Some of the genetic modifications performed on metabolic pathways related to lipid synthesis in microalgae and their effect on lipid enrichment.
Microalgae were originally considered as sources of long-chain polyunsaturated fatty acids (PUFAs), mainly for aquaculture purposes. However, based on the fact that their fatty acids (FA), stored as triacylglycerides (TAG), can be converted into biodiesel via a transesterification reaction, several microalgal species have emerged over the last decade as promising feedstocks for biofuel production. Elucidation of microalgae FA and TAG metabolic pathways is therefore becoming a cutting-edge field for developing transgenic algal strains with improved lipid accumulation ability. Furthermore, many of the biomolecules produced by microalgae can also be exploited. In this chapter, we describe recent advances in the field of FA and TAG pathways in microalgae, focusing in particular on the enzymes involved in FA and TAG synthesis, their accumulation in lipid droplets, and their degradation. Mention is made of potentially high-value products that can be obtained from microalgae, and possible molecular targets for enhancing FA and TAG production are outlined. A summary is provided of transcriptomics, proteomics, and metabolomics of the above-mentioned pathways in microalgae. Understanding the relation between anabolic and catabolic lipid enzyme pathways will provide new insights into biodiesel production and other valuable biomolecules obtained from microalgae.
- fatty acids
- lipid metabolism
Despite the drop in crude oil prices over the last few years, global efforts to develop alternative renewable energy sources continue to be driven by increasing air pollution and growing energy consumption. Extensive research is therefore being conducted in the field of biofuels , which are derived from renewable biological sources. Biodiesel is the main substitute for diesel fuel and can be produced from both edible and non-edible oils. The use of edible oils has generated controversy because of the negative impact on food availability and the environment [2, 3]. As a consequence of these ethical considerations, non-food crops have emerged as a viable alternative for the production of biodiesel [4–6]. However, since non-food crops do not produce sufficient biomatter to feasibly cover the fuel requirements of the world’s transport sector, attention is turning to oleaginous microalgae which are able to produce and accumulate large amounts of fatty acids (FA) in the form of triacylglycerides (TAG) that can be converted into biodiesel through a transesterification reaction [2, 3, 7]. Furthermore, some species of oleaginous microalgae can also produce high-value products such as long-chain polyunsaturated fatty acids (docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids), carbohydrates (cellulose, starch), proteins, and other high-value compounds, such as pigments, antioxidants (i.e., β-carotene, astaxanthin), and vitamins, which may have commercial application in various industrial sectors [2, 3, 8, 9]. In addition to their potential as biological factories, the advantage of these photosynthetic microorganisms is that their simple growing requirements (light, CO2, and nutrients) offer several environmental benefits such as high solar energy conversion efficiency, utilization of saline water, CO2 sequestration from the air and self-purification if coupled with wastewater treatment .
Despite the wide range of metabolites able to be synthesized by microalgae, little is known about the regulation of FA and TAG biosynthetic pathways and their storage and turnover in microalgae. In this chapter, we therefore describe recent advances in these fields and possible high-value co-products that could render the production of biodiesel from microalgae more sustainably. Recent studies on the transcriptomics, proteomics, and metabolomics of the above-mentioned pathways are also outlined. Understanding these metabolic pathways will accelerate the availability of biodiesel and other valuable biomolecules obtained from microalgae.
2. FA and TAG biosynthetic pathways in microalgae
Fatty acids are organic acids containing a carboxylic functional group with an aliphatic chain that can be saturated (SFA), monounsaturated (MUFA), or polyunsaturated (PUFA). The number of carbon atoms can vary, generating short-chain, medium-chain, or long-chain FA.
In plants, the FA biosynthetic pathway occurs in the chloroplasts (Figure 1).
As shown in Figure 1, the first step in the pathway involves the acetyl-CoA carboxylase (ACCase) which catalyzes the formation of malonyl-CoA from acetyl-CoA and bicarbonate . There is evidence suggesting the presence of genes encoding this enzyme (accA and accD) in
The next step in the FA synthesis is mediated by the malonyl-CoA:Acyl Carrier Protein (ACP) transacylase (MCAT) which transfers the malonyl group from malonyl-CoA to malonyl-ACP . A putative MCAT was identified as a part of the FA biosynthetic pathway in
Acyl-ACP is the carbon source or substrate for the elongation of FA. This reaction is catalyzed by enzymes known as ketoacyl-ACP synthases (KASIII, KASI, and KASII). After each condensation, a reduction, dehydration, and second reduction occur. These steps are catalyzed by enzymes known as the FAS complex: beta-ketoacyl-ACP reductase (KAR), hydroxyacyl-ACP dehydrase (HAD), and enoyl-ACP reductase (EAR), respectively . Transcriptome analysis of the diatom
Some reports suggest the presence of both enzyme types in microalgae. In the marine microalgae
Upon completion of elongation, FAs are transported to the cytoplasm to act as substrates of the acyl transferases involved in the TAG synthesis. TAG are neutral lipids formed by the esterification of one molecule of glycerol with three FAs. Because of their energy-rich acyl chains, they are the dominant form of stored energy in microalgae. Cellular stresses, such as nutrient deprivation (carbon dioxide, nitrogen, silica, and phosphorous), temperature fluctuation, or high light exposure trigger their formation [22–28]. It has been demonstrated that lipid biosynthetic pathways are induced under these conditions to potentiate the lipid storage (30–60% of dry cell weight), and this mechanism is thought to play a role in microalgae adaptation and survival [24, 29–39]. It has further been reported that multiple stressors have no additive effect on lipid accumulation [24, 40].
Data relating to plant FA and TAG metabolism provided the key to identifying possible molecular targets involved in lipid synthesis and accumulation in microalgae . As shown in Figure 2, in plants, the first step of the conventional Kennedy pathway involves the acylation of the glycerol-3-phosphate (G-3-P), catalyzed by the glycerol-3-phosphate acyltransferase (GPAT) to yield lysophosphatidic acid (LPA). GPAT is the rate-limiting step subject to many regulatory controls at the transcriptional and post-transcriptional level and to allosteric mechanisms [42, 43]. Recent studies have revealed the presence of this enzyme in microalgae. In the marine diatom
As described in Figure 2, lysophosphatidic acid acyltransferase (LPAAT) participates in the second step of the Kennedy pathway. This enzyme catalyzes the acylation of the LPA to yield phosphatidic acid (PA) . Candidate LPAATs have been found in some algal genomes including that of
Phosphatidic acid phosphohydrolase (PAP) uses PA as substrate to form diacylglycerol (DAG), a precursor of TAG (Figure 2) . In eukaryotes, PAP enzymes are the members of the evolutionarily conserved lipin protein family whose activity is related to TAG storage . In the green microalga
The last enzyme of the
As can be observed, much research has focused on the acyl-CoA-dependent reaction catalyzed by DGAT. However, the relative contribution of DGAT1 and DGAT2 isoenzymes to TAG accumulation appears to be species-dependent, so further studies should be performed to gain insight into this aspect.
TAG can be formed by the acyl-CoA-dependent pathway, detailed previously, or through acyl-CoA-independent reactions. Acyl-CoA-independent formation of TAG is mediated by the activities of two types of enzyme: the phospholipid:diacylglycerol acyltransferases (PDAT), which catalyze the formation of TAG using DAG and phosphatidylcholine (PC); and the DAG:DAG transacylases (DGTA) which utilize two molecules of DAG to form TAG and MAG [54, 65].
In fact, in
As already mentioned, many aspects of
3. Transcriptomics, proteomics, and metabolomics
A better understanding of the mechanisms involved in TAG enrichment under stress conditions will help to maximize microalgae productivity. However, many biochemical approaches for elucidating molecular pathways depend on the availability of genomic sequence data . Transcriptomics, proteomics, and metabolomics, however, are able to provide a detailed description of cell transcripts (RNA), proteins and metabolites, respectively while completely bypassing the requirement of genomic information [74, 75].
Transcriptome analysis helped to identify sequences of the enzymes involved in the biosynthesis and catabolism of FA, TAG, and starch in
The transcriptome of
In conclusion, these assembled transcriptomes, proteomes, and metabolomes offer valuable approaches for improving microalgal productivity, providing possible targets for molecular engineering that could enhance microalgae-derived products.
4. Molecular targets for enhancing lipid biosynthesis
Genetic strain modification to improve microalgal productivity and accelerate the industrialization of algal-derived products is a major challenge . Reflecting the fact that enhancement of the FA synthesis pathway had little effect on total lipid content in some plants [85, 86], a growing body of research now focuses on overexpression of the enzymes or heterologous expression of genes involved in the TAG biosynthetic pathway. Table 1 provides an outline of some of the genetic manipulations performed on several microalgal strains, leading to an improvement in their TAG content.
|Enzymes overexpressed or heterologously expressed||Organism||Effect on lipid production (changes over control condition)||References|
|ME of ||18.7%|||
|GPAT of ||Yeast GPAT-deficient mutant||Restored TAG formation|||
|GPAT of ||50%|||
|GPAT of ||2-fold|||
|G3PDH, GPAT, DGAT, LPAAT and PAP of ||2-fold|||
|LPAAT of ||20%|||
|DGAT2 of ||3.5-fold|||
|DGAT1 and DGAT2 of ||Re-stored TAG formation|||
|DGAT2 of ||Re-stored TAG formation|||
|DGAT 1 of ||Re-stored TAG synthesis and lipid body formation|||
|DGAT 2 of ||35%|||
|Various DGAT type 2||20–44%|||
5. TAG-accumulation in lipid droplets
Lipid droplets (LDs) are cell organelles that are currently the subject of in-depth study in various organisms. These lipid globules not only act as a reservoir of cell carbon and energy, they may also have a role in lipid homeostasis, signaling, trafficking, and interorganelle communications [96, 97]. As previously mentioned, under stress conditions microalgae synthesize TAG and store them as cytoplasmic LDs [22–28], which can vary in size, shape, and function depending on the cell type and the environmental conditions (Figure 3) . In eukaryotic cells, LD structure consists of a TAG-rich hydrophobic core surrounded by surface polar glycerolipids into which proteins of the perilipin (Plin) (animal cells) or oleosin and caleosin (plants) families are embedded [99–102]. In microalgae, LD structure is conserved from eukaryotes but different LD proteins have been identified. The analysis of
In the oleaginous diatom
6. TAG degradation pathways in microalgae
As previously mentioned, the economic feasibility of using microalgae as a source of FA for biodiesel depends to a great extent on improvements in the production process, one of the most significant challenges being to increase lipid yields. The selection of oleaginous strains and the search for different culture strategies to increase lipid biosynthesis constitute viable approaches; blocking the competing pathways of carbohydrate formation may be another. However, both the approaches give rise to a decrease in strain growth . Lipid catabolism has largely been ignored as a relevant pathway for engineering, despite being a competing pathway to lipid biogenesis . However, lipases were identified in
7. Microalgae-based biorefineries
In the context of improving the economic feasibility of microalgae-based biodiesel, a closer look should be taken at the large amounts of TAG produced in some oleaginous microalgae alongside high-value products such as carbohydrates (cellulose and starch); proteins and other high-value compounds like pigments, antioxidants (i.e., β-carotene, astaxanthin), and vitamins [2, 3, 8, 9], all of which may have commercial application in different industrial sectors. Some potentially high-value products found in microalgae are described in Table 2.
|Exopolysaccharides||Pharmaceutics and agronomics|||
|Starch, glucose, cellullose||Bioethanol production||[112, 113]|
|Sulfated extracellular polisaccharides||Pharmaceutics|||
|Phytosterols; linoleic (C18:2n6) and alpha linolenic (C18:3n3) fatty acids||Human dietary supplement, nutraceutics|||
|Omega-3 long chain-PUFA||Functional food|||
|Tocopherol||Human dietary supplement, nutraceutics|||
|Tocopherol, pigments, phenolic compounds||Human dietary supplement, nutraceutics|||
|Astaxanthin||Antioxidant, cosmetics, pharmaceutics|||
|Lutein||Functional food, animal feed|||
|Carotenoids||Cosmetics, pharmaceutics, animal feed|||
|Carotenoids||Cosmetics, pharmaceutics, animal feed|||
|Silica||Abrassive products, insecticides|||
Oleaginous microalgae grown under stress conditions can synthesize and accumulate large quantities of FA, mainly in the form of TAG, which can then be converted into biodiesel. Although microalgae constitute a promising source of clean energy, knowledge gaps continue to abound in almost all aspects of FA and TAG metabolism for these microorganisms, including the precise identity of enzymatic machinery, the relative contributions of each enzyme and their precise regulation. Further studies are therefore required to establish the exact metabolic pathways involved in FA and TAG synthesis, accumulation, and degradation in order to develop genetic engineering strategies to obtain microalgal strains with improved capacity to convert their biomass into TAG and other valuable co-products.
The authors are grateful for research funds provided by the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina (CONICET); Agencia Nacional de Promoción Científica y Tecnológica, PICTs 2014-0893, 2013-0987, and 2015-0800; and the Secretaría de Ciencia y Tecnología de la Universidad Nacional del Sur, PGIs 24/B226 and 24/B196. Paola Scodelaro Bilbao, Gabriela Salvador and Patricia Leonardi are Research Members of CONICET.