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An increase in the membrane lipids recycling by PDAT overexpression stimulates the accumulation of triacylglycerol in Nannochloropsis gaditana
Abstract
Oleaginous microalgae represent potential feedstocks for the sustainable production of lipids thanks to their ability to accumulate triacylglycerols (TAGs). TAG accumulation in several algal species is strongly induced under specific conditions such as nutrient deprivation and high light which, however, also negatively impact growth. Genetic modification of lipogenic pathways can potentially enhance TAG accumulation without negatively affecting growth, avoiding the trade-off between biomass and lipids productivity.
In this study, the phospholipid: diacylglycerol acyltransferase (PDAT), an enzyme involved in membrane lipid recycling, was overexpressed in the seawater alga Nannochloropsis gaditana. PDAT overexpression induced increased TAG content in actively growing algae cultures while no effects were observed in conditions naturally stimulating strong lipid accumulation such as high light and nitrogen starvation. The increase of TAG content was confirmed also in a strain cultivated in industrially relevant conditions even though PDAT overexpression, if too strong, the gene overexpression becomes detrimental for growth in the longer term. Results overall suggest that genetic modulation of the PDAT gene represents a promising strategy to increase microalgae lipids content by minimizing negative effects on biomass productivity.
... It would be interesting to investigate the subcellular location of the LD formed by these chloroplast-located enzymes. In term of biotechnology, overexpression of GPATs [32,33], DGAT [34,35], LPAAT [36] and PDAT [37] have all been shown to enhance oil content in microalgae. ...
Microalgae are eukaryotic photosynthetic micro-organisms that
convert CO2 into carbohydrates, lipids, and other valuable
metabolites. They are considered promising chassis for the
production of various bioproducts, including fatty acid–derived
biofuels. However, algae-based biofuels are not yet
commercially available, mainly because of their low yields and
high production cost. Optimizing strains to improve lipid
productivity using the principles of synthetic biology should
help move forward. This necessitates developments in the
following areas: (1) identiffcation of molecular bricks (enzymes,
transcription factors, regulatory proteins etc.); (2) development
of genetic tools; and (3) availability of high-throughput
phenotyping methods. Here, we highlight the most recent
developments in some of these areas and provide examples of
the use of genome editing tools to improve oil content.
... Genetic stacking of NoDGAT2D with MCFA-or DAG-supplying enzymes or regulators that include mCpTE, CnLPAAT, and AtWR1 elevates the MCT share in total TAG by 66-fold and MCT productivity by 65-fold, at the peak phase of oil production [16]. On the other hand, by overexpressing PDAT in N. gaditana, the TAG content was increased in conditions naturally stimulating strong lipid accumulation such as high light and nitrogen starvation [114]. Meanwhile, GC-MS quantification revealed that NoPDAT overexpression enhanced TAG by 28-33% in N. oceanica [115]. ...
Microalgae can convert carbon dioxide into organic matter through photosynthesis. Thus, they are considered as an environment-friendly and efficient cell chassis for biologically active metabolites. Microalgal lipids are a class of organic compounds that can be used as raw materials for food, feed, cosmetics, healthcare products, bioenergy, etc., with tremendous potential for commercialization. In this review, we summarized the commercial lipid products from eukaryotic microalgae, and updated the mechanisms of lipid synthesis in microalgae. Moreover, we reviewed the enhancement of lipids, triglycerides, polyunsaturated fatty acids, pigments, and terpenes in microalgae via environmental induction and/or metabolic engineering in the past five years. Collectively, we provided a comprehensive overview of the products, biosynthesis, induced strategies and genetic engineering in microalgal lipids. Meanwhile, the outlook has been presented for the development of microalgal lipids industries, emphasizing the significance of the accurate analysis of lipid bioactivity, as well as the high-throughput screening of microalgae with specific lipids.
Microalgae are photosynthetic microorganisms that show potential for biofuel production due to their ability to accumulate large quantities of neutral lipids. From a climate perspective, microalgae are valued as a biofuel feedstock because they use the greenhouse gas carbon dioxide as a carbon source. The production of microalgal-based drop-in biofuel requires four main steps: cultivation, harvesting and dewatering, extraction, and conversion. Over the years, the processes used to produce microalgal biofuels have improved, taking advantage of the unique characteristics of microalgae, but there is much improvement. The primary challenge in the implementation of industrial microalgal biofuel production systems is the cost of production, which cannot be solved simply.
In the acyl-CoA-independent pathway of triacylglycerol (TAG) synthesis unique to plants, fungi and algae, TAG formation is catalyzed by the enzyme phospholipid:diacylglycerol acyltransferase (PDAT). The unique PDAT gene of the model diatom Phaeodactylum tricornutum strain CCMP2561 boasts 47 single nucleotide variants within protein coding regions of the alleles. To deepen our understanding of TAG synthesis, we observed the allele-specific expression of PDAT by the analysis of 87 published RNA-seq data and experimental validation. The transcription of one of the two PDAT alleles, allele 2, could be specifically induced by decreasing nitrogen concentrations. Overexpression of allele 2 in P. tricornutum substantially enhanced the accumulation of TAG by 44∼74% under nutrient stress; however, overexpression of allele 1 resulted in little increase of TAG accumulation. Interestingly, a more serious growth inhibition was observed in the PDAT allele 1 overexpression strains compared with allele 2 counterparts. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that enzymes encoded by PDAT allele 2 but not allele 1 had TAG biosynthetic activity, and 7 N-terminal and 3 C-terminal amino acid variants between the two allele-encoded proteins substantially affected enzymatic activity. P. tricornutum PDAT, localized in the innermost chloroplast membrane, used monogalactosyldiacylglycerol and phosphatidylcholine as acyl-donors as demonstrated by the increase of the two lipids in PDAT knockout lines, which indicated a common origin in evolution with green algal PDATs. Our study reveals unequal roles among allele-encoded PDATs in mediating carbon storage and growth in response to nitrogen stress and suggests an unsuspected strategy towards lipid and biomass improvement for biotechnological purposes.
Background
Microalgae's low tolerance to high CO2 concentrations presents a significant challenge for its industrial application, especially when considering the utilization of industrial exhaust gas streams with high CO2 content—an economically and environmentally attractive option. Therefore, the objectives of this study were to investigate the metabolic changes in carbon fixation and lipid accumulation of microalgae under ambient air and high CO2 conditions, deepen our understanding of the molecular mechanisms driving these processes, and identify potential target genes for metabolic engineering in microalgae. To accomplish these goals, we conducted a transcriptomic analysis of the high CO2-tolerant strain, Chlorella sp. ABC-001, under two different carbon dioxide levels (ambient air and 10% CO2) and at various growth phases.
Results
Cells cultivated with 10% CO2 exhibited significantly better growth and lipid accumulation rates, achieving up to 2.5-fold higher cell density and twice the lipid content by day 7. To understand the relationship between CO2 concentrations and phenotypes, transcriptomic analysis was conducted across different CO2 conditions and growth phases. According to the analysis of differentially expressed genes and gene ontology, Chlorella sp. ABC-001 exhibited the development of chloroplast organelles during the early exponential phase under high CO2 conditions, resulting in improved CO2 fixation and enhanced photosynthesis. Cobalamin-independent methionine synthase expression was also significantly elevated during the early growth stage, likely contributing to the methionine supply required for various metabolic activities and active proliferation. Conversely, the cells showed sustained repression of carbonic anhydrase and ferredoxin hydrogenase, involved in the carbon concentrating mechanism, throughout the cultivation period under high CO2 conditions. This study also delved into the transcriptomic profiles in the Calvin cycle, nitrogen reductase, and lipid synthesis. Particularly, Chlorella sp. ABC-001 showed high expression levels of genes involved in lipid synthesis, such as glycerol-3-phosphate dehydrogenase and phospholipid-diacylglycerol acyltransferase. These findings suggest potential targets for metabolic engineering aimed at enhancing lipid production in microalgae.
Conclusions
We expect that our findings will help understand the carbon concentrating mechanism, photosynthesis, nitrogen assimilation, and lipid accumulation metabolisms of green algae according to CO2 concentrations. This study also provides insights into systems metabolic engineering of microalgae for improved performance in the future.
In this research, the gene expression of genes involved in lipid metabolism of the eustigmatophyte alga Nannochloropsis gaditana was measured by transcriptomic data. This microalga can be used as a source of triacylglycerol (TAG) and the omega-3 fatty acid eicosapentaenoic acid (EPA). Insight in TAG and EPA production and regulation are needed to improve their productivity. Nitrogen starvation induces TAG accumulation in N. gaditana. Previous research showed that during nitrogen starvation, EPA was translocated from the polar lipids to TAG and de novo synthesized in N. gaditana. Therefore, the expression levels of genes involved in fatty acid translocation and de novo TAG synthesis were measured. Furthermore, the genes involved in de novo EPA synthesis such as elongases and desaturases were studied. The expression levels were measured during the first hours of nitrogen starvation and the subsequent period of 14 days. One phospholipid:diacylglycerol acyltransferase (PDAT) gene involved in translocation of fatty acids from membrane lipids to TAG was upregulated. In addition, several lipases were upregulated, suggesting that these enzymes might be responsible for the translocation of EPA to TAG. Most desaturases and elongases involved in de novo EPA synthesis were downregulated during nitrogen starvation, except for Δ9 desaturase which was upregulated. This upregulation correlates with the increase in oleic acid. Due to the presence of many hypothetical genes, improvement in annotation is needed to increase our understanding of these pathways and their regulation.
The composition of polyunsaturated fatty acids (PUFAs) in triacylglycerols (TAGs) is key to health benefits and for oil applications, yet the underlying genetic mechanism remains poorly understood. In this study, by in silico, ex vivo, and in vivo profiling of type-2 diacylglycerol acyltransferases (DGAT2s) in Nannochloropsis oceanica we revealed two novel PUFA-preferring enzymes that discriminate individual PUFA species in TAG assembly, with NoDGAT2J for linoleic acid (LA) and NoDGAT2K for eicosapentaenoic acid (EPA). The LA and EPA composition of TAG molecules is mediated in vivo via the functional partitioning between NoDGAT2J and 2K, both of which are localized in the chloroplast envelope. By modulating transcript abundance of the DGAT2s, an N. oceanica strain bank was created, where proportions of LA and EPA in TAG vary by 18.7-fold (between 0.21% and 3.92% dry weight) and 34.7-fold (between 0.09% and 3.12% dry weight), respectively. These findings lay the foundation for producing designer TAG molecules with tailored health benefits or for biofuel applications in industrial microalgae and higher-plant crops.
Microalgae accumulate triacylglycerols (TAGs), a promising feedstock for biodiesel production, under unfavorable environmental or stress conditions for their growth. Our previous analyses revealed that only transcripts of CmGPAT1 and CmGPAT2, both encoding glycerol-3-phosphate acyltransferase, were increased among fatty acid and TAG synthesis genes under TAG accumulation conditions in the red alga Cyanidioschyzon merolae. In this study, to investigate the role of these proteins in TAG accumulation in C. merolae, we constructed FLAG-fused CmGPAT1 and CmGPAT2 overexpression strains. We found that CmGPAT1 overexpression resulted in marked accumulation of TAG even under normal growth conditions, with the maximum TAG productivity increased 56.1-fold compared with the control strain, without a negative impact on algal growth. The relative fatty acid composition of 18:2 in the TAGs and the sn-1/sn-3 positions were significantly increased compared with the control strain, suggesting that CmGPAT1 had a substrate preference for 18:2. Immunoblot analysis after cell fractionation and immunostaining analysis demonstrated that CmGPAT1 localizes in the endoplasmic reticulum (ER). These results indicate that the reaction catalyzed by the ER-localized CmGPAT1 is a rate-limiting step for TAG synthesis in C. merolae, and would be a potential target for improvement of TAG productivity in microalgae.
Microalgae are promising feedstocks for the production of biofuels thanks to their high biomass yield and the lack of competition with food crops for arable land. Algal industrial exploitation, however, still needs to address several technological challenges to reach energetic and economic sustainability. Genetic improvement of microalgae strains is seminal to fully exploit the potential of these organisms.
In this work, we investigated how key environmental parameters affected productivity of a promising genetically modified algal strain cultivated in industrially relevant conditions. We observed that intensive growth conditions strongly influenced algae biology as evidenced by molecular and functional analyses. We also showed that specific operational conditions significantly affected performances, enhancing or reducing the advantages of the strains genetic modification, such as the chlorophyll content per cell and the abundance of photosynthetic apparatus components.
This work demonstrates the presence of a strong influence of cultivation environment on the phenotype of improved strains, suggesting that operational parameters have a seminal influence on algae performances and must be taken into full account during strains development efforts.
The metabolism of glycerol-3-phosphate (G3P) is important for environmental stress responses by eukaryotic microalgae. G3P is an essential precursor for glycerolipid synthesis and the accumulation of triacylglycerol (TAG) in response to nutrient starvation. G3P dehydrogenase (GPDH) mediates G3P synthesis but the roles of specific GPDH isoforms are currently poorly understood. Of the five GPDH enzymes in the model alga Chlamydomonas reinhardtii, GPD2 and GPD3 were shown to be induced by nutrient starvation and/or salt stress. Heterologous expression of GPD2, a putative chloroplastic GPDH, and GPD3, a putative cytosolic GPDH, in a yeast gpd1Δ mutant demonstrated functionality of both enzymes. C. reinhardtii knockdown mutants for GPD2 and GPD3 showed no difference in growth but displayed significant reduction in TAG concentration compared to wild type in response to phosphorus or nitrogen starvation. Overexpression of GPD2 and GPD3 in C. reinhardtii gave distinct phenotypes. GPD2 overexpression lines showed only subtle metabolic phenotypes and no significant alteration in growth. In contrast, GPD3 overexpression lines displayed significantly inhibited growth and chlorophyll concentration, reduced glycerol concentration, and changes to lipid composition compared to wild type, including increased abundance of phosphatidic acids but reduced abundance of diglycerides, triglycerides and phosphatidylglycerol lipids. This may indicate a block in the downstream glycerolipid metabolism pathway in GPD3 overexpression lines. Thus, lipid engineering by GPDH modification may depend on the activities of other downstream enzyme steps. These results also suggest that GPD2 and GPD3 GPDH isoforms are important for nutrient starvation-induced TAG accumulation but have distinct metabolic functions.
Background:
Photosynthetic microalgae are considered a viable and sustainable resource for biofuel feedstocks, because they can produce higher biomass per land area than plants and can be grown on non-arable land. Among many microalgae considered for biofuel production, Nannochloropsis oceanica (CCMP1779) is particularly promising, because following nutrient deprivation it produces very high amounts of triacylglycerols (TAG). The committed step in TAG synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT). Remarkably, a total of 13 putative DGAT-encoding genes have been previously identified in CCMP1779 but most have not yet been studied in detail.
Results:
Based on their expression profile, six out of 12 type-2 DGAT-encoding genes (NoDGTT1-NoDGTT6) were chosen for their possible role in TAG biosynthesis and the respective cDNAs were expressed in a TAG synthesis-deficient mutant of yeast. Yeast expressing NoDGTT5 accumulated TAG to the highest level. Over-expression of NoDGTT5 in CCMP1779 grown in N-replete medium resulted in levels of TAG normally observed only after N deprivation. Reduced growth rates accompanied NoDGTT5 over-expression in CCMP1779. Constitutive expression of NoDGTT5 in Arabidopsis thaliana was accompanied by increased TAG content in seeds and leaves. A broad substrate specificity for NoDGTT5 was revealed, with preference for unsaturated acyl groups. Furthermore, NoDGTT5 was able to successfully rescue the Arabidopsis tag1-1 mutant by restoring the TAG content in seeds.
Conclusions:
Taken together, our results identified NoDGTT5 as the most promising gene for the engineering of TAG synthesis in multiple hosts among the 13 DGAT-encoding genes of N. oceanica CCMP1779. Consequently, this study demonstrates the potential of NoDGTT5 as a tool for enhancing the energy density in biomass by increasing TAG content in transgenic crops used for biofuel production.
The seawater microalga Nannochloropsis gaditana is capable of accumulating a large fraction of reduced carbon as lipids. To clarify the molecular bases of this metabolic feature, we investigated light-driven lipid biosynthesis in Nannochloropsis gaditana cultures combining the analysis of photosynthetic functionality with transcriptomic, lipidomic and metabolomic approaches. Light-dependent alterations are observed in amino acid, isoprenoid, nucleic acid and vitamin biosynthesis, suggesting a deep remodeling in the microalgal metabolism triggered by photoadaptation. In particular, high light intensity is shown to affect lipid biosynthesis, inducing the accumulation of diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and triacylglycerols (TAGs), together with the up-regulation of genes involved in their biosynthesis. Chloroplast polar lipids are instead decreased. This situation correlates with the induction of genes coding for a putative cytosolic fatty acid synthase of type 1 (FAS1) and polyketide synthase (PKS) and the downregulation of the chloroplast fatty acid synthase of type 2 (FAS2). Lipid accumulation is accompanied by the regulation of triose phosphate/inorganic phosphate transport across the chloroplast membranes, tuning the carbon metabolic allocation between cell compartments, favoring the cytoplasm, mitochondrion and endoplasmic reticulum at the expense of the chloroplast. These results highlight the high flexibility of lipid biosynthesis in Nannochloropsis gaditana and lay the foundations for an hypothetical mechanism of regulation of primary carbon partitioning by controlling metabolite allocation at the sub-cellular level.
In addition to the Kennedy pathway for de novo biosynthesis, triacylglycerol (TAG), the most important stock for microalgae-based biodiesel production, can be synthesized by phospholipid: diacylglycerol acyltransferase (PDAT) that transfers an acyl group from phospholipids (PLs) to diacylglycerol (DAG). This study presents a novel gene that encodes PDAT from the green microalga Myrmecia incisa Reisigl H4301 (designated MiPDAT ). MiPDAT is localized on the plasma membrane (PM) via the agroinfiltration of tobacco leaves with a green fluorescent protein-fused construct. MiPDAT synthesizes TAG based on functional complementary experiments in the mutant yeast strain H1246 and the membrane lipid phosphatidylcholine (PC) is preferentially used as substrates as revealed by in vitro enzyme activity assay. The gradually increased transcription levels of MiPDAT in M. incisa during the cultivation under nitrogen starvation conditions is proposed to be responsible for the decrease and increase of the PC and TAG levels, respectively, as detected by liquid chromatography-mass spectrometry after 4 d of nitrogen starvation. In addition, the mechanism by which MiPDAT in this microalga uses PC to yield TAG is discussed. Accordingly, it is concluded that this PM-located PDAT contributes to the conversion of membrane lipids into TAG in M. incisa during the nitrogen starvation stress.
The productivity of an algal culture depends on how efficiently it converts sunlight into biomass and lipids. Wild-type algae in their natural environment evolved to compete for light energy and maximize individual cell growth; however, in a photobioreactor, global productivity should be maximized. Improving light use efficiency is one of the primary aims of algae biotechnological research, and genetic engineering can play a major role in attaining this goal.
In this work, we generated a collection of Nannochloropsis gaditana mutant strains and screened them for alterations in the photosynthetic apparatus. The selected mutant strains exhibited diverse phenotypes, some of which are potentially beneficial under the specific artificial conditions of a photobioreactor. Particular attention was given to strains showing reduced cellular pigment contents, and further characterization revealed that some of the selected strains exhibited improved photosynthetic activity; in at least one case, this trait corresponded to improved biomass productivity in lab-scale cultures.
This work demonstrates that genetic modification of N. gaditana has the potential to generate strains with improved biomass productivity when cultivated under the artificial conditions of a photobioreactor.
Microalgal triacylglycerides (TAGs) are a promising sustainable feedstock for the biofuel, chemical and food industry. However, industrial production of microalgal products for commodity markets is not yet economically viable, largely because of low microalgal productivity. The latter is strictly dependent on initial-biomass-specific (IBS) light availability (i.e. ratio of light impinging on reactor ground area divided by initial biomass concentration per ground area). This study investigates the effect of IBS-light availability on batch TAG production for Nannochloropsis sp. cultivated in two outdoor tubular reactors (i.e. vertical and horizontal) at different initial biomass concentrations for the TAG accumulation phase, during two distinct seasons (i.e. high and low light conditions).
Increasing IBS-light availability led to both a higher IBS-TAG production rate and TAG content at the end of the batch, whereas biomass yield on light decreased. As a result, an optimum IBS-light availability was determined for the TAG productivity obtained at the end of the batch and several guidelines could be established. The vertical reactor (VR) should be operated at an initial biomass concentration of 1.5 g L(-1) to achieve high TAG productivities (1.9 and 3.2 g m(-2) day(-1) under low and high light, respectively). Instead, the horizontal reactor (HR) should be operated at 2.5 g L(-1) under high light (2.6 g m(-2) day(-1)), and at 1.5 g L(-1) under low light (1.4 g m(-2) day(-1)).
From this study, the great importance of IBS-light availability on TAG production can be deduced. Although maintaining high light availabilities in the reactor is key to reach high TAG contents at the end of the batch, considerable losses in TAG productivity were observed for the two reactors regardless of light condition, when not operated at optimal initial biomass concentrations (15-40% for VR and 30-60% for HR).
Microalgae are emerging platforms for production of a suite of compounds targeting several markets, including food, nutraceuticals, green chemicals, and biofuels. Many of these products, such as biodiesel or polyunsaturated fatty acids (PUFAs), derive from lipid metabolism. A general picture of lipid metabolism in microalgae has been deduced from well-characterized pathways of fungi and land plants, but recent advances in molecular and genetic analyses of microalgae have uncovered unique features, pointing out the necessity to study lipid metabolism in microalgae themselves. In the past 10 years, in addition to its traditional role as a model for photosynthetic and flagellar motility processes, Chlamydomonas reinhardtii has emerged as a model organism to study lipid metabolism in green microalgae. Here, after summarizing data on total fatty acid composition, distribution of acyl-lipid classes, and major acyl-lipid molecular species found in Chlamydomonas reinhardtii, we review the current knowledge on the known or putative steps for fatty acid synthesis, glycerolipid desaturation and assembly, membrane lipid turnover, and oil remobilization. A list of characterized or putative enzymes for the major steps of acyl-lipid metabolism in Chlamydomonas reinhardtii is included, and subcellular localizations and phenotypes of associated mutants are discussed. Biogenesis and composition of Chlamydomonas lipid droplets and the potential importance of lipolytic processes in increasing cellular oil content are also highlighted. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Phospholipid:diacylglycerol acyltransferase (PDAT) is an enzyme that catalyzes the transfer of a fatty acyl moiety from the sn-2-position of a phospholipid to the sn-3-position of sn-1, 2-diacylglyerol, thus forming triacylglycerol (TAG) and a lyso-phospholipid. Although the importance of PDAT in TAG biosynthesis has been illustrated in some previous studies, the evolutionary relationship of plant PDATs has not been studied in detail. In this study, we investigated the evolutionary relationship of the PDAT gene family across the green plants using a comparative phylogenetic framework. We found that the PDAT candidate genes are present in all examined green plants, including algae, lowland plants (a moss and a lycophyte), monocots and eudicots. Phylogenetic analysis revealed the evolutionary division of the PDAT gene family into seven major clades. The separation is supported by the conservation and variation in the gene structure, protein properties, motif patterns and/or selection constraints. We further demonstrated that there is a eudicot-wide PDAT gene expansion, which appears to have been mainly caused by the eudicot-shared ancient gene duplication and subsequent species-specific segmental duplications. In addition, selection pressure analyses show that different selection constraints have acted on three core eudicot clades, which might enable paleo-duplicated PDAT paralogs to either become non-functionalized or develop divergent expression pattern during evolution. Overall, our study provides important insights into the evolution of the plant PDAT gene family and explores the evolutionary mechanism underlying the functional diversification among the core eudicot PDAT paralogs.
Copyright © 2015, American Society of Plant Biologists.
Detection of living microalgae cells is very important for ballast water treatment and analysis. Chlorophyll fluorescence is an indicator of photosynthetic activity and hence the living status of plant cells. In this paper, we developed a novel microfluidic biosensor system that can quickly and accurately detect the viability of single microalgae cells based on chlorophyll fluorescence. The system is composed of a laser diode as an excitation light source, a photodiode detector, a signal analysis circuit, and a microfluidic chip as a microalgae cell transportation platform. To demonstrate the utility of this system, six different living and dead algae samples (Karenia mikimotoi Hansen, Chlorella vulgaris, Nitzschia closterium, Platymonas subcordiformis, Pyramidomonas delicatula and Dunaliella salina) were tested. The developed biosensor can distinguish clearly between the living microalgae cells and the dead microalgae cells. The smallest microalgae cells that can be detected by using this biosensor are 3 μm ones. Even smaller microalgae cells could be detected by increasing the excitation light power. The developed microfluidic biosensor has great potential for in situ ballast water analysis.
Microalgae have been emerging as an important source for the production of bioactive compounds. Marine diatoms can store high amounts of lipid and grow quite quickly. However, the genetic and biochemical characteristics of fatty acid biosynthesis in diatoms remain unclear. Glycerophospholipids are integral as structural and functional components of cellular membranes, as well as precursors of various lipid mediators. In addition, diacylglycerol acyltransferase (DGAT) is a key enzyme that catalyzes the last step of triacylglyceride (TAG) biosynthesis. However, a comprehensive sequence-structure and functional analysis of DGAT in diatoms is lacking. In this study, an isoform of diacylglycerol acyltransferase type 2 of the marine diatom Phaeodactylum tricornutum was characterized. Surprisingly, DGAT2 overexpression in P. tricornutum stimulated more oil bodies, and the neutral lipid content increased by 35%. The fatty acid composition showed a significant increase in the proportion of polyunsaturated fatty acids; in particular, EPA was increased by 76.2%. Moreover, the growth rate of transgenic microalgae remained similar, thereby maintaining a high biomass. Our results suggest that increased DGAT2 expression could alter fatty acid profile in the diatom, and the results thus represent a valuable strategy for polyunsaturated fatty acid production by genetic manipulation.
There is growing interest in engineering green biomass to expand the production of plant oils as feed and biofuels. Here, we show that PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1) is a critical enzyme involved in triacylglycerol (TAG) synthesis in leaves. Overexpression of PDAT1 increases leaf TAG accumulation, leading to oil droplet overexpansion through fusion. Ectopic expression of oleosin promotes the clustering of small oil droplets. Coexpression of PDAT1 with oleosin boosts leaf TAG content by up to 6.4% of the dry weight without affecting membrane lipid composition and plant growth. PDAT1 overexpression stimulates fatty acid synthesis (FAS) and increases fatty acid flux toward the prokaryotic glycerolipid pathway. In the trigalactosyldiacylglycerol1-1 mutant, which is defective in eukaryotic thylakoid lipid synthesis, the combined overexpression of PDAT1 with oleosin increases leaf TAG content to 8.6% of the dry weight and total leaf lipid by fourfold. In the plastidic glycerol-3-phosphate acyltransferase1 mutant, which is defective in the prokaryotic glycerolipid pathway, PDAT1 overexpression enhances TAG content at the expense of thylakoid membrane lipids, leading to defects in chloroplast division and thylakoid biogenesis. Collectively, these results reveal a dual role for PDAT1 in enhancing fatty acid and TAG synthesis in leaves and suggest that increasing FAS is the key to engineering high levels of TAG accumulation in green biomass.
Nannochloropsis is rapidly emerging as a model organism for the study of biofuel production in microalgae.Here we report a high quality genomic assembly of Nannochloropsis gaditana, consisting of large contigs, up to 500 kbp long, and scaffolds that in most cases span the entire length of chromosomes. We identified 10,646 complete genes and characterized possible alternative transcripts. The annotation of the predicted genes and the analysis of cellular processes revealed traits relevant for the genetic improvement of this organism such as genes involved in DNA recombination, RNA silencing and cell wall synthesis.We also analysed the modification of the transcriptional profile in nitrogen deficiency, a condition known to stimulate lipid accumulation. While the content of lipids increased, we did not detect major changes in expression of the genes involved in their biosynthesis. At the same time we observed a very significant down regulation of mitochondrial gene expression, suggesting that part of the Acetyl-CoA and NAD(P)H, normally oxidized through the mitochondrial respiration, would be made available for fatty acids synthesis, increasing the flux through the lipid biosynthetic pathway.Finally we released an information resource of the genomic data of N. gaditana, available at www.nannochloropsis.org.
The oil from flax (Linum usitatissimum L.) has high amounts of α-linolenic acid (ALA; 18:3cisΔ9,12,15) and is one of the richest sources of omega-3 polyunsaturated fatty acids (ω-3-PUFAs). To produce ∼57% ALA in triacylglycerol
(TAG), it is likely that flax contains enzymes that can efficiently transfer ALA to TAG. To test this hypothesis, we conducted
a systematic characterization of TAG-synthesizing enzymes from flax. We identified several genes encoding acyl-CoA:diacylglycerol
acyltransferases (DGATs) and phospholipid:diacylglycerol acyltransferases (PDATs) from the flax genome database. Due to recent
genome duplication, duplicated gene pairs have been identified for all genes except DGAT2-2. Analysis of gene expression indicated that two DGAT1, two DGAT2, and four PDAT genes were preferentially expressed in flax embryos. Yeast functional analysis showed that DGAT1, DGAT2, and two PDAT enzymes
restored TAG synthesis when produced recombinantly in yeast H1246 strain. The activity of particular PDAT enzymes (LuPDAT1
and LuPDAT2) was stimulated by the presence of ALA. Further seed-specific expression of flax genes in Arabidopsis thaliana indicated that DGAT1, PDAT1, and PDAT2 had significant effects on seed oil phenotype. Overall, this study indicated the existence
of unique PDAT enzymes from flax that are able to preferentially catalyze the synthesis of TAG containing ALA acyl moieties.
The identified LuPDATs may have practical applications for increasing the accumulation of ALA and other polyunsaturated fatty
acids in oilseeds for food and industrial applications.
The Nannochloropsis genus contains oleaginous microalgae that have served as model systems for developing renewable biodiesel. Recent genomic and transcriptomic studies on Nannochloropsis spp. have provided insights into regulation of lipid production in response to nitrogen stress. Previous studies have focused on the responses of Nannochloropsis to short term nitrogen stress, but the effect of long-term nitrogen deprivation remains largely unknown. In this study, physiological and proteomic approaches were combined to understand the mechanisms by which Nannochloropsis oceanica IMET1 is able to endure long-term nitrate deprivation and its ability to recover homeostasis when nitrogen is amended. Changes of the proteome during chronic nitrogen starvation espoused the physiological changes observed, and there was a general trend towards recycling nitrogen and storage of lipids. This was evidenced by a global down-regulation of protein expression, but a retained expression of proteins involved in glycolysis and synthesis of fatty acids as well as an up-regulation of enzymes used in nitrogen scavenging, and protein turnover. Also, lipid accumulation and autophagy of plastids may play a key role in maintaining cell vitality. Following addition of nitrogen there were proteomic changes and metabolic changes observed within 24 hours, which resulted in a return of the culture to steady state within 4 days. These results demonstrate the ability of N. oceanica IMET1 to recover from long periods of nitrate deprivation without apparent detriment to the culture as well as provide proteomic markers for genetic modification.
Microalgae of the genus Nannochloropsis are capable of accumulating triacylglycerols when exposed to nutrient limitation (in particular nitrogen, N) and are therefore considered promising organisms for biodiesel production. Here, after nitrogen removal from the medium, Nannochloropsis gaditana cells show extensive triacylglycerol accumulation (38%TAG on a dry-weight basis). Triacylglycerols accumulated during N deprivation harbor signatures indicating they mainly stem from freshly synthesized fatty acids with a small proportion originating from a recycling of membrane glycerolipids. The amount of chloroplast galactoglycerolipids, which are essential for the integrity of thylakoids, decreases while their fatty acid composition appears unaltered. In starved cells, galactolipids are kept at a sufficient level to maintain chloroplast integrity as confirmed by electron microscopy. Consistently, N starved Nannochloropsis cells contain less photosynthetic membranes but are still efficiently performing photosynthesis. N starvation leads to a modification of photosynthetic apparatus with a change in pigment composition and a decrease in the content of all the major electron flow complexes, including Photosystem II, Photosystem I and the Cytochrome b6f complex. The Photosystem II content is particularly affected, leading to the inhibition of linear electron flow from water to CO2. Such a reduction, however, is partially compensated for by activation of alternative electron pathways such as cyclic electron transport. Overall, these changes allow cells to modify their energetic metabolism in order to maintain photosynthetic growth.
Studies involving the cloning and disruption of the gene for acyl-CoA:diacylglycerol acyltransferase (DGAT) have shown that
alternative mechanisms exist for triglyceride synthesis. In this study, we cloned and characterized a second mammalian DGAT,
DGAT2, which was identified by its homology to a DGAT in the fungus Mortierella rammaniana. DGAT2 is a member of a gene family that has no homology with DGAT1 and includes several mouse and human homologues that
are candidates for additional DGAT genes. The expression of DGAT2 in insect cells stimulated triglyceride synthesis 6-fold
in assays with cellular membranes, and DGAT2 activity was dependent on the presence of fatty acyl-CoA and diacylglycerol,
indicating that this protein is a DGAT. Activity was not observed for acyl acceptors other than diacylglycerol. DGAT2 activity
was inhibited by a high concentration (100 mm) of MgCl2 in anin vitro assay, a characteristic that distinguishes DGAT2 from DGAT1. DGAT2 is expressed in many tissues with high expression levels
in the liver and white adipose tissue, suggesting that it may play a significant role in mammalian triglyceride metabolism.
Many unicellular microalgae produce large amounts (∼20 to 50% of cell dry weight) of triacylglycerols (TAGs) under stress (e.g., nutrient starvation and high light), but the synthesis and physiological role of TAG are poorly understood. We present detailed genetic, biochemical, functional, and physiological analyses of phospholipid:diacylglycerol acyltransferase (PDAT) in the green microalga Chlamydomonas reinhardtii, which catalyzes TAG synthesis via two pathways: transacylation of diacylglycerol (DAG) with acyl groups from phospholipids and galactolipids and DAG:DAG transacylation. We demonstrate that PDAT also possesses acyl hydrolase activities using TAG, phospholipids, galactolipids, and cholesteryl esters as substrates. Artificial microRNA silencing of PDAT in C. reinhardtii alters the membrane lipid composition, reducing the maximum specific growth rate. The data suggest that PDAT-mediated membrane lipid turnover and TAG synthesis is essential for vigorous growth under favorable culture conditions and for membrane lipid degradation with concomitant production of TAG for survival under stress. The strong lipase activity of PDAT with broad substrate specificity suggests that this enzyme could be a potential biocatalyst for industrial lipid hydrolysis and conversion, particularly for biofuel production.
Biofuels from algae are highly interesting as renewable energy sources to replace, at least partially, fossil fuels, but great research efforts are still needed to optimize growth parameters to develop competitive large-scale cultivation systems. One factor with a seminal influence on productivity is light availability. Light energy fully supports algal growth, but it leads to oxidative stress if illumination is in excess. In this work, the influence of light intensity on the growth and lipid productivity of Nannochloropsis salina was investigated in a flat-bed photobioreactor designed to minimize cells self-shading. The influence of various light intensities was studied with both continuous illumination and alternation of light and dark cycles at various frequencies, which mimic illumination variations in a photobioreactor due to mixing. Results show that Nannochloropsis can efficiently exploit even very intense light, provided that dark cycles occur to allow for re-oxidation of the electron transporters of the photosynthetic apparatus. If alternation of light and dark is not optimal, algae undergo radiation damage and photosynthetic productivity is greatly reduced. Our results demonstrate that, in a photobioreactor for the cultivation of algae, optimizing mixing is essential in order to ensure that the algae exploit light energy efficiently.
Algae have recently gained attention as a potential source for biodiesel; however, much is still unknown about the biological
triggers that cause the production of triacylglycerols. We used RNA-Seq as a tool for discovering genes responsible for triacylglycerol
(TAG) production in Chlamydomonas and for the regulatory components that activate the pathway. Three genes encoding acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression.
DGAT1 and DGTT1 also show increased mRNA abundance in other TAG-accumulating conditions (minus sulfur, minus phosphorus, minus zinc, and
minus iron). Insertional mutants, pdat1-1 and pdat1-2, accumulate 25% less TAG compared with the parent strain, CC-4425, which demonstrates the relevance of the trans-acylation
pathway in Chlamydomonas. The biochemical functions of DGTT1 and PDAT1 were validated by rescue of oleic acid sensitivity and restoration of TAG accumulation in a yeast strain lacking all acyltransferase
activity. Time course analyses suggest than a SQUAMOSA promoter-binding protein domain transcription factor, whose mRNA increases precede that of lipid biosynthesis genes like
DGAT1, is a candidate regulator of the nitrogen deficiency responses. An insertional mutant, nrr1-1, accumulates only 50% of the TAG compared with the parental strain in nitrogen-starvation conditions and is unaffected by
other nutrient stresses, suggesting the specificity of this regulator for nitrogen-deprivation conditions.
Ricinoleic acid (12-hydroxy-octadeca-9-enoic acid) is a major unusual fatty acid in castor oil. This hydroxy fatty acid is useful in industrial materials. This unusual fatty acid accumulates in triacylglycerol (TAG) in the seeds of the castor bean (Ricinus communis L.), even though it is synthesized in phospholipids, which indicates that the castor plant has an editing enzyme, which functions as a phospholipid:diacylglycerol acyltransferase (PDAT) that is specific to ricinoleic acid. Transgenic plants containing fatty acid Δ12-hydroxylase encoded by the castor bean FAH12 gene produce a limited amount of hydroxy fatty acid, a maximum of around 17% of TAGs present in Arabidopsis seeds, and this unusual fatty acid remains in phospholipids of cell membranes in seeds. Identification of ricinoleate-specific PDAT from castor bean and manipulation of the phospholipid editing system in transgenic plants will enhance accumulation of the hydroxy fatty acid in transgenic seeds. The castor plant has three PDAT genes; PDAT1-1 and PDAT2 are homologs of PDAT, which are commonly found in plants; however, PDAT1-2 is newly grouped as a castor bean-specific gene. PDAT1-2 is expressed in developing seeds and localized in the endoplasmic reticulum, similar to FAH12, indicating its involvement in conversion of ricinoleic acid into TAG. PDAT1-2 significantly enhances accumulation of total hydroxy fatty acid up to 25%, with a significant increase in castor-like oil, 2-OH TAG, in seeds of transgenic Arabidopsis, which is an identification of the key gene for oilseed engineering in production of unusual fatty acids.
We examined responses of batch cultures of the marine microalga Nannochloropsis sp. to combined alterations in salinity (13, 27, and 40 g/l NaCl) and light intensity (170 and 700 μmol photons/m(2)·s). Major growth parameters and lipid productivity (based on total fatty acid determination) were determined in nitrogen-replete and nitrogen-depleted cultures of an initial biomass of 0.8 and 1.4 g/l, respectively. On the nitrogen-replete medium, increases in light intensity and salinity increased the cellular content of dry weight and lipids due to enhanced formation of triacylglycerols (TAG). Maximum average productivity of ca. 410 mg TFA/l/d were obtained at 700 μmol photons/m(2)·s and 40 g/l NaCl within 7 days. Under stressful conditions, content of the major LC-PUFA, eicosapentaenoic acid (EPA), was significantly reduced while TAG reached 25% of biomass. In contrast, lower salinity tended to improve major growth parameters, consistent with less variation in EPA contents. Combined higher salinity and light intensity was detrimental to lipid productivity under nitrogen starvation; biomass TFA content, and lipid productivity amounted for only 33% of DW and ca. 200 mg TFA/l/day, respectively. The highest biomass TFA content (ca. 47% DW) and average lipid productivity of ca. 360 mg TFA/l/day were achieved at 13 g/l NaCl and 700 μmol photons/m(2)·s. Our data further support selecting Nannochloropsis as promising microalgae for biodiesel production. Moreover, appropriate cultivation regimes may render Nannochloropsis microalgae to produce simultaneously major valuable components, EPA, and TAG, while sustaining relatively high biomass growth rates.
Acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1.20) is a membrane protein present mainly in the endoplasmic reticulum. It
catalyzes the final and committed step in the biosynthesis of triacylglycerol, which is the principal repository of fatty
acids for energy utilization and membrane formation. Two distinct family members of acyl-CoA:diacylglycerol acyltransferase,
known as DGAT1 and DGAT2, have been characterized in different organisms, including mammals, fungi, and plants. In this study,
we characterized the functional role and topological orientation of signature motifs in yeast (Saccharomyces cerevisiae) DGAT2 using mutagenesis in conjunction with chemical modification. Our data provide evidence that both the N and C termini
are oriented toward the cytosol and have different catalytic roles. A highly conserved motif, 129YFP131, and a hydrophilic segment exclusive to yeast DGAT2 reside in a long endoplasmic reticulum luminal loop following the first
transmembrane domain and play an essential role in enzyme catalysis. In addition, the strongly conserved His195 within the motif HPHG, which may play a role in the active site of DGAT2, is likely embedded in the membrane. These results
indicate some similarities to the topology model of murine DGAT2 but also reveal striking differences suggesting that the
topological organization of DGAT2 is not ubiquitously conserved.
Unicellular photosynthetic algae (dinoflagellate) from the genus Symbiodinium live in mutualistic symbiosis with reef-building corals. Cultured Symbiodinium sp. (clade C) were exposed to a range of environmental stresses that included elevated temperatures (29°C and 32°C) under high (100 μmol quanta m(-2) s(-1) Photosynthetic Active Radiation) and low (10 μmol quanta m(-2) s(-1)) irradiances. Using real-time RT-PCR the stability of expression for the nine selected putative housekeeping genes (HKGs) was tested. The most stable expression pattern was identified for cyclophilin and S-adenosyl methionine synthetase (SAM) followed by S4 ribosomal protein (Rp-S4), Calmodulin (Cal), and Cytochrome oxidase subunit 1 (Cox), respectively. Thermal stress alone resulted in the highest expression stability for Rp-S4 and SAM, with a minimum of two reference genes required for data normalization. For Symbiodinium exposed to both, light and thermal stresses, at least five reference genes were recommended by geNorm analysis. In parallel, the expression of Hsp90 for Symbiodinium in culture and in symbiosis within coral host (Acropora millepora) was evaluated using the most stable HKGs. Our results revealed a drop in Hsp90 expression after an 18 h-period and a 24 h-period of exposure to elevated temperatures indicating the similar Hsp90 expression profile in symbiotic and non-symbiotic environments. This study provides the first list of the HKGs and will provide a useful reference in future gene expression studies in symbiotic dinoflagellates.
Triacylglycerol (TAG) biosynthesis is a principal metabolic pathway in most organisms, and TAG is the major form of carbon storage in many plant seeds. Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is the only acyltransferase enzyme that has been confirmed to contribute to TAG biosynthesis in Arabidopsis thaliana seeds. However, dgat1 null mutants display only a 20 to 40% decrease in seed oil content. To determine whether other enzymes contribute to TAG synthesis, candidate genes were expressed in TAG-deficient yeast, candidate mutants were crossed with the dgat1-1 mutant, and target genes were suppressed by RNA interference (RNAi). An in vivo role for phospholipid:diacylglycerol acyltransferase 1 (PDAT1; At5g13640) in TAG synthesis was revealed in this study. After failing to obtain double homozygous plants from crossing dgat1-1 and pdat1-2, further investigation showed that the dgat1-1 pdat1-2 double mutation resulted in sterile pollen that lacked visible oil bodies. RNAi silencing of PDAT1 in a dgat1-1 background or DGAT1 in pdat1-1 background resulted in 70 to 80% decreases in oil content per seed and in disruptions of embryo development. These results establish in vivo involvement of PDAT1 in TAG biosynthesis, rule out major contributions by other candidate enzymes, and indicate that PDAT1 and DGAT1 have overlapping functions that are essential for normal pollen and seed development of Arabidopsis.
The ease and effectiveness of colony polymerase chain reaction (PCR) has allowed rapid amplification of DNA fragments and screening of large number of colonies of interest including transformants and mutants with genetic manipulations. Here, we evaluated colony PCR in Chlamydomonas. Individual colonies were treated with 10 mM ethylenediaminetetraacetic acid (EDTA) or Chelex-100 and the resulting clear cell lysate was used for PCR reaction. Either genomic DNA or plasmid DNA incorporated into the genome was equally amplified. We found that the Chelex method is superior to EDTA method in certain cases. This colony PCR technique will bypass the tedious process of isolating genomic DNA for PCR reaction and will make it possible for rapid amplification of genomic DNA fragments as well as rapid large-scale screening of transformants.
The terminal step in triglyceride biosynthesis is the esterification of diacylglycerol. To study this reaction in the model
eukaryote, Saccharomyces cerevisiae, we investigated five candidate genes with sequence conservation to mammalian acyltransferases. Four of these genes are similar
to the recently identified acyl-CoA diacylglycerol acyltransferase and, when deleted, resulted in little or no decrease in
triglyceride synthesis as measured by incorporation of radiolabeled oleate or glycerol. By contrast, deletion of LRO1, a homolog of human lecithin cholesterol acyltransferase, resulted in a dramatic reduction in triglyceride synthesis, whereas
overexpression of LRO1yielded a significant increase in triglyceride production. In vitro microsomal assays determined that Lro1 mediated the esterification of diacylglycerol using phosphatidylcholine as the acyl
donor. The residual triglyceride biosynthesis that persists in theLRO1 deletion strain is mainly acyl-CoA-dependent and mediated by a gene that is structurally distinct from the previously identified
mammalian diacylglycerol acyltransferase. These mechanisms may also exist in mammalian cells.
Acyl CoA:diacylgycerol acyltransferase (EC2.3.1.20; DGAT) catalyzes the final step in the production of triacylglycerol. Two polypeptides, which co-purified with DGAT activity,
were isolated from the lipid bodies of the oleaginous fungusMortierella ramanniana with a procedure consisting of dye affinity, hydroxyapatite affinity, and heparin chromatography. The two enzymes had molecular
masses of 36 and 36.5 kDa, as estimated by gel electrophoresis, and showed a broad activity maximum between pH 6 and 8. Based
on partial peptide sequence information, polymerase chain reaction techniques were used to obtain full-length cDNA sequences
encoding the purified proteins. Expression of the cDNAs in insect cells conferred high levels of DGAT activity on the membranes
isolated from these cells. The two proteins share 54% homology with each other but are unrelated to the previously identified
DGAT gene family (designated DGAT1), which is related to the acyl CoA:cholesterol acyltransferase gene family, or to any other
gene family with ascribed function. This report identifies a new gene family, including members in fungi, plants and animals,
which encode enzymes with DGAT function. To distinguish the two unrelated families we designate this new class DGAT2 and refer
to the M. ramanniana genes asMrDGAT2A and MrDGAT2B.
Diacylglycerol esterification provides an excellent target for the pharmacological reduction of triglyceride accumulation
in several human disease states. We have usedSaccharomyces cerevisiae as a model system to study this critical component of triglyceride synthesis. Recent studies of an oleaginous fungus, Mortierella ramanniana, identified a new family of enzymes with in vitroacyl-CoA:diacylglycerol acyltransferase activity. We show here that DGA1, the sole member of this gene family in yeast, has a physiological role in triglyceride synthesis. Metabolic labeling of
DGA1 deletion strains with triglyceride precursors detected significant reductions in triglyceride synthesis. Triglyceride synthesis
was virtually abolished in four different growth conditions when DGA1 was deleted in concert with LRO1, an enzyme that esterifies diacylglycerol from a phospholipid acyl donor. The relative contributions of the two enzymes depended
on growth conditions. The residual synthesis was lost when ARE2, encoding an acyl-CoA:sterol acyltransferase, was deleted.In vitro microsomal assays verified that DGA1and ARE2 mediate acyl-CoA:diacylglycerol acyltransferase reactions. Three enzymes can thus account for diacylglycerol esterification
in yeast. Yeast strains deficient in both diacylglycerol and sterol esterification showed only a slight growth defect indicating
that neutral lipid synthesis is dispensable under common laboratory conditions.
A new pathway for triacylglycerol biosynthesis involving a phospholipid:diacylglycerol acyltransferase (PDAT) was recently described (Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S, [2000] Proc Natl Acad Sci USA 97: 6487-6492). The LRO1 gene that encodes the PDAT was identified in yeast (Saccharomyces cerevisiae) and shown to have homology with animal lecithin:cholesterol acyltransferase. A search of the Arabidopsis genome database identified the protein encoded by the At5g13640 gene as the closest homolog to the yeast PDAT (28% amino acid identity). The cDNA of At5g13640 (AtPDAT gene) was overexpressed in Arabidopsis behind the cauliflower mosaic virus promoter. Microsomal preparations of roots and leaves from overexpressers had PDAT activities that correlated with expression levels of the gene, thus demonstrating that this gene encoded PDAT (AtPDAT). The AtPDAT utilized different phospholipids as acyl donor and accepted acyl groups ranging from C10 to C22. The rate of activity was highly dependent on acyl composition with highest activities for acyl groups containing several double bonds, epoxy, or hydroxy groups. The enzyme utilized both sn-positions of phosphatidylcholine but had a 3-fold preference for the sn-2 position. The fatty acid and lipid composition as well as the amounts of lipids per fresh weight in Arabidopsis plants overexpressing AtPDAT were not significantly different from the wild type. Microsomal preparations of roots from a T-DNA insertion mutant in the AtPDAT gene had barely detectable capacity to transfer acyl groups from phospholipids to added diacylglycerols. However, these microsomes were still able to carry out triacylglycerol synthesis by a diacylglycerol:diacylglycerol acyltransferase reaction at the same rate as microsomal preparations from wild type.
Microalgae represent a potential sustainable source of molecules and materials and the improvement of the
knowledge of their metabolic regulation is essential to maximize their potential. Nutrients deprivation stimulates
the accumulation of reserve lipids, triacylglycerols but also inhibits photosynthesis with a negative impact on
biomass production and therefore overall productivity.
In this work, the seawater microalga Nannochloropsis gaditana was cultivated long term in a semi-continuous
system where the concentrations of nitrogen or phosphorus were limiting but still sufficient to sustain growth
indefinitely, to highlight the response to a long-term nutrient limitation and distinguishing it from the one to
acute short-term stress. N. gaditana cells can acclimate to chronic nutrients limitation maintaining photosynthetic
activity while also accumulating lipids. Both nitrogen and phosphorus limitation induced an increase of triacylglycerols
content, although not by the induction of the synthesis of fatty acids but rather by modulating the
fluxes of reduced carbon molecules toward lipid biosynthesis. Photosynthetic activity was maintained under P
limitation while this was strongly affected by nitrogen depletion, where proteins of photosynthetic apparatus
were largely reduced in content but still maintained their functionality and were able to achieve half of the
biomass productivity with 30% of the nitrogen supply.
Triacylglycerol (TAG) is the major storage lipid in most terrestrial plants and microalgae, and has great nutritional and industrial value. Since the demand for vegetable oil is consistently increasing, numerous studies have been focused on improving the TAG content and modifying the fatty‐acid compositions of plant seed oils. In addition, there is a strong research interest in establishing plant vegetative tissues and microalgae as platforms for lipid production. In higher plants and microalgae, TAG biosynthesis occurs via acyl‐CoA‐dependent or acyl‐CoA‐independent pathways. Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step in the acyl‐CoA‐dependent biosynthesis of TAG, which appears to represent a bottleneck in oil accumulation in some oilseed species. Membrane‐bound and soluble forms of DGAT have been identified with very different amino‐acid sequences and biochemical properties. Alternatively, TAG can be formed through acyl‐CoA‐independent pathways via the catalytic action of membrane‐bound phospholipid:diacylglycerol acyltransferase (PDAT). As the enzymes catalyzing the terminal steps of TAG formation, DGAT and PDAT play crucial roles in determining the flux of carbon into seed TAG and thus have been considered as the key targets for engineering oil production. Here, we summarize the most recent knowledge on DGAT and PDAT in higher plants and microalgae, with the emphasis on their physiological roles, structural features, and regulation. The development of various metabolic engineering strategies to enhance the TAG content and alter the fatty‐acid composition of TAG is also discussed.
Biofuels are regarded as one of the most viable options for reduction of CO2 emissions in the transport sector. However, conventional plant-based biofuels (e.g., biodiesel, bioethanol)’s share of total transportation-fuel consumption in 2016 was very low, about 4%, due to several major limitations including shortage of raw materials, low CO2 mitigation effect, blending wall, and poor cost competitiveness. Advanced biofuels such as drop-in, microalgal, and electro biofuels, especially from inedible biomass, are considered to be a promising solution to the problem of how to cope with the growing biofuel demand. In this paper, recent developments in oxy-free hydrocarbon conversion via catalytic deoxygenation reactions, the selection of and lipid-content enhancement of oleaginous microalgae, electrochemical biofuel conversion, and the diversification of valuable products from biomass and intermediates are reviewed. The challenges and prospects for future development of eco-friendly and economically advanced biofuel production processes also are outlined herein.
Oleaginous microalgae have received a considerable attention as potential biofuel feedstock. However, lack of industry-suitable strain with lipid rich biomass limits its commercial applications. Targeted engineering of lipogenic pathways represents a promising strategy to enhance the efficacy of microalgal oil production. In this study, a type 2 diacylglycerol acyltransferase (DGAT), a rate-limiting enzyme in triacylglycerol (TAG) biosynthesis, was identified and overexpressed in heterokont oleaginous microalga Nannochloropsis oceanica for the first time. Overexpression of DGAT2 in Nannochloropsis increased the relative transcript abundance by 3.48-fold in engineered microalgae cells. TAG biosynthesis was subsequently accelerated by DGAT2 overexpression and neutral lipid content was significantly elevated by 69% in engineered microalgae. The fatty acid profile determined by GC-MS revealed that fatty acid composition was altered in engineered microalgae. Saturated fatty acids and polyunsaturated fatty acids were found to be increased whereas monounsaturated fatty acids content decreased. Furthermore, DGAT2 overexpression did not show negative impact on algal growth parameters. The present investigation showed that the identified DGAT2 would be a potential candidate for enhancing TAG biosynthesis and might facilitate the development of promising oleaginous strains with industrial potential.
Global climate change linked to the accumulation of greenhouse gases has caused concerns regarding the use of fossil fuels as the major energy source. To mitigate climate change while keeping energy supply sustainable, one solution is to rely on the ability of microorganisms to use renewable resources for biofuel synthesis. In this Review, we discuss how microorganisms can be explored for the production of next-generation biofuels, based on the ability of bacteria and fungi to use lignocellulose; through direct CO2 conversion by microalgae; using lithoautotrophs driven by solar electricity; or through the capacity of microorganisms to use methane generated from landfill. Furthermore, we discuss how to direct these substrates to the biosynthetic pathways of various fuel compounds and how to optimize biofuel production by engineering fuel pathways and central metabolism.
In the study, the relationship between the quality and intensity of LED illumination with FAMEs produced were investigated. Nannochloropsis sp. was cultivated for 14days under different intensities of 100, 150 and 200μmol photonsm(-2)s(-1) of red, blue and mixed red blue LED. The findings revealed that suitable combination of LED wavelengths and intensity; (red LED: 150, blue: 100 and mixed red blue: 200μmol photonsm(-2)s(-1)) produced maximum biomass growth and lipid content. It was observed that the quality and intensity of LED significantly influenced the composition of FAMEs. FAMEs produced under blue LED has high degree of unsaturation (DU) and low cetane number while those under red LED has low DU but higher CN. The combination of red blue LED has produced FAMEs with high ignition and lubricating property and also good oxidation stability indicated by the DU and CN values which lies midway between the red and blue.
Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol
acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA
as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes
for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose
tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has
its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol
acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence
tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected
when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined;
during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in
DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism
and its regulation.
Polyunsaturated fatty acids (PUFAs) are essential components of higher eukaryotes. Single cell oils (SCO) are now widely accepted in the market place and there is a growing awareness of the health benefits of PUFAs, such as γ-linolenic acid (GLA), arachidonic acid (ARA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). ARA and DHA have also been used for fortification of infant formulae in many parts of the world. Fish oils are rich sources of DHA and EPA and a limited number of plant oilseeds are good sources of other PUFAs. Marine protists and dinoflagellates, such as species of Thraustochytrium, Schizochytrium and Crypthecodinium are the rich sources of DHA, whereas microalgae like Phaeodactylum and Monodus are good sources of EPA. Species of lower fungi Mortierella accumulate a high percentage of ARA in the lipid fraction. In this paper, various microbiological and enzymatic methods for synthesis of PUFAs are discussed.
Triacylglycerols are the most important storage lipids in most plants and animals. Acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1.20) catalyzes the final step of the pathway of triacylglycerol synthesis and is the only step which is unique to this process. Diacylglycerol acyltransferase is required for the synthesis of storage oil in a wide range of oil-bearing seeds and fruits and in floral structures such as petals, anthers and pollen. We describe the first cloning and functional expression of a cDNA encoding diacylglycerol acyltransferase from a plant. The cDNA, cloned from Arabidopsis thaliana, encodes a 520 amino acid protein with a predicted molecular mass of 59.0 kDa which shares 38% amino acid sequence identity with diacylglycerol acyltransferase from mouse. When expressed in insect cell cultures, the protein catalyzes the synthesis of [14C]triacylglycerol from [14C]diacylglycerol and acyl-CoA. Primer extension analysis revealed that the transcription begins 225 bases before the translation start site, yielding an unusually long 5′ untranslated region. The gene is expressed in a wide range of tissues but most strongly in developing embryos and petals of flowers.
Biofuels derived from marine algae are a potential source of sustainable energy that can contribute to future global demands. The realisation of this potential will require manipulation of the fundamental biology of algal physiology to increase the efficiency with which solar energy is ultimately converted into usable biomass. This 'photosynthetic solar energy conversion efficiency' sets an upper limit on the potential of algal-derived biofuels. In this review, we outline photosynthetic molecular targets that could be manipulated to increase the efficiency and yield of algal biofuel production. We also highlight modern 'omic' and high-throughput technologies that might enable identification, selection and improvement of algal cell lines on timescales relevant for achieving significant contributions to future energy solutions.
Microalgae have much higher lipid yields than those of agricultural oleaginosous crops, and they do not compromise arable land. Despite this, current microalga-based processes suffer from several constraints pertaining to the biocatalyst and the bioreactor, which hamper technologically and economically feasible scale-up. Here, we briefly review recent active research and development efforts worldwide, and discuss the most relevant shortcomings of microalgal biofuels. This review goes one step further relative to related studies, because it tackles otherwise scarcely mentioned issues - for example, heterotrophic versus autotrophic metabolism, alkane versus glyceride synthesis, conduction versus bubbling of CO(2), and excretion versus accumulation of lipids. Besides promising solutions that have been hypothesized and arise from multidisciplinary approaches, we also consider less conventional ones. Microalgae and biofuels hold indeed a promising partnership, but a fully competitive technology is not expected to be available before the end of this decade, because the need for one order of magnitude increase in productivity requires development of novel apparatuses and transformed cells.
During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation was 2-3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases. Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol acyltransferase-transformed plants.
Steryl esters and triacylglycerol (TAG) are the main storage lipids in eukaryotic cells. In the yeastSaccharomyces cerevisiae, these storage lipids accumulate during stationary growth phase within organelles known as lipid bodies. We have used single
and multiple gene disruptions to study storage lipid synthesis in yeast. Four genes, ARE1, ARE2, DGA1, and LRO1, were found to contribute to TAG synthesis. The most significant contribution is made byDGA1, which encodes a novel acyl-CoA:diacylglycerol acyltransferase. Two of the genes, ARE1 andARE2, are also involved in steryl ester synthesis. A yeast strain that lacks all four genes is viable and has no apparent growth
defects under standard conditions. The strain is devoid of both TAG and steryl esters, and fluorescence microscopy revealed
that it also lacks lipid bodies. We conclude that neither storage lipids nor lipid bodies are essential for growth in yeast.
Docosahexaenoic acid (C22:6, DHA) is an omega-3 fatty acid required for the normal development of the mammalian nervous and visual system. DHA is provided by the mother during pregnancy and lactating period. Mother's DHA supplementation during pregnancy, and even before pregnancy, has been suggested. DHA can be provided by marine oils, egg's yolk phospholipids, single cell algae oils, the pure fatty acid, or by the ethyl ester derivative (DHA-EE). Another way to provide DHA can be by sn-2 docosahexaenyl monoacylglyceride (DHA-MG), obtained by the treatment of fish oil with stereospecific lipases. sn-2 Fatty acid monoacylglycerides can be more easily absorbed at the intestine than other fatty acid derivatives.
Female rats fed with a synthetic, which provided essentially no DHA, received a 40-day supplementation of either DHA-EE or DHA-MG. Plasma and erythrocyte fatty acid composition were assessed by gas chromatography at day 0 and 40 of supplementation.
DHA-EE increased plasma and erythrocyte DHA by 15 and 11.9%, respectively, with no modification of arachidonic acid (AA) content. DHA-MG supplementation increased plasma and erythrocyte DHA by 24 and 23.8%, respectively, but reduced AA by 5.5 and 3%, respectively.
We conclude that in the rat, DHA-MG supplementation allows a higher plasma and erythrocyte DHA content than DHA-EE with minor modification of AA content.
To more fully understand the function of phospholipid: diacylglycerol acyltransferase (PDAT, EC 2.3.1.158) in plants we have isolated and characterized a knockout mutant of Arabidopsis thaliana L. which has a T-DNA insertion in PDAT locus At5g13640. Lipid analysis was conducted on these plants to assess the contribution of the PDAT gene to lipid composition. The fatty acid content and composition in seeds do not show significant changes in the mutant. This is a contrary situation to yeast where PDAT is a major contributor to triacylglycerol (TAG) accumulation in exponential growth phase. The results indicate that PDAT activity encoded by At5g13640 is not a major determinant of TAG synthesis in Arabidopsis seeds.