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Edible oils from microalgae: insights in TAG accumulation A.J. Klok1, P.P. Lamers1, D.E. Martens1, R.B. Draaisma2, and R.H. Wijffels1 1 2

AlgaePARC, Bioprocess Engineering, Wageningen University, PO Box 8129, 6700 EV Wageningen, The Netherlands Unilever Research and Development Vlaardingen, PO Box 114, 3133 AT Vlaardingen, The Netherlands

Microalgae are a promising future source for sustainable edible oils. To make microalgal oil a cost-effective alternative for common vegetable oils, increasing TAG productivity and TAG content are of high importance. Fulfilling these targets requires proper understanding of lipid metabolism in microalgae. Here, we provide an overview of our current knowledge on the biology of TAG accumulation as well as the latest developments and future directions for increasing oil production in microalgae, considering both metabolic engineering techniques and cultivation strategies. Microalgal oils: a sustainable and widely applicable feedstock The world is heading towards 9 billion inhabitants by 2050, which will lead to global challenges in terms of affordable food supply and sustainability [1–3]. For several years microalgae have been mentioned as promising candidates for the sustainable and affordable production of feed, fuels, and chemicals [4,5]. Microalgae, when grown photoautotrophically and outdoors, consume sunlight and CO2 in natural day–night cycles. They can thus be considered ‘microplants’ and their products can simply be labelled as vegetable products. In contrast to traditional food crops, microalgae can be grown on non-arable land and require less land area to obtain similar yields [6,7]. Similarly to higher plants, they are a potential source of edible protein [8], carbohydrate, and lipids. Many species produce lipids in the form of triacylglycerol (TAG), with fatty acid compositions similar to vegetable oils [9,10], and some produce the high-value fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [11–13]. Therefore, microalgae are an interesting option for sustainable production of natural edible oils for bulk and specialty applications in foods. In this review we will limit our discussion to the production of TAG as an alternative to plant-derived oil. TAG is typically produced in microalgae under adverse growth conditions and is accumulated in specialised organelles termed lipid bodies (LBs). After prolonged exposure to adverse growth conditions, such as nitrogen deprivation, the TAG fraction of microalgae ranges from 20–60% (weight/dry weight) [14]. Corresponding author: Klok, A.J. ([email protected]). Keywords: microalgae; vegetable oil; triacylglycerol (TAG); metabolism; cultivation strategies; strain development. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.07.004

Owing to the relatively low cost of commodity vegetable oils, that are marketed at 0.50–1.00 s/kg (http://faostat3.fao.org), the production costs of microalgal TAG, currently estimated at 8.30 s/kg [9], must be decreased considerably before production for the food market can become cost-efficient. However, competition with the low priced oils used in fuel production is even more difficult [15], which makes the realisation of edible oils from microalgae an interesting intermediate target. To make edible oils from microalgae cost-effective, increasing TAG productivity and TAG content are of high importance. This will therefore be the focus of this review. It should be noted, however, that biorefining of valuable coproducts such as pigments, proteins, and longchain v-3 polyunsaturated fatty acids can help to offset production costs [16]. Likewise, an increase in harvesting efficiency [17] and oil extraction efficiency [18] can substantially contribute to the required cost reduction for economical oil production. The following sections provide an overview of current efforts to optimise TAG content through strain selection and engineering (Figure 1) and bioprocess improvements to effectively increase the TAG content and productivity of the existing production strains and systems, and ultimately improving the cost-effectiveness of oil production in microalgae. Optimization of TAG accumulation in microalgae requires a thorough understanding of lipid metabolism and TAG accumulation in eukaryotic phototrophic microalgae (Box 1). To realise this fully, particular aspects that are not well understood in regards to TAG biosynthesis, such as metabolic and genetic regulation (Box 2), subcellular location (Box 3), and biological function (Box 4), must be further investigated. Selecting a microalgal species with maximal TAG accumulation capacity Microalgae exhibit different capacities to accumulate TAG under adverse growth conditions [19]. Because most of the algae species available in nature remain unexplored today, additional screening for promising strains remains a worthwhile option. Interspecies differences in TAG productivity may be explained by a preference to accumulate other compounds competing for carbon and energy, such as starch [20]. Another important factor is the extent to which different species are able to maintain photosynthetic rates under conditions unfavourable for growth. Microalgae constantly adapt photosynthesis to meet metabolic demands [21], and when these metabolic demands diminish under nitrogen depletion, excess absorbed photons can be dissipated as heat Trends in Biotechnology xx (2014) 1–8

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Box 1. Basics of TAG accumulation in microalgae

3

2a Starch

CO2 C3 1 2a TAG

4

–N TRENDS in Biotechnology

Figure 1. Targets for strain improvement for increased oil production. (1) Increasing lipid biosynthesis capacity by overexpression and heterologous expression. (2) Disabling of competing pathways (a) and reduction of TAG catabolism (b). (3) Increasing photosynthetic efficiency under nitrogen deprivation. (4) Decoupling TAG accumulation and nitrogen deprivation.

or fluorescence [22]. Furthermore, algae severely reduce their photosynthetic capacity by decreasing pigmentation, photosynthetic membranes, photosynthetic proteins, and active photosystem reaction centres [23–25]. The degree to which photosynthetic capacity is decreased under nutrient limitation is species-specific and can heavily influence the amount of energy that is available for TAG synthesis. For example, it was shown that only 8.6% of the potential excess electrons generated ended up in TAG in Neochloris oleoabundans exposed to nitrogen limitation, whereas most energy was dissipated in the photosystems or used in catabolic processes [26]. We expect that choosing a species that retains a high photosynthetic capacity under nitrogen deprivation will be beneficial for TAG productivity. Differences in TAG accumulation rates could point towards differences in the function of TAG accumulation between microalgal species (Box 4). This species-specific function should be kept in mind when determining the optimal approach for metabolic engineering as well as the optimal cultivation strategy for maximised TAG production. For example, N. oleoabundans primarily dissipates energy under nitrogen-limited conditions and disintegrates its plastid to reduce its energy intake, whereas Nannochloropsis gaditana decreases its plastid membrane only to a small extent and is able to retain its chloroplast structure and photosynthetic activity for a relatively long period of nitrogen starvation. This results in almost similar initial N. gaditana biomass production rates for both nitrogen-sufficient and nitrogen-starved conditions [25]. Combined with the observation that most of the TAG in this species is produced de novo, TAG synthesis in N. gaditana seems to be employed predominantly to scavenge electrons and store 2

Microalgae produce a large variety of lipid-like compounds, such as waxes, sterols, hydrocarbons, and glycerolipids, of which the latter are the most abundant and best-described of the algal lipid classes. Glycerolipids are characterised by a glycerol backbone with one, two, or three fatty acyl groups attached, and can be divided into two large subclasses based on their specific function, being storage oils or membrane lipids [56]. Membrane lipids contain two fatty acyl groups and generally have a polar side-group at the sn-3 position of their glycerol backbone. These lipids are the essential building blocks for cell and organelle membranes. Storage lipids have three fatty acyl groups attached to glycerol backbone, and these lipids are known as triacylglycerols (TAG). Until recently our knowledge of oil biosynthesis in microalgae was largely based on the assumption that plant and microalgal lipid biosynthesis pathways are similar. However, as more physiological studies on microalgae are being published, differences with plant lipid biosynthesis become apparent. Examples of these are distinct fatty acyl groups and differences in the overall subcellular organization of glycerolipid metabolism [57]. More recent developments in understanding glycerolipid and TAG metabolism in microalgae are mainly generated using the accepted reference for microalgal metabolism, Chlamydomonas reinhardtii. This is because knowledge on this alga is ample, its genome sequence is available, it can be easily genetically transformed, and a range of different mutants are available, and these facilitate further study of its metabolism and physiology [29,58,59]. However, this alga predominantly accumulates starch as a storage metabolite and has a relatively stable lipid content [60]. Starchless mutants that do accumulate TAG provided an opportunity to study storage lipid biosynthesis in this species [61,62]. Whether this artificial induction of TAG accumulation in C. reinhardtii is fully representative of TAG production in microalgae that are oleaginous by nature remains a question. Moreover, the term ‘microalgae’ defines a polyphyletic group [63], which means that the regulation and localisation of TAG accumulation in a chlorophyte such as C. reinhardtii may be completely different from that of a heterokont such as Nannochloropsis.

carbon and energy efficiently. Adapting cultivation conditions towards maximising the energy imbalance might therefore be more beneficial in electron-scavenging N. gaditana than in energy-dissipating N. oleoabundans. Likewise, metabolic engineering approaches targeting the capacity of de novo TAG production pathways could appear unsuccessful in typical energy dissipaters because in these algae a low photosynthetic capacity might limit TAG accumulation. Strain improvement for optimised TAG production A more targeted approach would be to enhance the currently available production species using genetic engineering in such a way that more carbon and energy is directed towards TAG synthesis. Genetic engineering approaches have been effectively applied in several plant species, increasing seed oil contents or inducing oil synthesis in tissue that normally does not produce oil (reviewed in [27]). Genetic engineering of microalgae is currently in its infancy because reliable nuclear transformation systems, such as those that are currently used in plants, are only available to some degree for microalgae [28]. Nuclear transformation has proven successful in only a few species, including the model green algae Chlamydomonas reinhardtii [29], the oleaginous diatom Phaeodactylum tricornutum [30], and the robust TAG producer Nannochloropsis sp. [31]. The molecular toolboxes necessary for transformation of each of these species are very distinct, and

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Box 2. Metabolic and genetic regulation of lipid biosynthesis In heterotrophic oleaginous microorganisms adverse growth conditions result in inhibition of the citric acid cycle and subsequent conversion of citric acid to acetyl-CoA, the precursor for fatty acid synthesis. The enzyme responsible, ATP:citrate lyase (ACL), is regarded as a key enzyme for lipid accumulation in these microorganisms because the enzyme is not present in their non-oleaginous counterparts [64]. Also in plants, ACL is responsible for acetyl-CoA formation for fatty acid production in the plastid [65]. Although ACL is present in some microalgae, its function and subcellular location remain unclear, as is the origin of the acetyl-CoA necessary for lipid biosynthesis. Fatty acid synthesis in green organisms takes place in the plastid and is initiated by conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACCase) [66], a highly regulated enzyme and therefore essential in controlling the initiation of fatty acid biosynthesis [67]. Subsequently, the malonyl group is transferred to an acyl carrier protein (ACP) by malonyl-CoA:ACP transacylase (MCT), after which the multisubunit fatty acid synthase (FAS) complex extends malonyl-ACP by two carbon atoms in successive reaction cycles. When the fatty-acyl chain reaches its full length, usually C16:0–ACP or C18:0–ACP, it is removed from the ACP group and either hydrolysed by acyl-ACP esterase (AAE) to produce free fatty acids or transferred to glycerol-3-

Cytosol

Chloroplast

ACCase AcCoA

phosphate by glycerol-3-phosphate acyltransferase (GPAT) to produce lysophosphatidic acid (LysoPA). Addition of a subsequent acyl chain by LysoPA acyltransferase (LPAT) yields phosphatidic acid (PA), which is dephosphorylated by PA phosphatase (PAP). This yields diacylglycerol (DAG), the common precursor of all algal glycerolipids, to which a third group can be added to the vacant position to produce either TAG or membrane lipids (Figure I). TAG formation from DAG can take place via two routes. In the first route, the fatty acyl group is transferred from acyl-ACP by diacylglycerol acyltransferase (DGAT) in the final step of the Kennedy pathway. This pathway entails the three sequential acylations of glycerol-3-phosphate. Alternatively, fatty acyl donors for production of TAG are derived from membrane lipids in the acyl-CoA-independent pathway, either directly using phospholipid:diacylglycerol transferase (PDAT), or by means of specific lipase activity on membrane lipids, which liberates fatty acyl groups for TAG synthesis [68]. For further reading on genes involved in lipid biosynthesis in microalgae we refer to [69]. Excellent reviews on the biosynthesis of long-chain and unsaturated fatty acids, the specificity of the acyltransferases, and targeted engineering of the fatty acid profile can be found in [70,71].

ER

MalCoA MCT MalACP

FAS complex GPAT LPAT PAP PA DGAT?

LCAS

Free FA

AAE

AcyIACP

Prokaryoc pathway

GLYC3P

LysoPA Membrane lipids

AcylCoA GPAT

LPAT?

GGL

DAG

PAP PA DGAT

DAG

PDAT TAG

GLYC3P

LysoPA

TAG PDAT

Eukaryoc pathway

Membrane lipids TRENDS in Biotechnology

Figure I. Schematic representation of lipid biosynthesis in microalgae. All enzymes are indicated in gray and their substrates in black. Broken lines and the use of ‘?’ indicate uncertainty about the occurrence of these enzymes in a specific organelle. Abbreviation: ER, endoplasmic reticulum. Enzyme abbreviations: ACCase, acetyl-CoA carboxylase; AAE, acyl-ACP esterase; DGAT, diacylglycerol acyltransferase; FAS complex, fatty acid synthase complex; GPAT, glycerol-3-phosphate acyltransferase; LCAS, long-chain acyl-CoA synthase; LPAT, lysophosphatidic acid acyltransferase; MCT, malonyl-CoA:ACP transacylase; PAP, phosphatidic acid phosphatase; PDAT, phospholipid:diacylglycerol transferase. Compound abbreviations: AcCoA, acetyl-CoA; AcylACP, acyl-ACP; acylCoA, Acyl-CoA; DAG, diacylglycerol; FA, fatty acid; Glyc3P, glycerol-3-phosphate; MalACP, malonyl-ACP; MalCoA, malonyl-CoA; LysoPA, lysophosphatidic acid; PA, phosphatidic acid; TAG, triacylglycerol.

therefore it is expected, despite a growing number of successful transformation techniques [32], that it will take considerable additional research to develop the proper techniques for transforming other promising algal species. Outdoor utilisation of strains improved by genetic modification requires additional considerations with respect to contained use. Effective biological containment can be achieved in closed photobioreactors, especially when using a strain that cannot survive outside the cultivation environment. In most countries, however, with the exception of the USA, there are no specific regulations that address the outdoor cultivation of genetically modified microalgae.

This illustrates the need to define regulations proactively before large-scale cultivation of GM microalgae becomes a reality [33]. Increasing lipid biosynthesis capacity by overexpression and heterologous expression Because the techniques for altering metabolic pathways in microalgae are limited, examples of targeted overexpression and heterologous expression of genes involved in lipid biosynthesis are sparse. Successful manipulation of lipid biosynthesis gene expression have shown mixed results (Table 1), which might be caused by differences in regulation 3

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Review Box 3. The subcellular location of lipid biosynthesis Currently two locations for de novo lipid biosynthesis are assigned (Box 2 Figure I), being the plastid (prokaryotic pathway) and the endoplasmic reticulum (ER) (eukaryotic pathway). The free fatty acids feeding the latter pathway are transported over the plastid and ER membranes and subsequently attached to CoA by long-chain acyl-CoA synthase (LCAS). From there, the same sequence of reactions is followed as in the plastid, using acyl-CoA as a substrate instead of acyl-ACP. In plants, the location of synthesis of a glycerolipid can be determined based on the fatty acyl group occupying the sn-2 position because plastidic and cytosolic LPAT enzymes show distinct substrate specificities. A 16-carbon acyl group at the sn-2 position indicates a plastidic origin, whereas an 18-carbon acyl group at this position only occurs in ER-derived glycerolipids [72]. Whether this specific signature is also maintained in microalgae cannot be confirmed because extraplastidic LPAT is absent in most algae and the reaction is possibly catalysed by a divergent protein, or by a GPAT with a broader substrate specificity [62]. This makes the localisation of lipid biosynthesis in microalgae difficult. Assuming the same signature for plastid-derived lipids in microalgae as in plants, Giroud et al. [73] found that plastidic membrane lipids in Chlamydomonas reinhardtii are exclusively of plastidic origin, whereas the extraplastidic lipids are solely synthesised in the ER. This would suggest that microalgae maintain a strict compartmentalisation of membrane lipid synthesis, whereas in plants each individual pathway (prokaryotic and eukaryotic) provides lipids for both the plastidic and extraplastidic membranes [74]. TAG biosynthesis in plants takes place exclusively outside the plastid [75]. In microalgae, this view was initially adopted and confirmed for C. reinhardtii because the six identified DGATencoding genes lack a plastid-targeting sequence [57]. By contrast, many LBs are found to be physically connected or merged with the plastid membrane [69,76], or are even present inside the plastid [77], which suggests the plastid as an alternative location for TAG synthesis. If the signature for plastid-derived lipids in algae is the same as it is in plants, the existence of a chloroplast pathway for TAG synthesis in microalgae is very likely because approximately 90% of the TAG produced in C. reinhardtii carried the signature of plastid-derived DAG [78].

(Box 2), subcellular location (Box 3), and function (Box 4) of TAG accumulation in these species. The overexpression of native cytosolic acetyl-CoA carboxylase (ACCase) in Cyclotella cryptica and Navicula saprophila did not affect TAG levels [34], which demonstrates that in these species ACCase activity is not a rate limiting step in TAG biosynthesis. It is very likely that homeostatic regulation, such as redox activation of ACCase under nitrogen stress [35], already ensures a sufficient production of de novo fatty acyl groups. Overexpression of acyl-ACP (acyl carrier protein) esterase (AAE), the enzyme responsible for termination of chain elongation during fatty acid biosynthesis, led to changes in fatty acid profiles in P. tricornutum [36] and C. reinhardtii [37], and increased lipid content only in P. tricornutum. Overexpression of a type 2 diacylglycerol acyltransferase (DGAT) in P. tricornutum increased TAG levels by 35% [38], and also in C. reinhardtii overexpression of some (but not all) type 2 DGAT enzymes triggered an increase in TAG content upon targeted overexpression [39,40]. Heterologous expression of yeast-derived LPAT (lysophosphatidic acid acyltransferase), PAP (phosphatidic acid phosphatase), GPAT (glycerol-3-phosphate acyltransferase), or DGAT (diacylglycerol acyltransferase), and also of G3PDH (glycerol-3-phosphate-dehydrogenase), the 4

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Box 4. Hypotheses for the function of TAG synthesis under adverse growth conditions TAG synthesis for carbon and/or energy storage In many organisms, including microalgae, TAG accumulation is recognised as a means for storage of carbon and energy [79]. When cell proliferation is limited by adverse growth conditions, TAG synthesis allows continuous capturing of energy and carbon which can be employed when favourable growth conditions are restored. TAG synthesis functions as an electron scavenger under adverse growth conditions Under adverse growth conditions microalgae are confronted with an excess of NADPH that, under normal conditions, would be employed for cell proliferation. Under these adverse circumstances, dangerous over-reduction of the photosynthetic electron transport chain is prevented by accumulation of highly reduced compounds such as TAG [4,69]. This hypothesis is supported by the fact that the first committed enzyme in de novo TAG synthesis, acetyl-CoA carboxylase (ACCase), is under redox regulatory control [35]. Moreover, it was shown that Chlamydomonas reinhardtii TAGdeficient mutants were unable to lower the reductive state of the photosynthetic electron transport chain and were less viable under nitrogen-depleted conditions than the wild type [68]. In addition, TAG synthesis in Neochloris oleoabundans correlated with exposure to an electron imbalance that was created by limiting anabolic rates while maintaining a constant light-absorption rate [53]. TAG-filled lipid bodies (LBs) as a temporary plastid component depository TAG accumulation serves as a means to store redundant plastid membranes temporarily when chloroplast size and structure are severely reduced under adverse growth conditions [23,24]. Many observations localise TAG synthesis in proximity to or inside the plastid [69,76,77], and it has been demonstrated that plastid membrane galactoglycerolipids are an important donor of fatty acids for TAG synthesis in C. reinhardtii [68]. Likewise, TAG-derived fatty acids are used in the production of chloroplast lipids in Parietochloris incisa when growth is restored [80]. It is likely that microalgal LBs have a much more dynamic and complex function. Unbound chlorophyll was discovered in the LBs of nitrogen-starved N. oleoabundans [81], suggesting that these organelles serve for storage of redundant photosynthetic pigments. The discovery of ‘refugee proteins’, which have no obvious connection to TAG metabolism or trafficking, in the LBs of several eukaryote cells suggests that LBs also function as a dynamic protein deposit [82,83]. Although the few published proteome studies of algal LBs have mostly revealed proteins involved in LB synthesis [84] and oil metabolism [85], the latter work revealed some stromal or thylakoid-associated proteins in the isolated LBs, although it remains unsure whether these proteins were contaminants in the isolated fraction.

enzyme responsible for producing the glycerol backbone of TAG, showed that overexpression of single genes had limited effect on the TAG content of Chlorella minutissima, whereas a construct that contained all five genes yielded a twofold increase in TAG content in this species [41]. Heterologous expression of D5-elongase and D6-elongase from the picoalga Ostreococcus tauri, resulted in an elevated content of DHA in TAGs of transgenic P. tricornutum [42]. In plants, increasing the oil content and/or lipid accumulation rate by targeting the enzymes responsible for lipid formation have also shown mixed results. In general, overexpression of more upstream enzymes, such as ACCase and those associated with the fatty acid synthase (FAS) complex, resulted only in marginal increases of seed oil content, whereas targeting some of the Kennedy pathway enzymes led to a substantial increase in seed oil, as extensively reviewed by Radakovits et al. [32] and Yu et al.

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Table 1. Overexpression and heterologous expression of genes involved in TAG biosynthesis in microalgae gives mixed resultsa Microalga Cyclotella cryptica Navicula saprophila Phaeodactylum tricornutum

Overexpression ACCase ACCase AAE

Chlamydomonas reinhardtii P. tricornutum C. reinhardtii C. reinhardtii Chlorella minutissima

AAE DGAT2 DGAT2-1; DGAT2-5 DGAT2-a,b,c Yeast-derived G3PDH, GPAT, LPAT, PAP, DGAT D5 Elo; D6 Elo

P. tricornutum

Observation No effect No effect Changed fatty acid profile and twofold increase in cellular lipid content, possibly due to decrease in growth rate Changed fatty acid profile Increase in neutral lipid content of 35%, increase in EPA of 76.2% Increase in lipid content of 20% and 44%, respectively, relative to wild type No effect Quintuple gene construct showed a twofold increase in TAG content

Refs [34] [34] [36]

Changed fatty acid profile with an eightfold increase in DHA, DHA accumulation in TAG fraction

[42]

[37] [38] [39] [40] [41]

a

Enzyme abbreviations: ACCase, acetyl-CoA carboxylase; AAE, acyl-ACP esterase; DGAT, diacylglycerol acyltransferase; D5 Elo, D5-elongase; D6 Elo, D6-elongase; G3PDH, glycerol-3-phosphate-dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase; LPAT, lysophosphatidic acid acyltransferase; PAP, phosphatidic acid phosphatase.

[27]. The most noticeable increase, however, was obtained in rapeseed, where overexpression of a yeast G3PDH led to a 40% increase in seed oil content [43]. Algal and plant lipid biosynthesis pathways exhibit some significant differences (Boxes 1 and 2), and the regulation of TAG synthesis in committed oil-accumulating organs might be profoundly different than in single cell microalgae. Therefore, we expect that the results obtained in plants cannot be directly translated to microalgae. More insight in transcriptional and (post)translational regulation of the algal lipid biosynthetic pathways is necessary, and the integration of disciplines, such as detailed pathway analysis by metabolic flux models [44] combined with gene expression analysis and proteomics techniques [45], will play an important role when identifying the future engineering targets for increasing TAG production rates in microalgae. Disabling of competing pathways and reduction of TAG catabolism A very successful approach in directing more carbon towards TAG biosynthesis has proved to be knocking down competing pathways. The best-known examples are the starch mutants that have been developed for C. reinhardtii [20] and, more recently, Scenedesmus obliquus [46,47], for which the TAG levels obtained under nitrogen starvation are significantly higher than in the wild type. Another pathway that is possibly worthwhile to knock down is lipid catabolism, which potentially decreases TAG losses due to turnover or respiration. This approach already proved successful in the diatom Thalassiosira pseudonana in which a targeted knockdown of a multifunctional lipase/ phospholipase/acyltransferase resulted in a fourfold higher TAG level under silicon starvation [48]. Moreover, the same knockdown also resulted in a threefold increase in TAG content during exponential growth. Increasing photosynthetic efficiency under nitrogen deprivation The loss in photosynthetic capacity under nitrogen limitation generally goes hand in hand with a loss in TAG productivity. When linear electron transport is gradually reduced [25], rates of carbon fixation also subside, and preventing this reduction in linear electron transport

might thus prove an efficient strategy for increasing TAG levels. For example, C. reinhardtii mutants unable to form violaxanthin from zeaxanthin, a conversion which normally plays an important role in thermal dissipation of excess energy in the wild type, showed less photosystem PSII inactivation and therefore higher linear electron transport rates under high-light conditions [49]. Such a mutation might provide increased linear electron transport rates under nitrogen starvation as well, and possibly also higher TAG accumulation rates. Decoupling TAG accumulation and nitrogen deprivation Although the regulating mechanisms behind TAG accumulation in microalgae remain obscure, it is very likely that the cause of this phenomenon is an imbalance between received energy and metabolic energy demands. Insight into how this energy imbalance is sensed under nitrogen starvation, and subsequently is translated into increased TAG synthesis rates, might allow the uncoupling of TAG synthesis from the use of adverse growth conditions and the resulting decline in photosynthetic capacity. Targeting regulators for TAG accumulation, such as the nitrogen response regulator identified in C. reinhardtii [50], could be the next step towards highly efficient TAG formation under nitrogen-replete conditions, where more energy can be channelled towards the product of interest. Cultivation strategies for optimised TAG production TAG synthesis rates can be increased by several cultivation strategies, of which nitrogen shortage is the most effective [4]. Two conditions with respect to nitrogen shortage can be distinguished: nitrogen limitation (generally a continuous cultivation mode), and nitrogen starvation (generally a batch process) [51] (Box 5). When reflecting upon the available data in the scientific literature, starvation processes yield the highest average TAG production rates and final TAG contents, provided that these cultures are harvested at the right time [52,53]. Surprisingly, a proper comparison between batch starvation and continuous limitation strategies using the same species, set-up, and light conditions under laboratory conditions is still lacking. This type of comparison is highly necessary to develop the proper cultivation strategies for outdoor, large-scale TAG production. 5

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Box 5. TAG production strategies Nitrogen starvation: can be considered as the classic approach to TAG production. The vast majority of scientific publications on TAG accumulation in microalgae uses this technique. The process is characterised by a biomass production phase followed by a starvation phase in which nitrogen is absent from, and not supplied to, the culture medium. In this latter stage the production of functional biomass stops and TAG is accumulated. After prolonged exposure to nitrogen-starved conditions cells stop accumulating TAG, start

degrading it, and eventually die. Therefore, nitrogen starvation is a finite, batch process (Figure IA). Nitrogen limitation: the situation where nitrogen is supplied to the culture in such a way that the production rate of functional biomass is limited by the rate at which nitrogen can be consumed. Light energy is thus supplied in excess, creating an energy imbalance, and TAG is accumulated while cell proliferation and division continue. Therefore, nitrogen limitation can be applied in a continuous process (Figure IB).

(A) Batch (N starvaon)

C

C

High TAG conte ent, produccvity and yield

N

Easy to op perate

Down-me Sep SSe ep e arate growth phase Limit i d control over outdoor ite it TAG accumulaon rates Flexible contrrol over outtdoor TAG accumulaon rates

(B) Connuous (N limitaon)

C

Custom mised biomaass composion

N

Lo er TAG content, producvity Low and yield, although not yet opmised Complex process TRENDS in Biotechnology

Figure I. (A) Batch cultivation with a growth phase followed by a nitrogen starvation phase. (B) Continuous cultivation with nitrogen limitation in a continuously operated system. Each has advantages (+) and disadvantages ( ) for commercial TAG production.

Besides yielding higher average TAG production rates and contents, batch systems are easy to operate and are therefore most often applied. As a result, the more complex approach of continuous nitrogen-limited cultivation remains relatively unexplored [53]. Often disregarded is that continuous cultivation could offer an important advantage in terms of operation flexibility under varying outdoor conditions. Because continuous cultivation implies the controlled dilution of a system, biomass concentrations and nutrient feed rates could be easily varied throughout the day in accordance with the received irradiation to create the optimal conditions for TAG accumulation. The advantage of this flexibility under continuous outdoor operation was already demonstrated for the production of astaxanthin, which is also accumulated under nitrogen shortage [54]. Another advantage of continuous operation over batch operation is that by adjusting cultivation conditions tailor-made biomass compositions could be obtained [26]. Biomass composition can even be varied during production, which increases the opportunities to make value of all cell components [16,55]. For these reasons, continuous cultivation for the production of microalgal oil on a commercial scale deserves further exploration. 6

Concluding remarks and future perspectives Increasing TAG productivity and TAG content in microalgae are important targets for making microalgal oil a cost-effective and sustainable alternative to common vegetable oils. Further optimisation requires proper understanding of the physiology and regulation of lipid metabolism in microalgae (Box 6). Important knowledge gaps are the compartmentalisation of TAG synthesis, the regulation of TAG accumulation, and the relation between TAG metabolism and photosynthesis. Filling these knowledge gaps is necessary to indicate potential bottlenecks in TAG production and targets for metabolic engineering.

Box 6. Outstanding questions  Which strain, next to Chlamydomonas reinhardtii, would be a good model to study TAG biosynthesis in oleaginous microalgae?  What is the subcellular location of TAG biosynthesis?  What is the biological function of TAG biosynthesis?  How is TAG synthesis metabolically and genetically regulated?  Which enzymes limit the TAG biosynthesis flux and thereby provide interesting targets for strain improvement?  Which cultivation strategy is the best-suited for TAG production in microalgae under large-scale, outdoor conditions?

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