PDAT knockout was evaluated in a Δvtc1 mutant, in which PDAT was upregulated under stress, resulting in a 90% increase in TAG content. Knockout of PDAT in the Δvtc1 background induced a 12.8‐fold upregulation of DGTT1 and a 272.3% increase in TAG content in Δvtc1/pdat1 cells, while remaining viable. These data suggest that genetic compensation contributes to the genetic robustness of microalgal TAG biosynthetic pathways, maintaining lipid and redox homeostasis in the knockout mutants under abiotic stress. This work demonstrates examples of genetic compensation in microalgae, implies the physiological relevance of genetic compensation in TAG biosynthesis under stress, and provides guidance for future genetic engineering and mutant characterization efforts.

13C-labeling reveals how membrane lipid components contribute to triacylglycerol accumulation in Chlamydomonas.

Lipid metabolism in microalgae has attracted much interest due to potential utilization of lipids as feedstocks for biofuels, nutraceuticals, and other high-value compounds. Chlamydomonas reinhardtii is a model organism for characterizing the synthesis of the neutral lipid triacylglycerol (TAG), from which biodiesel is made. While much of TAG accumulation under N-deprivation is the result of de novo fatty acid (FA) synthesis, recent work has revealed that approximately one-third of FAs, especially polyunsaturated FAs (PUFAs), come from preexisting membrane lipids. Here, we used 13C-isotopic labeling and mass spectrometry to analyze the turnover of glycerol backbones, headgroups, FAs, whole molecules, and molecular fragments of individual lipids. About one-third of the glyceryl backbones in TAG are derived from preexisting membrane lipids, as are approximately one-third of FAs. The different moieties of the major galactolipids turn over synchronously, while the FAs of diacylglyceryltrimethylhomoserine (DGTS), the most abundant extraplastidial lipid, turn over independently of the rest of the molecule. The major plastidic lipid monogalactosyldiacylglycerol (MGDG), whose predominant species is 18:3α/16:4, was previously shown to be a major source of PUFAs for TAG synthesis. This study reveals that MGDG turns over as whole molecules, the 18:3α/16:4 species is present in both DAG and TAG, and the positional distribution of these PUFAs is identical in MGDG, DAG, and TAG. We conclude that headgroup removal with subsequent acylation is the mechanism by which the major MGDG species is converted to TAG during N-deprivation. This has noteworthy implications for engineering the composition of microalgal TAG for food, fuel, and other applications.

Improving the co-production of triacylglycerol and isoprenoids in Chlamydomonas

Biodiesel and natural products derived from microalgae require a smaller land area and have higher production rates compared to plants and animals and has recently attracted considerable interest. However, biodiesel production from microalgal triacylglycerol is still far from commercial realization due to its high production cost. One way to overcome this obstacle is to improve the triacylglycerol accumulation and couple its production with other high-value compounds. Of particular interest is the sterol biosynthetic pathway with squalene as an intermediate due to its close relationship with triacylglycerol and carotenoid biosynthetic pathways. Besides, both squalene and carotenoids are isoprenoid lipids that have health benefits. Perturbation of one pathway has been suggested to affect other pathways. Three terbinafine-sensitive mutants of the green microalga Chlamydomonas reinhardtii were isolated using terbinafine, a drug that inhibits squalene epoxidase, leading to squalene accumulation. One of the mutants, tfs2, accumulated twice the amount of wild-type triacylglycerol. As well as squalene accumulation, the presence of terbinafine further increased the triacylglycerol content. The level of prenyl lipid carotenoid and chlorophyll was also more significant than that of the wild type. Growth and photosynthesis were not compromised in this mutant. This is the first study that has demonstrated a mutant screening method to improve the co-production of TAG and isoprenoid lipids in a green microalgae.

Nitrogen starvation-inducible promoter of microalga Neochloris oleoabundans lipogenic gene encoding diacylglycerol acyltransferase 2

Microalgal lipid triacylglycerol (TAG) is a promising source for sustainable production of biofuels and edible oils. TAG biosynthesis in microalgae can be induced by nitrogen starvation (−N); however, regulation of the genes involved in this process is poorly known. To explore the regulation of gene encoding diacylglycerol acyltransferase 2 of oleaginous microalga Neochloris oleoabundans (NeoDGAT2) responsible for TAG biosynthesis, regulatory sequence of NeoDGAT2 gene (RDG) was cloned, and its functional regions were mapped by deletion analysis using the modified cyan fluorescent protein gene (CFP) as a reporter. The efficiency of CFP gene mTurquoise2 (Tq) without any intron, Tq1 and Tq2 with one and two copies of Chlamydomonas reinhardtii rbcS2 intron 1, respectively, was evaluated; Tq2 exhibited the highest CFP fluorescence activity in N. oleoabundans was therefore used as reporter for RDG deletion analysis. Deletion analysis of RDG revealed that the −N inducible region contained the predicted binding site of MYB transcription factor (MYB-bs). Specific binding between MYB-bs of RDG and the DNA-binding domain of MYB-related transcription factor ROC40 from C. reinhardtii was observed using electrophoretic mobility shift assay. Therefore, the corresponding MYB transcription factor in N. oleoabundans is probably the transcription factor regulating NeoDGAT2. The interaction between MYB transcription factor and the MYB-bs may play a role in regulating −N induced expression of NeoDGAT2, affecting TAG accumulation. MYB transcription factors can be the potential targets for engineering to increase TAG content. Increasing TAG content is essential for products derived from microalgal TAG to achieve economic viability.

Enhanced triacylglycerol production in oleaginous microalga Neochloris oleoabundans by co-overexpression of lipogenic genes: Plastidial LPAAT1 and ER-located DGAT2

Microalgae accumulate lipid triacylglycerol (TAG), a promising feedstock for production of natural edible oils and biofuels. To make products derived from microalgal TAG economically viable, increasing TAG content and productivity are of high importance. To increase TAG content, two endogenous key enzymes of TAG biosynthesis: plastidial lysophosphatidic acid acyltransferase (NeoLPAAT1) and endoplasmic reticulum-located diacylglycerol acyltransferase 2 (NeoDGAT2) were co-overexpressed in oleaginous microalga Neochloris oleoabundans. The neutral lipid content in NeoLPAAT1-NeoDGAT2 co-overexpressing transformant detected by Nile red staining increased 2-fold without compromising cell growth. The transcriptional levels of NeoLPAAT1 and NeoDGAT2 levels were 1.9-fold higher in the transformant than wild type. Considerably higher lipid accumulation was found in the transformant than wild type: total lipid content (73.72 ± 4.17 % DCW) increased 1.6-fold, TAG content (50.63 ± 6.15 % DCW) increased 2.1-fold, total lipid productivity (16.84 ± 0.66mg/L/day) increased 1.9-fold, and TAG productivity (11.68 ± 0.90mg/L/day) increased 2.1-fold. Fatty acid composition was slightly altered in the transformant compared to wild type; saturated fatty acid C16:0 increased to 26% from 20%, whereas C18:0 was reduced to 7% from 14%. Long-term stability of NeoLPAAT1-NeoDGAT2 co-overexpression was observed in the transformant continuously maintained on solid medium in a period of 4 years. The results suggested that targeted engineering of genes in pathway located at different organelles should be possible in microalgal lipid metabolism.

Galactolipid DGDG and Betaine Lipid DGTS Direct De Novo Synthesized Linolenate into Triacylglycerol in a Stress-Induced Starchless Mutant of Chlamydomonas reinhardtii.

The increasing demand for triacylglycerol (TAG) enriching polyunsaturated fatty acids (PUFAs) has led to a surge of interest in microalgal TAG metabolism. Polar membrane lipids serve as the desaturation carrier for PUFA, and the functional group of PUFA can be incorporated into TAG. Monogalactoglycerolipid has been found to provide the de novo synthesized oleate acyl group or the nascent polyunsaturated diacylglycerol backbone for TAG biosynthesis in the model green alga, Chlamydomonas reinhardtii. However, whether other membrane lipids take part in the formation of PUFA-attached TAG has not been clearly discovered. A time course study of glycerolipidomics in the starchless mutant of C. reinhardtii, BAFJ5, which hyper-accumulates TAG, revealed that digalactosyldiacylglycerol (DGDG) and diacylglycerol-N,N,N-trimethylhomoserine (DGTS) turned into the main components of membrane lipids, accounting for 62% of the total polar lipids, under nitrogen deprivation combined with high light conditions. In addition, the membrane lipid molecules DGDG 18:3n3/16:0 and DGTS 16:0/18:3n6 were presumed to be involved in the consecutive integration of the de novo synthesized linolenates into TAG. Based on the stoichiometry calculation, DGDG and DGTS were demonstrated to provide a major contribution to the accumulation of linolenate-attached TAG. Our study gives insights into the potential PUFA-attached TAG formation pathway mediated by the turnover of de novo synthesized DGDG and DGTS in the starchless mutant of Chlamydomonas.

Increased triacylglycerol production in oleaginous microalga Neochloris oleoabundans by overexpression of plastidial lysophosphatidic acid acyltransferase

BackgroundMicroalgae are promising sources of lipid triacylglycerol (TAG) for sustainable production of natural edible oils and biofuels. Nevertheless, products derived from microalgal TAG are not yet economically feasible; increasing TAG content via targeted genetic engineering of genes in TAG biosynthesis pathway are important to achieve economic viability. To increase TAG content, oleaginous microalga Neochloris oleoabundans was genetically engineered with the endogenous enzyme lysophosphatidic acid acyltransferase (NeoLPAAT1) responsible for plastidial TAG biosynthesisResultsNeoLPAAT1 was found to contain all canonical motifs attributed to LPAAT proteins, two hypothetical membrane-spanning domains and a putative chloroplast transit peptide, indicating as a member of plastidial LPAAT type 1 subfamily. The NeoLPAAT1-expression cassette integrated in N. oleoabundans transformant was confirmed by PCR. The neutral lipid content in the transformant detected by Nile red staining was 1.6-fold higher than in wild type. The NeoLPAAT1 transcript was twofold higher in the transformant than wild type. Considerably higher lipid quantity was found in the transformant than wild type: total lipid content increased 1.8- to 1.9-fold up to 78.99 ± 1.75% dry cell weight (DCW) and total lipid productivity increased 1.8- to 2.4-fold up to 16.06 ± 2.68 mg/L/day; while TAG content increased 2.1- to 2.2-fold up to 55.40 ± 5.56% DCW and TAG productivity increased 1.9- to 2.8-fold up to 10.67 ± 2.37 mg/L/day. A slightly altered fatty acid composition was detected in the transformant compared to wild type; polyunsaturated fatty acid (C18:2) increased to 19% from 11%. NeoLPAAT1-overexpression stability was observed in the transformant continuously maintained in solid medium over 150 generations in a period of about 6 years.ConclusionsOur results demonstrate the considerably increased TAG content and productivity in N. oleoabundans by overexpression of plastidial NeoLPAAT1 that are important for products derived from microalgal TAG to achieve economic viability. Plastidial LPAAT1 can be a candidate for target genetic manipulation to increase TAG content in other microalgal species with desired characteristics for production of natural edible oils and biofuels.

Dual expression of plastidial GPAT1 and LPAT1 regulates triacylglycerol production and the fatty acid profile in Phaeodactylum tricornutum

BackgroundMetabolic 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.ResultsEngineered 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.ConclusionOverexpression 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.

Assessment of Microalgal Triacylglycerol as Environmental Benign Approach for Biodiesel Production

Energy plays a vital role for the development for the human welfare. The global economy totally runs on energy, or we can say that all living things depends on it, for example, plants takes energy from the sunlight, water and minerals, and this process is called photosynthesis, animals takes energy from plants and from one another, whereas humans takes energy from both. As the population increases, the uses of fossil fuels increased rapidly and because of this supply of renewable resources get affected and global warming makes earth more complicated due to increase in carbon dioxide. The production of biodiesel using microalgae biomass appears to be more viable and cheaper. Microalgae are a good source of lipid and triacylglycerol (TAG) that are sustainable feedstock for biodiesel. The present paper is an attempt to know the potential of microalgae for the biodiesel production.<div><br></div>

An Extended Approach to Quantify Triacylglycerol in Microalgae by Characteristic Fatty Acids.

Microalgae represent a third generation biofuel feedstock due to their high triacylglycerol (TAG) content under adverse environmental conditions. Microalgal TAG resides in a single cell and serves as a lipid class mixed with complicated compositions. We previously showed that TAG possessed characteristic fatty acids (CFAs) for quantification and was linearly correlated with the relative abundance of CFA within certain limits in microalgae. Here, we defined the application range of the linear correlation between TAG and CFA in the oleaginous microalgae Chlamydomonas reinhardtii and Phaeodactylum tricornutum. In addition, TAG quantification was further expanded to a wide range of levels and the absolute amounts of saturated or monounsaturated CFAs, 16:0 and 18:1n9 of C. reinhardtii and 16:0 and 16:1n7 of P. tricornutum, instead of polyunsaturated CFAs, were verified to be linearly correlated to TAG levels throughout the entire period of nitrogen stress. This approach utilizes a single fatty acid to quantify TAG mixtures, and is rapid, simple and precise, which provides a useful tool for monitoring TAG accumulation of distinct microalgal species and facilitating high-throughput mutant screening for microalgae.

Sorting cells of the microalga Chlorococcum littorale with increased triacylglycerol productivity.

BackgroundDespite extensive research in the last decades, microalgae are still only economically feasible for high valued markets. Strain improvement is a strategy to increase productivities, hence reducing costs. In this work, we focus on microalgae selection: taking advantage of the natural biological variability of species to select variations based on desired characteristics. We focused on triacylglycerol (TAG), which have applications ranging from biodiesel to high-value omega-3 fatty-acids. Hence, we demonstrated a strategy to sort microalgae cells with increased TAG productivity.Results1. We successfully identified sub-populations of cells with increased TAG productivity using Fluorescence assisted cell sorting (FACS). 2. We sequentially sorted cells after repeated cycles of N-starvation, resulting in five sorted populations (S1–S5). 3. The comparison between sorted and original populations showed that S5 had the highest TAG productivity [0.34 against 0.18 g l−1 day−1 (original), continuous light]. 4. Original and S5 were compared in lab-scale reactors under simulated summer conditions confirming the increased TAG productivity of S5 (0.4 against 0.2 g l−1 day−1). Biomass composition analyses showed that S5 produced more biomass under N-starvation because of an increase only in TAG content and, flow cytometry showed that our selection removed cells with lower efficiency in producing TAGs.ConclusionsAll combined, our results present a successful strategy to improve the TAG productivity of Chlorococcum littorale, without resourcing to genetic manipulation or random mutagenesis. Additionally, the improved TAG productivity of S5 was confirmed under simulated summer conditions, highlighting the industrial potential of S5 for microalgal TAG production.Graphical abstract

Nitrogen starvation-induced accumulation of triacylglycerol in the green algae: evidence for a role for ROC40, a transcription factor involved in circadian rhythm.

Microalgal triacylglycerol (TAG), a promising source of biofuel, is induced upon nitrogen starvation (-N), but the proteins and genes involved in this process are poorly known. We performed isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative proteomics to identify Chlorella proteins with modulated expression under short-term -N. Out of 1736 soluble proteins and 2187 membrane-associated proteins identified, 288 and 56, respectively, were differentially expressed under -N. Gene expression analysis on select genes confirmed the same direction of mRNA modulation for most proteins. The MYB-related transcription factor ROC40 was the most induced protein, with a 9.6-fold increase upon -N. In a previously generated Chlamydomonas mutant, gravimetric measurements of crude total lipids revealed that roc40 was impaired in its ability to increase the accumulation of TAG upon -N, and this phenotype was complemented when wild-type Roc40 was expressed. Results from radiotracer experiments were consistent with the roc40 mutant being comparable to the wild type in recycling membrane lipids to TAG but being impaired in additional de novo synthesis of TAG during -N stress. In this study we provide evidence to support the hypothesis that transcription factor ROC40 has a role in -N-induced lipid accumulation, and uncover multiple previously unknown proteins modulated by short-term -N in green algae.

Regulation of the initial events in microalgal triacylglycerol (TAG) synthesis: hypotheses

Possible scenarios for the regulation of the early events in triacylglycerol accumulation by microalgae are discussed. It is hypothesized that buildup of α-ketoglutarate (α-KG) in the cell as a result of nitrogen limitation is involved in the stimulation of the first events in fatty acid synthesis. It is proposed that only cells unable to complete a cell cycle will accumulate lipid.

Diacylglycerol acyltransferase type 2 cDNA from the oleaginous microalga Neochloris oleoabundans: cloning and functional characterization

Currently, biodiesel production from microalgae is technically, but not yet economically, feasible. Increasing microalgal triacylglycerol (TAG) content via genetic engineering could be a potential approach to improve the efficiency of biodiesel production. In all organisms studied so far, diacylglycerol acyltransferase (DGAT) is the primary enzyme of TAG biosynthesis. In this study, we cloned the cDNA encoding DGAT type 2 (NeoDGAT2) from the oleaginous microalga Neochloris oleoabundans. The open reading frame of NeoDGAT2 cDNA was 978 bp encoding a protein of 325 amino acids. Although N. oleoabundans has been classified in the class Chlorophyceae, NeoDGAT2 was closely related to Treboxiophycean DGAT2 as it shared 80 % amino acid identity with CvDGAT2-2 of Chlorella variabilis, but distantly related to Chlorophycean DGAT2 as it shared only 34 % identity with CrDGAT2A (DGTT4) of Chlamydomonas reinhardtii. As suggested by hydropathy analysis, NeoDGAT2 might contain 4 to 5 transmembrane domains. We tested whether NeoDGAT2 cDNA encoded a protein with DGAT activity by functional complementation assay in yeast Saccharomyces cerevisiae mutant H1246. NeoDGAT2 was able to compensate the endogenous DGAT2 activity of mutant H1246 and restore the lipid body formation and TAG synthesis. The successful cloning of cDNA encoding a protein with DGAT activity provides a candidate for genetic manipulation in microalgae to increase TAG content for biodiesel production.

Effect of light intensity, pH, and temperature on triacylglycerol (TAG) accumulation induced by nitrogen starvation in Scenedesmus obliquus

Microalgae-derived lipids in the form of triacylglycerols (TAGs) are considered an alternative resource for the production of biofuels and food commodities. Large scale production of microalgal TAGs is currently uneconomical. The cost price could be reduced by improving the areal and volumetric TAG productivity. The economic value could be increased by enhancing the TAG quality. To improve these characteristics, the impact of light intensity, and the combined impact of pH and temperature on TAG accumulation were studied for Scenedesmus obliquus UTEX 393 under nitrogen starved conditions. The maximum TAG content was independent of light intensity, but varied between 18% and 40% of dry weight for different combinations of pH and temperature. The highest yield of fatty acids on light (0.263 g/mol photon) was achieved at the lowest light intensity, pH 7 and 27.5 °C.