Characterization of major lipid droplet proteins from Dunaliella

Many green algal species can accumulate large amounts of triacylglycerides (TAG) under nutrient deprivation, making them a potential source for production of biodiesel. TAG are organized in cytoplasmic lipid bodies, which contain a major lipid droplet protein termed MLDP. Green algae MLDP differ in sequence from plant oleosins and from animal perilipins, and their structure and function are not clear. In this study, we describe the isolation of MLDP from three species of the extreme halotolerant green algae Dunaliella. Sequence alignment with other green algae MLDP proteins identified a conserved 4-proline domain that may be considered as a signature domain of Volvocales green algae MLDP. Gold immunolabeling localized MLDP at the surface of lipid droplets in D. salina. The induction of MLDP by nitrogen deprivation is kinetically correlated with TAG accumulation, and inhibition of TAG biosynthesis impairs MLDP accumulation suggesting that MLDP induction is co-regulated with TAG accumulation. These results can lead to a better understanding of the structure and function of Volvocales green algae MLDP proteins.     

Large fluxes of fatty acids from membranes to triacylglycerol and back during N-deprivation and recovery in Chlamydomonas

Microalgae accumulate triacylglycerol (TAG) during nutrient deprivation and break it down after nutrient resupply, and these processes involve dramatic shifts in cellular carbon allocation. Due to the importance of algae in the global carbon cycle, and the potential of algal lipids as feedstock for chemical and fuel production, these processes are of both ecophysiological and biotechnological importance. However, the metabolism of TAG is not well understood, particularly the contributions of fatty acids (FAs) from different membrane lipids to TAG accumulation and the fate of TAG FAs during degradation. Here, we used isotopic labeling time course experiments on Chlamydomonas reinhardtii to track FA synthesis and transfer between lipid pools during nitrogen (N)-deprivation and resupply. When cells were labeled before N-deprivation, total levels of label in cellular FAs were unchanged during subsequent N-deprivation and later resupply, despite large fluxes into and out of TAG and membrane lipid pools. Detailed analyses of FA levels and labeling revealed that about one-third of acyl chains accumulating in TAG during N-deprivation derive from preexisting membrane lipids, and in total, at least 45% of TAG FAs passed through membrane lipids at one point. Notably, most acyl chains in membrane lipids during recovery after N-resupply come from TAG. Fluxes of polyunsaturated FAs from plastidic membranes into TAG during N-deprivation were particularly noteworthy. These findings demonstrate a high degree of integration of TAG and membrane lipid metabolism and highlight a role for TAG in storage and supply of membrane lipid components.

In Chlamydomonas, about a third of triacylglycerol (TAG) made during nitrogen deprivation is derived from preexisting membranes, and most membranes made after resupply are derived from TAG.     

Rapid Induction of Lipid Droplets in Chlamydomonas reinhardtii and Chlorella vulgaris by Brefeldin A

Algal lipids are the focus of intensive research because they are potential sources of biodiesel. However, most algae produce neutral lipids only under stress conditions. Here, we report that treatment with Brefeldin A (BFA), a chemical inducer of ER stress, rapidly triggers lipid droplet (LD) formation in two different microalgal species, Chlamydomonas reinhardtii and Chlorella vulgaris. LD staining using Nile red revealed that BFA-treated algal cells exhibited many more fluorescent bodies than control cells. Lipid analyses based on thin layer chromatography and gas chromatography revealed that the additional lipids formed upon BFA treatment were mainly triacylglycerols (TAGs). The increase in TAG accumulation was accompanied by a decrease in the betaine lipid diacylglyceryl N,N,N-trimethylhomoserine (DGTS), a major component of the extraplastidic membrane lipids in Chlamydomonas, suggesting that at least some of the TAGs were assembled from the degradation products of membrane lipids. Interestingly, BFA induced TAG accumulation in the Chlamydomonas cells regardless of the presence or absence of an acetate or nitrogen source in the medium. This effect of BFA in Chlamydomonas cells seems to be due to BFA-induced ER stress, as supported by the induction of three homologs of ER stress marker genes by the drug. Together, these results suggest that ER stress rapidly triggers TAG accumulation in two green microalgae, C. reinhardtii and C. vulgaris. A further investigation of the link between ER stress and TAG synthesis may yield an efficient means of producing biofuel from algae.     

Mechanism and challenges in commercialisation of algal biofuels

Biofuels made from algal biomass are being considered as the most suitable alternative energy in current global and economical scenario. Microalgae are known to produce and accumulate lipids within their cell mass which is similar to those found in many vegetable oils. The efficient lipid producer algae cell mass has been reported to contain more than 30% of their cell weight as lipids. According to US DOE microalgae have the potential to produce 100 times more oil per acre land than any terrestrial plants. This article reviews up to date literature on the composition of algae, mechanism of oil droplets, triacylglycerol (TAG) production in algal biomass, research and development made in the cultivation of algal biomass, harvesting strategies, and recovery of lipids from algal mass. The economical challenges in the production of biofuels from algal biomass have been discussed in view of the future prospects in the commercialisation of algal fuels.     

The ins and outs of algal metal transport

Metal transporters are a central component in the interaction of algae with their environment. They represent the first line of defense to cellular perturbations in metal concentration, and by analyzing algal metal transporter repertoires, we gain insight into a fundamental aspect of algal biology. The ability of individual algae to thrive in environments with unique geochemistry, compared to non-algal species commonly used as reference organisms for metal homeostasis, provides an opportunity to broaden our understanding of biological metal requirements, preferences and trafficking. Chlamydomonas reinhardtii is the best developed reference organism for the study of algal biology, especially with respect to metal metabolism; however, the diversity of algal niches necessitates a comparative genomic analysis of all sequenced algal genomes. A comparison between known and putative proteins in animals, plants, fungi and algae using protein similarity networks has revealed the presence of novel metal metabolism components in Chlamydomonas including new iron and copper transporters. This analysis also supports the concept that, in terms of metal metabolism, algae from similar niches are more related to one another than to algae from the same phylogenetic clade. This article is part of a Special Issue entitled: Cell Biology of Metals.     

微藻三酰甘油合成途径及其最新研究进展

三酰甘油(triacylglycerols,TAGs)是动物、植物、微生物和微藻细胞主要的储藏性脂类,它可应用于食品、轻工业和生物燃料等方面,是一种新型可再生能源——生物柴油生产的重要原料。与高等油料作物相比,微藻具有光合作用效率高、生长速度快、油脂产量高、不占用农业耕地和适应多种生长环境等优势,是一种潜在的新型生物柴油生产原料。然而,目前人们对有机体,尤其是微藻细胞内TAG合成与积累的分子机制及细胞的代谢调控机制还知之甚少。对TAG合成的一系列重要过程,包括脂肪酸的合成,TAG生物合成的主要途径和旁路途径,以及与TAG合成相关的关键酶和重要基因等进行了综述,特别对微藻细胞中与TAG合成相关的关键基因的最新研究进展进行了总结,旨在更好地了解油脂代谢的调控途径,为最大限度地供应生物柴油的生产原料提供理论基础。     

A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii

Neutral lipid metabolism has been extensively studied in yeast, plants and mammals. In contrast, little information is available regarding the biochemical pathway, enzymes and regulatory factors involved in the biosynthesis of triacylglycerol (TAG) in microalgae. In the conventional TAG biosynthetic pathway widely accepted for yeast, plants and mammals, TAG is assembled in the endoplasmic reticulum (ER) from its immediate precursor diacylglycerol (DAG) made by ER-specific acyltransferases, and is deposited exclusively in lipid droplets in the cytosol. Here, we demonstrated that the unicellular microalga Chlamydomonas reinhardtii employs a distinct pathway that uses DAG derived almost exclusively from the chloroplast to produce TAG. This unique TAG biosynthesis pathway is largely dependent on de novo fatty acid synthesis, and the TAG formed in this pathway is stored in lipid droplets in both the chloroplast and the cytosol. These findings have wide implications for understanding TAG biosynthesis and storage and other areas of lipid metabolism in microalgae and other organisms.     

A Framework to Report the Production of Renewable Diesel from Algae

Recently, algae have received significant interest as a potential feedstock for renewable diesel (such as biodiesel), and many researchers have attempted to quantify this potential. Some of these attempts are less useful because they have not incorporated specific values of algal lipid content, have not included processing inefficiencies, or omitted processing steps required for renewable diesel production. Furthermore, the associated energy, materials, and costs requirements are sometimes omitted. The accuracy and applicability of these estimates can be improved by using data that are more specific, including all relevant information for renewable diesel production, and by presenting information with more relevant metrics. To determine whether algae are a viable source for renewable diesel, three questions that must be answered are (1) how much renewable diesel can be produced from algae, (2) what is the financial cost of production, and (3) what is the energy ratio of production? To help accurately answer these questions, we propose an analytical framework and associated nomenclature system for characterizing renewable diesel production from algae. The three production pathways discussed in this study are the transesterification of extracted algal lipids, thermochemical conversion of algal biomass, and conversion of secreted algal oils. The nomenclature system is initially presented from a top-level perspective that is applicable to all production pathways for renewable diesel from algae. Then, the nomenclature is expanded to characterize the production of renewable diesel (specifically, biodiesel) from extracted algal lipids in detail (cf. Appendix 2). The analytical framework uses the presented nomenclature system and includes three main principles: using appropriate reporting metrics, using symbolic notation to represent unknown values, and presenting results that are specific to algal species, growth conditions, and product composition.     

Microorganism lipid droplets and biofuel development

Lipid droplet (LD) is a cellular organelle that stores neutral lipids as a source of energy and carbon. However, recent research has emerged that the organelle is involved in lipid synthesis, transportation, and metabolism, as well as mediating cellular protein storage and degradation. With the exception of multi-cellular organisms, some unicellular microorganisms have been observed to contain LDs. The organelle has been isolated and characterized from numerous organisms. Triacylglycerol (TAG) accumulation in LDs can be in excess of 50% of the dry weight in some microorganisms, and a maximum of 87% in some instances. These microorganisms include eukaryotes such as yeast and green algae as well as prokaryotes such as bacteria. Some organisms obtain carbon from CO2 via photosynthesis, while the majority utilizes carbon from various types of biomass. Therefore, high TAG content generated by utilizing waste or cheap biomass, coupled with an efficient conversion rate, present these organisms as bio-tech ‘factories’ to produce biodiesel. This review summarizes LD research in these organisms and provides useful information for further LD biological research and microorganism biodiesel development. [BMB Reports 2013; 46(12): 575-581]     

Potential of mixed microalgae to harness biodiesel from ecological water-bodies with simultaneous treatment

Biodiesel as an eco-friendly fuel is gaining much acceptance in recent years. This communication provides an overview on the possibility of using mixed microalgae existing in ecological water-bodies for harnessing biodiesel. Microalgal cultures from five water-bodies are cultivated in domestic wastewater in open-ponds and the harvested algal-biomass was processed through acid-catalyzed transesterification. Experiments evidenced the potential of using mixed microalgae for harnessing biodiesel. Presence of palmitic acid (C16:0) in higher fraction and physical properties of algal oil correlated well with the biodiesel properties. Functional characteristics of water-bodies showed to influence both species diversity and lipid accumulation. Microalgae from stagnant water-bodies receiving domestic discharges documented higher lipid accumulation. Algal-oil showed to consist 33 types of saturated and unsaturated fatty acids having wide food and fuel characteristics. Simultaneous wastewater treatment was also noticed due to the syntrophic association in the water-body microenvironment. Diversity studies visualized the composition of algae species known to accumulate higher lipids.     

Differential effects of nitrogen and sulfur deprivation on growth and biodiesel feedstock production of Chlamydomonas reinhardtii

Biodiesel production from microalgae is a promising approach for energy production; however, high cost of its process limits the use of microalgal biodiesel. Increasing the levels of triacylglycerol (TAG) levels, which is used as a biodiesel feedstock, in microalgae has been achieved mainly by nitrogen starvation. In this study, we compared effects of sulfur (S) and nitrogen (N) starvation on TAG accumulation and related parameters in wild-type Chlamydomonas reinhardtii CC-124 mt(-) and CC-125 mt(+) strains. Cell division was interrupted, protein and chlorophyll levels rapidly declined while cell volume, total neutral lipid, carotenoid, and carbohydrate content increased in response to nutrient starvation. Cytosolic lipid droplets in microalgae under nutrient starvation were monitored by three-dimensional confocal laser imaging of live cells. Infrared spectroscopy results showed that relative TAG, oligosaccharide and polysaccharide levels increased rapidly in response to nutrient starvation, especially S starvation. Both strains exhibited similar levels of regulation responses under mineral deficiency, however, the degree of their responses were significantly different, which emphasizes the importance of mating type on the physiological response of algae. Neutral lipid, TAG, and carbohydrate levels reached their peak values following 4 days of N or S starvation. Therefore, 4 days of N or S starvation provides an excellent way of increasing TAG content. Although increase in these parameters was followed by a subsequent decline in N-starved strains after 4 days, this decline was not observed in S-starved ones, which shows that S starvation is a better way of increasing TAG production of C. reinhardtii than N starvation.     

Medium pH and nitrate concentration effects on accumulation of triacylglycerol in two members of the chlorophyta

Algal-derived biodiesel is of particular interest because of several factors including: the potential for a near-carbon-neutral life cycle, the prospective ability for algae to capture carbon dioxide generated from coal, and algae’s high per acre yield potential. Our group and others have shown that in nitrogen limitation, and for a single species of Chlorella, a rise in culture medium pH yields triacylglycerol (TAG) accumulation. To solidify and expand on these triggers, the influence and interaction of pH and nitrogen concentration on lipid production was further investigated on Chlorophyceae Scenedesmus sp. and Coelastrella sp. Growth was monitored optically and TAG accumulation was monitored by Nile red fluorescence and confirmed by gas chromatography. Both organisms grew in all treatments and TAG accumulation was observed by two distinct conditions: high pH and nitrogen limitation. The Scenedesmus sp. was shown to grow and produce lipids to a larger degree in alkaliphilic conditions (pH >9) and was used to further investigate the interplay between TAG accumulation from high pH and/or nitrate depletion. Results given here indicate that TAG accumulation per cell, monitored by Nile red fluorescence, correlates with pH at the time of nitrate depletion.     

Microplate assay for quantitation of neutral lipids in extracts from microalgae

Lipid quantitation is widespread in the algae literature, but popular methods such as gravimetry, gas chromatography and mass spectrometry (GC–MS), and Nile red cell staining suffer drawbacks, including poor quantitation of neutral lipids, expensive equipment, and variable results among algae species, respectively. A high-throughput microplate assay was developed that uses Nile red dye to quantify neutral lipids that have been extracted from algae cells. Because the algal extracts contained pigments that quenched Nile red fluorescence, a mild bleach solution was used to destroy pigments, resulting in a nearly linear response for lipid quantities in the range of 0.75 to 40 μg. Corn oil was used as a standard for quantitation, although other vegetable oils displayed a similar response. The assay was tested on lipids extracted from three species of Chlorella and resulted in close agreement with triacylglycerol (TAG) levels determined by thin layer chromatography. The assay was found to more accurately measure algal lipids conducive to biodiesel production and nutrition applications than the widely used gravimetric assay. Assay response was also consistent among different species, in contrast to Nile red cell staining procedures.     

Bioprocess engineering of microalgae to optimize lipid production through nutrient management

Microalgae have been used commercially as a feedstock for the production of high-value compounds, pigments, cosmetics, and nutritional supplements. In addition, because of their rapid growth rates, high photosynthetic efficiency, and high lipid and protein content, commodity products including biodiesel, feed supplements, and polyunsaturated fatty acids derived from algal biomass are of current interest. Since microalgae lack non-photosynthetic structures and float in water, they do not need massive amounts of structural cellulose found in land plants. Thus, under optimal culture conditions, some oleaginous species can allocate up to 70 % of their biomass to lipids. Lipid production and its regulation in microalgae are species-specific and influenced by environmental conditions. Various strategies have been developed to improve lipid productivity and fatty acid composition to meet specific production goals. Manipulation of physiochemical parameters, trophic modes, and nutrient levels, known as process engineering, is a simple approach that leads to desired alterations in the biochemical composition of algal biomass, including lipid quantity and quality. In this paper, we review the effects of manipulating biochemical parameters such as necessary nutrients (C, N, P, S, Fe, and Si), NaCl concentration, and pH of culture medium to optimize lipid content and profile in some algae strains with commercial potential.     

RNA Interference Silencing of a Major Lipid Droplet Protein Affects Lipid Droplet Size in Chlamydomonas reinhardtii

Eukaryotic cells store oils in the chemical form of triacylglycerols in distinct organelles, often called lipid droplets. These dynamic storage compartments have been intensely studied in the context of human health and also in plants as a source of vegetable oils for human consumption and for chemical or biofuel feedstocks. Many microalgae accumulate oils, particularly under conditions limiting to growth, and thus have gained renewed attention as a potentially sustainable feedstock for biofuel production. However, little is currently known at the cellular or molecular levels with regard to oil accumulation in microalgae, and the structural proteins and enzymes involved in the biogenesis, maintenance, and degradation of algal oil storage compartments are not well studied. Focusing on the model green alga Chlamydomonas reinhardtii, the accumulation of triacylglycerols and the formation of lipid droplets during nitrogen deprivation were investigated. Mass spectrometry identified 259 proteins in a lipid droplet-enriched fraction, among them a major protein, tentatively designated major lipid droplet protein (MLDP). This protein is specific to the green algal lineage of photosynthetic organisms. Repression of MLDP gene expression using an RNA interference approach led to increased lipid droplet size, but no change in triacylglycerol content or metabolism was observed.Triacylglycerols (TAGs) are stored in lipid droplets which are subcellular structures in specialized cells ubiquitous to eukaryotes but have more recently also been identified in some prokaryotes (26). In plants and animals, lipid droplets are surrounded by cytosol and are believed to bud off the endoplasmic reticulum (ER) (15, 26). While traditionally considered merely as storage compartments, recent studies suggest that lipid droplets in animals play important additional roles in lipid homeostasis and protein storage (8). In oilseed plants, TAG accumulated in seeds is used as a reservoir of energy and membrane lipid building blocks to support rapid growth after germination (15). Many green algae are capable of accumulating large amounts of TAG in lipid droplets, particularly as a result of abiotic stresses, such as nutrient deprivation or high-light exposure. Although TAG metabolism in algae has not yet been extensively studied at the biochemical or molecular level, it is proposed that TAG turnover contributes primarily to the assembly of membrane lipids to facilitate rapid cell division after the cessation of nutrient limitation (14, 38).The general structure of lipid droplets is conserved in different species with a globular neutral lipid core enclosed by a membrane lipid monolayer (26). In addition, specific proteins are associated with lipid droplets and play important roles in lipid droplet structure and function. A number of recent proteomic studies of lipid droplets from different animals and tissues (8, 40), Saccharomyces cerevisiae (4), and plants (16, 17) have revealed that the lipid droplet-associated proteins of these organisms are quite distinct. For example, the abundant lipid droplet proteins in animals—the so-called “PAT” family of proteins comprised of perilipin, adipose differentiation-related protein (ADRP), and TIP47 (21)—have no apparent orthologs in the desiccating seed plant Arabidopsis thaliana; conversely, the oleosins which coat the oil bodies of Arabidopsis and many other seed plants are not found in animals (26). Reverse genetic studies of these proteins have helped to elucidate the role of A. thaliana oleosins in regulating lipid droplet size and preventing droplet fusion (35, 36) or that of mouse adipocyte perilipin in regulating lipolytic activity at the lipid droplet surface (37). Moreover, recent genomewide RNAi screens in Drosophila cells implicated 1.5 to 3.0% of all genes as directly or indirectly involved in lipid droplet formation and/or regulation and resulted in the identification of a new role for the Arf1-COPI vesicular transport machinery in regulating droplet morphology and lipid utilization (7, 12). In contrast, few molecular details are known about algal lipid droplet biogenesis although many TAG-rich algal species have been described (14).Our efforts to identify proteins related to the PAT protein family or oleosins in the Chlamydomonas reinhardtii genome (24) or genomes of other green algal and diatom species, including Thalassiosira pseudonana, Volvox carteri, and Chlorella sp. NC64A, revealed no putative algal orthologs. In order to identify both potentially novel and conserved proteins which function in algal lipid droplet biogenesis, we studied the accumulation of TAG in lipid droplets of nitrogen-limited C. reinhardtii cells and identified candidate lipid droplet-associated proteins by mass spectrometry.     

Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances

Microalgae represent an exceptionally diverse but highly specialized group of micro-organisms adapted to various ecological habitats. Many microalgae have the ability to produce substantial amounts (e.g. 20–50% dry cell weight) of triacylglycerols (TAG) as a storage lipid under photo-oxidative stress or other adverse environmental conditions. Fatty acids, the building blocks for TAGs and all other cellular lipids, are synthesized in the chloroplast using a single set of enzymes, of which acetyl CoA carboxylase (ACCase) is key in regulating fatty acid synthesis rates. However, the expression of genes involved in fatty acid synthesis is poorly understood in microalgae. Synthesis and sequestration of TAG into cytosolic lipid bodies appear to be a protective mechanism by which algal cells cope with stress conditions, but little is known about regulation of TAG formation at the molecular and cellular level. While the concept of using microalgae as an alternative and renewable source of lipid-rich biomass feedstock for biofuels has been explored over the past few decades, a scalable, commercially viable system has yet to emerge. Today, the production of algal oil is primarily confined to high-value specialty oils with nutritional value, rather than commodity oils for biofuel. This review provides a brief summary of the current knowledge on oleaginous algae and their fatty acid and TAG biosynthesis, algal model systems and genomic approaches to a better understanding of TAG production, and a historical perspective and path forward for microalgae-based biofuel research and commercialization.     

Structural correlates of cytoplasmic and chloroplast lipid body synthesis in Chlamydomonas reinhardtii and stimulation of lipid body production with acetate boost

Light microscopy and deep-etch electron microscopy were used to visualize triacylglyceride (TAG)-filled lipid bodies (LBs) of the green eukaryotic soil alga Chlamydomonas reinhardtii, a model organism for biodiesel production. Cells growing in nitrogen-replete media contain small cytoplasmic lipid bodies (α-cyto-LBs) and small chloroplast plastoglobules. When starved for N, β-cyto-LB formation is massively stimulated. β-Cyto-LBs are intimately associated with both the endoplasmic reticulum membrane and the outer membrane of the chloroplast envelope, suggesting a model for the active participation of both organelles in β-cyto-LB biosynthesis and packaging. When sta6 mutant cells, blocked in starch biosynthesis, are N starved, they produce β-cyto-LBs and also chloroplast LBs (cpst-LBs) that are at least 10 times larger than plastoglobules and eventually engorge the chloroplast stroma. Production of β-cyto-LBs and cpst-LBs under the conditions we used is dependent on exogenous 20 mM acetate. We propose that the greater TAG yields reported for N-starved sta6 cells can be attributed to the strain's ability to produce cpst-LBs, a capacity that is lost when the mutant is complemented by a STA6 transgene. Provision of a 20 mM acetate "boost" during N starvation generates sta6 cells that become so engorged with LBs-at the expense of cytoplasm and most organelles-that they float on water even when centrifuged. This property could be a desirable feature for algal harvesting during biodiesel production.