Yonghua Li-Beisson
email: yonghua.li@cea.fr

Jay J Thelen
email: thelenj@missouri.edu

Eric Fedosejevs
email: fedosejevse@missouri.edu

John L Harwood
email: harwood@cardiff.ac.uk

Dr Kent Chapman

The lipid biochemistry of eukaryotic algae

Keywords: Algal lipid metabolism, Acetyl-CoA carboxylase, β-oxidation, Mitochondrial respiration, Reducing equivalents, Triacylglycerols, Environmental effects, Commercial exploitation

Algal lipid metabolism fascinates both scientists and entrepreneurs due to the large diversity of fatty acyl structures that algae produce. Algae have therefore long been studied as sources of genes for novel fatty acids; and, due to their superior biomass productivity, algae are also considered a potential feedstock for biofuels. However, a major issue in a commercially viable "algal oil-to-biofuel" industry is the high production cost, because most algal species only produce large amounts of oils after being exposed to stress conditions. Recent studies have therefore focused on the identification of factors involved in TAG metabolism, on the subcellular organization of lipid pathways, and on interactions between organelles. This has been accompanied by the development of genetic/genomic and synthetic biological tools not only for the reference green alga Chlamydomonas reinhardtii but also for Nannochloropsis spp.

and Phaeodactylum tricornutum. Advances in our understanding of enzymes and regulatory proteins of acyl lipid biosynthesis and turnover are described herein with a focus on carbon and energetic aspects. We also summarize how changes in environmental factors can impact lipid metabolism and describe present and potential industrial uses of algal lipids.

Introduction

Algae, sensu lato, are a large, diverse, and polyphyletic group of photosynthetic organisms.

They range from unicellular microalgae (including Ostreococcus, the smallest known freeliving eukaryote [START_REF] Courties | Smallest eukaryotic organism[END_REF]) to the giant kelp, which can reach 45 m in length [START_REF] Steneck | Kelp forest ecosystems: biodiversity, stability, resilience and future[END_REF]. Most scientists no longer consider prokaryotes, such as cyanobacteria, amongst the algae, but it is from prokaryotes that algal plastids are derived [START_REF] Keeling | Diversity and evolutionary history of plastids and their hosts[END_REF]. While green and red algae contain primary chloroplasts of endosymbiotic origin, heterokont algae (diatoms and brown algae, or more properly Phaeophyceae) contain secondary plastids most commonly derived from endosymbiotic red algae [START_REF] Palmer | The plant tree of life: an overview and some points of view[END_REF]. The three-membraned secondary plastids of the Euglenophyceae and the four-membraned secondary plastids of the Chlorarachniophyta are exceptions however, being derived from endosymbiotic green algae. Algae have a range of reproductive strategies and, as implied earlier, can be unicellular organisms or possess complex multicellularity [START_REF] Brodie | The algal revolution[END_REF].

Although there is as yet no accurate tally of the total number of algal species, a recent estimate is that there are 72,500 species in the biosphere [START_REF] Guiry | How many species of algae are there?[END_REF].

As befits their diversity, algae can use sunlight for photosynthesis or can exist as mixotrophs or facultative heterotrophs. Some of the latter have lost their ability to photosynthesize and have become obligate heterotrophic parasites, such as Plasmodium and Toxoplasma [START_REF] Brodie | The algal revolution[END_REF]. There are also those species of algae that form important symbiotic relationships with other organisms such as in coral reefs [START_REF] Taylor | The coral-algal symbiosis[END_REF], lichens [START_REF] Pearson | The diversity and evolution of plants[END_REF] and sea sponges. The complexity of algae is manifested in the origins and functions of algal genes [START_REF] Brodie | The algal revolution[END_REF] as well as in their lipid biochemistry [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF].

Algae are prominent in bodies of water (both freshwater and marine) but are also found in unusual environments such as snow and ice or hot springs. In most cases they are at the base of food chains and provide core ecosystem functions such as supplying half the oxygen we breathe [START_REF] Gualtieri | Algae: anatomy, biochemistry and biotechnology[END_REF]. In high densities, such as algal blooms, algae can outcompete other life forms and cause a health hazard. In other situations, algae can act as indicator organisms to monitor pollution [START_REF] Omar | Perspectives on the use of algae as biological indicators for monitoring and protecting aquatic environments, with special reference to malaysian freshwater ecosystems[END_REF].

Algae have been exploited by humans for hundreds of years and are currently used to produce agar and other alginates, fertilizers, nutritional products and pigments, in addition to their use in bioremediation. With our increasing knowledge of algal genomes and availability of algal transcriptomes [START_REF] Keeling | The marine microbial eukaryote transcriptome sequencing project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing[END_REF], there are more opportunities to exploit algae for biotechnological purposes such as for biofuels, nutraceuticals, and pharmaceuticals [START_REF] Brodie | The algal revolution[END_REF]. Opportunities related to lipids are discussed in section 7.

For background information on algal lipid biochemistry, please refer to [START_REF] Hitchcock | Plant lipid biochemistry: the biochemistry of fatty acids and acyl lipids with particular reference to higher plants and algae[END_REF][START_REF] Harwood | Lipid metabolism in algae[END_REF][START_REF] Pohl | Fatty acids and lipids of marine algae and the control of their biosynthesis by environmental factors, marine algae in pharmaceutical science[END_REF][START_REF] Nakamura | Lipids in plant and algae development[END_REF]. In this review we will concentrate on literature following the review by Guschina and Harwood [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF].

As a special note, the term "lipid" is used in a more strict sense in this review, i.e. refers mainly to "glycerolipid".

Lipids in algae

The major lipid classes in algae are the membrane lipids (glycosylglycerides, phosphoglycerides, betaine ether lipids) and the storage lipids (in the form of triacylglycerol) [START_REF] Li-Beisson | Lipids: from chemical structures, biosynthesis, and analyses to industrial applications[END_REF]. Algae also possess small amounts of other lipid classes such as terpenoids, sphingolipids, hydrocarbons, sterols and, of course, pigments that are present in different percentages depending on the class of alga.

There are a number of 'unusual' compounds which have been detected in a limited number of species. No doubt many more will be found. For example, phosphatidylsulphocholine (the sulphonium analogue of phosphatidylcholine) has been identified in diatoms and Euglena [START_REF] Harwood | Lipid metabolism in algae[END_REF], halogenated fatty acids (FAs) and their derivatives [START_REF] Dembitsky | Natural halogenated fatty acids: their analogues and derivatives[END_REF] in various algae and novel hydrocarbons in Botryococcus braunii [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF].

Table 1 shows the acyl lipid composition of a variety of algae. In keeping with the diverse structure of different algae, the quantitative and qualitative compositions of lipids varies considerably. While the three glycosylglycerides (monogalactosyldiacylglycerol, MGDG; digalactosyldiacylglycerol, DGDG; sulphoquinovosyldiacyglycerol, SQDG) are major components, their % contributions are distinct. In general there is more MGDG than DGDG, as in higher plants [START_REF] Li-Beisson | Lipids: from chemical structures, biosynthesis, and analyses to industrial applications[END_REF] but, in contrast to the latter, SQDG is often a major constituent of algae.

Although there has been little effort to examine the subcellular distribution of SQDG, one presumes that in those algae with a high content, it is not just localized to thylakoids (unlike in higher plants). As in land plants, the MGDG of algae tends to contain a higher proportion of polyunsaturated fatty acids (PUFA) than DGDG. Both galactolipids are more unsaturated than SQDG [START_REF] Kalisch | DGDG and glycolipids in plants and algae[END_REF]. There is an acylated derivative of SQDG, 2'-O-acylsulphoquinovosyldiacylglycerol, which is found in algae of both primary and secondary endosymbionts, such as Chlamydomonas reinhardtii [START_REF] Riekhof | The sulfolipids 2′-O-Acylsulfoquinovosyldiacylglycerol and sulfoquinovosyldiacylglycerol are absent from a Chlamydomonas reinhardtii mutant deleted in SQD1[END_REF] and Phaeodactylum tricornutum [START_REF] Abida | Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum[END_REF]. There has been considerable interest in analyzing the molecular species of the glycosylglycerides, especially MGDG. This interest is in relation to the so-called 'prokaryotic' and 'eukaryotic' pathways of acyl lipid synthesis (see [START_REF] Gurr | Lipid biochemistry[END_REF]) and is discussed in section 4.

However, it is also relevant to a study in diatoms where MGDG and DGDG molecular species were compared in two centric species (Skeletonema marinoi, Thalassiosira weissflogii) with pennate species (Phaeodactylum tricornutum, Haslea ostrearia, Navicula perminuta) [START_REF] Dodson | Mono-and digalactosyldiacylglycerol composition of the marennine-producing diatom, Haslea ostrearia: Comparison to a selection of pennate and centric diatoms[END_REF].

Although monoacyl-glycosylglycerides have been reported in algae [START_REF] Logvinov | Examination of the structures of several glycerolipids from marine macroalgae by NMR and GC-MS[END_REF], artifactual formation during extraction is possible if careful precautions to inhibit any endogenous lipases are not taken.

Several betaine lipids are important components of algae. DGTS (diacylglyceryl-O-(N,N,N-trimethyl)-homoserine is the most common in nature and is found in green algae (Table 1). DGTA (1,2-diacylglyceryl-3-O-2'(hydroxymethyl)-(N,N,N-trimethyl)-beta-alanine) is typically found in many brown algae. The third betaine lipid is DGCC (diacylglycerylcarboxylhydroxymethylcholine) and was first discovered in the marine genus Haptophyceae, such as in Pavlova luthera. The distribution of these betaine lipids in many different species of algae have been reported [START_REF] Eichenberger | Betain lipids in lower plants -distribution of DGTS, DGTA and phospholipids, and the intracellular-localization and site of biosynthesis of DGTS[END_REF][START_REF] Kato | Distribution of betaine lipids in marine algae[END_REF][START_REF] Kunzler | Betaine lipids and zwitterionic phospholipids in plants and fungi[END_REF]. A recent evaluation of the occurrence and molecular diversity of betaine lipids in marine microalgae has been published [START_REF] Canavate | Exploring occurrence and molecular diversity of betaine lipids across taxonomy of marine microalgae[END_REF].

With a few exceptions, the amount of phosphoglycerides in algae is much less than that of the glycosylglycerides (Table 1). All the usual phosphoglycerides are found even in minor amounts but phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn) and phosphatidylglycerol (PtdGro) are the main ones. Even in brown algae there is a wide variation in the percentages of different phosphoglycerides [START_REF] Vyssotski | Phospholipids of New Zealand edible brown algae[END_REF]. Phosphatidyl-O-N-(2hydroxyethyl)glycine (PHEG: preciously called N-CAPE), a ceramidephosphoinositol and an arsenic-containing phospholipid were also detected in a variety of brown algal species [START_REF] Vyssotski | Phospholipids of New Zealand edible brown algae[END_REF].

Because of the current interest in algae as sources of particular FAs or in the use of their accumulated triacylglycerol (TAG) for biofuel (section 7), there has been much research on evaluating TAG by mass spectrometry [START_REF] Danielewicz | Triacylglycerol profiling of marine microalgae by mass spectrometry[END_REF]. Such research has revealed the evolutionary divergence of the main TAG synthesis pathways in green microalgae [START_REF] Allen | Triglyceride quantification by catalytic saturation and LC-MS/MS reveals an evolutionary divergence in regioisometry among green microalgae[END_REF]. Since TAG is the main lipid accumulated, it may be necessary to rapidly screen many species (or lines) in order to pinpoint those which could be usefully considered by industry. This has led to evaluation of FAs as biomarkers or 'characteristic' components for the quantitation of TAG in algae [START_REF] Liu | Lipid metabolism in microalgae distinguishes itself[END_REF][START_REF] Wang | Identification of a putative patatin-like phospholipase domain-containing protein 3 (PNPLA3) ortholog involved in lipid metabolism in microalga Phaeodactylum tricornutum[END_REF][START_REF] Yang | An extended approach to quantify triacylglycerol in microalgae by characteristic fatty acids[END_REF][START_REF] Shen | Identification of characteristic fatty acids to quantify triacylglycerols in microalgae[END_REF].

Remarks about the overall FA composition of algae as well as their location in different lipids have been summarized in [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF][START_REF] Harwood | Lipid metabolism in algae[END_REF][START_REF] Pohl | Fatty acids and lipids of marine algae and the control of their biosynthesis by environmental factors, marine algae in pharmaceutical science[END_REF]. Specific comments in relation to single cell oils are in [START_REF] Ratledge | Single Cell Oils : Microbial and algal oils[END_REF]. A recent important and informative survey by Lang et al [START_REF] Lang | Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection[END_REF] has examined the stationary phase compositions of algae within the SAG culture collection. A selection of their analyses are shown in Table 2. What is immediately apparent is that the FA compositions of different species vary widely, as noted before [START_REF] Harwood | Lipid metabolism in algae[END_REF]. Moreover, even within the same class, there is no very consistent patternfor example, palmitate concentrations vary widely in Haptophyceae while linoleate concentration varies widely in Conjugatophyceae (Table 2). A recent review of diatoms, as the most abundant phytoplankton species, has noted that they tend to have 14:0, 16:0. 16:1 and 20:5 as their main FAs [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF]. The presence of 14:0 and a low amount of 18C acids is rather characteristic, as can be seen from the diatom representative, Phaeodactylum tricornutum in Table 2. For the commercially-important very long chain PUFAs (VLC-PUFAs), such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, only certain algal classes are productive (see section 7). Such FAs also tend to be more prevalent in marine or salt-tolerant algae rather than, for example, freshwater green algae (Table 3). Moreover, marine algae are also sometimes notable for their high concentrations of arachidonic acid (ARA) as well as EPA (Table 4). It should also be noted that different lipid classes will almost invariably have distinct FA compositions as mentioned above and discussed further in [START_REF] Harwood | Lipid metabolism in algae[END_REF][START_REF] Kalisch | DGDG and glycolipids in plants and algae[END_REF][START_REF] Jouhet | LC-MS/MS versus TLC plus GC methods: Consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation[END_REF]. Furthermore, growth conditions, seasonal variations and developmental stages will all play a role in influencing the FA contents of algae and their individual lipid classes [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF][START_REF] Harwood | Lipid metabolism in algae[END_REF][START_REF] Pohl | Fatty acids and lipids of marine algae and the control of their biosynthesis by environmental factors, marine algae in pharmaceutical science[END_REF][START_REF] Kendel | Non-methylene interrupted and hydroxy fatty acids in polar lipids of the alga Grateloupia turuturu over the four seasons[END_REF].

Evaluation of methodology for the extraction of lipids (and FAs) [START_REF] Kumari | Comparative evaluation and selection of a method for lipid and fatty acid extraction from macroalgae[END_REF][START_REF] Zheng | Purification of extracted fatty acids from the microalgae spirulina[END_REF][START_REF] Wang | Characterization of lipid components in two microalgae for biofuel application[END_REF] and, especially, in their further analysis have continued to be active areas of research. Special attention has been paid to the increasing use of mass spectrometry (MS) [START_REF] Vieler | The lipid composition of the unicellular green alga Chlamydomonas reinhardtii and the diatom Cyclotella meneghiniana investigated by MALDI-TOF MS and TLC[END_REF] which, of course, is sensitive and can provide information about molecular species and confirmation of identities. Nevertheless, some inherent problems with quantification using MS should be considered [START_REF] Han | Lipid analysis[END_REF]. The use of MS methods versus the more traditional TLC plus GC techniques have been compared for two microalgae (and Arabidopsis) by [START_REF] Jouhet | LC-MS/MS versus TLC plus GC methods: Consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation[END_REF]. They point out the difficulties of using MS for quantification but suggest a way of reducing the possible bias of MS data by using an external standard.

De novo FA synthesis in the chloroplast

The pathway and organization of de novo FA synthesis in algae is mostly inferred from that of plants wherein the steps and regulatory mechanisms of lipid synthesis have been better

characterized [START_REF] Li-Beisson | Acyl-lipid metabolism[END_REF]. The first in silico analysis of the genes encoding proteins of FA synthesis in algae was carried out for the model green alga Chlamydomonas reinhardtii in 2005, which allowed a reconstruction of the FA synthetic pathway [START_REF] Riekhof | Annotation of genes involved in glycerolipid biosynthesis in Chlamydomonas reinhardtii: Discovery of the betaine lipid synthase BTA1(Cr)[END_REF][START_REF] Moellering | Molecular genetics of lipid metabolism in the model green alga Chlamydomonas reinhardtii[END_REF]. Later on, with the advent of affordable genome sequencing and high sensitive RNAseq technologies, many more algal genomes have been sequenced and subjected to in silico analyses of metabolic pathways. Up to the time of this writing, ~30 algal genomes have been sequenced and annotated, allowing for bioinformatic analyses of algal lipid metabolism and a scaffold for synthetic biological studies.

In all known eukaryotic species with a chloroplast (derived either from primary or secondary endosymbiosis), de novo FA synthesis is known to occur in the stroma. For example, genome and expressed sequence tags (ESTs) analyses of some algal species from diverse evolutionary origins, including the diatom Phaeodactylum tricornutum, the eustigmatophytes Nannochloropsis sp., the red alga Galdieria sulphuraria and Cyanidioschyzon merolae [START_REF] Khozin-Goldberg | Unraveling algal lipid metabolism: Recent advances in gene identification[END_REF][START_REF] Khozin-Goldberg | LC-PUFA from photosynthetic microalgae: occurrence, biosynthesis, and prospects in biotechnology[END_REF][START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids[END_REF][START_REF] Banerjee | Metabolic pathways for lipid synthesis under nitrogen stress in Chlamydomonas and Nannochloropsis[END_REF][START_REF] Vieler | Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779[END_REF][START_REF] Sato | Lipid metabolism and potentials of biofuel and high added-value oil production in red algae[END_REF][START_REF] Sayanova | Modulation of lipid biosynthesis by stress in diatoms[END_REF],

revealed that FA synthesis is likely similar to that of the green lineage namely green algae (Chlorophyta) and higher plants (Embryophyta) [START_REF] Liu | Lipid metabolism in microalgae distinguishes itself[END_REF][START_REF] Li-Beisson | Acyl-lipid metabolism[END_REF][START_REF] Merchant | You're it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation[END_REF][START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF]. A simplified scheme for de novo FA synthesis, highlighting sources for carbon, ATP, and reducing equivalents is outlined in Figure 1.

Reactions and enzymes of FA synthesis

Acetyl-CoA carboxylase (ACCase)

The first committed step for de novo FA synthesis is the ATP-dependent carboxylation of acetyl-CoA to produce malonyl-CoA; a two-step reaction catalyzed by the biotin-containing enzyme ACCase. In nature, ACCase occurs in two forms: one is a heteromeric, multisubunit complex containing four different polypeptides including biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP) and α-and β-carboxyltransferase (α-and β-CT); while the other is a homomeric form wherein each of the four aforementioned components are fused in tandem on a single, large polypeptide. The subcellular location of ACCase varies between organisms. Animal ACCase, which is of the homomeric form, is present in both the cytoplasm and mitochondria, while plant and algal ACCase, which is of both homomeric and heteromeric forms, is located in the cytoplasm and plastid [START_REF] Abu-Elheiga | The subcellular localization of acetyl-CoA carboxylase 2[END_REF][START_REF] Hu | Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances[END_REF][START_REF] Ohlrogge | Lipid biosynthesis[END_REF]. The heteromeric form of ACCase, present in all plants except graminaceous monocots, is plastid-localized, while the homomeric form is either cytosolic or plastid-localized, depending on the species [START_REF] Sasaki | The compartmentation of acetyl-Coenzyme A carboxylase in plants[END_REF][START_REF] Parker | Analysis of Arabidopsis accessions hypersensitive to a loss of chloroplast translation[END_REF]. Orthologues to plant ACCase have been identified in all algal species with sequenced genomes [START_REF] Sayanova | Modulation of lipid biosynthesis by stress in diatoms[END_REF][START_REF] Radakovits | Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana[END_REF]. In silico sequence analyses of the genome of C. reinhardtii identified the occurrence of both types of ACCase [START_REF] Riekhof | Annotation of genes involved in glycerolipid biosynthesis in Chlamydomonas reinhardtii: Discovery of the betaine lipid synthase BTA1(Cr)[END_REF]. However their subcellular locations have not been verified experimentally. A comprehensive sequence analysis of ACCase across many algal species of diverse evolutionary origin revealed that most algae of primary symbiotic origin (i.e. Chlorophyta and Rhodophyta with their chloroplast surrounded by two envelope membranes) contain heteromeric ACCase in their chloroplasts, as evidenced by the presence of heteromeric BCCP in these taxa (Figure 2). In contrast, algal and apicoplast-containing species which possess plastids derived from secondary endosymbiosis (i.e. Heterokontophyta and Haptophyta), contain only the homomeric ACCase in their chloroplast [START_REF] Huerlimann | Comprehensive guide to acetyl-carboxylases in algae[END_REF][START_REF] Haq | Characterization of acetyl-CoA carboxylases in the basal dinoflagellate Amphidinium carterae[END_REF][START_REF] Jinkerson | Genomic insights from the oleaginous model alga Nannochloropsis gaditana[END_REF]. Thus, in general, the presence of heteromeric or homomeric ACCase is dependent on chloroplast origin. The cytosolic ACCase, which is always of homomeric form, is mostly known for its role in supplying malonyl-CoA for FA elongation or polyketide synthesis.

ACCase plays an important role in the regulation of carbon flux into FA biosynthesis [START_REF] Page | Acetyl-CoA carboxylase exerts strong flux control over lipid synthesis in plants[END_REF], including in algae, wherein a positive correlation between ACCase activity and FA amount has been observed in Chlorella vulgaris [START_REF] Leyva | Accumulation of fatty acids in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoAcarboxylase, temperature, and coimmobilization with Azospirillum brasilense[END_REF]. In plants, plastid targeting of a homomeric ACCase to rapeseed plastids produced a 5% increase in seed lipid content [START_REF] Roesler | Targeting of the Arabidopsis homomeric acetyl-coenzyme A carboxylase to plastids of rapeseeds[END_REF]. In contrast, overexpression of heteromeric ACCase in the diatom Cyclotella cryptica did not produce a measurable increase in total FAs, despite a 2-to 3-fold increase in ACCase activity in vitro [START_REF] Dunahay | Manipulation of microalgal lipid production using genetic engineering[END_REF]. Heterologous overexpression of the homomeric ACCase of the diatom P. tricornutum in Escherichia coli resulted in a 2-fold increase in neutral lipid production based on Nile red staining, although the identities of these neutral lipids remain unknown due to the absence of diacylglycerol acyltransferase (DGAT) and hence triacylglycerol (TAG) accumulation in most bacteria [START_REF] Xie | Functional characterization of an ACCase subunit from the diatom Phaeodactylum tricornutum expressed in Escherichia coli[END_REF].

The heteromeric form of ACCase is an equally attractive target for metabolic engineering as the homomeric form: overexpression of all four subunits of the heteromeric

ACCase in E. coli has been shown to result in a >100 fold increase in malonyl-CoA production followed by a six-fold increase in the rate of de novo FA synthesis [START_REF] Davis | Overproduction of acetyl-CoA carboxylase activity increases the rate of fatty acid biosynthesis in Escherichia coli[END_REF]. Although such an experiment has not been performed in plants or algae, individual subunits of heteromeric

ACCase have been overexpressed in plants including the subunits β-CT [START_REF] Madoka | Chloroplast transformation with modified accD operon increases acetyl-CoA carboxylase and causes extension of leaf longevity and increase in seed yield in tobacco[END_REF], BC [START_REF] Shintani | Antisense expression and overexpression of biotin carboxylase in tobacco leaves[END_REF] and BCCP [START_REF] Thelen | Both antisense and sense expression of biotin carboxyl carrier protein isoform 2 inactivates the plastid acetyl-coenzyme A carboxylase in Arabidopsis thaliana[END_REF]. In spite of successful overexpression, none of these transformants yielded increased ACCase activity and one (BCCP) produced lower activity due to incomplete biotinylation [START_REF] Thelen | Both antisense and sense expression of biotin carboxyl carrier protein isoform 2 inactivates the plastid acetyl-coenzyme A carboxylase in Arabidopsis thaliana[END_REF][START_REF] Chen | System analysis of an Arabidopsis mutant altered in de novo fatty acid synthesis reveals diverse changes in seed composition and metabolism[END_REF]. Collectively, these results suggest these three subunits are not limiting to ACCase activity in planta. This was recently confirmed by absolute quantitation of each of the subunits of the Arabidopsis ACCase during seed development [START_REF] Wilson | In vivo quantitative monitoring of subunit stoichiometry for metabolic complexes[END_REF]. Surprisingly, the α-CT subunit (which has never been over-expressed in plants) is between 3-10-fold less abundant than its partner β-CT, which is the only plastid-encoded subunit of heteromeric ACCase. Recently, a new plant subunit to the heteromeric ACCase was identified [START_REF] Salie | A family of negative regulators targets the committed step of de novo fatty acid biosynthesis[END_REF]. This subunit, termed BADC (an abbreviation for the original, tentative annotation as "biotin attachment domain-containing"), resembles the BCCP subunit but is not biotinylated, acting as a negative regulator of ACCase rather than as a carboxyl carrier. The regulation of ACCase by BADC and other factors is discussed in brief in section 3.8.1 and more comprehensively in a recent review [START_REF] Salie | Regulation and structure of the heteromeric acetyl-CoA carboxylase[END_REF].

Malonyl-CoA: ACP malonyltransferase (MCMT)

The malonyl-CoA generated by ACCase enters into the steps dedicated to de novo FA synthesis.

Malonyl-CoA is first converted to malonyl-acyl carrier protein (ACP) by MCMT. Overexpression of the native gene encoding MCMT in Nannochloropsis oceanica resulted in an 31% increase in neutral lipids together with a modified FA composition with eicosapentaenoic acid (20:5, EPA) increased by 8% [START_REF] Chen | Identification of a malonyl CoA-acyl carrier protein transacylase and its regulatory role in fatty acid biosynthesis in oleaginous microalga Nannochloropsis oceanica[END_REF]. This finding is of particular interest because neutral lipid content is increased together with an increased growth rate and photosynthetic performance. However, it is worth noting here that only one transgenic event is evaluated.

Therefore further efforts in generating large number of transgenes with better expression levels might be worthwhile.

The FA synthase (FAS) complex

Malonyl-ACP is then ligated to an acetyl-CoA molecule to form a 3-ketoacyl-ACP by ketoacyl-ACP synthase, while releasing a molecule of CO2. The 4-carbon 3-ketoacyl-ACP is subsequently reduced (by ketoacyl-ACP reductase, KAR), dehydrated (by hydoxyacyl-ACP dehydrase, HD), reduced again (by enoyl-ACP reductase, ER) until finally a 6-carbon-ACP is formed. The enzymes involved (KAS, KAR, HD, ER) collectively form the multi-subunit bacterial type II FA synthase (FAS) complex [START_REF] Li-Beisson | Acyl lipid metabolism in[END_REF]. In most algal or plant species, the FAS reaction repeats for 7 cycles until the formation of a C16-ACP. The C16-ACP has three fates:

it can be acylated to glycerol by chloroplast-resident acyltransferases to produce chloroplast lipids; it can also be further elongated to C18-ACP by a KASII; or it can be converted to a C16 free FA by acyl-ACP thioesterase (FAT). C18-ACP is either desaturated by stearoyl-ACP desaturase (SAD) or converted to free FA by FAT. The saturated and unsaturated C18 fatty acyl ACPs are substrates of FAT and their metabolic products (i.e. non-esterified (free) FAs) are exported out of the chloroplast. Expression of the cyanobacteria KAR in the chloroplast of the red alga Cyanoidioschyzon merolae resulted in strains over-accumulating TAG while maintaining cellular growth; transcriptome and metabolome analysis of the overexpressing lines suggest that KAR over-expression and N starvation, although both led to increased TAG accumulation, likely employed different metabolic routes for TAG accumulation [START_REF] Sumiya | Expression of cyanobacterial Acyl-ACP reductase elevates the triacylglycerol level in the red alga Cyanidioschyzon merolae[END_REF]. The only algal SAD studied to date is that from Chlorella zofingiensis [START_REF] Liu | Stearoyl-acyl carrier protein desaturase gene from the oleaginous microalga Chlorella zofingiensis: cloning, characterization and transcriptional analysis[END_REF], which exhibited a substrate preference for 18:0 similar to the plant enzyme.

In addition to the multi-component type II FAS, some algae also contain a cytosolic type I FAS, which normally is involved in FA elongations (>C18 FA), or may complement type II FAS when the demand for FA synthesis is high. For instance, it was observed that transcription of type I FAS was increased in cells of Nannochloropsis gaditana exposed to high light (HL), which is mirrored by a decrease in transcription of type II FAS, suggesting a shift in FA synthetic activities from chloroplast to cytoplasm [START_REF] Alboresi | Light remodels lipid biosynthesis in Nannochloropsis gaditana by modulating carbon partitioning between organelles[END_REF].

Acyl-ACP thioesterase (FAT or TE)

During the FAS extension cycles, the acyl chains are covalently bound via a thioester linkage to the prosthetic group of a soluble ACP. Termination of the chain elongation is thus carried out via the action of FAT which hydrolyzes acyl-ACP to form non-esterified FA and ACP. This step determines quantity and type of FAs that are exported. In 16:3 plants such as Arabidopsis, the acyl-flux through FAT has been determined to be ~60% of total FAs made in the chloroplast, and the flux can reach 90% in 18:3 plants [START_REF] Bonaventure | Disruption of the FATB gene in Arabidopsis demonstrates an essential role of saturated fatty acids in plant growth[END_REF]. FAT represents a key enzyme in the partitioning of de novo synthesized FAs between the prokaryotic and eukaryotic pathways (see section 4), and from a biotechnology perspective, FAT is therefore an important target for genetic engineering studies aiming to tailor FA production.

Based on sequence alignments and substrate specificities, FATs have been classified into two major families, FatA and FatB [START_REF] Jones | Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases[END_REF][START_REF] Salas | Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases[END_REF]. FatAs from diverse plant/algal species show strict substrate preference towards 18:1-ACP, whereas FatBs primarily hydrolyze saturated acyl-ACPs with 8 to 18 carbons [START_REF] Jones | Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases[END_REF][START_REF] Voelker | Fatty acid biosynthesis redirected to medium chains in transgenic oilseed plants[END_REF][START_REF] Kim | Toward production of jet fuel functionality in oilseeds: identification of FatB acyl-acyl carrier protein thioesterases and evaluation of combinatorial expression strategies in Camelina seeds[END_REF]. Several medium-chain specific FatBs have been cloned from Umbellularia california (California bay) and from several species of the genus Cuphea known to produce oils rich in medium chain FAs (MCFA, C6-12) in their seeds [START_REF] Dehesh | Production of high levels of 8:0 and 10:0 fatty acids in transgenic canola by overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana[END_REF].

Heterologous expression of MCFA-specific FatBs have been shown to produce MCFAs in transgenic oilseed crops [START_REF] Kim | Toward production of jet fuel functionality in oilseeds: identification of FatB acyl-acyl carrier protein thioesterases and evaluation of combinatorial expression strategies in Camelina seeds[END_REF][START_REF] Napier | Understanding and manipulating plant lipid composition: Metabolic engineering leads the way[END_REF][START_REF] Jaworski | Industrial oils from transgenic plants[END_REF]. Lately, transgenic expression of some of these specialized plant FatBs have resulted in production of MCFAs in algae including P. tricornutum [START_REF] Radakovits | Genetic engineering of fatty acid chain length in Phaeodactylum tricornutum[END_REF],

Dunaliella tertiolecta [START_REF] Lin | Genetic engineering of medium-chain-length fatty acid synthesis in Dunaliella tertiolecta for improved biodiesel production[END_REF] and C. reinhardtii [START_REF] Inaba | Alteration of fatty acid chain length of Chlamydomonas reinhardtii by simultaneous expression of medium-chain-specific thioesterase and acyl carrier protein[END_REF]. Interestingly, heterologous expression of a thioesterase of Duanliella tertiolecta in C. reinhardtii has resulted in a 50% increase in total FA production [START_REF] Tan | Expression of the heterologous Dunaliella tertiolecta fatty acyl-ACP thioesterase leads to increased lipid production in Chlamydomonas reinhardtii[END_REF]. A first report on characterization of algal FAT is the study of a novel thioesterase from P. tricornutum where PtTE showed no similarity to characterized plant and bacterial thioesterases [START_REF] Gong | Characterization of a novel thioesterase (PtTE) from Phaeodactylum tricornutum[END_REF], but its endogenous overexpression in P. tricornutum led to a 72% increase in FA content without altering FA composition [START_REF] Gong | Characterization of a novel thioesterase (PtTE) from Phaeodactylum tricornutum[END_REF]. Strikingly, P. tricornutum strains where PtTE was silenced accumulated 1.7-fold more TAG than native strains with marked change in fatty acid profile [START_REF] Hao | Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis[END_REF]. Moreover, recent work in C. reinhardtii has identified the important role of protein-protein (ACP-FAT) interaction in chloroplast FA synthesis [START_REF] Blatti | Manipulating fatty acid biosynthesis in microalgae for biofuel through protein-protein interactions[END_REF],

implying the importance of subcellular context in genetic engineering studies. In summary, current evidence show that FATs play not only a role in determining FA chain length but can also impact FA total amount.

FA export

Currently, no direct evidence is available regarding how the nascent FAs assembled in the stroma pass through the two, three or sometimes four envelope membranes of algal chloroplasts. Genes encoding known protein components of transport pathway in plants, including the fatty acid export 1 (FAX1) [START_REF] Li | FAX1, a novel membrane protein mediating plastid fatty acid export[END_REF] and long-chain acyl-CoA synthetase 9 (LACS9) [START_REF] Jessen | Two activities of long-chain acyl-coenzyme A synthetase are involved in lipid trafficking between the endoplasmic reticulum and the plastid in Arabidopsis[END_REF], can be identified in algal genomes, but the putative orthologues and their functions in FA export have not been examined in algae. Various similarities and differences in lipid transport between plants and algae are reviewed recently in [START_REF] Liu | Lipid metabolism in microalgae distinguishes itself[END_REF][START_REF] Li | Fatty acid and lipid transport in plant cells[END_REF].

FA modifications: elongation and desaturation

Neo-synthesized FAs (C16:0, C18:0; and C18:1) are usually further elongated or desaturated to finally constitute the lipid makeup of a given organism. FA elongations are mostly known to uniquely occur in the endoplasmic reticulum (ER) [START_REF] Leonard | Elongation of long-chain fatty acids[END_REF]; while FA desaturations occur both inside the chloroplast and in extra-chloroplast compartments. Except for SAD, mostly known desaturases are membrane-bound [START_REF] Shanklin | Desaturation and related modifications of fatty acids[END_REF][START_REF] Los | Structure and expression of fatty acid desaturases[END_REF]. Steps and enzymes required for desaturation of FA in C. reinhardtii have mostly been identified and are recently reviewed in [START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF]. One interesting feature is the occurrence of only one plastidial -3 FA desaturase (CrFAD7) in C. reinhardtii [START_REF] Nguyen | The green microalga Chlamydomonas reinhardtii has a single ω-3 fatty acid desaturase that localizes to the chloroplast and impacts both plastidic and extraplastidic membrane lipids[END_REF], which often occurs in multiple isoforms present in both chloroplast and extrachloroplast of plant cells [START_REF] Sayanova | Modulation of lipid biosynthesis by stress in diatoms[END_REF].

A survey of VLCPUFA synthesis in algae was included in the previous review by [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF] and here we provide an update on the pathways involved in the model diatom Phaeodactylum tricornutum where high amount of polyunsaturated fatty acids are made (Figure 3) [108] [55].

For both the n-3 and n-6 pathways, metabolism begins with 6-desaturation in most organisms.

However, 9-elongation from LA or from LNA provides an alternative route which has been found in Parietochloris incisa [START_REF] Bigogno | Biosynthesis of arachidonic acid in the oleaginous microalga Parietochloris incisa (Chlorophyceae): radiolabeling studies[END_REF], Isochrysis galbana [START_REF] Qi | Identification of a cDNA encoding a novel C18-Delta(9) polyunsaturated fatty acid-specific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana[END_REF], Pavlova salina [START_REF] Petrie | Metabolic engineering of omega-3 long-chain polyunsaturated fatty acids in plants using an acyl-CoA Δ6-desaturase with ω3preference from the marine microalga Micromonas pusilla[END_REF], Emiliania huxleyi [START_REF] Sayanova | Identification and functional characterisation of genes encoding the omega-3 polyunsaturated fatty acid biosynthetic pathway from the coccolithophore Emiliania huxleyi[END_REF] and Euglena gracilis [START_REF] Wallis | The Delta8-desaturase of Euglena gracilis: an alternate pathway for synthesis of 20-carbon polyunsaturated fatty acids[END_REF]. As shown in Figure 3, 3-desaturation can convert n-6 into n-3 PUFAs and such a conversion for ARA into EPA has been shown in Nannochloropsis sp. [START_REF] Vieler | Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779[END_REF], Monodus subterraneus [START_REF] Khozin-Goldberg | Biosynthesis of eicosapentaenoic acid (EPA) in the freshwater eustigmatophyte Monodus subterraneus (Eustigmatophyceae)[END_REF] and Porphyridium cruentum [START_REF] Khozin | Elucidation of the biosynthesis of eicosapentaenoic acid in the microalga Porphyridium cruentum (II. Studies with Radiolabeled Precursors)[END_REF]. In some marine eukaryotes of the Thraustochytriaceae, a polyketide synthase (PKS) pathway is used to make VLCPUFA. The pathway is used by Schizochytrium but in Thraustochytrium a desaturation/elongation pathway is utilised [START_REF] Metz | Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes[END_REF]. Due to the nutritional importance of very long chain PUFAs (see section 7), elongases and desaturases in algae have been intensively researched, and several reviews cover this area [START_REF] Khozin-Goldberg | LC-PUFA from photosynthetic microalgae: occurrence, biosynthesis, and prospects in biotechnology[END_REF][START_REF] Sayanova | Modulation of lipid biosynthesis by stress in diatoms[END_REF][START_REF] Harwood | The versatility of algae and their lipid metabolism[END_REF].

Carbon sources for acetyl-CoA synthesis

Increasing evidence suggests a positive link between the rate of FA synthesis and the amount of carbon precursors in plants and algae [START_REF] Avidan | Enhanced acetyl-CoA production is associated with increased triglyceride accumulation in the green alga Chlorella desiccata[END_REF][START_REF] Fan | Oil accumulation is controlled by carbon precursor supply for fatty acid synthesis in Chlamydomonas reinhardtii[END_REF][START_REF] Goodenough | The path to triacylglyceride obesity in the sta6 strain of Chlamydomonas reinhardtii[END_REF][START_REF] Ramanan | Lipid droplet synthesis is limited by acetate availability in starchless mutant of Chlamydomonas reinhardtii[END_REF][START_REF] Bourgis | Comparative transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically in carbon partitioning[END_REF], implying that enhancing the rate of carbon flux into chloroplasts might be a worthwhile approach for genetic engineering attempts to improve FA amount. This finding highlights the importance of understanding the potential sources and their contributions to chloroplast acetyl-CoA production. Various sources and use of acetyl-CoA in plants has been summarized in [START_REF] Harwood | Recent advances in the biosynthesis of plant fatty acids[END_REF]. In addition to the chloroplast acetyl-CoA pool, acetyl-CoA is also made by reactions inside the mitochondria and peroxisomes [START_REF] Leonardi | Coenzyme A: back in action[END_REF]. Activation of the pyruvate dehydrogenase complex (PDH) protein while silencing the pyruvate dehydrogenase kinase (PDK) has boosted acetyl-CoA production and, therefore, neutral lipid content in Phaeodactylum tricornutum [START_REF] Ma | Antisense knockdown of pyruvate dehydrogenase kinase promotes the neutral lipid accumulation in the diatom Phaeodactylum tricornutum[END_REF]. Carbons contained in acetyl-CoA can be shuttled to other compartments, but must first be converted into malate or pyruvate, which are transported across membranes through malate shuttles or by other solute transporters [START_REF] Scheibe | Malate valves to balance cellular energy supply[END_REF][START_REF] Weber | Solute transporters as connecting elements between cytosol and plastid stroma[END_REF]. Thus far, no known acetyl-CoA transporter is reported in any organism. Acetyl-CoA is thus considered not directly imported by chloroplasts [START_REF] Roughan | Acetate is the preferred substrate for long-chain fatty acid synthesis in isolated spinach chloroplasts[END_REF], but rather generated by chloroplastic enzymes. Four possible routes can lead to acetyl-CoA production, as discussed below. The relative importance of these possible sources varies between species, and between phototrophic or heterotrophic tissues, or trophic style of a given species. Moreover, these carbon sources are by no means exclusive, for instance, through a chemical-genetic screen for oil inducers in P. tricornutum, the authors have suggested that sterol metabolism contributes to TAG synthesis probably by providing acetyl-CoA [START_REF] Conte | Screening for biologically annotated drugs that trigger triacylglycerol accumulation in the diatom Phaeodactylum[END_REF].

Chloroplast pyruvate dehydrogenase (PDH)

In plants and algae grown photoautotrophically, acetyl-CoA is mostly produced via the oxidative decarboxylation of pyruvate by the chloroplast PDH enzyme [128]. In addition to acetyl-CoA, this reaction generates CO2 and NADH [START_REF] Johnston | Cloning and molecular analyses of the Arabidopsis thaliana plastid pyruvate dehydrogenase subunits[END_REF]. In turn, pyruvate can be made from glycolysis, malate (through malic enzyme, ME), as a side reaction of RuBisCO, or pentose phosphate pathway (PPP) linked to photosynthesis or to sugar oxidation (oxidative PPP = OPPP) (Figure 1). The proportion of their contribution to pyruvate formation has not been worked out, but it most likely varies by species and trophic style. The temporal expression of plastid PDH upon N starvation, i.e. during high rate of oil synthesis, is consistent with its role in FA synthesis in several algal species including C. reinhardtii [START_REF] Avidan | Enhanced acetyl-CoA production is associated with increased triglyceride accumulation in the green alga Chlorella desiccata[END_REF][START_REF] Miller | Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism[END_REF]. Indeed, silencing of a gene encoding a putative chloroplast E1α subunit of PDH using microRNA in C.

reinhardtii has resulted in strains producing >40% less total FAs than control strains expressing only an empty vector during photoautotrophic N starvation, but there was little or no impact on lipid accumulation during photoheterotrophic growth (i.e. with the presence of acetate) [START_REF] Shtaida | Downregulation of a putative plastid PDC E1α subunit impairs photosynthetic activity and triacylglycerol accumulation in nitrogen-starved photoautotrophic Chlamydomonas reinhardtii[END_REF].

In addition, photosynthetic parameters and growth of PDH-E1α silenced strains were also negatively affected, implying the importance of chloroplast PDH not only in FA synthesis but also in general algal physiology and development. Indeed, acetyl-CoA is a key intermediate in a number of different metabolic pathways [START_REF] Oliver | Acetyl-CoA-Life at the metabolic nexus[END_REF]. Moreover, high CO2 supply has been observed

to increase FA synthesis in several algal species [START_REF] Jose | High carbon (CO2) supply leads to elevated intracellular acetyl CoA levels and increased lipid accumulation in Chlorella vulgaris[END_REF][START_REF] Peng | Regulation of carbon metabolic fluxes in response to CO2 supplementation in phototrophic Chlorella vulgaris: a cytomic and biochemical study[END_REF][START_REF] Wang | Biomass, total lipid production, and fatty acid composition of the marine diatom Chaetoceros muelleri in response to different CO2 levels[END_REF][START_REF] Wu | Enzyme activity highlights the importance of the oxidative pentose phosphate pathway in lipid accumulation and growth of Phaeodactylum tricornutum under CO2 concentration[END_REF] (see also section 6.1.), suggesting the importance of photosynthesis in carbon supply. However, the picture could be different if acetate is present as already observed in the PDH-silenced strains. This is consistent with the recent finding that when acetate is present, C. reinhardtii employs principally the CO2 carbon fixation pathway for starch synthesis whereas acetate is used mainly for FA synthesis [START_REF] Juergens | The relationship of triacylglycerol and starch accumulation to carbon and energy flows during nutrient deprivation in Chlamydomonas reinhardtii[END_REF].

Acetyl-CoA synthetase (ACS)

Acetyl-CoA can also be produced from acetate: a direct conversion through ACS or through a two-step reaction catalyzed by acetate kinase (ACK) and phosphate acetyltransferase (PAT).

This feature enables radio-tracer studies of lipid metabolism in algae via simply feeding cells with radio-labelled C14-acetate. The ACS route is widely present in plants and algae, whereas the ACK/PAT route occur mostly in prokaryotes and some eukaryotic microalgae [START_REF] Ingram-Smith | Acetate kinase: not just a bacterial enzyme[END_REF], with both routes requiring ATP. The idea of "acetate-to-acetyl-CoA" for FA synthesis has been discarded in plants due to extensive in vivo flux analyses demonstrating the actual source of acetyl-CoA is from PDH [START_REF] Schwender | A flux model of glycolysis and the oxidative pentosephosphate pathway in developing Brassica napus embryos[END_REF]. Nevertheless, the ACS route is known to play a major role in heterotrophically or mixotrophically grown algae, because it has been shown recently that de novo FA synthesis is boosted by increased acetate supply in C. reinhardtii in three independent studies [START_REF] Fan | Oil accumulation is controlled by carbon precursor supply for fatty acid synthesis in Chlamydomonas reinhardtii[END_REF][START_REF] Ramanan | Lipid droplet synthesis is limited by acetate availability in starchless mutant of Chlamydomonas reinhardtii[END_REF][START_REF] Goodson | Structural correlates of cytoplasmic and chloroplast lipid body synthesis in Chlamydomonas reinhardtii and stimulation of lipid body production with acetate boost[END_REF], and also in other heterokont species as reviewed in [START_REF] Mühlroth | Pathways of lipid metabolism in marine algae, co-expression network, bottlenecks and candidate genes for enhanced production of EPA and DHA in species of Chromista[END_REF]. Furthermore, heterogeneous expression of a bacteria ACS in a marine alga Schizochytrium sp. increased its FA proportion by 11.3% [START_REF] Yan | Overexpression of acetyl-CoA synthetase increased the biomass and fatty acid proportion in microalga Schizochytrium[END_REF]. Nevertheless, it remains to be determined what the relative contribution of ACS is versus that of the ACK/PAT pathway for acetyl-CoA formation in those algae where both routes are present.

ATP: citrate lyase (ACL)

Another possible source of acetyl-CoA for de novo FAS is the cleavage of citrate by ATPcitrate lyase (ACL). Cytosolic ACL has been shown to play a critical role in determining the oleaginicity of animals and yeasts where de novo FAS occurs in cytoplasm [START_REF] Botham | A biochemical explanation for lipid accumulation in Candida 107 and other oleaginous micro-organisms[END_REF][START_REF] Ratledge | Regulation of lipid accumulation in oleaginous micro-organisms[END_REF][START_REF] Ratledge | The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms[END_REF], and in plants, cytosolic ACL has been shown to play a critical role in FA elongation [START_REF] Fatland | Reverse genetic characterization of cytosolic acetyl-CoA generation by ATP-citrate lyase in Arabidopsis[END_REF]. The involvement of ACL in FA synthesis in algae remains to be demonstrated. The ACL enzymatic activities from the glaucocystophyte alga Cyanophora paradoxa were found associated with the cytosol; however its involvement in FA synthesis has not been addressed. A single gene (Cre05.g241850) encoding a putative ACL homolog has been identified in the genome of C.

reinhardtii. The putative protein does not contain any transit peptide based on analyses using Predalgo [START_REF] Tardif | PredAlgo, a new subcellular localization prediction tool dedicated to green algae[END_REF], and is likely cytosolic and therefore it is anticipated to provide a role in filling the cytosolic acetyl-CoA pool for FA elongation in the cytoplasm, as C. reinhardtii does contain 20:1n-9 and 22:1n-9 [START_REF] Sakurai | Detailed identification of fatty acid isomers sheds light on the probable precursors of triacylglycerol accumulation in photoautotrophically grown Chlamydomonas reinhardtii[END_REF]. Nevertheless, the contribution of plastidial ACL, if it occurs, to de novo FAS remains to be examined in algae.

Sources of reducing equivalents in the chloroplast

As lipids are highly reduced compounds, lipid synthesis requires large quantities of NAD(P)H supplied in a stoichiometric ratio with respect to acetyl-CoA and ATP [START_REF] Li-Beisson | Acyl-lipid metabolism[END_REF][START_REF] Ohlrogge | Lipid biosynthesis[END_REF]. Indeed, a positive correlation between the level of reducing equivalents and FA synthesis has been established in fungi, algae and plants [START_REF] Zhang | Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation[END_REF][START_REF] Xue | The pivotal role of malic enzyme in enhancing oil accumulation in green microalga Chlorella pyrenoidosa[END_REF]. For instance, recent transcriptomic studies in P. tricornutum found that a buildup of precursors such as acetyl-CoA and reducing equivalents may provide a more significant contribution to TAG accumulation than an increase in ACCase activity alone [START_REF] Valenzuela | Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum[END_REF]152]. Neither NADH nor NADPH are permeable to the chloroplast envelope; therefore they have to be produced by chloroplast-localized reactions including photosynthesis, ME, PDH, pentose phosphate pathway (PPP) and glycolysis.

Chloroplast PDH

As detailed in section 3.2.1 considering its contribution as a carbon source, the plastidial PDH also produces one NADH for each molecule of acetyl-CoA produced. The trans 2-enoyl-ACP reductase of de novo FAS complex is shown to require one mole of NAD(P)H to ensure de novo FAS [START_REF] Mou | Deficiency in fatty acid synthase leads to premature cell death and dramatic alterations in plant morphology[END_REF][START_REF] Zhao | Malate transported from chloroplast to mitochondrion triggers production of ROS and PCD in Arabidopsis thaliana[END_REF]. This provides a compelling argument for acetyl-CoA coming from the plastid PDH since this is one of only a few enzymes that produces NADH in plastids.

Photosynthesis

In photosynthetically active cells (algae or plants), photochemical reactions are believed to provide a significant part of reducing equivalents (NADPH) for anabolic reactions inside chloroplast. The finding that cells exposed to HL possessed higher amount of total FAs could be considered an evidence to support this [START_REF] Alboresi | Light remodels lipid biosynthesis in Nannochloropsis gaditana by modulating carbon partitioning between organelles[END_REF][START_REF] Ruuska | The capacity of green oilseeds to utilize photosynthesis to drive biosynthetic processes[END_REF][START_REF] Goold | Saturating light induces sustained accumulation of oil in plastidal lipid droplets in Chlamydomonas reinhardtii[END_REF][START_REF] He | Effect of light intensity on physiological changes, carbon allocation and neutral lipid accumulation in oleaginous microalgae[END_REF]. However, upon HL exposure the entire photosynthetic chain is upregulated: increased NADPH production occurs together with enhanced CO2 fixation. Therefore the observed effect on increased lipid amount could be due to a combinatory effect of the increase in both carbon precursors and reducing equivalents.

Glycolysis

Glycolysis is defined as the set of reactions that lead to the generation of pyruvate from glucose.

In addition to its obvious role as a carbon source, glycolysis produces two (net) ATP and two NADH. Glycolysis can occur in both cytoplasm and chloroplast, and parallel pathways operate in both compartments in A. thaliana [START_REF] Plaxton | The organization and regulation of plant glycolysis[END_REF]. In C. reinhardtii a single pathway operates: the "upper half" of the pathway occurs in the chloroplast, and the 'lower half', i.e. reactions after 3-phosphoglycerate (3-PGA) occurs in the cytoplasm [START_REF] Ball | Regulation of starch biosynthesis[END_REF]. This compartmentation of glycolysis can have implications on the chloroplast redox state and subcellular energetics. For example, in C. reinhardtii glycolysis produces two NADHs inside the chloroplast and two ATPs in the cytoplasm. NADH produced by glycolysis could contribute substantially to FA synthesis especially when starch degradation is high.

Malic enzyme (ME)

Malic enzyme catalyzes the reversible conversion of malate to pyruvate while producing NADPH. The contribution of ME to de novo FA synthesis has been demonstrated through an overexpression study where strains over-expressing ME possessed improved FA synthesis in the diatom P. tricornutum and also in the green alga Chlorella pyrenoidosa [START_REF] Xue | The pivotal role of malic enzyme in enhancing oil accumulation in green microalga Chlorella pyrenoidosa[END_REF][START_REF] Xue | Genetic improvement of the microalga Phaeodactylum tricornutum for boosting neutral lipid accumulation[END_REF].

Furthermore, reducing NADPH supply via inhibition of ME activities using sesamol in

Haematococcus pluvialis and Nannochloropsis sp led to reduction in total FAs [START_REF] Recht | Patterns of carbohydrate and fatty acid changes under nitrogen starvation in the microalgae Haematococcus pluvialis and Nannochloropsis sp[END_REF].

Moreover, a high docosahexaneoic acid (DHA) production and total lipid content in the marine alga Schizochytrium sp has been found to correlate positively with the cultivation stages when the activities of ME is high [START_REF] Ren | Enhanced docosahexaenoic acid production by reinforcing acetyl-CoA and NADPH supply in Schizochytrium sp. HX-308[END_REF]. When Schizochytrium sp was fed with malate, DHA production is increased by 47% [START_REF] Perez-Garcia | Heterotrophic cultures of microalgae: Metabolism and potential products[END_REF]. In addition to supplying the FAS complex with NADPH, the reaction catalyzed by ME also produces pyruvate, which is a substrate for acetyl-CoA synthesis using PDH. Therefore, the contribution of ME to FA synthesis could be two-fold:

providing both carbon and reducing equivalents.

Pentose phosphate pathway (PPP)

Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the conversion of glucose-6phosphate (G6P) to 6-phospho-D-glucono-1,5-lactonate and while generating NADPH for anabolic reactions. G6PDH is a key enzyme of the PPP pathway, and its over-expression led to a 3.7-fold increase in lipid content in P. tricornutum, highlighting the critical role of G6PDH in algal lipid accumulation by enhancing NADPH supply [START_REF] Xue | Glucose-6phosphate dehydrogenase as a target for highly efficient fatty acid biosynthesis in microalgae by enhancing NADPH supply[END_REF]. The contribution of OPPP pathway to supply NADPH for FA synthesis has also been observed in the oleaginous diatom

Fistulifera solaris [START_REF] Osada | Enhanced NADPH production in the pentose phosphate pathway accelerates lipid accumulation in the oleaginous diatom Fistulifera solaris[END_REF].

ATP supply for FA synthesis

For every two NAD(P)H molecules supplied to the FAS complex, one molecule of ATP is required (2:1 NAD(P)H:ATP). While sources of acetyl-CoA and NAD(P)H for FA synthesis have been studied intensively [START_REF] Liu | Lipid metabolism in microalgae distinguishes itself[END_REF][START_REF] Khozin-Goldberg | Unraveling algal lipid metabolism: Recent advances in gene identification[END_REF][START_REF] Merchant | You're it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation[END_REF][START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF][START_REF] Tan | The dilemma for lipid productivity in green microalgae: importance of substrate provision in improving oil yield without sacrificing growth[END_REF], the supply of ATP for the FAS complex is often overlooked. It is generally considered that most of the ATP needed for biosynthetic reactions in algae is provided by photophosphorylation (i.e. photosynthesis) or respiratory oxidative phosphorylation (mitochondrial respiration), but the re-partition of ATP produced between various anabolic reactions has not been investigated, and most current observations in this area remain correlative.

Linear and cyclic electron pathway (LEF and CEF)

When algae are starved for N (i.e. during active TAG synthesis), it has been observed that although the LEF rate and the respiration rate drop, the CEF pathway (as well as the activity of thylakoid membrane-located ATP synthase) increase significantly [START_REF] Zhang | Nitrogen starvation induced oxidative stress in an oilproducing green alga Chlorella sorokiniana C3[END_REF]. The CEF pathway around photosystem I cycles electrons back to the lumen therefore generating a proton motive force to drive ATP synthesis but without O2 evolution or NADPH production. The CEF pathway has been suggested to supply ATP for lipid production during N starvation in C.

reinhardtii mutants impaired in the proton gradient regulation 5 like 1 protein (PGRL1), which nonetheless accumulates significantly less neutral lipids under N starvation [START_REF] Chen | Ca2+-regulated cyclic electron flow supplies ATP for nitrogen starvation-induced lipid biosynthesis in green alga[END_REF].

Mitochondrial respiration

In the context of lipid metabolism, mention of mitochondrial respiration is reminiscent of the respiration of acetyl-CoA during peroxisomal FA β-oxidation [START_REF] Eastmond | Arabidopsis uses two gluconeogenic gateways for organic acids to fuel seedling establishment[END_REF][START_REF] Graham | Seed storage oil mobilization[END_REF][START_REF] Kong | Lipid catabolism in microalgae[END_REF]. Although less known and often ignored, the interaction between mitochondrial respiration and lipid synthesis has started to be appreciated, mostly due to its role in supplying ATP for FA synthesis in algae [START_REF] Lemaire | Restoration of phototrophic growth in a mutant of Chlamydomonas reinhardtii in which the chloroplast atpB gene of the ATP synthase has a deletion: an example of mitochondria-dependent photosynthesis[END_REF]. Studies of the ATPase mutant (FUD50) of C. reinhardtii have revealed that photosynthetic ATP production is not essential during heterotrophic growth as long as mitochondrial respiration is functioning, implying the occurrence of active ATP transport from mitochondria to chloroplasts [START_REF] Lemaire | Restoration of phototrophic growth in a mutant of Chlamydomonas reinhardtii in which the chloroplast atpB gene of the ATP synthase has a deletion: an example of mitochondria-dependent photosynthesis[END_REF]. Put another way, defects in ATP synthesis in the chloroplast can be compensated for by ATP derived from mitochondrial respiration. Furthermore, it has been reported that Chlamydomonas mutants of complex I or complex III were impaired in their lipid production activity during sulfur deprivation [START_REF] Lecler | Characterization of an internal type-II NADH dehydrogenase from Chlamydomonas reinhardtii mitochondria[END_REF][START_REF] Lecler | Functional analysis of hydrogen photoproduction in respiratory-deficient mutants of Chlamydomonas reinhardtii[END_REF]. Interplay between mitochondrial metabolism and lipid synthesis could occur at two levels: metabolic (Krebs cycle) as well as energetic (supply of NADH and ATP) (detailed in section 3.5). Nevertheless, detailed interactions between these pathways are yet to be characterized. With the increasing number of respiratory mutants available for C. reinhardtii [START_REF] Massoz | Isolation of Chlamydomonas reinhardtii mutants with altered mitochondrial respiration by chlorophyll fluorescence measurement[END_REF][START_REF] Salinas | Respiratory-deficient mutants of the unicellular green alga Chlamydomonas: A review[END_REF], a systematic analysis of the interaction between FA synthesis and mitochondrial respiration should be possible.

Chloroplast redox poise and FA synthesis

Photosynthesis converts light energy into ATP and NADPH, and this energy is subsequently used to drive CO2 assimilation through the Calvin-Benson-Bassham (CBB) cycle. In the absence of exogenous carbon supply, photosynthesis is the only provider of energy and carbon skeletons for all anabolic reactions in the cell. In addition to the CBB cycle, NAD(P)H is a ubiquitous electron carrier also required for starch and FA synthesis. The flux along these pathways is mostly determined by the ratio of NAD(P)H versus NAD(P + ) or ATP levels.

Photoautotrophs live in an ever-changing environment where metabolic demand for NAD(P)H and ATP is constantly changing. Multiple strategies have evolved to fine tune photosynthesis to meet downstream metabolic needs because excess production of reducing power may result in an over-reduction of the photosynthetic electron transport chain and consequent photooxidative damage [START_REF] Niyogi | Safety valves for photosynthesis[END_REF][START_REF] Dietz | Redox-and reactive oxygen species-dependent signaling into and out of the photosynthesizing chloroplast[END_REF]. The processes known to poise chloroplast redox balance could have an impact on FA synthesis.

During N starvation, photosynthetic electron transport chain complexes are reduced [START_REF] Juergens | The regulation of photosynthetic structure and function during nitrogen deprivation in Chlamydomonas reinhardtii[END_REF][START_REF] Terashima | Chlamydomonas: triacylglycerol accumulation[END_REF]. Lipid production during N starvation has often been thought to act as an electron sink to accommodate an over-reduced chloroplast [START_REF] Hu | Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances[END_REF][START_REF] Li | A galactoglycerolipid lipase is required for triacylglycerol accumulation and survival following nitrogen deprivation in Chlamydomonas reinhardtii[END_REF], but this theory has recently been partly challenged by the observation that heterotrophic cultures accumulate TAGs and starch during N starvation [START_REF] Juergens | The relationship of triacylglycerol and starch accumulation to carbon and energy flows during nutrient deprivation in Chlamydomonas reinhardtii[END_REF]. Nevertheless, this accumulation does occur to a lesser extentthe amount of TAG accumulated during heterotrophic N starvation (in the dark) is four times less than that accumulated in N-starved autotrophic cultures. TAG may thus at least partly serve as an electron sink for N-starved autotrophic algae.

Chloroplast-located alternative electron dissipation pathways

Several chloroplast-located alternative electron pathways (notably the cyclic electron flow, O2 photoreduction processes, chlororespiration and the water-to-water cycle) are known to play roles in dissipation of photo-reductant [START_REF] Curien | The water to water cycles in microalgae[END_REF][START_REF] Saroussi | Nutrient scavenging and energy management: acclimation responses in nitrogen and sulfur deprived Chlamydomonas[END_REF]. The importance of CEF in metabolism of Nstarved cells is further supported by the observation that in N-starved cells of C. reinhardtii, LEF fell approximately 15% more than CEF over the first 24 h of N starvation [START_REF] Juergens | The regulation of photosynthetic structure and function during nitrogen deprivation in Chlamydomonas reinhardtii[END_REF]. This is further supported by the observation that the rate of CEF increased while LEF decreased during the cell's adaptation to N starvation in Chlorella sorokiniana [START_REF] Chen | Ca2+-regulated cyclic electron flow supplies ATP for nitrogen starvation-induced lipid biosynthesis in green alga[END_REF]. Perturbation of the above pathways should have impact on FA synthesis. Thus, for instance, the pgrl1 mutant defected in the proton gradient regulation 5 like 1 (PGRL1) accumulated 30% less neutral lipids based on BODIPY staining than its corresponding wild type. This finding could be interpreted as indicating that CEF supplies ATP for FA synthesis (discussed in section 3.4.1), or could also imply a possible competition for NADPH between CEF and FA synthesis [START_REF] Chen | Ca2+-regulated cyclic electron flow supplies ATP for nitrogen starvation-induced lipid biosynthesis in green alga[END_REF]. A systematic analysis of the interaction between various chloroplast electron dissipation pathways and FA synthesis remains to be conducted. With the large number of Chlamydomonas mutants available (Chlamydomonas Mutant Library -CLiP library: https://www.chlamylibrary.org/ ) [START_REF] Li | An indexed, mapped mutant library enables reverse genetics studies of biological processes in Chlamydomonas reinhardtii[END_REF], it should be possible to comprehensively address questions related to the interaction between FA synthesis and various electron dissipation mechanisms.

Electron dissipation through collaboration with mitochondria

Recently, it has also been shown that chloroplast redox poise can be achieved through export of excess reducing equivalents to mitochondria in green algae [START_REF] Dang | Combined increases in mitochondrial cooperation and oxygen photoreduction compensate for deficiency in cyclic electron flow in Chlamydomonas reinhardtii[END_REF] and diatoms [START_REF] Bailleul | Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms[END_REF].

Structural components involved in the energy trafficking between chloroplasts and mitochondria have not been identified, but malate shuttles and triose phosphate transporters (TPTs) are strong candidates. Once inside mitochondria, reducing equivalents (NADH) are consumed by oxidative respiration operating through two pathways in plants and algae, i.e.

cytochrome oxidase (COX) pathway and alternative oxidase (AOX) pathway [START_REF] Lecler | Characterization of an internal type-II NADH dehydrogenase from Chlamydomonas reinhardtii mitochondria[END_REF][START_REF] Salinas | Respiratory-deficient mutants of the unicellular green alga Chlamydomonas: A review[END_REF][START_REF] Cardol | Photosynthesis and state transitions in mitochondrial mutants of Chlamydomonas reinhardtii affected in respiration[END_REF].

The COX pathway couples consumption of NADH to ATP synthesis, while they are uncoupled in the AOX pathway. For a long time, the latter was considered a wasteful process and its physiological significance was uncertain. However, lately it has been observed that expression as well as translation of AOX genes in a number of algal species are upregulated in conditions when photosynthetically-produced reducing equivalents should be attenuated [START_REF] Miller | Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism[END_REF][START_REF] Zhang | Enhanced fatty acid accumulation in Isochrysis galbana by inhibition of the mitochondrial alternative oxidase pathway under nitrogen deprivation[END_REF][START_REF] Shaun | Manipulation of an alternative respiratory pathway in photoautotrophs[END_REF][START_REF] Schmollinger | Nitrogen-sparing mechanisms in Chlamydomonas affect the transcriptome, the proteome, and photosynthetic metabolism[END_REF].

Indeed, inhibition of the AOX pathway using salicylhydroxamic acid (SHAM) under N starvation led to a 23% increase in FA amount in the marine alga Isochrysis galbana [START_REF] Zhang | Enhanced fatty acid accumulation in Isochrysis galbana by inhibition of the mitochondrial alternative oxidase pathway under nitrogen deprivation[END_REF].

These authors suggest that the increase in FA production is likely a result of an increase in chloroplast NADPH (although not measured in above study) due to a defect in its dissipation through the mitochondria AOX pathway. Taken together, these studies suggest that the chloroplast reduction state can be increased, via blocking the mechanisms of NADPH dissipation, therefore boosting FA synthesis.

Energy interactions between peroxisomes and chloroplasts impact FA synthesis

Energetic exchanges between mitochondria and chloroplast have been evidenced, and the impact on FA synthesis has also been evaluated in some cases (discussed in section 3.5.2). Until lately, little was known about the energetic interactions between chloroplast and peroxisome, although these two organelles are often seen located in close proximity [START_REF] Hu | Plant peroxisomes: biogenesis and function[END_REF][START_REF] Hayashi | Visualization of microbodies in Chlamydomonas reinhardtii[END_REF]. We recently showed that, during acclimation of C. reinhardtii to N starvation or HL exposure, extensive energetic exchanges occur from peroxisome to chloroplast employing the peroxisomal malate dehydrogenase (MDH2) and redox-based signaling [START_REF] Kong | Interorganelle communication: peroxisomal MALATE DEHYDROGENASE2 connects lipid catabolism to photosynthesis through redox coupling in Chlamydomonas[END_REF]. This interorganelle communication between peroxisome and chloroplast is essential in maintaining chloroplast redox poise, and in its absence, the chloroplast is over-reduced, therefore activating FA and starch syntheses.

Relationship between FA synthesis and starch accumulation

During a diurnal cycle, most green algae, like plant leaves, accumulate starch during the day and degrade it to provide cells with carbon and energy at night [START_REF] Ball | Physiology of starch storage in the monocellular alga Chlamydomonas reinhardtii[END_REF]. Lipid synthesis also follows a similar cycle [START_REF] Juppner | Dynamics of lipids and metabolites during the cell cycle of Chlamydomonas reinhardtii[END_REF][START_REF] Kong | Chlamydomonas carries out fatty acid beta-oxidation in ancestral peroxisomes using a bona fide acyl-CoA oxidase[END_REF]. Upon stress (for example N starvation or HL) massive amount of starch and neutral lipids, mostly TAGs, accumulate [START_REF] Liu | Lipid metabolism in microalgae distinguishes itself[END_REF][START_REF] Merchant | You're it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation[END_REF][START_REF] Goold | Saturating light induces sustained accumulation of oil in plastidal lipid droplets in Chlamydomonas reinhardtii[END_REF]. Because glyceraldehyde 3-phosphate is a common precursor for both FA and starch synthesis, it has sometimes been suggested that there occurs a competition for carbon precursors between biosynthesis of starch and lipid. Following this idea, the carbon partitioning between starch and TAG upon N starvation has been intensively studied in the past 10 years, mostly based on the starchless Chlamydomonas mutant bafJ5 defected in the small catalytic subunit of ADP-glucose pyrophosphorylase (AGPase) [START_REF] Zabawinski | Starchless mutants of Chlamydomonas reinhardtii lack the small subunit of a heterotetrameric ADPglucose pyrophosphorylase[END_REF]. Some studies reported that there occurs a competition for carbon precursors between starch and lipid synthesis under N starvation [START_REF] Li | Inhibition of starch synthesis results in overproduction of lipids in Chlamydomonas reinhardtii[END_REF][START_REF] Li | Chlamydomonas starchless mutant defective in ADP-glucose pyrophosphorylase hyper-accumulates triacylglycerol[END_REF] yet the competition does not occur during N replete growth [START_REF] Krishnan | Metabolic and photosynthetic consequences of blocking starch biosynthesis in the green alga Chlamydomonas reinhardtii sta6 mutant[END_REF], while others reported that this competition is minor, if at all, even under N starvation [START_REF] Siaut | Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves[END_REF][START_REF] Vonlanthen | Evaluation of novel starch-deficient mutants of Chlorella sorokiniana for hyper-accumulation of lipids[END_REF].

The contrasting conclusions (above) could be due partly to the way the oil content is expressed (per cell versus per dry biomass), or due to the use of control strains (with or without cell wall), or due to cell culture conditions (light, CO2 level, cell growth stage). The reported difference could also be due to the fact that interplays between starch and lipids go beyond a mere competition for carbon allocation, and likely also involve a competition for energy (i.e.

ATP) and reducing equivalents. Cellular redox context varies depending on genetic as well as environmental factors (such as light quality/quantity, autotrophy versus mixotrophy, CO2

versus air, growth phases). Increasing evidence suggests the simultaneous occurrence of starch synthesis and turnover in the light [START_REF] Sparla | Redox regulation of a novel plastid-targeted beta-amylase of Arabidopsis[END_REF][START_REF] Baslam | Genetic and isotope ratio mass spectrometric evidence for the occurrence of starch degradation and cycling in illuminated Arabidopsis leaves[END_REF][START_REF] Daloso | Metabolism within the specialized guard cells of plants[END_REF], and starch breakdown eventually generates reduced carbons, phosphorylating power and reducing equivalent, impacting stromal redox balance and therefore anabolic reactions of starch and FA synthesis. Thus, a much more complex relationship between starch and lipid synthesis likely occurs at both the carbon and energetic level. The energetic aspects of interaction between starch and lipid accumulation have so far mostly been ignored and definitely beg further examination.

It is worth noting that, in addition to the defects at the AGPase locus, the bafj5 mutant harbors two additional mutations: i.e. a defect in cell wall synthesis and a defect in a gene encoding a respiratory burst oxidase [START_REF] Blaby | Systems-level analysis of nitrogen starvation-induced modifications of carbon metabolism in a Chlamydomonas reinhardtii starchless mutant[END_REF]. These additional mutations could impact lipid metabolism, either via competition for carbon precursors (in the case of the cell wall mutations), or via their effect on redox metabolism (in the case of the respiratory burst oxidase). But these hypotheses remain to be tested.

Relationship between FA synthesis and chrysolaminarin accumulation

Instead of starch, most photosynthetic heterokonts, including P. tricornutum and Thalassiosira pseudonana, accumulate chrysolaminarin, another type of storage polysaccharide [START_REF] Caballero | Quantification of chrysolaminarin from the model diatom Phaeodactylum tricornutum[END_REF][START_REF] Hildebrand | Effects of chrysolaminarin synthase knockdown in the diatom Thalassiosira pseudonana: Implications of reduced carbohydrate storage relative to green algae[END_REF][START_REF] Roessler | Changes in the activities of various lipid and carbohydrate biosynthetic enzymes in the diatom Cyclotella cryptica in response to silicon deficiency[END_REF].

Similar to starch, chrysolaminarin accumulates during the day and is mobilized at night, supporting its role as a source of carbohydrates for heterotrophic metabolism in the dark. Two differences are observed between starch and chrysolaminarin: i) starch is stored in chloroplasts, while chrysolaminarin is found in vacuoles [START_REF] Schreiber | The central vacuole of the diatom Phaeodactylum tricornutum: identification of new vacuolar membrane proteins and of a functional dileucine-based targeting motif[END_REF], and ii) in contrast to massive starch accumulation in N-starved green algal cells, N starvation does not seem to stimulate chrysolaminarin over-accumulation. Silencing the chrysolaminarin synthase gene in Thalassiosira pseudonana resulted only in a transient accumulation of TAG [START_REF] Hildebrand | Effects of chrysolaminarin synthase knockdown in the diatom Thalassiosira pseudonana: Implications of reduced carbohydrate storage relative to green algae[END_REF]. Considering the widespread interest in the use of diatoms for biofuel and biochemical applications, the development of a routine method for quantification of chrysolaminarin [START_REF] Caballero | Quantification of chrysolaminarin from the model diatom Phaeodactylum tricornutum[END_REF], and genome editing technologies for diatoms [START_REF] Huang | Genetic and metabolic engineering in diatoms[END_REF][START_REF] Daboussi | Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology[END_REF], we should expect many more studies on chrysolaminarin metabolism and its relation to lipid synthesis in the near future.

Regulation of FA synthesis

Due to the central importance of lipids in cell metabolism, physiology, and its interaction with the environment, FA synthesis is subjected to multi-level control.

Regulation at the level of ACCase

ACCase catalyzes the first and committed step in de novo FA synthesis and is known to be regulated by a myriad of mechanisms at both the transcriptional and post-transcriptional level.

Plant heteromeric ACCase is activated by light, chloroplast redox status (thioredoxin), and precursor supply, and is inhibited by acyl-ACP; as recently summarized [START_REF] Salie | Regulation and structure of the heteromeric acetyl-CoA carboxylase[END_REF]. It remains to be determined which of these regulatory mechanisms are conserved in various algae, and whether algae possess other unique ACCase regulatory mechanisms. We currently know very little about the regulation of homomeric ACCase, which is the exclusive ACCase isoform present in many algal taxa (see section 3.1.1). A better understanding of ACCase regulation promises to significantly advance algal lipid biotechnology. Here we will discuss the recent discovery of two new classes of proteins identified as negative regulators of plant ACCase: BADC and PII.

BADC is an abbreviation for the prior (but rather unfortunate) annotation of this protein as a "biotin attachment domain-containing" protein [START_REF] Salie | A family of negative regulators targets the committed step of de novo fatty acid biosynthesis[END_REF]. As mentioned in section 3.1.1, BADC is derived from BCCP but lacks the latter's conserved biotinylation motif and biotinyl-Lys residue. As a consequence, rather than acting as a carboxyl carrier, BADC acts as a negative regulator of heteromeric ACCase through its competition with BCCP for binding to the holo-ACCase complex [START_REF] Salie | A family of negative regulators targets the committed step of de novo fatty acid biosynthesis[END_REF]. Additionally, the BADC gene family may be partially responsible for the feedback regulation of ACCase [START_REF] Keereetaweep | Biotin attachment domain-containing proteins irreversibly inhibit acetyl CoA carboxylase[END_REF]. In Arabidopsis, BADCs are represented by three genes, and all three lack the conserved biotinylation motif and biotinyl-Lys residue. Gene silencing of BADC isoform 1 results in a slight, but significant increase in oil content in seeds of A. thaliana [START_REF] Salie | A family of negative regulators targets the committed step of de novo fatty acid biosynthesis[END_REF]. BADC is present in higher plants and in a limited subset of green algae (including Chlorella spp., Volvox spp. and Coccomyxa spp.) but is otherwise absent from eukaryotic algae with a single exception (Figure 2a), suggesting that it diverged from BCCP in green algae. The only non-chlorophyte BADC representative is found in the red alga Galdieria sulphuraria, the most extremophilic red algal species, which has obtained large numbers of archaeal and bacterial genes through horizontal gene transfer [START_REF] Schönknecht | Gene transfer from bacteria and archaea facilitated evolution of an extremophilic eukaryote[END_REF]. The BADC in G. sulphuraria appears likely to have resulted from such a horizontal transfer (perhaps from an ancestral green alga).

The second negative regulator of ACCase recently identified is PII (At4g01900), a small homotrimeric protein that acts at the interface of C and N metabolism [START_REF] Ninfa | PII signal transduction proteins: sensors of α-ketoglutarate that regulate nitrogen metabolism[END_REF]. PII, acting as a 2oxoglutarate sensor, inhibits ACCase via binding to the biotin portion of BCCP, and this inhibition can also be relieved by high pyruvate concentrations. The implication is that PII connects carbon and N metabolism by sensing 2-oxoglutarate, pyruvate, and possibly the broader energy and nitrogen (N) status of the chloroplast [START_REF] Feria Bourrellier | Chloroplast acetyl-CoA carboxylase activity is 2-oxoglutarate-regulated by interaction of PII with the biotin carboxyl carrier subunit[END_REF]. Algal PII, which is of bacterial origin, is, like heteromeric ACCase, present in algae with chloroplasts of primary endosymbiotic origin (i.e. Chlorophyta, Rhodophyta), the Paulinellidae, as well as in higher plants, but is absent from other eukaryotic algal taxa (Figure 2b). PII appears, however, to have been lost from many red algal taxa. As N-starvation has been shown to induce transcription of genes encoding algal ACCase subunits [START_REF] Miller | Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism[END_REF][START_REF] Schmollinger | Nitrogen-sparing mechanisms in Chlamydomonas affect the transcriptome, the proteome, and photosynthetic metabolism[END_REF][START_REF] Boyle | Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas[END_REF], it seems likely that additional mechanisms exist to coordinate N-status and ACCase activity in algae. It is worth noting that PII shares distant orthology with the N-fixation-related nifH from archeae, but plants have not retained a nifH-derived ortholog.

Transcriptional regulation

Although genes encoding the enzymes of lipid metabolism in microalgae can be predicted from those of plants (based on amino acid sequence identity) [START_REF] Liu | Lipid metabolism in microalgae distinguishes itself[END_REF][START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF], the regulatory mechanisms of lipid synthesis in algae cannot be as easily inferred. Drastic changes in transcript levels of many putative transcription factors (TFs) have been observed in N-deplete versus N-replete

Chlamydomonas cells, implying their potential involvement in regulation of lipid metabolism [START_REF] Miller | Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism[END_REF][START_REF] Schmollinger | Nitrogen-sparing mechanisms in Chlamydomonas affect the transcriptome, the proteome, and photosynthetic metabolism[END_REF][START_REF] Gargouri | Identification of regulatory network hubs that control lipid metabolism in Chlamydomonas reinhardtii[END_REF]. However, only a couple of them have been experimentally validated to play such a role. A putative zinc-finger protein (Cre14.g624800) has been identified as a regulator of stress-induced lipid synthesis, and overexpression or silencing of the corresponding gene results in altered lipid content [START_REF] Yohn | Stress-induced lipid trigger[END_REF]. The regulatory mechanisms and downstream molecular targets of this protein remain, however, to be deciphered. A SQUAMOSA promoter-binding protein domain transcription factor was recently identified in C. reinhardtii, and it was named as N response regulator 1 (NRR1) and the insertional knockout mutants accumulated only half amount of the TAG usually found in WT strains upon N starvation, but not under other nutrient stresses (S, P, or Zn) [START_REF] Boyle | Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas[END_REF]. A correlation between the level of transcription of NRR1 and that of a major DGAT1 has been observed, but several uncertainties remain. At a molecular mechanistic level, it remains to be determined what the targets of the NRR1 are; and from a biotechnological perspective, it is unknown if overexpression of NRR1 could impact lipid production.

Another relatively well-studied transcription factor implicated in lipid metabolism is the phosphorus starvation response 1 (PSR1). PSR1 belongs to the MYB-CC (MYB coiled-coil domain) transcription factor family and was originally described as a component of the phosphate starvation pathway [220]. Two recent studies have suggested the role of PSR1 in regulation of lipid metabolism in C. reinhardtii [START_REF] Ngan | Lineage-specific chromatin signatures reveal a regulator of lipid metabolism in microalgae[END_REF][START_REF] Bajhaiya | PSR1 is a global transcriptional regulator of phosphorus deficiency responses and carbon storage metabolism in Chlamydomonas reinhardtii[END_REF]. Ngan et al [START_REF] Ngan | Lineage-specific chromatin signatures reveal a regulator of lipid metabolism in microalgae[END_REF] showed that oil content is positively correlated to the expression level of PSR1, which were altered by creating knock-out or overexpressor lines. Bajhaiya et al [START_REF] Bajhaiya | PSR1 is a global transcriptional regulator of phosphorus deficiency responses and carbon storage metabolism in Chlamydomonas reinhardtii[END_REF] further showed that PSR1 is not only regulating lipid synthesis but also starch synthesis. PSR1 overexpression lines showed increased starch content but reduced neutral lipid content under P starvation, and the phenotype is persistent regardless of the acetate status. The reason for the contradictory changes in lipid content in PSR1 overexpressors is not clear, but could be due to the use of different nutrient stress (N versus P). In summary, PSR1 likely plays a role in the regulation of global metabolism, but not specifically limited to lipid synthesis.

In addition to the studies carried out in C. reinhardtii, a basic helix-loop-helix (bHLH)

TF and a basic leucine zipper (bZIP)-domain containing TF have been identified from N. salina, and their overexpression led to an increase in both growth and lipid productivity [START_REF] Kang | Effects of overexpression of a bHLH transcription factor on biomass and lipid production in Nannochloropsis salina[END_REF][START_REF] Kwon | Enhancement of biomass and lipid productivity by overexpression of a bZIP transcription factor in Nannochloropsis salina[END_REF].

Therefore those TFs identified here could serve as genetic engineering targets for improving the production of biofuels and biomaterials in algae. Furthermore, overexpression of known plant TFs, for example the Dof-type (DNA binding with one finger) TF in C. reinhardtii [START_REF] Ibanez-Salazar | Over-expression of Dof-type transcription factor increases lipid production in Chlamydomonas reinhardtii[END_REF] and the Arabidopsis AtWRI1 in N. salina [START_REF] Kang | Increased lipid production by heterologous expression of AtWRI1 transcription factor in Nannochloropsis salina[END_REF], have resulted in transgenic strains with increased lipid production. These studies suggest the conserved nature of some of these regulatory mechanisms between plants and algae.

Regulation by kinases and other subcellular processes

Alongside TFs, a given pathway can also be regulated by other mechanisms [START_REF] Ngan | Lineage-specific chromatin signatures reveal a regulator of lipid metabolism in microalgae[END_REF]. For example, alterations of lipid content have also been observed in knock-out mutants of C. reinhardtii for two members of the dual-specificity tyrosine-phosphorylation-regulated (DYRK) kinase, i.e.

the plant specific DYRKP [START_REF] Schulz-Raffelt | Hyperaccumulation of starch and oil in a Chlamydomonas mutant affected in a plant-specific DYRK kinase[END_REF] and the Chlamydomonas triacylglycerol accumulation regulator1 (TAR1) -an orthologue of the yeast Yet another kinase1 (Yak1) subfamily [START_REF] Kajikawa | Algal dual-specificity tyrosine phosphorylation-regulated kinase, triacylglycerol accumulation regulator1, regulates accumulation of triacylglycerol in nitrogen or sulfur deficiency[END_REF].

Lipid production in N. gaditana is doubled by knocking out a homolog of fungal Zn(II)2Cys6 encoding a transcriptional regulator of N assimilation pathways [START_REF] Ajjawi | Lipid production in Nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator[END_REF]. Furthermore, manipulation of the target of rapamycin (TOR) or nitric oxide (NO) signaling pathway is also shown to impact lipid production in algae [START_REF] Imamura | Target of rapamycin (TOR) plays a critical role in triacylglycerol accumulation in microalgae[END_REF][START_REF] Dolch | Nitric oxide mediates nitrite-sensing and acclimation and triggers a remodeling of lipids[END_REF][START_REF] Prioretti | The inhibition of TOR in the model diatom Phaeodactylum tricornutum promotes a get-fat growth regime[END_REF]. It is worth noting that molecular targets or the regulatory circuits of the above regulatory proteins related to lipid metabolism have not yet been worked out, and biochemical or molecular research in this direction is needed. For additional details on the relation between autophagy and lipid synthesis, readers are referred to [START_REF] Kong | Lipid catabolism in microalgae[END_REF][START_REF] Wang | Algal lipid bodies: stress induction, purification, and biochemical characterization in wild-type and starchless Chlamydomonas reinhardtii[END_REF][START_REF] Perez-Perez | Autophagy in the model alga Chlamydomonas reinhardtii[END_REF][START_REF] Couso | Autophagic flux is required for the synthesis of triacylglycerols and ribosomal protein turnover in Chlamydomonas[END_REF].

Glycerolipid synthesis

As noted in section 2, eukaryotic algae contain phosphoglycerides, glycosylglycerides and, often, betaine lipids in significant amounts. Because algae carry out oxygen-evolving photosynthesis, their thylakoid membranes contain four lipids also typical of plants and cyanobacteria -MGDG, DGDG, SQDG and PtdGro. When present, betaine lipids are in extrachloroplast membranes. DGTS is found in many green algae while DGTA and DGCC are found in different algal species such as brown algae (Phaeophyceae). Algae with significant betaine lipids usually have little or no phosphatidylcholine (PtdCho) [START_REF] Kalisch | DGDG and glycolipids in plants and algae[END_REF]. Of the phosphoglycerides apart from phosphatidylglycerol (PtdGro), these are in extraplastidial membranes. PtdCho and phosphatidylethanolamine (PtdEtn) are usually the most significant while phosphatidylinositol (PtdIns) and phosphatidylserine (PtdSer) are minor components.

A brief discussion of the origins of algal chloroplasts and the differences between primary plastids (where the two plastid membranes were derived from the cyanobacterial endosymbiont) and complex plastids, which are surrounded by three or four membranes, is

given in [START_REF] Kalisch | DGDG and glycolipids in plants and algae[END_REF]. Genes involved in glycosylglyceride synthesis often show strong similarity to those of higher plants. These include MGD1, DGD1, SQD1 and SQD2. Indeed, DGDG synthases in eukaryotic algae have been shown to be similar to the plant type enzymes [START_REF] Riekhof | The sulfolipids 2′-O-Acylsulfoquinovosyldiacylglycerol and sulfoquinovosyldiacylglycerol are absent from a Chlamydomonas reinhardtii mutant deleted in SQD1[END_REF][START_REF] Sato | Isolation and characterization of mutants affected in lipid-metabolism of Chlamydomonas reinhardtii[END_REF]. However, analysis of chloroplasts is often complicated by the extra membranes in secondary plastids [START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids[END_REF].

The Glaucophyta are a small group of rare freshwater algae. Only 13 species are known, none of which is particularly common. Along with red algae, they harvest light through phycobilisomes and also store fixed carbon in the cytosol. There is rather little information about their lipids and, indeed, of their classification---mainly because they have been little studied. They contain the three glycosylglycerides (MGDG, DGDG, SQDG) [START_REF] Leblond | Mono-and digalactosyldiacylglycerol composition of glaucocystophytes (Glaucophyta): A modern interpretation using positive-ion electrospray ionization/mass spectrometry/mass spectrometry[END_REF] but there is little consistent information about genes encoding enzymes for MGDG and DGDG formation.

It is thought, however, that SQDG synthesis is similar to that in plants and Chlamydomonas [START_REF] Kalisch | DGDG and glycolipids in plants and algae[END_REF].

Glycosylglyceride synthesis has been studied most in red and green algae, especially

Chlamydomonas. Overall the lipid and fatty acid composition of Chlorophyta resembles that of higher plants. Interestingly, C. reinhardtii has some unusual fatty acids -16:44,7,10,13, pinolenic acid (18:35,12) and coniferonic acid (18:45,9,12,15) [START_REF] Sato | Isolation and characterization of mutants affected in lipid-metabolism of Chlamydomonas reinhardtii[END_REF]. An 13-desaturase is responsible for the 5 double bonds introduced into linoleic and α-linolenic acids [START_REF] Kajikawa | A front-end desaturase from Chlamydomonas reinhardtii produces pinolenic and coniferonic acids by omega 13 desaturation in methylotrophic yeast and tobacco[END_REF]. A 4desaturase uses 16:3 (7,10,13) bound to MGDG as a substrate to form 16:4 [START_REF] Zauner | A cytochrome b(5)-containing plastid-located fatty acid desaturase from Chlamydomonas reinhardtii[END_REF] and may be important in controlling overall MGDG synthesis. On the other hand, VLCPUFAs such as arachidonic acid (ARA) or EPA are usually only present in small amounts or absent from green freshwater algae. An exception is Lobosphaera (formerly Parietochloris) incisa which is an oleaginous species that has high amounts of ARA in its membrane lipids and TAG (see section 7). In contrast, both ARA and EPA are often present in low proportions in marine green algae.

For example, in Ulva fenestrata they are found in significant quantities in DGTS but not in glycosylglycerides [START_REF] Thompson | Lipids and membrane function in green algae[END_REF].

Diatoms are the most abundant phytoplankton species and, consequently, are major producers at the bottom of food chains. They occur ubiquitously in freshwater and marine habitats. Their metabolism, including the production of VLCPUFAs (mainly EPA) through lipid-linked desaturases has been detailed by [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF].

One matter which should be borne in mind when describing acyl lipid synthesis in algae is that the biochemistry of the reactions has lagged behind the identification of genes encoding the putative enzymes involved. Thus, in review papers describing metabolism of acyl lipids in diatoms [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF] or in C. reinhardtii [START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF] it will be seen that, while genes for most of the enzymes concerned have been identified, functional proof of their activity (and substrate specificity) is much less clear. Nevertheless, for now it can be assumed that the pathways identified in higher plants [START_REF] Bates | Biochemical pathways in seed oil synthesis[END_REF][START_REF] Weselake | Increasing the flow of carbon into seed oil[END_REF][START_REF] Lu | New frontiers in oilseed biotechnology: meeting the global demand for vegetable oils for food, feed, biofuel, and industrial applications[END_REF] are on the whole followed in eukaryotic algae though, perhaps, in a simpler form with less genetic redundancy [START_REF] Liu | Lipid metabolism in microalgae distinguishes itself[END_REF](Figure 4). In an overall survey by comparative genomics and subcellular localisation, it was concluded that the pathways for acyl lipid metabolism in the unicellular red alga Cyanidioschyzon merolae were essentially similar to Arabidopsis [START_REF] Mori | Construction of global acyl lipid metabolic map by comparative genomics and subcellular localization analysis in the red alga Cyanidioschyzon merolae[END_REF].

Although, as noted above, there has been relatively little biochemistry carried out on the algal enzymes used for acyl lipid formation and the reader is referred to previous work to serve as a background [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF]. For example, when studying the distribution of different acyl lipids (and their molecular species as being 'prokaryotic' or 'eukaryotic' in origin: see [245]), Eichenberger's group found that MGDG, DGDG, SQDG and PtdGro in C. reinhardtii were clearly of plastidic ('prokaryotic') origin while DGTS and PtdEtn, enriched in 18:3(5,9,12) and 18:4(5,9,12,15) were of 'eukaryotic' origin [START_REF] Giroud | Lipids of Chlamydomonas reinhardtii -analysis of molecular species and intracellular site(s) of biosynthesis[END_REF]. These data were followed by radiolabelling experiments to study the time-course of their metabolism which identified, for example, that lipid-linked desaturation on DGTS could give rise to PUFA formation on the sn-2 position while, for MGDG and DGDG, desaturation was at the sn-1 position [START_REF] Giroud | Lipids of CHlamydomonas reinhardtii -incorporation of C-14 acetate, C-14 palmitate and C-14 oleate into different lipids and evidence for lipid-linked desaturation of fatty acids[END_REF]. Similar radiolabelling experiments in other eukaryotic algae are summarised by [START_REF] Harwood | Lipid metabolism in algae[END_REF] and, later, by [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF].

The Kennedy Pathway

Glycerolipids are synthesized using what is commonly known as the Kennedy pathway (Figure 5). This is named after Eugene P. Kennedy who discovered and characterised many of the individual reactions for phosphoglyceride formation, although the first two (acylation) reactions were originally reported by Kornberg and Pricer [245]). The penultimate intermediate of the Kennedy pathway, DAG, is used to form zwitterionic phosphoglycerides (PtdCho, PtdEtn), TAG and the galactosylglycerides MGDG and DGDG (Figure 5).

The relative contributions of 'eukaryotic' and 'prokaryotic' pathways of glycerolipid synthesis has been discussed further by [START_REF] Xu | Cellular organization of triacylglycerol biosynthesis in microalgae[END_REF]. They point to the use of the 'prokaryotic'

pathway by Chlamydomonas because it lacks thylakoid lipids with 18C acids at the sn-2 position [START_REF] Harwood | The versatility of algae and their lipid metabolism[END_REF][START_REF] Giroud | Lipids of Chlamydomonas reinhardtii -analysis of molecular species and intracellular site(s) of biosynthesis[END_REF][START_REF] Giroud | Lipids of CHlamydomonas reinhardtii -incorporation of C-14 acetate, C-14 palmitate and C-14 oleate into different lipids and evidence for lipid-linked desaturation of fatty acids[END_REF]. But this has recently been challenged by the discovery of an ERlocated lysophosphatidate acyltransferase (LPAAT) with substrate preference of 16C rather than an 18:0 FA at its sn-2 position in C. reinhardtii [START_REF] Kim | Endoplasmic reticulum acyltransferase with prokaryotic substrate preference contributes to triacylglycerol assembly in Chlamydomonas[END_REF]. In addition, a chloroplast pathway for TAG formation in C. reinhardtii was recently reported [START_REF] Fan | A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii[END_REF]. In contrast, galactolipids of Dictyopteris mambranacea [START_REF] Hofmann | Lipid and fatty acid composition of the marine brown alga dictyopteris membranacea[END_REF] and several other brown algal species [START_REF] Jones | Lipid composition of the brown algae fucus vesiculosus and Ascophyllum nodosum[END_REF] are almost completely of the 'eukaryotic' type. On the other hand, green algae such as Chlorella kessleri and Acetabularia mediterranea and some red and brown algae seem to employ two parallel pathways of lipid formation [START_REF] Xu | Cellular organization of triacylglycerol biosynthesis in microalgae[END_REF].

Because Chlamydomonas lacks PtdCho, which is known to be intimately involved in the 'eukaryotic' pathway in plants, the lack of such synthesis in C. reinhardtii has been suggested to be explained by the lack of PtdCho [START_REF] Riekhof | Chapter 2 -Glycerolipid biosynthesis[END_REF]. However, as pointed out by [START_REF] Xu | Cellular organization of triacylglycerol biosynthesis in microalgae[END_REF],

several brown algae that lack PtdCho have thylakoid lipids made by the 'eukaryotic' pathway.

This suggests that PtdCho is not essential for the 'eukaryotic' pathway of chloroplast lipid formation.

Three pathways have been suggested for the accumulation of a 'prokaryotic' type of TAG in the cytosol of algae such as Chlamydomonas or Dunaliella bardawil [START_REF] Xu | Cellular organization of triacylglycerol biosynthesis in microalgae[END_REF]. The first pathway forms TAG at the chloroplast envelope while, in the second pathway, TAG is assembled in the ER using DAG exported from the plastid. In the third pathway, both DAG and TAG are formed on the ER. For the latter, specific mechanisms are needed to channel 16C fatty acids onto the sn-2 position of DAG [START_REF] Xu | Cellular organization of triacylglycerol biosynthesis in microalgae[END_REF].

Before describing the Kennedy pathway and its constituent enzymes in detail, it is timely to mention the recent use of metabolomics for the study of lipid synthesis in algae. For example, Juppner et al [START_REF] Juppner | Dynamics of lipids and metabolites during the cell cycle of Chlamydomonas reinhardtii[END_REF] used GC-MS to study polar metabolites and lipids in synchronous cultures of C. reinhardtii. Although this paper was mainly a technical advance, this proof-of-concept study has the potential to be used for further in depth metabolic phenotyping and the identification of biomarkers for various cellular processes, at least in C. reinhardtii.

The Kennedy pathway in detail and phosphoglyceride formation

The Kennedy pathway (Figure 5) begins with glycerol 3-phosphate (G3P). This intermediate is produced by reduction of dihydroxyacetone phosphate (DHAP) derived from photosynthesis and/or starch degradation [START_REF] Xue | Glucose-6phosphate dehydrogenase as a target for highly efficient fatty acid biosynthesis in microalgae by enhancing NADPH supply[END_REF]. Under normal growth conditions the supply of G3P can be one controlling factor that may influence total lipid synthesis (and oil accumulation). However, under osmotic stress, hydrolysis of glycerolipids can also take place, leading to an accumulation of glycerol [START_REF] Borowitzka | The salt relations of Dunaliella. Further observations on glycerol production and its regulation[END_REF]. Moreover, the conversion of G3P back to DHAP under salt stress is also well known [START_REF] Xue | Glucose-6phosphate dehydrogenase as a target for highly efficient fatty acid biosynthesis in microalgae by enhancing NADPH supply[END_REF][START_REF] Borowitzka | The salt relations of Dunaliella. Further observations on glycerol production and its regulation[END_REF]. The synthesis and degradation of G3P in Dunaliella salina under extracellular salt stress has been well studied (see [START_REF] Goyal | Osmoregulation in Dunaliella, Part II: Photosynthesis and starch contribute carbon for glycerol synthesis during a salt stress in Dunaliella tertiolecta[END_REF][START_REF] Cai | Molecular clone and expression of a NAD+-dependent glycerol-3phosphate dehydrogenase isozyme gene from the halotolerant alga Dunaliella salina[END_REF]).

Proteomic [START_REF] Song | Proteomic analysis in nitrogen-deprived Isochrysis galbana during lipid accumulation[END_REF] and transcriptome analysis [START_REF] Goodenough | The path to triacylglyceride obesity in the sta6 strain of Chlamydomonas reinhardtii[END_REF][START_REF] Schmollinger | Nitrogen-sparing mechanisms in Chlamydomonas affect the transcriptome, the proteome, and photosynthetic metabolism[END_REF][START_REF] Lv | Transcriptome analysis of Chlamydomonas reinhardtii during the process of lipid accumulation[END_REF][START_REF] Zhang | High-throughput genotyping of green algal mutants reveals random distribution of mutagenic insertion sites and endonucleolytic cleavage of transforming DNA[END_REF] have shown that the activity of cytosolic glycerol 3-phosphate dehydrogenase (GPDH) is positively correlated with TAG accumulation in algae. Moreover, overexpression of GPDH in P. tricornutum [START_REF] Yao | Glycerol and neutral lipid production in the oleaginous marine diatom Phaeodactylum tricornutum promoted by overexpression of glycerol-3phosphate dehydrogenase[END_REF] and C. reinhardtii [START_REF] Herrera-Valencia | Naturally occurring fatty acid methyl esters and ethyl esters in the green microalga Chlamydomonas reinhardtii[END_REF] increased lipid production. A multiple gene engineering strategy, including overexpression of glycerol kinase, GPDH and acetyl-CoA carboxylase, in Scenedesmus quadriauela increased the G3P pool and was paralleled by an increase in oil content [START_REF] Gomma | Improvement in oil production by increasing Malonyl-CoA and glycerol-3-phosphate pools in Scenedesmus quadricauda[END_REF].

These data suggest that augmentation of G3P levels may be important to enhance lipid accumulation [START_REF] Xue | Implications of glycerol metabolism for lipid production[END_REF].

An interesting recent observation with C. reinhardtii is the regulation of a gene GPD2 that encodes a multi-domain enzyme with GPDH and phosphoserine phosphatase activities.

This enzyme is, therefore, capable of synthesising either G3P or glycerol, depending on environmental conditions and/or metabolic demands [START_REF] Morales-Sanchez | A multidomain enzyme, with glycerol-3-phosphate dehydrogenase and phosphatase activities, is involved in a chloroplastic pathway for glycerol synthesis in Chlamydomonas reinhardtii[END_REF].

Candidate genes for the expression of chloroplast-localised glycerol 3-phosphate acyltransferase (GPAT) in C. reinhardtii were listed by [START_REF] Riekhof | Chapter 2 -Glycerolipid biosynthesis[END_REF] and by [START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF]. For diatoms, a gene for GPAT was predicted from the Phaeodactylum tricornutum genome [START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF] and molecular characterisation carried out [START_REF] Niu | Molecular characterization of a glycerol-3-phosphate acyltransferase reveals key features essential for triacylglycerol production in Phaeodactylum tricornutum[END_REF]. The gene was overexpressed in P. tricornutum and resulted in a two-fold increase in non-polar lipids (presumably TAG). This altered the overall fatty acid composition in the transgenics with a significant decrease in saturated acids and an increase in PUFA. This was interesting since PUFA such as EPA are thought to be synthesised on the ER [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF] even though the GPAT was a chloroplast enzyme [START_REF] Niu | Molecular characterization of a glycerol-3-phosphate acyltransferase reveals key features essential for triacylglycerol production in Phaeodactylum tricornutum[END_REF]. A membrane-bound GPAT was characterised from the marine diatom Thalassiosira pseudonana and found to regulate the acyl composition of glycerolipids when expressed in a GPAT-lacking mutant of yeast [START_REF] Xu | A membrane-bound glycerol-3-phosphate acyltransferase from Thalassiosira pseudonana regulates acyl composition of glycerolipids[END_REF] with increases in 16:0 and decreases in 16:1 and 18:1 in both TAG and phosphoglycerides.

A GPAT-like gene (with sequence similarity to Arabidopsis GPAT9), was identified in the freshwater trebouxiophyte Lobosphaera (formerly Parietochloris) incisa which, when overexpressed in C. reinhardtii, increased TAG production [268]. The same alga was examined by

Ouyang et al. [START_REF] Ouyang | Site-directed mutagenesis from Arg195 to His of a microalgal putatively chloroplastidial glycerol-3-phosphate acyltransferase causes an increase in phospholipid levels in yeast[END_REF], who considered it localised to chloroplasts. Substitution of Arg to His in the glycerol 3-phosphate binding site increased the enzyme's activity and led to raised phospholipid levels when expressed in yeast.

Two candidate genes for LPAAT were noted in C. reinhardtii [START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF]. The CrLPAAT1 was reported to be located the chloroplast with a preference for 16C at its sn-2 position [270],

whereas LPAAT2 has recently been identified as the ER isoform but also with a preference of 16C at its sn-2 position [START_REF] Kim | Endoplasmic reticulum acyltransferase with prokaryotic substrate preference contributes to triacylglycerol assembly in Chlamydomonas[END_REF]. Both are found to be implicated in TAG synthesis, because plastidial over-expression of CrLPAAT1 increased oil content [270], and silencing of CrLPAAT2 reduced oil content during N starvation [START_REF] Kim | Endoplasmic reticulum acyltransferase with prokaryotic substrate preference contributes to triacylglycerol assembly in Chlamydomonas[END_REF]. The potential regulatory role of AGPAT (acylglycerolphosphate acyltransferase) in Phaeodactylum tricornutum for TAG synthesis has been discussed [START_REF] Balamurugan | Occurrence of plastidial triacylglycerol synthesis and the potential regulatory role of AGPAT in the model diatom Phaeodactylum tricornutum[END_REF]. A genome-wide analysis of LPAAT genes has been carried out in algae [START_REF] Misra | Genome-wide identification and evolutionary analysis of algal LPAT genes involved in TAG biosynthesis using bioinformatic approaches[END_REF] and in Nannochloropsis two differently-localised enzymes have been found, both of which are needed for TAG formation [START_REF] Nobusawa | Differently localized lysophosphatidic acid acyltransferases crucial for triacylglycerol biosynthesis in the oleaginous alga Nannochloropsis[END_REF].

Phosphatidic acid, the product of LPAAT activity, is at a branch-point of the Kennedy pathway (Figure 5) where it can be converted to CDP-DAG for the formation of anionic phosphoglycerides, PtdIns, PtdGro and DiPtdGro (diphosphatidylglycerol, cardiolipin).

Putative genes for phosphatidate cytidylyltransferase, phosphatidylglycerolphosphate (PGP) synthase and phosphatidylinositol synthase were described in C. reinhardtii [START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF][START_REF] Riekhof | Chapter 2 -Glycerolipid biosynthesis[END_REF] and one for phosphatidylglycerol synthase noted in P. tricornutum [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF] but, until recently, there has been a dearth of work on the synthesis of such anionic phospholipids in eukaryotic algae except for indirect studies on inoculum size in altering phosphoglyceride profiling [START_REF] Lu | Phospholipid metabolism in an industry microalga Chlorella sorokiniana: the impact of inoculum sizes[END_REF] and the work on both PGP synthase and PGP phosphatase 1 from C. reinhardtii [START_REF] Hung | Characterization of Chlamydomonas reinhardtii phosphatidylglycerophosphate synthase in Synechocystis sp. PCC 6803[END_REF][START_REF] Hung | Isolation and characterization of a phosphatidylglycerophosphate phosphatase1, PGPP1, in Chlamydomonas reinhardtii[END_REF]. Two homologues of PGP synthase (CrPGP1 and CrPGP2) were isolated and characterised from C. reinhardtii [START_REF] Hung | Characterization of Chlamydomonas reinhardtii phosphatidylglycerophosphate synthase in Synechocystis sp. PCC 6803[END_REF]. Their function was demonstrated by complementation of a mutant of Synechocystis sp.

PCC 6803. In nutrient-starved algae, expression of both homologues was decreased by Pdepletion at 4h but restored after 5 days. In contrast, while CrPGP1 was reduced by N-depletion, expression of CrPGP2 was unaffected.

For PGP phosphatase (PGPP), the gene was isolated from C. reinhardtii and its function shown using a yeast mutant [START_REF] Hung | Isolation and characterization of a phosphatidylglycerophosphate phosphatase1, PGPP1, in Chlamydomonas reinhardtii[END_REF]. While two aspartate residues were essential in yeast PGPP, only the first was needed for function in the algal phosphatase, despite conservation of the putative catalytic motif.

When C. reinhardtii was grown in sulphur-deficient medium, synthesis of PtdGro was enhanced and the accumulating phospholipid compensated for the loss of negatively-charged SQDG. Similar activation of PtdGro synthesis was also observed in a SQDG-deficient mutant under S-replete growth conditions. The data were suggested to indicate a critical role for PtdGro under S-starved conditions in the maintenance of Photosystem I activity [START_REF] Sugimoto | Upregulation of PG synthesis on sulfurstarvation for PS I in Chlamydomonas[END_REF]. In addition, a role for PtdGro in Photosystem II was suggested in two PtdGro-deficient mutants of C.

reinhardtii [START_REF] Pineau | A single mutation that causes phosphatidylglycerol deficiency impairs synthesis of photosystem II cores in Chlamydomonas reinhardtii[END_REF]. In these mutants there was a marked reduction of PtdGro and a complete loss of its 3-16:1 component. This unique fatty acid is confined to PtdGro in higher plants where there have been studies on its enigmatic function and localisation (see [START_REF] Unitt | Sidedness studies of thylakoid phosphatidylglycerol in higher plants[END_REF][START_REF] Trémolières | Restoration of thylakoid appression by Δ3-trans-hexadecenoic acid-containing phosphatidylglycerol in a mutant of Chlamydomonas reinhardtii. Relationships with the regulation of excitation energy distribution[END_REF]). In algae, it is likely to have similar properties but more research is needed to define these.

Diphosphatidylglycerol (DiPtdGro, cardiolipin) is a characteristic lipid component of the inner mitochondrial membrane, as originally demonstrated in plants (see [START_REF] Harwood | Plant mitochondrial lipids: structure, function and biosynthesis[END_REF]. Cardiolipin synthase (CLS) was identified in C. reinhardtii [START_REF] Hung | Functional specificity of cardiolipin synthase revealed by the identification of a cardiolipin synthase CrCLS1 in Chlamydomonas reinhardtii[END_REF] when it rescued a CLS-mutant of yeast.

The sequence for the gene was similar to that from other eukaryotes suggesting that the C.

reinhardtii CLS catalyses a reaction using CDP-DAG as opposed to the E.coli enzyme which uses two molecules of PtdGro [245].

For the formation of the zwitterionic phosphoglycerides, PtdCho and PtdEtn, phosphatidate needs to be dephosphorylated to DAG. Genes for eleven putative plastid phosphatidate phosphatases (PAP) were identified in C. reinhardtii [START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF] and two in P.

tricornutum [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF]. The mRNA level of the CrPAP2 isoform was found to increase in C.

reinhardtii grown in nitrogen-limiting conditions. RNA interference of the gene reduced total lipid by up to 17% while overexpression caused an increase, indicating that PAP activity can regulate lipid accumulation. The CrPAP2 enzyme showed PAP activity when expressed in E.coli [START_REF] Deng | Involvement of phosphatidate phosphatase in the biosynthesis of triacylglycerols in Chlamydomonas reinhardtii[END_REF].

Once DAG has been formed in the Kennedy pathway it can be used for PtdCho and PtdEtn production as well as the synthesis of glycosylglycerides and TAG (Figure 5). For the biosynthesis of PtdCho and PtdEtn by the Kennedy pathway, the cytidylyltransferase reaction is considered to show the most flux control, at least in higher plants [START_REF] Price-Jones | The control of CTP:choline-phosphate cytidylyltransferase activity in pea (Pisum sativum L.)[END_REF] and animals [245].

The CTP: phosphoethanolamine cytidylyltransferase has been studied in C. reinhardtii [START_REF] Yang | Membrane lipid biosynthesis in Chlamydomonas reinhardtii: ethanolaminephosphotransferase is capable of synthesizing both phosphatidylcholine and phosphatidylethanolamine[END_REF].

It showed a typical signature peptide sequence for the cytidylyltransferase family and was probably localised to mitochondria. It showed cell cycle fluctuations with high activity in the dark. The same group also identified a cDNA encoding CDP-ethanolamine phosphotransferase and expressed it in a yeast mutant deficient in both choline-and ethanolamine phosphotransferase activity and found that it had both activities. This was notable since C.

reinhardtii only contains PtdEtn and the PtdCho is replaced by DGTS in this alga. Other kinetic properties of the expressed enzyme were measured and parallels with the higher plant enzyme noted [START_REF] Yang | Membrane lipid biosynthesis in Chlamydomonas reinhardtii: expression and characterization of CTP : phosphoethanolamine cytidylyltransferase[END_REF]. In Chlamydomonas, no gene for PtdSer decarboxylase has been found and, therefore, it can be assumed that PtdEtn is made exclusively by the Kennedy pathway using CDP-Etn [START_REF] Riekhof | Chapter 2 -Glycerolipid biosynthesis[END_REF].

As noted in section 2, PtdCho is found in most algae but not in a few species, of which C. reinhardtii is one. However, some species of the Chlamydomonas genus do contain PtdCho [START_REF] Sakurai | Detection and characterization of phosphatidylcholine in various strains of the genus Chlamydomonas (Volvocales, Chlorophyceae)[END_REF]. Accordingly, Sato et al [START_REF] Sato | Diverse pathways of phosphatidylcholine biosynthesis in algae as estimated by labeling studies and genomic sequence analysis[END_REF] studied the biosynthesis of PtdCho in these algae. In plants PtdCho can be made by the CDP-base pathway as well as by methylation in three steps from PtdEtn [245]. There are also some extra methylation routers (see e.g. [START_REF] Williams | Alternative pathways for phosphatidylcholine synthesis in olive (Olea europaea L.) callus cultures[END_REF] Sato et al [START_REF] Sato | Diverse pathways of phosphatidylcholine biosynthesis in algae as estimated by labeling studies and genomic sequence analysis[END_REF] used radiolabelling studies in conjunction with comparative genomics to elucidate the pathways in three Chlamydomonas species together with the red alga Cyanidioschyzon merolae. Their results are shown in Figure 6 and revealed that both C. sphaeroides and C. merolae form PtdCho from PtdEtn by methyl transfers. In Chlamydomonas asymmetrica, PtdCho can be made by the CDP-base pathway or by methylation, as well as the intermediate conversion ofphosphoethanolamine to phosphocholine. These data revealed an unexpected diversity in the ability of Chlamydomonas strains to synthesise PtdCho [START_REF] Sato | Diverse pathways of phosphatidylcholine biosynthesis in algae as estimated by labeling studies and genomic sequence analysis[END_REF]. Presumably, this will also be reflected in other algae.

Transcriptional analysis (during N stress) of N. oceanica indicated that PtdEtn is synthesised be two distinct pathways (PtdSer decarboxylation as well as the Kennedy pathway).

In fact, PtdSer was below detection levels in this alga, indicating that it mainly served as a precursor for PtdEtn formation [START_REF] Han | Metabolic remodeling of membrane glycerolipids in the microalga Nannochloropsis oceanica under nitrogen deprivation[END_REF]. Such observations make the need for some biochemistry (enzymology) even more urgent.

One subject that should be emphasised when discussing acyl lipid biosynthesis is the fact that individual classes have distinct and usually well-preserved fatty acid compositions.

This was eluded to in section 2 but a particular example in C. reinhardtii is shown in Figure 7.

Other algae also show distinct patterns and, of course, these may be quite different to C.

reinhardtii [START_REF] Liu | Triacylglycerol profiling of microalgae Chlamydomonas reinhardtii and Nannochloropsis oceanica[END_REF]. So far as the three main phosphoglycerides are concerned, PtdEtn is more unsaturated than PtdGro, which often contains species with the unique trans-3-hexadecenoic acid. For algae containing both PtdCho and PtdEtn, then these classes tend to have a rather similar lipid composition [START_REF] Jones | Lipid composition of the brown algae fucus vesiculosus and Ascophyllum nodosum[END_REF]. Presumably this reflects either the formation of both PtdCho and PtdEtn from the same DAG pool or the conversion of PtdEtn to PtdCho by methylation, as discussed above.

Formation of DAG also allows biosynthesis of glycosylglycerides (Figure 4), which are the main membrane constituents of chloroplast thylakoids.

Biosynthesis of glycosylglycerides

The enzyme synthesising MGDG uses DAG and UDP-galactose substrates and is referred to as MGDG synthase. Only one enzyme is found in Chlorophyta (e.g. C. reinhardtii) in contrast to higher plants where there are three MGD genes [START_REF] Riekhof | Annotation of genes involved in glycerolipid biosynthesis in Chlamydomonas reinhardtii: Discovery of the betaine lipid synthase BTA1(Cr)[END_REF][START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids[END_REF]. Similarly, one DGDG synthase is found in C. reinhardtii [START_REF] Riekhof | Annotation of genes involved in glycerolipid biosynthesis in Chlamydomonas reinhardtii: Discovery of the betaine lipid synthase BTA1(Cr)[END_REF] and many other green algae [START_REF] Kalisch | DGDG and glycolipids in plants and algae[END_REF]. Although DGDG synthase also uses a UDP-galactose substrate the galactose link on DGDG is α-anomeric whereas the first galactose (on MGDG) is in the β-anomeric configuration. The DGD in C. reinhardtii resembles DGD1 of higher plants [START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids[END_REF][START_REF] Ulvskov | Classification, naming and evolutionary history of glycosyltransferases from sequenced green and red algal genomes[END_REF] but a second isoform is additionally found in Ostreococcus tauri, which resembles the plant DGD2 [START_REF] Ulvskov | Classification, naming and evolutionary history of glycosyltransferases from sequenced green and red algal genomes[END_REF]. An interesting short review discusses the pathways for glycosylglyceride synthesis in cyanobacteria and different algae with reference to the endosymbiotic origin of chloroplasts [START_REF] Sato | Diversity in biosynthetic pathways of galactolipids in the light of endosymbiotic origin of chloroplasts[END_REF]. The authors noted that, even within the red algae, the situation is complicated with Cyanidioschyzon merolae differing from, say,

Porphridium purpureum.

A complication in the formation of MGDG and DGDG in green algae is the variable contribution of the 'prokaryotic' (exclusively in the plastid) and the 'eukaryotic' pathways (where the ER also participates). Green algae can be divided into the first group (e.g. Chlorella)

where the DAG is derived from PtdCho on the ER and a second, more common, group (e.g. C.

reinhardtii, Dunaliella sp.) where only the 'prokaryotic' pathway is used [START_REF] Kalisch | DGDG and glycolipids in plants and algae[END_REF]. In Dunaliella two routes exist for the formation of DGDG. The first uses sequential desaturation of 18:1/16:0-DGDG to form 18:2/16:0-and 18:3/16:0-DGDG and the second uses more unsaturated species of MGDG to form DGDG species where further desaturation can lead to 18:3/16:3-or 18:3/16:4-DGDG [START_REF] Kalisch | DGDG and glycolipids in plants and algae[END_REF].

For algal species where 20C or 22C fatty acids are important an 'omega pathway' has been suggested [START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids[END_REF]. In this, VLCPUFA are formed by elongation and desaturation reactions on the ER. Apart from the involvement of elongase(s), the 'omega' pathway is superficially similar to the 'eukaryotic' pathway (as defined above). Our knowledge (or lack of!) of the detailed biochemistry of the formation of lipids with VLCPUFA has been covered in detail [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF][START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids[END_REF] The situation in diatoms has been summarised recently by [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF]. Since VLCPUFA are characteristic of diatoms (section 2), the contribution of the ER is very important (see above).

For the red alga, Cyanidioschyzon merolae, desaturation does not occur in the plastids and PUFA have to be imported, resulting in a coupled pathway for galactolipid synthesis [START_REF] Sato | Genomic and biochemical analysis of lipid biosynthesis in the unicellular rhodophyte Cyanidioschyzon merolae: Lack of a plastidic desaturation pathway results in the coupled pathway of galactolipid synthesis[END_REF].

A thorough review of the evolution of MGDG and DGDG biosynthetic pathways has been published [START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids[END_REF]. This compares these pathways based on both molecular and biochemical data and highlights enzyme reactions that have been conserved and those which have diverged.

In addition, the Chlamydomonas genome encodes an orthologue of the trigalactosyldiacylglycerol (TGD) transport protein, needed for ER to chloroplast lipid trafficking [START_REF] Warakanont | Chloroplast lipid transfer processes in Chlamydomonas reinhardtii involving a TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2) orthologue[END_REF]. In a tgd mutant, MGDG synthase was strongly stimulated but with TAG accumulation due to the defective lipid trafficking.

The pathway for SQDG synthesis in green algae is similar to that in higher plants. The pathway was first proposed by [START_REF] Pugh | A new pathway for the synthesis of the plant sulpholipid, sulphoquinovosyldiacylglycerol[END_REF] and the genes involved (SQD1, SQD2) were later isolated by Benning's group [START_REF] Essigmann | Phosphate availability affects the thylakoid lipid composition and the expression of SQD1, a gene required for sulfolipid biosynthesis in Arabidopsis thaliana[END_REF][START_REF] Yu | Arabidopsis disrupted in SQD2 encoding sulfolipid synthase is impaired in phosphate-limited growth[END_REF] to provide independent confirmation (see [START_REF] Shimojima | Biosynthesis and functions of the plant sulfolipid[END_REF]). SQD1 catalyses a complex overall reaction to generate UDP-sulfoquinovose before the transfer of sulfoquinovose to DAG by SQD2 to generate SQDG. SQD1 sequences are highly conserved in plants and algae. They form three distinct clustersin green algae, in red algae (and some cyanobacteria e.g. Synochocystis) and in Archaea. The gene was first identified in C. reinhardtii by [START_REF] Riekhof | The sulfolipids 2′-O-Acylsulfoquinovosyldiacylglycerol and sulfoquinovosyldiacylglycerol are absent from a Chlamydomonas reinhardtii mutant deleted in SQD1[END_REF]. In addition, two further putative genes for SQDG synthase have been identified (see [START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF]).

In general, SQDG is thought to play an important role in photosynthesis as discussed by [START_REF] Riekhof | Chapter 2 -Glycerolipid biosynthesis[END_REF]. It is important for the structural integrity and heat-tolerance of Photosystem II [START_REF] Sato | Organization, developmental dynamics, and evolution of plastid nucleoids[END_REF].

Also of note is the presence in C. reinhardtii of 2-O-acyl-SQDG [START_REF] Riekhof | The sulfolipids 2′-O-Acylsulfoquinovosyldiacylglycerol and sulfoquinovosyldiacylglycerol are absent from a Chlamydomonas reinhardtii mutant deleted in SQD1[END_REF], a lipid of unknown function. Various SQDGs or monoacyl derivatives (SQMG) have been suggested to be potential anti-neoplastic agents, which inhibit DNA polymerase [START_REF] Mizushina | Sulfo-quinovosyl-acylglycerol (SQAG), a eukaryotic DNA polymerase inhibitor and anti-cancer agent, Current medicinal chemistry[END_REF]. Moreover, while the galactosylglycerides can often serve to supply fatty acids during stress-induced TAG accumulation (see later), SQDG can also provide a major sulphur source for protein synthesis during early phases of sulphur starvation in C. reinhardtii [START_REF] Sugimoto | Utilization of a chloroplast membrane sulfolipid as a major internal sulfur source for protein synthesis in the early phase of sulfur starvation in Chlamydomonas reinhardtii[END_REF].

Red algae (Rhodophyta) differ from green algae in often having ARA and EPA as major fatty acids (Tables 2 and5) [START_REF] Sanina | Fatty acid composition of individual polar lipid classes from marine macrophytes[END_REF][START_REF] Khotimchenko | Distribution of glyceroglycolipids in marine algae and grasses[END_REF][START_REF] Li | Fatty acids of some algae from the Bohai Sea[END_REF]. Such acids accumulate in the glycosylglycerides as well as other lipid classes. In red algae (e.g. Porphyridium cruentum) galactolipids are formed by both the 'prokaryotic' and 'eukaryotic' pathways which give rise to 20C/16C or 20C/20C species, respectively. In the case of P. cruentum or Porphyra yezoensis the main MGDG species will be 20:5/16:0 and 20:5/20:5 [START_REF] Khozin | Elucidation of the biosynthesis of eicosapentaenoic acid in the microalga Porphyridium cruentum (II. Studies with Radiolabeled Precursors)[END_REF][START_REF] Araki | Positional distribution of fatty acids in glycerolipids of the marine red alga, Porphyra yezoensis[END_REF]. PtdCho is the source of the 'eukaryotic' moieties [START_REF] Khozin | Elucidation of the biosynthesis of eicosapentaenoic acid in the microalga Porphyridium cruentum (II. Studies with Radiolabeled Precursors)[END_REF] while 20C fatty acids can also come from any TAG reserves [START_REF] Khozin-Goldberg | Triacylglycerols of the red microalga Porphyridium cruentum can contribute to the biosynthesis of eukaryotic galactolipids[END_REF]. In contrast, the DGDG in different Rhodophyta appears to be mainly 20:5/16:0 (i.e. 'prokaryotic') as is the SQDG [START_REF] Araki | Positional distribution of fatty acids in glycerolipids of the marine red alga, Porphyra yezoensis[END_REF].

MGDG synthases and DGDG synthases of red algae form a clade separated from green algae [START_REF] Botte | Identification of plant-like galactolipids in Chromera velia, a photosynthetic relative of malaria parasites[END_REF]. Rhodophyta have one MGDG synthase and most of them have one or two plantlike DGDG synthase. DGD2-like isoforms are found in Chondrus crispus and many other red algae [START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids[END_REF] while the SQDG genes (SQD1, SQD2) are highly related to higher plant orthologues [START_REF] Kalisch | DGDG and glycolipids in plants and algae[END_REF].

There is a small group of primitive red algae of the order Cyanidiales, which have a very simple fatty acid composition, lacking the usual 20C fatty acids found in most red algae [START_REF] Sato | Genomic and biochemical analysis of lipid biosynthesis in the unicellular rhodophyte Cyanidioschyzon merolae: Lack of a plastidic desaturation pathway results in the coupled pathway of galactolipid synthesis[END_REF].

The formation of their glycosylglycerides is discussed in [START_REF] Kalisch | DGDG and glycolipids in plants and algae[END_REF].

Betaine lipids

As mentioned in section 2, algae often contain betaine lipids as major membrane constituents.

DGTS is a significant component of C. reinhardtii while DGTA is found in most brown algae as well as some other species. DGTS is formed by reaction of DAG with S-adenosylmethionine and the gene has been identified in Chlamydomonas reinhardtii. It codes for a single betaine synthase (BTA1Cr) protein whose function was confirmed by expression in E. coli [START_REF] Riekhof | Annotation of genes involved in glycerolipid biosynthesis in Chlamydomonas reinhardtii: Discovery of the betaine lipid synthase BTA1(Cr)[END_REF]. The reaction contrasts with the two enzymes needed for DGTS biosynthesis in the photosynthetic bacterium, Rhodobacter spaeroides [START_REF] Riekhof | Annotation of genes involved in glycerolipid biosynthesis in Chlamydomonas reinhardtii: Discovery of the betaine lipid synthase BTA1(Cr)[END_REF]. In algae the original pathway proposed involved transfer of a 4C amino acid moiety from methionine (in S-adenosylmethionine) followed by three methylations [START_REF] Sato | Dual role of methionine in the biosynthesis of diacylglyceryltrimethylhomoserine in Chlamydomonas reinhardtii[END_REF][START_REF] Hofmann | Biosynthesis of diacylglyceryl-N,N,N-trimethylhomoserine in Rhodobacter sphaeroides and evidence for lipid-linked N methylation[END_REF]. DGTS has been proposed as a major source of fatty acids during TAG accumulation following N-starvation in P. tricornutum [START_REF] Popko | Metabolome analysis reveals betaine lipids as major source for triglyceride formation, and the accumulation of sedoheptulose during nitrogen-starvation of Phaeodactylum tricornutum[END_REF]. However, since P.

tricornutum contains DGTA as a major lipid [START_REF] Abida | Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum[END_REF], this may (also) be a source of fatty acids.

DGTS can be converted to DGTA in brown algae [START_REF] Vogel | Betaine lipids in lower plants. Biosynthesis of DGTS and DGTA in Ochromonas danica (Chrysophyceae) and the possible role of DGTS in lipid metabolism[END_REF] and in P. tricornutum [START_REF] Popko | Metabolome analysis reveals betaine lipids as major source for triglyceride formation, and the accumulation of sedoheptulose during nitrogen-starvation of Phaeodactylum tricornutum[END_REF].

For the other betaine lipids, despite being important components of many marine algae, only a little is known of the biosynthetic pathway for DGTA. Early labelling experiments identifying DGTA (then an unidentified lipid) as a rapidly metabolised lipid are summarised in [START_REF] Harwood | Lipid metabolism in algae[END_REF]. Experiments using differentially radiolabelled methionine isomers in Ochromonas danica suggested that DGTS could be converted to DGTA by decarboxylation and recarboxylation of the polar part (and simultaneous deacylation and reacylation of the glycerol moiety) [START_REF] Vogel | Betaine lipids in lower plants. Biosynthesis of DGTS and DGTA in Ochromonas danica (Chrysophyceae) and the possible role of DGTS in lipid metabolism[END_REF]. The same workers suggested that DGTA could act as a substrate for desaturation. Using a different brown alga, Ectocarpus fasciculatus, Eichenberger's group found that, after labelling with

[14C]oleate, label rapidly appeared in phosphoglycerides such as PtdGro, PtdEtn and PtdCho but little in DGTA [START_REF] Makewicz | Lipids of Ectocarpus fasciculatus (Phaeophyceae). Incorporation of [l-14C]oleate and the role of TAG and MGDG in lipid metabolism[END_REF]. In contrast, when another brown alga, Dictyopteris membranacea, was labelled with 14C-acetate or 14C-oleate [START_REF] Hofmann | Radiolabelling studies on the lipid metabolism in the marine brown alga Dictyopteris membranacea[END_REF] label rapidly appeared in DGTA (and PtdGro) and was then transferred to glycosylglycerides during a 6-day chase period. These data confirmed experiments with other brown algae such as Fucus serratus [START_REF] Smith | Changes in the lipid metabolism of fucus serratus and fucus vesiculosus caused by copper[END_REF] or A. nodosum [START_REF] Jones | Lipid composition of the brown algae fucus vesiculosus and Ascophyllum nodosum[END_REF] in showing that DGTA was actively metabolised and could supply fatty acids to chloroplast glycosylglycerides. They also emphasise that generalisations about metabolism or, indeed, betaine lipid distributions in brown algae cannot be made.

Pavlova lutheri contains not only DGTA and DGCC but also a diacylglycerylglucuronide (DGGA). Radiolabelling experiments in this alga showed that while both DGTA and DGCC were extra-plastidic lipids, only DGCC was important for redistribution of fatty acids back to plastid components such as MGDG [START_REF] Eichenberger | Lipids of Pavlova lutheri: Cellular site and metabolic role of DGCC[END_REF].

Triacylglycerol biosynthesis and accumulation

Recent commercial interest in using oleaginous microalgae for VLCPUFA-enriched oils or for biofuels (see section 7) has led to a surge in interest in TAG metabolism. During normal growth, algae usually contain only small amounts of TAG but this is increased remarkably under stress conditions, such as N or P deficiency, elevated temperature or light intensity [START_REF] Du | Triacylglycerol accumulation in photosynthetic cells in plants and algae[END_REF][START_REF] Hu | Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances[END_REF]. C.

reinhardtii has often been used in studies because this model green alga accumulates TAG rapidly under various stresses [START_REF] Merchant | You're it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation[END_REF] such as N-deficiency which also halts cell division and then causes quiescence [START_REF] Tsai | The protein Compromised Hydrolysis of Triacylglycerols 7 (CHT7) acts as a repressor of cellular quiescence in Chlamydomonas[END_REF]. Key genes in the process have been identified by forward or reverse genetic approaches [START_REF] Merchant | You're it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation[END_REF][START_REF] Du | Triacylglycerol accumulation in photosynthetic cells in plants and algae[END_REF]. These include forward genetic screening by insertional mutagenesis [START_REF] Khozin-Goldberg | Unraveling algal lipid metabolism: Recent advances in gene identification[END_REF][START_REF] Li | A galactoglycerolipid lipase is required for triacylglycerol accumulation and survival following nitrogen deprivation in Chlamydomonas reinhardtii[END_REF][START_REF] Cagnon | Development of a forward genetic screen to isolate oil mutants in the green microalga Chlamydomonas reinhardtii[END_REF], deep transcriptome analysis by RNA sequencing [START_REF] Juergens | The regulation of photosynthetic structure and function during nitrogen deprivation in Chlamydomonas reinhardtii[END_REF][START_REF] Schmollinger | Nitrogen-sparing mechanisms in Chlamydomonas affect the transcriptome, the proteome, and photosynthetic metabolism[END_REF][START_REF] Blaby | Systems-level analysis of nitrogen starvation-induced modifications of carbon metabolism in a Chlamydomonas reinhardtii starchless mutant[END_REF][START_REF] Park | The response of Chlamydomonas reinhardtii to nitrogen deprivation: a systems biology analysis[END_REF] and proteomics [START_REF] Schmollinger | Nitrogen-sparing mechanisms in Chlamydomonas affect the transcriptome, the proteome, and photosynthetic metabolism[END_REF][START_REF] Wang | Algal lipid bodies: stress induction, purification, and biochemical characterization in wild-type and starchless Chlamydomonas reinhardtii[END_REF][START_REF] Hemme | Systems-wide analysis of acclimation responses to long-term heat stress and recovery in the photosynthetic model organism Chlamydomonas reinhardtii[END_REF]. In the latter case, a major lipid droplet protein (MLDP) was identified in Chlamydomonas, proposed to play a similar role as plant oil crop oleosin [START_REF] James | Fatty acid profiling of Chlamydomonas reinhardtii under nitrogen deprivation[END_REF][START_REF] Moellering | RNA interference silencing of a major lipid droplet protein affects lipid droplet size in Chlamydomonas reinhardtii[END_REF][START_REF] Nguyen | Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii: With focus on proteins involved in lipid metabolism[END_REF]. In order to evaluate TAG accumulation and, hence, the usefulness of particular algae or growth conditions, efficient analytical methods (especially high-throughput) are needed. Two such procedures are in [START_REF] Danielewicz | Triacylglycerol profiling of marine microalgae by mass spectrometry[END_REF][START_REF] Liu | Triacylglycerol profiling of microalgae Chlamydomonas reinhardtii and Nannochloropsis oceanica[END_REF].

For TAG synthesis by the Kennedy pathway, four reaction steps are required (Figure 5). In addition it is known from work in higher plants that phospholipid:diacylglycerol acyltransferase (PDAT) [START_REF] Dahlqvist | Phospholipid : diacylglycerol acyltransferase: An enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants[END_REF] can have a prominent role, depending on the plant species [START_REF] Banas | Activities of acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) in microsomal preparations of developing sunflower and safflower seeds[END_REF] [328]. In Chlamydomonas there is one PDAT [START_REF] Boyle | Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas[END_REF][START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF] but six diacylglycerol:acyl-CoA acyltransferases (DGATs) of two types, type 1 DGAT and type 2 DGTT (an abbreviation has been adopted for algal researchers). The DGAT genes are called DGAT1 and DGTT1-5 [START_REF] Miller | Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism[END_REF][START_REF] Boyle | Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas[END_REF][START_REF] La Russa | Functional analysis of three type-2 DGAT homologue genes for triacylglycerol production in the green microalga Chlamydomonas reinhardtii[END_REF]. There is a little confusion in the literature regarding nomenclature of the type 2 DGATs. For example, in C. reinhardtii these are referred to as DGTT1-5 or DGAT2 A-E (see [START_REF] Hung | Functional study of diacylglycerol acyltransferase type 2 family in Chlamydomonas reinhardtii[END_REF]) or even as CrDGAT2-1 to CrDGAT2-5 [START_REF] Deng | The roles of acyl-CoA: diacylglycerol acyltransferase 2 genes in the biosynthesis of triacylglycerols by the green algae Chlamydomonas reinhardtii[END_REF]. Nevertheless, regardless of nomenclature, there is a pattern across algal species with, in general, most having a single DGAT1 but multiple DGAT2 genes. Three picoplankton species (M. pusilla, O. taura, O. lucimarinus) did not contain a putative DGAT1 but this could be due to incomplete genome sequences or because their DGAT1 sequences were too divergent to be detected by similarity searches [START_REF] Chen | A look at diacylglycerol acyltransferases (DGATs) in algae[END_REF]. These authors speculate as to why algae contain multiple copies of DGATs which suggest multiple origins rather than gene duplication events. The same conclusion was reached from a study of DGATs in a wide variety of different organisms from mammals through to fungi and yeasts but including Chlorella and Coccomyxa sp. [START_REF] Turchetto-Zolet | Evolutionary view of acyl-CoA diacylglycerol acyltransferase (DGAT), a key enzyme in neutral lipid biosynthesis[END_REF].

While most attention has been paid to the multiple DGAT2 genes/enzymes, a putative sequence for DGAT1 in C. reinhardtii [START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF] has been reported. For P. tricornutum, a DGAT1

was cloned and its activity demonstrated in a yeast mutant [START_REF] Guiheneuf | Cloning and molecular characterization of a novel acyl-CoA:diacylglycerol acyltransferase 1-like gene (PtDGAT1) from the diatom Phaeodactylum tricornutum[END_REF] where a preference for saturated 16 or 18C fatty acids was displayed.

For the type-2 DGATs in C. reinhardtii, CrDGAT2A, B and C were investigated in overexpressing strains but total TAG accumulation was not changed significantly from wildtype under normal growth or after N-or S-depletion [START_REF] La Russa | Functional analysis of three type-2 DGAT homologue genes for triacylglycerol production in the green microalga Chlamydomonas reinhardtii[END_REF]. The substrate selectivity of CrDGTT1, 2 and 3 (DGAT2B, E and D, respectively) was assessed. CrDGTT1 preferred PUFAs, CrDGTT2 preferred monounsaturated acyl-CoAs and DGTT3 preferred 16C acyl-CoAs [START_REF] Liu | Characterization of type 2 diacylglycerol acyltransferases in Chlamydomonas reinhardtii reveals their distinct substrate specificities and functions in triacylglycerol biosynthesis[END_REF]. Knock-down of each of the three genes caused a 20-35% decrease in TAG together with a change in TAG fatty acids. Hung et al. [START_REF] Hung | Functional study of diacylglycerol acyltransferase type 2 family in Chlamydomonas reinhardtii[END_REF] performed heterologous complementation assays for C. reinhardtii DGTT1-4 in yeast mutants and showed that DGTT1, 2 and 3 but not 4 complemented the TAG deficiency phenotype. Complementation with DGTT2 was the most effective. In agreement with previous reports, the authors could not detect transcripts for DGTT-5.

In the green alga, Ostreococcus tauri, three putative DGAT2 genes were identified. No homologues to DGAT1 or DGAT3 could be detected and two of the three DGAT2 sequences (OtDGAT2A and OtDGAT2B) gave enzyme activity in TAG-free yeast mutants. OtDGAT2B

was shown to have a broad substrate specificity [START_REF] Wagner | Identification and characterization of an acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) gene from the microalga O. tauri[END_REF]. Gong et al. [START_REF] Gong | Identification and characterization of PtDGAT2B, an acyltransferase of the DGAT2 acyl-coenzyme A: diacylglycerol acyltransferase family in the diatom Phaeodactylum tricornutum[END_REF] identified four putative type 2 DGAT genes in P. tricornutum (now considered to have five such genes; [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF]). The PtDGAT2B was expressed in yeast mutants to restore TAG formation. Moreover, up-regulation of PtDGAT2A and PtDGAT2B preceded TAG synthesis in the alga. However, PtDGAT2B was not regulated by N-starvation [START_REF] Gong | Identification and characterization of PtDGAT2B, an acyltransferase of the DGAT2 acyl-coenzyme A: diacylglycerol acyltransferase family in the diatom Phaeodactylum tricornutum[END_REF].

Instead of the Kennedy pathway, using DGAT, TAG can also be produced by phospholipid: diacylglycerol acyltransferase (PDAT) (Figure 4). The relative contributions of PDAT versus DGAT in plants is still a matter of debate (see [START_REF] Woodfield | Using lipidomics to reveal details of lipid accumulation in developing seeds from oilseed rape (Brassica napus L.)[END_REF]). A gene encoding PDAT was found in C. reinhardtii and its activity demonstrated by expression in a TAG-deficient yeast mutant. MicroRNA silencing of PDAT in C. reinhardtii altered membrane lipid composition and reduced the growth rate [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF]. The CrPDAT also had a strong lipase activity and was suggested to be functional during the log phase of growth under normal conditions but not during the large induction of TAG deposition on N-depletion [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF]. It is noteworthy that this conclusion contrasts with the increase in PDAT expression following N starvation observed by [START_REF] Boyle | Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas[END_REF]. In higher plants PDAT is thought to generally use PtdCho, which is not present in C.reinhardtii although Boyle et al. [START_REF] Boyle | Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas[END_REF] reported that the CrPDAT complemented TAGdeficient yeast (where it may well have used PtdCho). Moreover, the gene for CrPDAT is predicted to be chloroplast-located [START_REF] Terashima | The chloroplast proteome: a survey from the Chlamydomonas reinhardtii perspective with a focus on distinctive features[END_REF] so the ability of the enzyme to use MGDG (but not DGDG or SQDG) as acyl donor in this alga is important [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF]. It will be interesting to look at the characteristics of other algal 'PDAT' enzymes such as in P. tricornutum where a putative gene has been identified [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF].

Regulation of triacylglycerol accumulation

Because of the commercial interest in enhancing TAG accumulation (section 7), the regulation of TAG biosynthesis is an active area of research. Recently, the use of transcriptional engineering has been reported [START_REF] Chen | Hacking an algal transcription factor for lipid biosynthesis[END_REF]. By identifying transcription factors whose expression was enhanced during the TAG accumulation caused by N-deprivation in N. gaditana, ZnCys was shown to be a key factor whose expression could be fine-tuned to increase TAG formation with only a minimal reduction in total carbon productivity [START_REF] Ajjawi | Lipid production in Nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator[END_REF]. These initial experiments are encouraging, although still fall short of commercial requirements [START_REF] Chen | Hacking an algal transcription factor for lipid biosynthesis[END_REF][START_REF] Posewitz | Algal oil productivity gets a fat bonus[END_REF]. An additional survey about gaps in our knowledge about the biochemistry of TAG accumulation and possible avenues for engineering has been made [START_REF] Goncalves | Metabolic regulation of triacylglycerol accumulation in the green algae: identification of potential targets for engineering to improve oil yield[END_REF]. Other aspects that impinge on lipid accumulation in green algae are chemical activators [START_REF] Wase | Identification and metabolite profiling of chemical activators of lipid accumulation in green algae[END_REF] and carbon precursor supply [START_REF] Fan | Oil accumulation is controlled by carbon precursor supply for fatty acid synthesis in Chlamydomonas reinhardtii[END_REF]. Because TAG biosynthesis is increased by nutrient deprivation, overexpression of important enzymes such as the type 2 DGAT (DGTT4) can be elicited with a P-starvation inducible promoter [345].

Parallel studies have also been made with the commercially-promising P. tricornutum (e.g. [START_REF] Valenzuela | Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum[END_REF]).

Stress (usually nutrient-deprivation) induction of TAG production is a widely employed method to increase algal oil accumulation. However, it has been noted that our knowledge of the process is largely based on genome predictions which have yet to be experimentally verified [START_REF] Zienkiewicz | Stress-induced neutral lipid biosynthesis in microalgae -Molecular, cellular and physiological insights[END_REF]. The impact of N-starvation has been examined in nine algal strains (chosen as promising for oil production from 96 candidates) where aspects such as biomass production and the duration of productivity were documented [START_REF] Breuer | The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains[END_REF]. As an alternative to N-starvation, an RNAi knock-down of nitrate reductase can also enhance lipid biosynthesis in P. tricornutum [START_REF] Levitan | Remodeling of intermediate metabolism in the diatom Phaeodactylum tricornutum under nitrogen stress[END_REF].

The authors also noted the changed expression (and binding) of transcription factors, thus heralding their recent use, referred to above [START_REF] Chen | Hacking an algal transcription factor for lipid biosynthesis[END_REF].

In another oleaginous alga (Nannochloropsis gaditana), the availability of detailed genetic information allowed predictions of lipid metabolism to be made [START_REF] Jinkerson | Genomic insights from the oleaginous model alga Nannochloropsis gaditana[END_REF]. When this alga was grown under N-starvation, the formation of TAG was accompanied by a decrease in galactosylglycerides (and a reorganisation of the photosynthetic apparatus) [START_REF] Simionato | The response of Nannochloropsis gaditana to nitrogen starvation includes de novo biosynthesis of triacylglycerols, a decrease of chloroplast galactolipids, and reorganization of the photosynthetic apparatus[END_REF]. Similarly, in N. oceanica under N-stress, the amounts of MGDG (as well as DGTS and PtdCho) decreased dramatically and expression analysis accompanying the changes identified a number of genes that seemed to be involved [START_REF] Han | Metabolic remodeling of membrane glycerolipids in the microalga Nannochloropsis oceanica under nitrogen deprivation[END_REF]. In Chlorella also, N-stress caused decreases in membrane lipids which accompanied the increase in TAG and this was confirmed by radiolabelling experiments [START_REF] Goncalves | Conversion of membrane lipid acyl groups to triacylglycerol and formation of lipid bodies upon nitrogen starvation in biofuel green algae Chlorella UTEX29[END_REF]. As an adjunct to increasing TAG accumulation by increased biosynthesis, disrupting lipid catabolism has also been used in the diatom Thalassiosira pseudonana [START_REF] Trentacoste | Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth[END_REF].

The consistent mobilisation of membrane acyl groups for TAG formation in various algae under N stress begs the question about which enzymes might be responsible. In N.

oceanica, a phospholipase was upregulated [START_REF] Han | Metabolic remodeling of membrane glycerolipids in the microalga Nannochloropsis oceanica under nitrogen deprivation[END_REF] while in C. reinhardtii a galactoglyceride lipase is involved [START_REF] Li | A galactoglycerolipid lipase is required for triacylglycerol accumulation and survival following nitrogen deprivation in Chlamydomonas reinhardtii[END_REF]. The unusual substrate selectivity of the so-called 'PDAT' in C.

reinhardtii should also be noted, together with its lipase activity [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF]. In Coccomyxa subellipsoida, N stress caused extensive chain remodelling of membrane lipids as well as TAG.

Over 2/3rds of the chloroplast lipids were lost during TAG accumulation, which was produced by the prokaryotic pathway [START_REF] Allen | Triacylglycerol synthesis during nitrogen stress involves the prokaryotic lipid synthesis pathway and acyl chain remodeling in the microalgae Coccomyxa subellipsoidea[END_REF].

Because TAG (especially when accumulated under nutrient stress) is found in lipid droplets, several groups have focussed on these organelles. Most of the work has been with C.

reinhardtii and the overall composition [START_REF] Wang | Algal lipid bodies: stress induction, purification, and biochemical characterization in wild-type and starchless Chlamydomonas reinhardtii[END_REF] and a major lipid droplet protein (MLDP) that affects droplet size [START_REF] Moellering | RNA interference silencing of a major lipid droplet protein affects lipid droplet size in Chlamydomonas reinhardtii[END_REF] reported. The MLDP was described by others (see [START_REF] Nguyen | Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii: With focus on proteins involved in lipid metabolism[END_REF]354]) but, in the studies by Huang et al [START_REF] Huang | Oleosin of subcellular lipid droplets evolved in green algae[END_REF] was found to only interact with the lipid droplet surface intermittently. The whole topic of microalgal lipid droplets, including their composition, formation and function has been reviewed by Goold et al [354].

For further details of the overall subjects of TAG biosynthesis and its elevation under nutrient stress see the reviews [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF][START_REF] Merchant | You're it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation[END_REF][START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF][START_REF] Xu | Cellular organization of triacylglycerol biosynthesis in microalgae[END_REF][START_REF] Du | Triacylglycerol accumulation in photosynthetic cells in plants and algae[END_REF] as well as the later sections 6 and 7.

TAG synthesis under nutrient, temperature or chemical stress is thought to be a protective mechanism to reduce reactive oxidant damage to photosynthetic membranes, as discussed by Du and Benning [START_REF] Du | Triacylglycerol accumulation in photosynthetic cells in plants and algae[END_REF]. It will occur whenever carbon supply outstrips the capacity for starch synthesis [START_REF] Fan | Oil accumulation is controlled by carbon precursor supply for fatty acid synthesis in Chlamydomonas reinhardtii[END_REF] and will be observed commonly in other algae (e.g. in the marine alga Desmodesmus sp.) [START_REF] Gorelova | Coordinated rearrangements of assimilatory and storage cell compartments in a nitrogen-starving symbiotic chlorophyte cultivated under high light[END_REF].

In contrast to Chamydomonas, the oleaginous algae Nannochloropsis sp. accumulate TAG not only under stress conditions but during normal growth [START_REF] Wang | Nannochloropsis genomes reveal evolution of microalgal oleaginous traits[END_REF]. Under such growth, TAG is around 10% of the dry weight [START_REF] Taleb | Development and validation of a screening procedure of microalgae for biodiesel production: Application to the genus of marine microalgae Nannochloropsis[END_REF]. Nevertheless, following stress (nutrient deprivation), Nannochloropsis will produce substantial amounts of carbohydrates, although TAG is always the major reserve compound [START_REF] Du | Triacylglycerol accumulation in photosynthetic cells in plants and algae[END_REF]. Nannochloropsis differs also from C.

reinhardtii in containing PtdCho as well as DGTS (C. reinhardtii only has DGTS; [START_REF] Jia | Molecular mechanisms for photosynthetic carbon partitioning into storage neutral lipids in Nannochloropsis oceanica under nitrogen-depletion conditions[END_REF] and in the high amounts of VLCPUFA (EPA) in its lipids [START_REF] Taleb | Development and validation of a screening procedure of microalgae for biodiesel production: Application to the genus of marine microalgae Nannochloropsis[END_REF].

The genomes of several Nannochloropsis species have been sequenced including N.

oceanica CCMP1779 [START_REF] Vieler | Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779[END_REF], N. oceanica IMET1 [START_REF] Wang | Nannochloropsis genomes reveal evolution of microalgal oleaginous traits[END_REF] and N. gaditana [START_REF] Radakovits | Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana[END_REF]. The first of these has a relatively small genome (28.7 Mb) coding for around 12,000 genes [START_REF] Vieler | Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779[END_REF]. Some 10 putative genes probably involved in TAG synthesis were identified including those for all the reactions of the Kennedy Pathway (Figure 5). There were no less than 13 putative DGAT genes and two for PDAT. In addition, a major lipid droplet protein (LDSP) was identified in Nannochloropsis [START_REF] Du | Triacylglycerol accumulation in photosynthetic cells in plants and algae[END_REF]. Such studies provide important background information for the potential industrial exploitation of Nannochlopsis (section 7).

Glycerolipid breakdown and β-oxidation of FAs

Cells require the ability to degrade storage TAGs and cell membranes when needed, or to digest extraneously supplied FAs as food source. In laboratories, this condition can be easily mimicked by manipulating the N content in the culture medium. As shown in Figure 8, TAGs are made upon N starvation and then degraded rapidly when N is added back. This process is accompanied by a degradation and then re-synthesis of membrane lipids [START_REF] Siaut | Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves[END_REF]. Based on this observation, several forward genetic screens have been performed aiming to isolate mutants defective in lipid catabolism [START_REF] Li | A galactoglycerolipid lipase is required for triacylglycerol accumulation and survival following nitrogen deprivation in Chlamydomonas reinhardtii[END_REF][START_REF] Kong | Chlamydomonas carries out fatty acid beta-oxidation in ancestral peroxisomes using a bona fide acyl-CoA oxidase[END_REF][START_REF] Tsai | The protein Compromised Hydrolysis of Triacylglycerols 7 (CHT7) acts as a repressor of cellular quiescence in Chlamydomonas[END_REF][START_REF] Cagnon | Development of a forward genetic screen to isolate oil mutants in the green microalga Chlamydomonas reinhardtii[END_REF]. Accompanying this process, many genes of lipid hydrolysis exhibit drastic alterations in their expression [START_REF] Miller | Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism[END_REF][START_REF] Schmollinger | Nitrogen-sparing mechanisms in Chlamydomonas affect the transcriptome, the proteome, and photosynthetic metabolism[END_REF]. The process of lipid breakdown is collectively called lipolysis and requires highly specialized enzymes called lipases. Most lipases act at the interface of hydrophobic and hydrophilic phases and are membrane proteins, making them very difficult to study; therefore despite intensive research on algal lipid metabolism and several forward genetic screens carried out in the past 10 years [START_REF] Li | A galactoglycerolipid lipase is required for triacylglycerol accumulation and survival following nitrogen deprivation in Chlamydomonas reinhardtii[END_REF][START_REF] Boyle | Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas[END_REF][START_REF] Kajikawa | Algal dual-specificity tyrosine phosphorylation-regulated kinase, triacylglycerol accumulation regulator1, regulates accumulation of triacylglycerol in nitrogen or sulfur deficiency[END_REF][START_REF] Tsai | The protein Compromised Hydrolysis of Triacylglycerols 7 (CHT7) acts as a repressor of cellular quiescence in Chlamydomonas[END_REF][START_REF] Cagnon | Development of a forward genetic screen to isolate oil mutants in the green microalga Chlamydomonas reinhardtii[END_REF][START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF], only six algal lipases have been identified and studied in more or less detail; yet the major TAG lipase remains to be identified in the most studied algal model C. reinhardtii.

Known algal lipases

The known lipases from algae include two orthologues of the Arabidopsis major TAG lipase (Sugar dependent 1, SDP1) [START_REF] Barka | Identification of a triacylglycerol lipase in the diatom Phaeodactylum tricornutum[END_REF][START_REF] Siegler | Analysis of the lipid body proteome of the oleaginous alga Lobosphaera incisa[END_REF][START_REF] Eastmond | SUGAR-DEPENDENT1 encodes a patatin domain triacylglycerol lipase that initiates storage oil breakdown in germinating Arabidopsis seeds[END_REF], a DAG lipase (CrLIP1) from C. reinhardtii [START_REF] Li | Rapid triacylglycerol turnover in Chlamydomonas reinhardtii requires a lipase with broad substrate specificity[END_REF], a sn-2 MGDG lipase from C. reinhardtii (PGD1) [START_REF] Li | A galactoglycerolipid lipase is required for triacylglycerol accumulation and survival following nitrogen deprivation in Chlamydomonas reinhardtii[END_REF], an orthologue of the Arabidopsis CGI58 found in the diatom Thalassiosira pseudonana [START_REF] Trentacoste | Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth[END_REF], a putative patatin-like phospholipase domain-containing protein 3 (PNPLA3) from P. tricornutum [START_REF] Wang | Identification of a putative patatin-like phospholipase domain-containing protein 3 (PNPLA3) ortholog involved in lipid metabolism in microalga Phaeodactylum tricornutum[END_REF], including a recently identified envelop-located TAG lipase OmTGL from P. tricornutum [START_REF] Li | Identification of the triacylglycerol lipase in the chloroplast envelope of the diatom Phaeodactylum tricornutum[END_REF]. . Moreover the Chlamydomonas PDAT was observed to also possess lipase activity in vitro [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF]. Expressional manipulation of the above-mentioned genes has often resulted in strains with modified TAG amount. The lipolytic processes and enzymes involved have recently been reviewed in [START_REF] Kong | Lipid catabolism in microalgae[END_REF] for microalgae. In this section, we therefore chose to focus on the oxidation of FAs, the major products of lipolysis.

The β-oxidation of FAs

All living organisms have developed the capacity to breakdown FAs to produce acetyl-CoAs, which are further metabolized either for energy production when coupled to mitochondrial electron transport chain, or for synthesis of sugars when coupled to glyoxylate and gluconeogenesis pathways [START_REF] Graham | Seed storage oil mobilization[END_REF][START_REF] Plancke | Lack of isocitrate lyase in Chlamydomonas leads to changes in carbon metabolism and in the response to oxidative stress under mixotrophic growth[END_REF]. With the exception of cyanobacteria [START_REF] Ruffing | Physiological effects of free fatty acid production in genetically engineered Synechococcus elongatus PCC 7942[END_REF], β-oxidation of FAs is universally present and has been intensively studied in mammals [START_REF] Eaton | Mammalian mitochondrial beta-oxidation[END_REF], oleaginous yeast [START_REF] Poirier | Peroxisomal beta-oxidation -A metabolic pathway with multiple functions[END_REF][START_REF] Hiltunen | The biochemistry of peroxisomal beta-oxidation in the yeast Saccharomyces cerevisiae[END_REF] and in germinating oilseeds [START_REF] Graham | Seed storage oil mobilization[END_REF] and senescing leaves [START_REF] Troncoso-Ponce | Lipid turnover during senescence[END_REF]. (ACAD). It was previously believed that ACOX is exclusively peroxisomal, while ACAD is mitochondrial, but peroxisomal ACAD has since been reported from humans and from the fungus Ustilago mays [START_REF] Camões | New insights into the peroxisomal protein inventory: Acyl-CoA oxidases and -dehydrogenases are an ancient feature of peroxisomes[END_REF]. The β-oxidation of FAs occurs mostly in peroxisomes in yeast and plant cells, in contrast to mammalian cells wherein it occurs principally in mitochondria, with a small peroxisomal contribution [START_REF] Eaton | Mammalian mitochondrial beta-oxidation[END_REF][START_REF] Farr | Peroxisome biogenesis in mammalian cells: The impact of genes and environment[END_REF][START_REF] Eaton | Control of mitochondrial beta-oxidation flux[END_REF]. FA degradation has been studied in various algal species, although by no means comprehensively, and the location of FA β-oxidation in algae varies extensively, occurring either in the peroxisome, the mitochondrion or both [START_REF] Stabenau | beta-oxidation in algal peroxisomes of the leaf and unspecialized type[END_REF][START_REF] Stabenau | Enzymes of beta-oxidation in different types of algal microbodies[END_REF][START_REF] Stabenau | Microbodies in different algae[END_REF][START_REF] Stabenau | Compartimentation of enzymes of the beta-oxidation pathway in different types of algae[END_REF][START_REF] Winkler | Beta-oxidation of fatty acids in algae -localization of thiolase and acyl-CoA oxidizing enzymes in 3 different organisms[END_REF]. A better understanding of ACOX/ACAD evolution and localization is essential for the successful engineering (and control) of algal lipid catabolic pathways.

Subcellular location of β-oxidation and phylogenetics of acyl-

Based on a comprehensive phylogenetic analysis (Figure 9), ACOXs and ACADs appear to have diverged and diversified during early prokaryote evolution, as previously reported [START_REF] Swigonova | Acyl-CoA dehydrogenases: Dynamic history of protein family evolution[END_REF]. Eukaryotes then inherited over twenty distinct ACOXs/ACADs, either via endosymbiosis or via lateral gene transfer, and these inherited prokaryotic genes in turn gave rise to eukaryotic subfamilies. Additional archaeal sequences made available since previous analyses of ACAD evolution [START_REF] Swigonova | Acyl-CoA dehydrogenases: Dynamic history of protein family evolution[END_REF] now suggest that the major eukaryotic ACAD lineages are of both archaeal and bacterial ancestry rather than solely arising from the latter, while ACOX appears to be exclusively of bacterial origin (Figure 9).

As a result of the distinct prokaryotic ancestry of the different eukaryotic ACOX/ACAD subfamilies, inter-subfamily inferences regarding enzyme localization are fraught with peril.

Furthermore, the presence of contrasting mitochondrial and peroxisomal members within the same ACAD subfamily indicates that even intra-subfamily localization is not always conserved (e.g. group D-II in Figure 9). Nonetheless, some common trends are apparent: all ACOXs thus far characterized are peroxisomal, with peroxisomal localization (and accompanying catalase activity) perhaps being essential for efficient activity of these peroxide-producing enzymes. By contrast, ACADs are either mitochondrial or peroxisomal.

The conservation of eukaryotic members of the various ACOX/ACAD subfamilies varies extensively by taxonomic group [START_REF] Swigonova | Acyl-CoA dehydrogenases: Dynamic history of protein family evolution[END_REF], particularly in the case of eukaryotic algae, and may be in part responsible for establishing the organellar location of β-oxidation. Green, heterokont and haptophyte algae possess a variety of ACADs and ACOXs, while red algae Below we discuss current literature on the known steps of peroxisomal FA β-oxidation in Chlamydomonas, which is thus far the best studied model alga for lipid catabolism [START_REF] Kong | Lipid catabolism in microalgae[END_REF][START_REF] Kong | Chlamydomonas carries out fatty acid beta-oxidation in ancestral peroxisomes using a bona fide acyl-CoA oxidase[END_REF].

Core reactions of peroxisomal β-oxidation

The core peroxisomal pathway requires the acyl-CoA oxidase (ACOX), multifunctional protein (MFP) and 3-ketoacyl-CoA thiolase (KAT) to catalyze the sequential oxidation, hydration and dehydrogenation, and thiolytic cleavage of the acyl-CoA molecule (Figure 10). Although genes encoding putative orthologues to known proteins of FA β-oxidation can be identified in algal species [START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF][START_REF] Boyle | Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas[END_REF][START_REF] Miller | Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism[END_REF], only the ACOX2 catalyzing the first step in the FA β-oxidation spiral has been characterized experimentally at both genetic and biochemical levels. ACOX2, which is closely related to Arabidopsis ACOX2 (Figure 9) exhibited high activity toward a broad range of acyl-chains, and showed highest activity toward C16 and C18 acyl-CoAs [START_REF] Kong | Chlamydomonas carries out fatty acid beta-oxidation in ancestral peroxisomes using a bona fide acyl-CoA oxidase[END_REF]. The acox2 (or acx2) mutants lost >50% of the wild-type capacity in remobilization of TAGs upon N resupply following a period of N starvation; and moreover, the acox2 mutants accumulated 30% more TAG during photoheterotrophic N starvation and with a modified TAG composition [START_REF] Kong | Chlamydomonas carries out fatty acid beta-oxidation in ancestral peroxisomes using a bona fide acyl-CoA oxidase[END_REF]. The occurrence of five ACOX isozymes is not surprising [START_REF] Kong | Lipid catabolism in microalgae[END_REF][START_REF] Merchant | The Chlamydomonas genome reveals the evolution of key animal and plant functions[END_REF], since FA β-oxidation is a chain-shortening reaction, and different isozymes should be required to shorten acyl-CoA of various chain lengths [245]. It is worth noting here that the above core enzymatic activities of FA β-oxidation are not sufficient for the oxidation of unsaturated FAs whose degradation normally requires the participation of additional enzymatic reactions. Two alternative pathways are known in plants [START_REF] Li-Beisson | Acyl-lipid metabolism[END_REF] (Aralip: http://aralip.plantbiology.msu.edu/pathways/pathways), of which only the Arabidopsis enoyl-CoA isomerase (ECI) has been studied in detail [START_REF] Goepfert | Peroxisomal Δ3,Δ2-enoyl CoA isomerases and evolution of cytosolic paralogues in embryophytes[END_REF], and none of which have been characterized in algae.

Metabolism of hydrogen peroxide (H2O2)

In addition to acetyl-CoAs, peroxisomal FA β-oxidation produces hydrogen peroxide and reducing equivalents in a molar ratio of approximately 1:1:1. In higher plants, the highly oxidative H2O2 is usually decomposed to water by peroxisome-resident catalase; which is a major protein in plant peroxisomes and is at the origin for the formation of a crystalloid core apparent under transmission electron microscope [START_REF] Beevers | Microbodies in higher-plants[END_REF]. The crystalloid core is however often absent in peroxisomes of C. reinhardtii [START_REF] Hayashi | Visualization of microbodies in Chlamydomonas reinhardtii[END_REF]. Based on subcellular fractionation studies, Kato et al [START_REF] Kato | Characterization of catalase from green algae Chlamydomonas reinhardtii[END_REF] showed that catalase activities in C. reinarhdtii could be associated to mitochondria fractions. Nevertheless, homology searches using known plant catalase have identified at least two genes encoding putative catalases in the genome of C. reinhardtii [START_REF] Merchant | The Chlamydomonas genome reveals the evolution of key animal and plant functions[END_REF], but their subcellular localization and biological function remain to be determined. Indeed, it has long been questioned whether C. reinhardtti contains any H2O2-producing activities [START_REF] Hagemann | Evolution of photorespiration from cyanobacteria to land plants, considering protein phylogenies and acquisition of carbon concentrating mechanisms[END_REF][START_REF] Aboelmy | Enzymatic characterization of Chlamydomonas reinhardtii glycolate dehydrogenase and its nearest proteobacterial homologue[END_REF]. We have lately shown that the primitive peroxisomes or microbodies in C. reinhardtti do indeed contain reactions that generate H2O2, at the first step of FA β-oxidation using ACOX2. It is observed that in vitro, the recombinant Chlamydomonas ACOX2 catalyzes the conversion of acyl-CoA to trans-2-enoyl-CoA while producing H2O2. This study was the first to demonstrate that C. reinhardtii uses a peroxisomal pathway for FA degradation, and that H2O2 producing activities had already evolved in green microalgae.

From an evolutionary perspective, it is not clear if there is any advantage of housing FA β-oxidation in peroxisomes instead of mitochondria. The mitochondrial pathway employs an acyl-CoA dehydrogenase at its first step, and this reaction is directly coupled to the mitochondrial respiratory pathway for ATP production, therefore energy is conserved; whereas in the peroxisomal pathway, energy is transferred to O2 with the production of H2O2 and subsequently H2O; therefore energy is lost. This loss of energy could potentially be advantageous under some conditions, allowing FA degradation to occur without affecting cellular energy/redox status. This also raises questions regarding the possible physiological roles or significance of peroxisome-derived H2O2. A study in Arabidopsis has shown that in mutant plants defective in the ascorbate peroxidase (APX)/monodehydroascorbate reductase (MDAR) electron transfer system, the escaped H2O2 inhibited the activities of the major TAG lipase SDP1 and this slowed TAG hydrolysis [START_REF] Eastmond | MONODEHYROASCORBATE REDUCTASE4 is required for seed storage oil hydrolysis and postgerminative growth in Arabidopsis[END_REF]. This study implies therefore a possible role of H2O2 in coordinating lipolysis to β-oxidation of FAs. H2O2 is the most stable form of reactive oxygen species (ROS), and is known to play dual roles in cellular physiologyin excess it can cause oxidative damage, but in sub-lethal levels, chloroplast-derived H2O2 is known to play a signaling role [START_REF] Dietz | Redox-and reactive oxygen species-dependent signaling into and out of the photosynthesizing chloroplast[END_REF]. Through characterization of two mutants defected in the peroxisomal malate dehydrogenase 2 (MDH2), we have recently provided evidence that peroxisome-derived H2O2 likely plays a role in transmitting the redox state of the peroxisome to the chloroplast, thereby impacting photosynthesis, de novo FA synthesis and starch metabolism (discussed in detail in section 3.5.3) [START_REF] Kong | Interorganelle communication: peroxisomal MALATE DEHYDROGENASE2 connects lipid catabolism to photosynthesis through redox coupling in Chlamydomonas[END_REF]. However the mechanisms by which peroxisome-derived H2O2 passes through the peroxisomal membranes remains largely unknown (i.e. is it by free diffusion or an aquaporin mediated process?).

NADH re-oxidation in peroxisomes

Similar to most oxidative reactions, β-oxidation of FAs also produces NADH through the reaction catalyzed by the 3-hydroxylacyl-CoA dehydrogenase (MFP-DH). De novo NAD + synthesis occurs in the cytoplasm [START_REF] Noctor | NAD(P) synthesis and pyridine nucleotide cycling in plants and their potential importance in stress conditions[END_REF][START_REF] Bernhardt | A peroxisomal carrier delivers NAD+ and contributes to optimal fatty acid degradation during storage oil mobilization[END_REF], yet it is required by reactions present in almost every subcellular compartment. Its transport and homeostasis therefore can play a key role in regulation of metabolic pathways [START_REF] Scheibe | Malate valves to balance cellular energy supply[END_REF][START_REF] Mettler | Oxidation of NADH in glyoxysomes by a malate-aspartate shuttle[END_REF]. Newly-synthesized NAD + is imported from the cytoplasm into the peroxisome by the peroxisomal NAD + carrier (PXN) [START_REF] Van Roermund | The peroxisomal NAD carrier from Arabidopsis imports NAD in exchange with[END_REF][START_REF] Bernhardt | A peroxisomal carrier delivers NAD(+) and contributes to optimal fatty acid degradation during storage oil mobilization[END_REF]. However, studies in yeasts and plants show that re-oxidation of peroxisomal NADH must occur inside the organelle because the peroxisomal membrane is not permeable to NAD + [START_REF] Vanroermund | The membrane of peroxisomes in Saccharomyces-cerevisiae is permeable to NAD(H) and acetyl-CoA under in vivo conditions[END_REF]. Lately it has been shown that the peroxisomal malate dehydrogenase 2 (MDH2) in C. reinhardtii plays a major role in NADH re-oxidation because the mdh2 mutants defected in MDH2 protein are impaired by 80% in their capacity to reutilize TAGs following N resupply [START_REF] Kong | Interorganelle communication: peroxisomal MALATE DEHYDROGENASE2 connects lipid catabolism to photosynthesis through redox coupling in Chlamydomonas[END_REF]. MDHs are ubiquitous enzymes, and each subcellular compartment usually contains at least one isoform [START_REF] Scheibe | Malate valves to balance cellular energy supply[END_REF][START_REF] Merchant | The Chlamydomonas genome reveals the evolution of key animal and plant functions[END_REF][START_REF] Mettler | Oxidation of NADH in glyoxysomes by a malate-aspartate shuttle[END_REF]. Alongside the peroxisomal MDH2, Chlamydomonas genome encodes one NADP + -dependent chloroplast MDH5 [START_REF] Lemaire | NADP-malate dehydrogenase from unicellular green alga Chlamydomonas reinhardtii. A first step toward redox regulation?[END_REF], and three other NAD + -dependent MDHs (MDH1, MDH3 and MDH4) [START_REF] Merchant | The Chlamydomonas genome reveals the evolution of key animal and plant functions[END_REF].

In addition to MDH2, two other mechanisms are known to play a role in NADH reoxidation in plant peroxisomes. These additional pathways of NADH re-oxidation employ either a peroxisomal hydroxypyruvate reductase (HPR) [START_REF] Pracharoenwattana | Fatty acid beta-oxidation in germinating Arabidopsis seeds is supported by peroxisomal hydroxypyruvate reductase when malate dehydrogenase is absent[END_REF], or the APX/MDAR electron transfer system [START_REF] Eastmond | MONODEHYROASCORBATE REDUCTASE4 is required for seed storage oil hydrolysis and postgerminative growth in Arabidopsis[END_REF]. Genes encoding putative orthologues to these proteins can be identified in algal genomes, but their function remains unknown. The serious impairment in oil reutilization observed in the mdh2 mutants suggest that other mechanisms, if they occur, are not expected to play major roles in C. reinhardtii, at least not under the conditions tested (i.e.

N resupply following a period of N starvation [START_REF] Kong | Interorganelle communication: peroxisomal MALATE DEHYDROGENASE2 connects lipid catabolism to photosynthesis through redox coupling in Chlamydomonas[END_REF]).

Additional roles for the β-oxidation spiral

Mostly through phenotypic analyses of Arabidopsis mutants defected in various steps of FA βoxidation, it has become obvious that the β-oxidation spiral does not only play a role in FA breakdown, but also plays a role i) in the production of lipid-based signaling molecules such as jasmonic acid, ii) in the conversion of indole butyric acid to the phytohormone indole acetic acid, and iii) in the later steps of BCAA degradation, therefore impacting plant physiology and development [START_REF] Hu | Plant peroxisomes: biogenesis and function[END_REF][START_REF] Graham | Pathways of straight and branched chain fatty acid catabolism in higher plants[END_REF][START_REF] Penfield | Storage reserve mobilisation and seedling establishment in Arabidopsis[END_REF][START_REF] Theodoulou | Seed storage oil catabolism: a story of give and take[END_REF]. Thus unlike many other metabolic pathways, peroxisomal oxidation is multi-functional, and is sometimes called "a pathway with multiple functions" [START_REF] Poirier | Peroxisomal beta-oxidation -A metabolic pathway with multiple functions[END_REF]. Jasmonic acid has been identified in Euglena gracilis, Chlorella and Spirulina [START_REF] Ueda | Identification of jasmonic acid in Chlorella and Spirulina[END_REF] and also a variety of marine algae [START_REF] Arnold | Evidence for methyl jasmonate induced phlorotannin production in Fuus vesiculosus (Phaeophyceae)[END_REF] but nothing is known about the enzymes or subcellular locations for their biosynthesis in microalgae.

Environmental effects

Early studies of the effects of the environment on lipid metabolism in algae were summarised by Pohl and Zurheide [START_REF] Pohl | Fatty acids and lipids of marine algae and the control of their biosynthesis by environmental factors, marine algae in pharmaceutical science[END_REF]. In our previous review on algal lipids we also discussed effects of nutrition (especially N, P and S limitation), other growth conditions (temperature, light, pH) and some pollutants (e.g. heavy metals) [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF]. It is noteworthy that many of these chemical or non-chemical stresses can induce TAG accumulation, which may also be accompanied by alterations in FA and lipid composition [START_REF] Du | Triacylglycerol accumulation in photosynthetic cells in plants and algae[END_REF]. Induction of TAG biosynthesis in algae is also relevant to industrial uses of algal oils (section 7).

Nutrients

Algal species vary in their nutritional requirements although the basic macro-nutrients for all species are carbon, N and phosphorus (P). Some marine microalgae (e.g. many diatoms) need silicon [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF]. For a common freshwater green alga like Chlorella, growth declines once N and P concentrations are below 31.5 and 10.5 mg/l, respectively [START_REF] Aslan | Batch kinetics of nitrogen and phosphorus removal from synthetic wastewater by algae[END_REF]. For Chlorella vulgaris, nitrogen deficiency below 0.5 mg/l gives an optimal induction of lipid production [START_REF] Daliry | Investigation of optimal condition for Chlorella vulgaris microalgae growth[END_REF]. Indeed, N is the usual stress used to induce TAG production [START_REF] Hu | Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances[END_REF]. Under these conditions, green microalgae or diatoms will accumulate TAG at 20-50% dry weight [START_REF] Hu | Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances[END_REF]. Similarly, phosphorus deficiency has major effects on lipid metabolism and, hence, algal oil content [START_REF] Abida | Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum[END_REF][START_REF] Khozin-Goldberg | Unraveling algal lipid metabolism: Recent advances in gene identification[END_REF][START_REF] Khozin-Goldberg | The effect of phosphate starvation on the lipid and fatty acid composition of the fresh water eustigmatophyte Monodus subterraneus[END_REF].

Naturally, because of the interest in using algae for industrial purposes (especially postinduction of oil accumulation), most attention has focussed on species which offer commercial possibilities. Nevertheless, examination of a variety of microalgae showed that most of them increased TAG production when grown in N-deficient conditions. There were, however, significant differences in biomass production, %TAG accumulated and the duration of productivity [START_REF] Breuer | The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains[END_REF][START_REF] Breuer | Effect of light intensity, pH, and temperature on triacylglycerol (TAG) accumulation induced by nitrogen starvation in Scenedesmus obliquus[END_REF]. For promising feedstocks such as P. tricornutum, Nannochloropsis spp. and Chlorella pyrenoidosa, a variety of laboratories have examined their productivity under N-starvation [START_REF] Abida | Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum[END_REF][START_REF] Han | Metabolic remodeling of membrane glycerolipids in the microalga Nannochloropsis oceanica under nitrogen deprivation[END_REF][START_REF] Simionato | The response of Nannochloropsis gaditana to nitrogen starvation includes de novo biosynthesis of triacylglycerols, a decrease of chloroplast galactolipids, and reorganization of the photosynthetic apparatus[END_REF][START_REF] Fan | Lipid accumulation and biosynthesis genes response of the oleaginous Chlorella pyrenoidosa under three nutrition stressors[END_REF][406][START_REF] Wan | Effects of nitrogen concentration and media replacement on cell growth and lipid production of oleaginous marine microalga Nannochloropsis oceanica DUT01[END_REF]. As mentioned in section 4, accumulation of TAG is allowed by de novo synthesis accompanied by a decrease in plastid galactolipids and consequent re-organisation of the photosynthetic apparatus in Nannochloropsis gaditana [START_REF] Simionato | The response of Nannochloropsis gaditana to nitrogen starvation includes de novo biosynthesis of triacylglycerols, a decrease of chloroplast galactolipids, and reorganization of the photosynthetic apparatus[END_REF].

A detailed examination of glycerolipid classes and their molecular species was made in P.

tricornutum. Most lipids were relatively unaffected although large decreases in MGDG and PtdGro were noted. For the accumulating TAG there was an enrichment of 16:1 which correlated with its synthesis whereas 20:5 seemed to be transferred from MGDG [START_REF] Abida | Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum[END_REF]. In Nannochloropsis oceanica, the increase in TAG was accompanied not only by a decrease in MGDG but also of the extra-chloroplastic lipids DGTS and PtdCho [START_REF] Han | Metabolic remodeling of membrane glycerolipids in the microalga Nannochloropsis oceanica under nitrogen deprivation[END_REF]. Changes in FA profiles were also reported for Phaeodactylum tricornutum [START_REF] Alonso | Acyl lipid composition variation related to culture age and nitrogen concentration in continuous culture of the microalga Phaeodactylum tricornutum[END_REF] and for other algae (Pavlova viridis, Tetraselmis subcordiformis) [START_REF] Huang | Effects of nitrogen supplementation of the culture medium on the growth, total lipid content and fatty acid profiles of three microalgae (Tetraselmis subcordiformis, Nannochloropsis oculata and Pavlova viridis)[END_REF]. During the increased TAG accumulation on Nstarvation in Chlorella pyrenoidosa, expression levels of genes for acetyl-CoA carboxylase and DGAT were increased [START_REF] Fan | Lipid accumulation and biosynthesis genes response of the oleaginous Chlorella pyrenoidosa under three nutrition stressors[END_REF]. The latter was also shown to be raised in C. reinhardtii along with other acyltransferases and a nitrogen responsive regulator [START_REF] Boyle | Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas[END_REF]. Overall, it can be concluded that TAG formation is by a combination of de novo synthesis as well as transfer of FAs from membrane lipids.

Only limited information is available regarding the mechanisms controlling TAG accumulation during N-limitation. Early on Boyle et al [START_REF] Boyle | Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas[END_REF] showed that enhanced expression of a nitrogen response regulator accompanied N-starvation induction of TAG production.

Furthermore, RNAi knock-down of nitrate reductase can enhance lipid biosynthesis in

Phaeodactylum tricornutum [START_REF] Levitan | Remodeling of intermediate metabolism in the diatom Phaeodactylum tricornutum under nitrogen stress[END_REF]. Recently, ROC40, a transcription factor involved in circadian rhythm, was found to increase markedly on N-starvation. Further information from mutant analysis supported a role for ROC40 in N-starvation induction of TAG synthesis [START_REF] Goncalves | Metabolic regulation of triacylglycerol accumulation in the green algae: identification of potential targets for engineering to improve oil yield[END_REF].

Please see section 3.8 for more regulatory factors involved in novo FA synthesis.

The effect of different N levels in the growth media for N. oceanica has been examined with a view to finding the best concentrations for lipid production [START_REF] Wan | Effects of nitrogen concentration and media replacement on cell growth and lipid production of oleaginous marine microalga Nannochloropsis oceanica DUT01[END_REF]. The strain studied, DUT01 produced an unusually high amount of 16:2. In a separate study with three different microalgae, the effect of different N concentrations between zero and 1.76 mmol/L were examined. All three algae showed highest lipid accumulation with 0.22 mmol N/L and Nannochloropsis oculata and Pavlova viridis showed promise for biodiesel production because of the changes in their FA patterns [START_REF] Huang | Effects of nitrogen supplementation of the culture medium on the growth, total lipid content and fatty acid profiles of three microalgae (Tetraselmis subcordiformis, Nannochloropsis oculata and Pavlova viridis)[END_REF].

A second major nutrient needed for algal growth is phosphorus which, of course, is essential for phosphoglyceride biosynthesis. Riekhof et al [START_REF] Riekhof | The sulfolipids 2′-O-Acylsulfoquinovosyldiacylglycerol and sulfoquinovosyldiacylglycerol are absent from a Chlamydomonas reinhardtii mutant deleted in SQD1[END_REF] showed that C. reinhardtii had reduced levels of all phosphoglycerides on P-starvation. The 50% reduction in PtdGro, an essential thylakoid (photosynthetic) constituent, was critical but it could be replaced by another anionic membrane lipid, SQDG. In a low-phosphate bleaching mutant of C. reinhardtii (lpb1)

it was shown that normal responses to P-deprivation (and S-deprivation) appeared as usual but that the lpb1 mutant lacked critical acclimation ability [START_REF] Chang | The LPB1 gene is important for acclimation of Chlamydomonas reinhardtii to phosphorus and sulfur deprivation[END_REF]. In the fresh water eustigmatophyte, Monodus subterraneus, P-starvation caused increases in DGDG and DGTS (and TAG) which accompanied the loss of phosphoglycerides. The increase in DGDG (but not MGDG) resembled the response of higher plants to P-deprivation. There were also some changes in the FA contents of individual lipid classes [START_REF] Khozin-Goldberg | The effect of phosphate starvation on the lipid and fatty acid composition of the fresh water eustigmatophyte Monodus subterraneus[END_REF]. As noted above, P-starvation triggers lipid (TAG) accumulation in the same way as N-deprivation does [START_REF] Fan | Lipid accumulation and biosynthesis genes response of the oleaginous Chlorella pyrenoidosa under three nutrition stressors[END_REF], but not to the same extent [START_REF] Abida | Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum[END_REF]. When considering the time-course of changes in Phaeodactylum tricornutum, it was noted that there was a step-wise adaptive response. The authors suggested that phosphoglycerides provided emergency P following their catabolism and that there was some replacement with non-phosphorus lipids, which included SQDG for PtdGro and DGTA for PtdCho [START_REF] Abida | Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum[END_REF]. The effect of P-starvation in diatoms is discussed thoroughly by [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF].

Other macronutrients, such as carbon dioxide [START_REF] Prathima Devi | CO2 supplementation to domestic wastewater enhances microalgae lipid accumulation under mixotrophic microenvironment: Effect of sparging period and interval[END_REF] or sulphur can alter lipid metabolism. In the latter case, S-starvation can increase non-polar lipids in Chlorella ellipsoidea [START_REF] Otsuka | Changes of lipid and carbohydrate contents in Chlorella cells during the sulfur starvation, as studied by the technique of synchronous cutlure[END_REF] or C. reinhardtii [START_REF] Matthew | The metabolome of Chlamydomonas reinhardtii following induction of anaerobic H2 production by sulfur depletion[END_REF]. Sulphur is utilised for the synthesis of proteins and a wide variety of metabolites critical for growth. When C. reinhardtii was transferred to Sdepleted conditions, some 85% of the SQDG was broken down to yield a major pool of S for protein synthesis [START_REF] Sugimoto | Utilization of a chloroplast membrane sulfolipid as a major internal sulfur source for protein synthesis in the early phase of sulfur starvation in Chlamydomonas reinhardtii[END_REF]. To an extent this utilisation of SQDG is consistent with its role as an important contributor to the global S cycle [START_REF] Harwood | The plant sulpholipid--a major component of the sulphur cycle[END_REF][START_REF] Roy | Glycolytic breakdown of sulfoquinovose in bacteria: a missing link in the sulfur cycle[END_REF].

Silicon depletion was also noted to induce TAG formation in the diatom Cyclotella cryptica [START_REF] Traller | High throughput imaging to the diatom Cyclotella cryptica demonstrates substantial cell-to-cell variability in the rate and extent of triacylglycerol accumulation[END_REF]. This TAG had a modified FA composition (less unsaturated) compared to cells grown in adequate silicon concentrations. In fact, as noted previously, silicon is a macronutrient for many diatoms [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF], including oleaginous species. Thus, silicon depletion will enhance TAG production in those diatoms that need it [START_REF] Adams | Enhancing lipid production of the marine diatom Chaetoceros gracilis: synergistic interactions of sodium chloride and silicon[END_REF][START_REF] Jiang | Coupled effect of silicate and nickel on the growth and lipid production in the diatom Nitzschia perspicua[END_REF] and, for example, Thalassiosira pseudonana will accumulate an average of 24% more lipids than it does under N-starvation [406,[START_REF] Zendejas | Characterization of the acylglycerols and resulting biodiesel derived from vegetable oil and microalgae (Thalassiosira pseudonana and Phaeodactylum tricornutum)[END_REF].

Phaeodactylum tricornutum, in contrast, has little, if any, requirement for silicon [START_REF] Zendejas | Characterization of the acylglycerols and resulting biodiesel derived from vegetable oil and microalgae (Thalassiosira pseudonana and Phaeodactylum tricornutum)[END_REF].

However, although silicon does not seem to be required for laboratory or factory cultures of Phaeodactylum tricornutum, it seems to be needed for normal expression of miRNAs and growth [START_REF] Zhao | Silicon enhances the growth of Phaeodactylum tricornutum Bohlin under green light and low temperature[END_REF].

Clearly, carbon is a macronutrient, although it will normally be sourced from the atmosphere. Nevertheless, with culture conditions in mind, there has been some attention paid to different concentrations of CO2 or to various regimes. In Chlorella kessleri, CO2

concentrations have a dramatic effect on lipid metabolism and on the incorporation of [14C]acetate into FAs ( and lipids). Part of the changes were due to adjustment of the 'prokaryotic' versus 'eukaryotic' pathways and one result was elevated 18:3 at low CO2 levels [START_REF] Sato | Glycerolipid synthesis in Chlorella kessleri 11h. I. Existence of a eukaryotic pathway[END_REF]. Likewise, glycerol feeding in batch cultures of Schizochytrium sp. [START_REF] Chang | Fatty acid shifts and metabolic activity changes of Schizochytrium sp. S31 cultured on glycerol[END_REF] or alterations in inorganic carbon regimes supplied to C. reinhardtii changed FA patterns [START_REF] Lohman | Carbon partitioning in lipids synthesized by Chlamydomonas reinhardtii when cultured under three unique inorganic carbon regimes[END_REF]. Carbon metabolism in diatoms, including the impact of environmental factors, has been discussed recently [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF][START_REF] Sayanova | Modulation of lipid biosynthesis by stress in diatoms[END_REF]. Furthermore, a general commentary on growth and lipid accumulation by nutrient depletion and supplementation in C. reinhardtii has been recently published [START_REF] Yang | Growth and lipid accumulation by different nutrients in the microalga Chlamydomonas reinhardtii[END_REF].

Micronutrients, needed in trace amounts (e.g. Co, Cu, Fe, Mg, Mn, Mo, Zn) may have a strong influence on algal growth since they can alter normal enzyme activity [START_REF] Khan | The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products[END_REF]. Elements such as Fe and Zn have been shown to influence TAG accumulation in Chlamydomonas [START_REF] Kropat | A revised mineral nutrient supplement increases biomass and growth rate in Chlamydomonas reinhardtii[END_REF][START_REF] Urzica | Remodeling of membrane lipids in iron-starved Chlamydomonas[END_REF]. Of course, for many such elements there is a fine line between nutrient deficiency, sufficiency, and toxicity [START_REF] Hanikenne | Chapter 10 -Transition metal nutrition: A balance between deficiency and toxicity[END_REF]. Iron seems to be a key factor in regulating phytoplankton biomass. When FeCl3 was added to fresh medium at 1.2 x 10 -5 mol/l in late exponential phase cultures of Chlorella vulgaris, it boosted biomass and % lipid composition whereas lower concentrations led to lower lipid levels [START_REF] Liu | Effect of iron on growth and lipid accumulation in Chlorella vulgaris[END_REF]. In Fe-starved C. reinhardtii, lipid droplets and TAG accumulated. An increased saturation index was noted, suggesting that desaturase activity was compromised. The FA profiles of DGDG and DGTS (but not MGDG) were changed and gene expression of enzymes or proteins involved in TAG accumulation (e.g. the major lipid droplet protein or DGAT) was increased [START_REF] Urzica | Remodeling of membrane lipids in iron-starved Chlamydomonas[END_REF]. Effects of Fe-deficiency in Phaeodactylum tricornutum were coincident with a partial deficiency of photosynthetic transport and a high sensitivity to light [START_REF] Roncel | Iron deficiency induces a partial inhibition of the photosynthetic electron transport and a high sensitivity to light in the diatom Phaeodactylum tricornutum[END_REF].

Like iron, copper is needed for certain enzyme activities [START_REF] Hanikenne | Chapter 10 -Transition metal nutrition: A balance between deficiency and toxicity[END_REF]. However, toxic effects have been noted in a variety of algae as well as changes to lipid metabolism (see [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF][START_REF] Harwood | Lipid metabolism in algae[END_REF]). Copper response regulator1-dependent and -independent responses of C. reinhardtii to dark anoxia were shown to be important. Under hypoxic conditions this alga accumulates TAG which, in contrast to during N-depletion, was enriched in unsaturated FAs [START_REF] Hemschemeier | COPPER RESPONSE REGULATOR1-dependent and -independent responses of the Chlamydomonas reinhardtii transcriptome to dark anoxia[END_REF]. When chromium effects were studied in Euglena gracilis obtained from a culture or collected from a polluted river, PUFA levels were most affected. Electron microscope examination revealed thylakoid disorganisation in treated cells [START_REF] Rocchetta | Effect of chromium on the fatty acid composition of two strains of Euglena gracilis[END_REF]. Another toxic metal is cadmium, which affects lipid metabolism [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF]. As noted for other algae, susceptibility to heavy metal toxicity varies considerably with species (see [START_REF] Harwood | Lipid metabolism in algae[END_REF]) and Phaeodactylum tricornutum is relatively resistant to Cd probably because it has protective transport and detoxification processes [START_REF] Brembu | Genome-wide profiling of responses to cadmium in the diatom Phaeodactylum tricornutum[END_REF].

Light

Naturally, for photosynthetic organisms, light is a major controlling factor for algal growth [START_REF] Pohl | Fatty acids and lipids of marine algae and the control of their biosynthesis by environmental factors, marine algae in pharmaceutical science[END_REF] [9]. Its duration and intensity influence both biochemical composition and algal mass yield [START_REF] Krzeminska | Influence of photoperiods on the growth rate and biomass productivity of green microalgae[END_REF]. Of course, algal species vary in their requirements [START_REF] Khan | The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products[END_REF]. Increasing light intensity raises the photosynthetic rate and growth until the latter levels off as photosynthesis is balanced by photorespiration and photoinhibition [START_REF] Ye | Photosynthetic performance in aquatic and terrestrial colonies of Nostoc flagelliforme (Cyanophyceae) under aquatic and aerial conditions[END_REF]. Intensities for maximum growth (and lipid production) have been reported for a number of algal species recently [START_REF] Daliry | Investigation of optimal condition for Chlorella vulgaris microalgae growth[END_REF][START_REF] Kitaya | Effects of temperature, CO2/O2 concentrations and light intensity on cellular multiplication of microalgae, Euglena gracilis[END_REF] as well as the effect of different light intensities in combination with CO2 levels [START_REF] Carvalho | Optimization of omega-3 fatty acid production by microalgae: crossover effects of CO2 and light intensity under batch and continuous cultivation modes[END_REF] or salinity and N [START_REF] Pal | The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp[END_REF].

These can affect both qualitative and quantitative aspects of lipid metabolism.

In Pavlova lutheri, the percentage of EPA and DHA in polar lipids was highest at low light intensities whereas their synthesis was best at intermediate (19 w/m -2 ) intensities [START_REF] Guedes | Changes in lipid class and fatty acid composition of cultures of Pavlova lutheri, in response to light intensity[END_REF].

For the red alga, Trichocarpus crinitus, low light conditions favoured an increase in membrane components especially SQDG, PtdGro and PtdCho. However, in contrast to Pavlova lutheri there were no differences in total FAs under different light regimes. Nevertheless, there were some changes in individual lipid classes. For example, the percentage of 20:5 in MGDG decreased but in PtdGro it increased. HL increased the proportion of trans-16:1 in PtdGro [START_REF] Khotimchenko | Lipid composition of the red alga Tichocarpus crinitus exposed to different levels of photon irradiance[END_REF].

Differences in how individual algae respond to light was also emphasised in a study of four freshwater phytoplankton species (including rarely examined Chrysophyceae and Zygnematophyceaea) under different light intensities. While there were significant changes in all algae examined, no generalisations about these alterations could be made [START_REF] Wacker | Light-induced changes in fatty acid profiles of specific lipid classes in several freshwater phytoplankton species[END_REF].

HL intensity tends to increase TAG levels together with decreases in polar lipid classes.

This has been observed in the diatom, Thallassiosira pseudonana [START_REF] Brown | Effects pf harvest stage and light on the biochemical composition of the diatom Thalassiosira pseudonana[END_REF], the red alga

Trichocarpus crinitus [START_REF] Khotimchenko | Lipid composition of the red alga Tichocarpus crinitus exposed to different levels of photon irradiance[END_REF] and various freshwater species [START_REF] Napolitano | The relationship of lipids with light and chlorphyll measurements in freshwater algae and periphyton 1[END_REF]. For C. reinhardtii saturating light induces sustained accumulation of TAG in lipid droplets. Interestingly, some of these droplets appeared to be located in plastids in contrast to N-deprived growth wherein 60% are of ER-origin [START_REF] Goold | Saturating light induces sustained accumulation of oil in plastidal lipid droplets in Chlamydomonas reinhardtii[END_REF]. Also in Chlamydomonas, extended (24h) dark periods cause TAG accumulation [START_REF] Hemschemeier | COPPER RESPONSE REGULATOR1-dependent and -independent responses of the Chlamydomonas reinhardtii transcriptome to dark anoxia[END_REF]. Prolonged darkness in Phaeodactylum tricornutum has also been studied in detail and information about nuclear transcriptional activity, pigment content and photosynthesis reported during darkness and following re-illumination [START_REF] Heydarizadeh | Response of CO2-starved diatom Phaeodactylum tricornutum to light intensity transition[END_REF].

Finally, the effect of UV-B irradiation in various algae has been examined. The extent of changes depended on whether the algae were UV sensitive or tolerant. In particular, reduction in VLCPUFAs, such as EPA and DHA, were noted and also that nutrient-deprived cells were more sensitive [START_REF] Wang | Reduction in omega-3 fatty acids by UV-B irradiation in microalgae[END_REF].

Temperature

Previous studies indicated that temperature can influence not only FA proportions but also the lipid class content in different algae [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF][START_REF] Pohl | Fatty acids and lipids of marine algae and the control of their biosynthesis by environmental factors, marine algae in pharmaceutical science[END_REF]. Optimal algal growth usually occurs at 20-30°C [START_REF] Singh | Effect of temperature and light on the growth of algae species: A review[END_REF] but each species has its optimal value [START_REF] Béchet | Modeling the impact of high temperatures on microalgal viability and photosynthetic activity[END_REF]. This has important implications for outdoor culture systems [START_REF] Khan | The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products[END_REF]. Of course, some specialised algae can endure temperatures of 40°C while those growing in hot springs can tolerate temperatures of 80°C.

Naturally, given the diversity of algal species and the temperature range of their habitats, it is unsurprising that lipid analyses have revealed differences. Anesi et al. [START_REF] Anesi | Comparative analysis of membrane lipids in psychrophilic and mesophilic freshwater dinoflagellates[END_REF] examined ten dinoflagellates from different freshwater habitats with 4, 13 or 20°C temperatures. They could be grouped depending on the molecular species of their lipid classes. The glycosylglycerides (MGDG, DGDG, SQDG) seemed particularly useful for classification. The range of dinoflagellate tolerance was concluded to be best reflected in thylakoid glycolipids while phylogeny could be better revealed by the distribution of non-thylakoid lipids and their species [START_REF] Anesi | Comparative analysis of membrane lipids in psychrophilic and mesophilic freshwater dinoflagellates[END_REF]. A study of a lipid-producing, cold-tolerant yellow green alga (Xanthophyceae) isolated from the Rocky Mountains showed that it produced the highest amount of lipids when grown with HL at 4°C. Under these conditions Heterococcus sp. DN1, produced enhanced amounts of PUFA (especially EPA) at the expense of 16:1 [START_REF] Nelson | New lipid-producing, cold-tolerant yellow-green alga isolated from the Rocky Mountains of Colorado[END_REF].

Temperature can be used as a stressor to encourage the production of valuable metabolites [START_REF] Møller | Carotenoid-dependent signals: indicators of foraging efficiency, immunocompetence or detoxification ability?[END_REF] and can be used in combination with other parameters to increase lipid yields [START_REF] Converti | Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production[END_REF]. Increased growth temperature can be used to elevate lipid contents in several species [317] [9] including Chlamydomonas [START_REF] Hemme | Systems-wide analysis of acclimation responses to long-term heat stress and recovery in the photosynthetic model organism Chlamydomonas reinhardtii[END_REF][START_REF] Légeret | Lipidomic and transcriptomic analyses of Chlamydomonas reinhardtii under heat stress unveil a direct route for the conversion of membrane lipids into storage lipids[END_REF]. The broad effects of culture temperature on growth, lipid composition and FA quality have been studied fairly extensively (see [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF]). With the important application of algae in biotechnology (section 7), species of commercial interest have formed the main recent focus. Thus, the thrustochytrid, Aurantiochytrium, that forms appreciable DHA, was examined in the range 10-30°C and optimised at 10°C [453]. In addition, the combination of N-stress with different growth temperatures has been studied in C.

reinhardtii in connection with potential biofuel production. In these experiments growth at 32°C seemed optimal for FA content and composition in connection with potential use as biofuel [START_REF] James | Temperature modulation of fatty acid profiles for biofuel production in nitrogen deprived Chlamydomonas reinhardtii[END_REF]. With increased global warming some relevant studies have been conducted

recently with regard to heat stress (in Chlamydomonas) [START_REF] Légeret | Lipidomic and transcriptomic analyses of Chlamydomonas reinhardtii under heat stress unveil a direct route for the conversion of membrane lipids into storage lipids[END_REF][START_REF] Schroda | The Chlamydomonas heat stress response[END_REF] and for climate warming in Scenedesmus obliquus [START_REF] Fuschino | Rising water temperatures alter lipid dynamics and reduce n-3 essential fatty acid concentrations in cenedesmus obliquus (Chlorophyta)[END_REF]. In the latter case, even a relatively moderate increase in ambient temperature (20 to 28°C) resulted in significant changes in endogenous lipids and their metabolism. In particular, the decrease in unsaturation and, consequently, essential PUFAs has implications concerning food quality for higher trophic levels.

Other factors

Production of lipids in algae can be influenced by such factors as dehydration, salinity, culture age and pH. For more general details on these factors please refer to [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF][START_REF] Du | Triacylglycerol accumulation in photosynthetic cells in plants and algae[END_REF][START_REF] Khan | The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products[END_REF].

Supplementary information for hyper salinity is provided for Chlamydomonas where PtdOH seems to be involved as a second messenger [START_REF] Arisz | The salt stress-induced LPA response in Chlamydomonas is produced via PLA(2) hydrolysis of DGK-generated phosphatidic acid[END_REF], and for various stressors (salinity, N-or Pdeprivation, temperature) in combination with elevated carbon dioxide. For Nannochloropsis, EPA yields were increased by nitrate, low salinity and low temperature [START_REF] Hu | Response of growth and fatty acid compositions of Nannochloropsis sp. to environmental factors under elevated CO2 concentration[END_REF]. Lipid accumulation in 50 strains of microalgae during fluctuating brackish and sea water locations has also been examined. From these studies, some promising algae for biodiesel or -3 FA production were found [START_REF] Duong | Growth and lipid accumulation of microalgae from fluctuating brackish and sea water locations in South East Queensland-Australia[END_REF].

Finally, two special conditions for changes in acyl lipid production have been found for Chlorella spp. In Chlorella sorokiniana, the impact of inoculum sizes on phospholipid metabolism revealed that PtdGro, PtdEtn and several molecular species of PtdCho may be changed under the experimental conditions. The authors suggested that their data may help in providing potential targets for engineering to improve biofuel production [START_REF] Lu | Phospholipid metabolism in an industry microalga Chlorella sorokiniana: the impact of inoculum sizes[END_REF]. Additionally, air drying of cells was found to stimulate TAG synthesis in Chlorella kessleri by 2.7-fold. The same conditions also stimulated oil accumulation in C. reinhardtii but to a much smaller extent [START_REF] Shiratake | Air-drying of cells, the novel conditions for stimulated synthesis of triacylglycerol in a green alga, Chlorella kessleri[END_REF].

Algae for industry

Commercial applications of algae have been reviewed extensively over the years since the article by Guschina and Harwood [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF] see [START_REF] Brodie | The algal revolution[END_REF][START_REF] Harwood | The versatility of algae and their lipid metabolism[END_REF][START_REF] Khan | The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products[END_REF][START_REF] Spolaore | Commercial applications of microalgae[END_REF][START_REF] Raja | A perspective on the biotechnological potential of microalgae[END_REF][START_REF] Harun | Bioprocess engineering of microalgae to produce a variety of consumer products[END_REF][START_REF] Rawat | Dual role of microalgae: Phycoremediation of domestic wastewater and biomass production for sustainable biofuels production[END_REF][START_REF] Bellou | Microalgal lipids biochemistry and biotechnological perspectives[END_REF][START_REF] Bajhaiya | Transcriptional engineering of microalgae: Prospects for high-value chemicals[END_REF][START_REF] Yeh | Commercializing algae -challenges and opportunities[END_REF][START_REF] Lane | Hottest trends in algae[END_REF]. Apart from uses in the food or aquaculture industries, algae can be used for pigments, various useful chemicals and, of course potentially for biofuel. While being used for these purposes, algae can also serve environmental applications [START_REF] Harun | Bioprocess engineering of microalgae to produce a variety of consumer products[END_REF] such as in bioremediation [START_REF] Rawat | Dual role of microalgae: Phycoremediation of domestic wastewater and biomass production for sustainable biofuels production[END_REF]. While much of recent research has concentrated on biotechnological products for wealthy countries [START_REF] Arrieta | What lies underneath: Conserving the oceans' genetic resources[END_REF], the data are also applicable to developing countries where the use of macroalgae, such as seaweeds is growing significantly [START_REF] Colombo | Marine macroalgae as sources of polyunsaturated fatty acids, Plant foods for human nutrition[END_REF][START_REF] Mazarrasa | Rapid growth of seaweed biotechnology provides opportunities for developing nations[END_REF].

Before discussing uses of algae, some comments should be made about important technical aspects. One of the most expensive stages in the industrial use of algae is extraction of lipids (and other products). This subject is covered in many of the above reviews but is specifically highlighted in others (e.g. [START_REF] Chen | A look at diacylglycerol acyltransferases (DGATs) in algae[END_REF][START_REF] Halim | Extraction of oil from microalgae for biodiesel production: A review[END_REF][START_REF] Ryckebosch | Optimization of an analytical procedure for extraction of lipids from microalgae[END_REF][START_REF] Yao | Neutral lipid accumulation at elevated temperature in conditional mutants of two microalgae species[END_REF] and, importantly, the use of a forward genetic screen to identify useful oil mutants [START_REF] Cagnon | Development of a forward genetic screen to isolate oil mutants in the green microalga Chlamydomonas reinhardtii[END_REF] is notable. Some useful technical aspects include LC-MS technology [START_REF] Samburova | Analysis of triacylglycerols and free fatty acids in algae using ultra-performance liquid chromatography mass spectrometry[END_REF], optimisation of productivity using FTIR analysis [START_REF] Liu | Optimization of lipid production for algal biodiesel in nitrogen stressed cells of Dunaliella salina using FTIR analysis[END_REF] and high throughput analysis with MS [START_REF] Lee | High-throughput analysis of algal crude oils using high resolution mass spectrometry[END_REF].

Furthermore, there have been important developments in the molecular biology of algae, particularly species which have been earmarked as commercially important. Following publication of a diatom EST database [START_REF] Maheswari | The diatom EST database[END_REF], the Phaeodactylum genome [START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF] and that for

Nannochloropsis gaditana [START_REF] Radakovits | Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana[END_REF], further comments about diatoms have been up-dated [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF].

Additional technical aspects include stable nuclear transformation of P. tricornutum [START_REF] Apt | Stable nuclear transformation of the diatom Phaeodactylum tricornutum[END_REF], gene silencing in the same species [START_REF] De Riso | Gene silencing in the marine diatom Phaeodactylum tricornutum[END_REF], RNAi-based gene knockdown in N. oceanica [START_REF] Wei | RNAi-based targeted gene knockdown in the model oleaginous microalgae Nannochloropsis oceanica[END_REF] and the use of CRISPR technology for genome editing [START_REF] Wang | Genome editing of model oleaginous microalgae Nannochloropsis spp. by CRISPR/Cas9[END_REF].

Of the various uses of eukaryotic algae for industry, two topics have attracted particular attention over the last decade. These are, respectively, algae as sources of VLCPUFAs and for biofuel production.

Algae as sources of very long chain fatty acids (VLCPUFAs)

Humans and most animals require essential fatty acids of the n-3 and n-6 series [245]. The basic acids of these series are, respectively, α-linolenic (LNA) and linoleic (LA) acids. The usual sources of such are vegetable oils either in the form of spreads or cooking oils but they are also contained in most food products. However, current diets (especially 'Western diets') provide excessive amounts of n-6 PUFA so that a typical ratio of dietary n-6/n-3 PUFA is around 15, whereas the best nutritional advice suggests that a ratio of 3-4 would be more appropriate [START_REF] Hibbeln | Healthy intakes of n-3 and n-6 fatty acids: estimations considering worldwide diversity[END_REF][START_REF] Lands | Historical perspectives on the impact of n-3 and n-6 nutrients on health[END_REF][485]. This is because the main role of the essential fatty acids is to be converted to various 20C or 22C PUFAs which are then oxidised to powerful signalling compounds such eicosanoids, resolvins and protectins. Those signalling molecules derived from n-3 PUFAs are generally anti-inflammatory while those from n-6 PUFAs are generally pro-inflammatory [245,[START_REF] Schmitz | The opposing effects of n-3 and n-6 fatty acids[END_REF].

This is believed to have led to an increase in chronic inflammation and associated diseases (e.g. arthritis, cardio-vascular complaints) in Western societies [START_REF] Calder | Marine omega-3 fatty acids and inflammatory processes: Effects, mechanisms and clinical relevance[END_REF][START_REF] Calder | Very long-chain n-3 fatty acids and human health: fact, fiction and the future[END_REF][START_REF] Harwood | Dietary omega-3 polyunsaturated fatty acids and inflammation[END_REF].

Although LA and LNA are the main PUFAs in the diet and are thought to be appropriate for normal health requirements [START_REF] Barcelo-Coblijn | Alpha-linolenic acid and its conversion to longer chain n-3 fatty acids: benefits for human health and a role in maintaining tissue n-3 fatty acid levels[END_REF], they are poorly converted (especially by men) to ARA, EPA and DHA which are the immediate precursors of signalling compounds. Thus, under certain situations there is a 'conditional requirement' for ARA and/or EPA and DHA in the diet [START_REF] Cunnane | Problems with essential fatty acids: time for a new paradigm?[END_REF]. In fact, the perceived need for more n-3 PUFA in diets has led to an increased consumption of fish oils as a convenient source of EPA and DHA. However, given the advised human (and animal) daily requirements [START_REF] Kris-Etherton | Dietary reference intakes for DHA and EPA[END_REF] and perceived over-fishing in the World, this is not a sustainable situation [START_REF] Hutchings | Marine fish population collapses: consequences for recovery and extinction risk[END_REF][START_REF] Tocher | Issues surrounding fish as a source of omega-3 long-chain polyunsaturated fatty acids[END_REF][START_REF] Adarme-Vega | Towards sustainable sources for omega-3 fatty acids production[END_REF].

Following the undeniable demonstration of the importance of VLCPUFA for good health, the commercial market for such products has increased considerably. This has led to a search for algal sources to supplement the obvious limitation to fish oil supplies [START_REF] Ratledge | Single Cell Oils : Microbial and algal oils[END_REF][START_REF] Yongmanitchai | Growth of and omega-3 fatty acid production by Phaeodactylum tricornutum under different culture conditions[END_REF]. Such algal oils have proven useful in infant milk formulations, adult nutraceuticals and in fish feeds.

The first of such oils was 'DHASCO' from Crypthecodinium cohnii [START_REF] Breivik | Long-Chain omega-3 specialty oils[END_REF] and, later, those from Schizochytrium spp. have proven commercially successful (Table 5). Other sources of DHA were discussed in [START_REF] Ratledge | Single Cell Oils : Microbial and algal oils[END_REF][START_REF] Kuratko | Docosahexaenoic acid from algal oil[END_REF], where some of the advantages of algal oils are described. For example, algal oils are usually enriched in a particular VLCPUFA whereas fish oils often have a variable ratio of EPA/DHA (depending on the algae that the fish consumed). Furthermore, the potential problem of toxic compounds in fish oils (or in fish themselves) is obviated by culturing algae [START_REF] Ratledge | Single Cell Oils : Microbial and algal oils[END_REF].

The primary producers of PUFAs are photosynthetic organisms and, in the case of EPA and DHA, are marine algae [START_REF] Spolaore | Commercial applications of microalgae[END_REF]. In fact, ironically, fish are often as poor as humans in converting LA to ARA or LNA to EPA and DHA [START_REF] Tocher | Effects of dietary vegetable oil on Atlantic salmon hepatocyte fatty acid desaturation and liver fatty acid compositions[END_REF]. This has led to the increasing use of algae as direct sources of VLCPUFAs in human diets [START_REF] Adarme-Vega | Microalgal biofactories: a promising approach towards sustainable omega-3 fatty acid production[END_REF] as well as for farmed fish feeds [START_REF] Bell | Arachidonic acid in aquaculture feeds: current status and future opportunities[END_REF][START_REF] Patil | Fatty acid composition of 12 microalgae for possible use in aquaculture feed[END_REF][START_REF] Ganuza | Crypthecodinium cohnii and Schizochytrium sp. as potential substitutes to fisheries-derived oils from seabream (Sparus aurata) microdiets[END_REF]. Thus, for example, Nannochloropsis spp. and Phaeodactylum tricornutum can have an EPA content of up to 40% total fatty acids under autotrophic conditions [START_REF] Yongmanitchai | Growth of and omega-3 fatty acid production by Phaeodactylum tricornutum under different culture conditions[END_REF][START_REF] Sukenik | Ecophysiological considerations in the optimization of eicosapentaenoic acid production by Nannochloropsis sp. (Eustigmatophyceae)[END_REF].

Similarly, Thraustochytrium and Schizochytrium limacinum, when grown under heterotrophic fermentation conditions, can accumulate 30-40%DHA [START_REF] Adarme-Vega | Microalgal biofactories: a promising approach towards sustainable omega-3 fatty acid production[END_REF]. One comment that should be made here is that EPA and DHA (despite being metabolically interconvertible) seem to have independent effects on humans [START_REF] Mori | The independent effects of eicosapentaenoic acid and docosahexaenoic acid on cardiovascular risk factors in humans[END_REF][START_REF] Wei | Effects of eicosapentaenoic acid versus docosahexaenoic acid on serum lipids: a systematic review and meta-analysis[END_REF].

Wen and Chen [START_REF] Wen | 8 -Production of eicosapentaenoic acid using heterotrophically grown microalgae[END_REF] have discussed the production of EPA by microorganisms in some detail. Although considerable interest has been focussed on Shewanella spp. (a marine bacterium), factors affecting production in microalgae (e.g. Nannochloropsis) including diatoms (e.g. Phaeodactylum tricornutum) have been described. These include the use of different systems as well as various environmental factors [START_REF] Wen | 8 -Production of eicosapentaenoic acid using heterotrophically grown microalgae[END_REF]. Because fish oils contain a mixture of EPA and DHA, the question of a need for EPA per se is important (see [START_REF] Babcock | Eicosapentaenoic acid (EPA): an antiinflammatory omega-3 fat with potential clinical applications[END_REF][START_REF] Calder | n-3 polyunsaturated fatty acids and cytokine production in health and disease[END_REF]; and remarks above). Moreover, in terms of nutraceuticals, the subject is complicated by the known ability of dietary DHA to be retro-converted to EPA [START_REF] Schacky | Metabolism and effects on platelet function of the purified eicosapentaenoic and docosahexaenoic acids in humans[END_REF].

The other important VLCPUFA is ARA which, together with DHA, is a prominent component of brain and other nervous tissues [START_REF] Hall | Braine lipids in health and disease[END_REF]. This acid is accumulated in many bryophytes and some marine algae, such as Phaeophyceae [START_REF] Pohl | Fatty acids and lipids of marine algae and the control of their biosynthesis by environmental factors, marine algae in pharmaceutical science[END_REF]. On the other hand, it rarely accumulates to any great extent in most microalgae [START_REF] Lang | Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection[END_REF]. However, a freshwater microalga, Lobosphaera incisa (formerly Parietochloris incisa), accumulates ARA at around 50% of total fatty acids in TAG under N-starvation conditions [START_REF] Bigogno | Lipid and fatty acid composition of the green oleaginous alga Parietochloris incisa, the richest plant source of arachidonic acid[END_REF]. To convert oleic acid into ARA requires three desaturases (5, 6 and 12) which have been cloned from L. incisa [START_REF] Iskandarov | Identification and characterization of Delta12, Delta6, and Delta5 Desaturases from the green microalga Parietochloris incisa[END_REF] as well as an elongase [START_REF] Iskandarov | Cloning and characterization of the a dagger 6 polyunsaturated fatty acid elongase from the green microalga Parietochloris incisa[END_REF]. In addition, overexpression of a 'GPAT-like' gene from L. incisa was shown to increase TAG synthesis [268]. The accumulation of TAG in microalgae, stimulated by nutrient stress (see section 6) is associated not only with increased synthesis but also mobilisation of carbon from membrane lipids, for example those in chloroplasts [START_REF] Abida | Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum[END_REF][START_REF] Miller | Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism[END_REF][START_REF] Schmollinger | Nitrogen-sparing mechanisms in Chlamydomonas affect the transcriptome, the proteome, and photosynthetic metabolism[END_REF].

This may involve autophagy [START_REF] Davey | Triacylglyceride production and autophagous responses in Chlamydomonas reinhardtii depend on resource allocation and carbon source[END_REF][START_REF] Bassham | Autophagy in plants and algae[END_REF]. Moreover, since N-starvation is a reversible process, the transient production of TAG-enriched lipid bodies gives rise to the transfer of fatty acids back into chloroplast membranes during recovery in L. incisa [517].

The commercial production of DHA by Schizochytrium has been thoroughly discussed by [START_REF] Barclay | 4 -Development of a docosahexaenoic acid production technology using Schizochytrium: historical perspective and update[END_REF]. These organisms are Thraustochytrids which make up a significant proportion of phytoplankton, although they are often under-reported [START_REF] Raghukumar | An epifluoresence method for direct enumeration of the fungilike marine protists, the Thraustochytrids[END_REF]. (It should be noted that, although most researchers will call Thraustochytrids algae, their classification is a little controversial).

The PKS system for making VLCPUFAs (sometimes called PUFA synthetase) first identified by Metz et al. [START_REF] Metz | Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes[END_REF] has been studied further with respect to its acyl carrier protein (ACP) domains [START_REF] Jiang | The role of tandem acyl carrier protein domains in polyunsaturated fatty acid biosynthesis[END_REF], expression in E. coli to make DPAn-6 and DHA [START_REF] Hauvermale | Fatty acid production in Schizochytrium sp.: involvement of a polyunsaturated fatty acid synthase and a type I fatty acid synthase[END_REF] and formation of nonesterified fatty acids as end products [START_REF] Metz | Biochemical characterization of polyunsaturated fatty acid synthesis in Schizochytrium: release of the products as free fatty acids[END_REF]. Commercial uses of Schizochytrium oils have been evaluated for feeds (fish, poultry and cattlewhere DHA-enriched milks have been produced), breads, milk drinks, nutritional bars, margarines etc. [START_REF] Barclay | 4 -Development of a docosahexaenoic acid production technology using Schizochytrium: historical perspective and update[END_REF]. A review of how lipid metabolism could be manipulated in Schizochytrium and other Chromista, especially P. tricornutum, to increase EPA and DHA has been made [START_REF] Mühlroth | Pathways of lipid metabolism in marine algae, co-expression network, bottlenecks and candidate genes for enhanced production of EPA and DHA in species of Chromista[END_REF].

A background to the use of algae for producing high-value products, like VLCPUFAs, is given in [START_REF] Stefan | Advances in the production of high-value products by microalgae[END_REF] while for specific aspects of the latter see [START_REF] Armenta | Single-cell oils as a source of omega-3 fatty acids: an overview of recent advances[END_REF]. A variety of algae from different classes have been or are of interest either as species for basic research or for biotechnological development (Table 5).

Several freshwater or marine species of the genus Nannochloropsis (e.g. N.gaditana, N. oculata) contain high concentrations of EPA in their DGTS as well as chloroplast glycerolipids [525] [349] which can be transferred to TAG. Another eustigmatophyte, Trachydiscus minatus, also contains a high % of EPA in storage lipids and may be a potential industrial source [START_REF] Rezanka | Trachydiscus minutus, a new biotechnological source of eicosapentaenoic acid[END_REF][START_REF] Rezanka | Effect of nitrogen and phosphorus starvation on the polyunsaturated triacylglycerol composition, including positional isomer distribution, in the alga Trachydiscus minutus[END_REF]. Likewise, diatoms (in particular Phaeodactylum tricornutum) have attracted attention not only as a source of EPA but also DHA [START_REF] Khozin-Goldberg | Microalgae as a source for VLC-PUFA production[END_REF][START_REF] Draaisma | Food commodities from microalgae[END_REF]. P. tricornutum has been genetically modified to enhance productivity of n-3 VLCPUFAs [START_REF] Hamilton | Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids[END_REF] and its biotechnological economics assessed [START_REF] Chauton | A techno-economic analysis of industrial production of marine microalgae as a source of EPA and DHA-rich raw material for aquafeed: Research challenges and possibilities[END_REF]. Additionally, haptophytes like Isochrysis galbana or Pavlova lutheri, are important potential industrial species. Marine macroalgae often contain high amounts of LCPUFAs [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF][START_REF] Harwood | Lipid metabolism in algae[END_REF][START_REF] Pohl | Fatty acids and lipids of marine algae and the control of their biosynthesis by environmental factors, marine algae in pharmaceutical science[END_REF] and their use in nutrition has been considered [START_REF] Colombo | Marine macroalgae as sources of polyunsaturated fatty acids, Plant foods for human nutrition[END_REF].

As mentioned previously, culture conditions are critical in ensuring good growth and productivity. Some relevant studies are [532-534] [535]. Moreover, some aspects of genetic modification of algae to enhance productivity are discussed by Khozin-Goldberg et al [START_REF] Khozin-Goldberg | Microalgae as a source for VLC-PUFA production[END_REF]. In particular, information about TAG accumulation in Chlamydomonas has been applied to the oleaginous microalgae Nannochloropsis sp. which is often considered one of the best species for industrial utility [START_REF] Taleb | Development and validation of a screening procedure of microalgae for biodiesel production: Application to the genus of marine microalgae Nannochloropsis[END_REF] especially for the production of EPA [START_REF] Vieler | A lipid droplet protein of Nannochloropsis with functions partially analogous to plant oleosins[END_REF]. Knowledge of its genome and RNA-sequencing of samples from N-replete and -deprived growth, have revealed that many of the genes involved in the Kennedy pathway, as well as PDAT1 and PDAT2 are up-regulated on N-deprivation [START_REF] Du | Triacylglycerol accumulation in photosynthetic cells in plants and algae[END_REF].

As a result of such studies, several commercial companies have exploited algae for the production of EPA and/or DHA. These include the use of Nannochloropsis [START_REF] Winwood | Recent developments in the commercial production of DHA and EPA rich oils from micro-algae[END_REF] or Odontella aurita [START_REF] Guiheneuf | Effect of UV stress on the fatty acid and lipid class composition in two marine microalgae Pavlova lutheri (Pavlovophyceae) and Odontella aurita (Bacillariophyceae)[END_REF][START_REF] Haimeur | The role of Odontella aurita, a marine diatom rich in EPA, as a dietary supplement in dyslipidemia, platelet function and oxidative stress in high-fat fed rats[END_REF] for EPA production. For DHA, Crypthecodinium cohnii (Dynophyta) and Schizochytrium (Thraustochytriaceae) have been used for several years in the infant formula market [START_REF] Winwood | Recent developments in the commercial production of DHA and EPA rich oils from micro-algae[END_REF] (Table 6). Such algal oils have been shown to be efficacious, non-toxic and of high nutritional value [START_REF] Khozin-Goldberg | Microalgae as a source for VLC-PUFA production[END_REF].

As mentioned previously, the first commercial SCO (single cell oil) containing DHA was from C. cohnii, a dinoflagellate. There are over 2000 identified dinoflagellates, of which only about half are photosynthetic. The production of DHA in such species and, especially, in C. cohnii is well discussed in [START_REF] Wynn | Apt, 6 -Production of single cell oils by dinoflagellates[END_REF]. This has included the use of substrates other than glucose to boost or extend production [START_REF] Sijtsma | 7 -alternative carbon sources for heterotrophic production of docosahexaenoic acid by the marine alga Crypthecodinium cohnii[END_REF]. For the latter, it was demonstrated that higher yields of DHA (compared to glucose) could be obtained using acetic acid or ethanol. Glycerol is also a potential substrate [START_REF] Pyle | Producing docosahexaenoic acid (DHA)-rich algae from biodieselderived crude glycerol: effects of impurities on DHA production and algal biomass composition[END_REF] although that produced in surplus from biofuel manufacture is not of food grade [START_REF] Sijtsma | 7 -alternative carbon sources for heterotrophic production of docosahexaenoic acid by the marine alga Crypthecodinium cohnii[END_REF]. In addition, there have been studies of lipid productivity (in Chlorella and N.

salina) using a lab-scale open pond simulating reactor [START_REF] Bellou | Biochemical activities in Chlorella sp. and Nannochloropsis salina during lipid and sugar synthesis in a lab-scale open pond simulating reactor[END_REF].

General considerations used for the commercial processing of algal oils, particularly as applied to VLCPUFA products, are described by Ratledge et al [START_REF] Ratledge | 1 -Single cell oils for the 21st century[END_REF]. Not only are algal VLCPUFAs important in the human nutritional food industry, they are also increasingly used in feed to modify meat, milk or egg characteristics, in pet formulations [START_REF] Abril | 18 -Applications of single cell oils for animal nutrition[END_REF] and in aquaculture [START_REF] Velasco-Escudero | 19 -Applications of single cell oils for aquaculture[END_REF].

Biofuels

A second major area for the applied use of algae is in their potential as sources of biofuels [START_REF] Khan | The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products[END_REF].

As fossil fuels are diminishing, sustainable replacement sources are required. Moreover such sources would not elevate atmospheric carbon dioxide and, hence, contribute to climate change.

General reviews on the production and uses of algal oils (TAG) for biofuel production (and other purposes) are given by [START_REF] Hu | Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances[END_REF][START_REF] Wijffels | An outlook on microalgal biofuels[END_REF][START_REF] Mata | Microalgae for biodiesel production and other applications: A review[END_REF][START_REF] Jones | Algae biofuels: versatility for the future of bioenergy[END_REF][START_REF] Rittmann | Opportunities for renewable bioenergy using microorganisms[END_REF][START_REF] Robertson | A new dawn for industrial photosynthesis[END_REF][START_REF] Ratledge | Microbial and algal oils: Do they have a future for biodiesel or as commodity oils?[END_REF] in addition to those given in the introduction to section 7.

Microalgal-based fuels are eco-friendly and non-toxic and, of course, are formed by fixing atmospheric carbon dioxide [START_REF] Schenk | Second generation biofuels: high-efficiency microalgae for biodiesel production[END_REF]. Microalgae grow rapidly and have been estimated to have the potential of transforming 9-10% of solar energy into biomass with a theoretical yield of around 77g/biomass/m2/day (equivalent to 280 tonne/hectare/year) [START_REF] Formighieri | Regulation of the pigment optical density of an algal cell: filling the gap between photosynthetic productivity in the laboratory and in mass culture[END_REF][START_REF] Melis | Solar energy conversion efficiencies in photosynthesis: Minimizing the chlorophyll antennae to maximize efficiency[END_REF]. It is also very important that the growth of algae does not use agricultural land and, therefore, does not compete with food/feed production, unlike most plant crops. Moreover, algae can often use saline or waste water or can even be employed to simultaneously remove pollutants [START_REF] Zamora-Castro | A novel approach for bioremediation of a coastal marine wastewater effluent based on artificial microbial mats[END_REF][START_REF] Park | Theoretical calculations on the fasibility of microalgal biofuels: utilization of marine resources could help realizing the potential of microalgae[END_REF][START_REF] Pandley | Microalgae biomass production for carbon dioxide mitigation and biodiesel production[END_REF],

including phytoremediation of domestic wastewater [START_REF] Rawat | Dual role of microalgae: Phycoremediation of domestic wastewater and biomass production for sustainable biofuels production[END_REF]. In addition, the great diversity of microalgae provides opportunities for selection of species and strains that can produce oils which can yield biofuels with specific properties. Furthermore, there are also possibilities of using genetic manipulation to enhance productivity and/or modified oil properties [START_REF] Borowitzka | 13 -Algae oils for biofuels: chemistry, physiology, and production[END_REF].

From the above discussion, it can be concluded that algae appear to be one (some would argue the only [START_REF] Chisti | Biodiesel from microalgae[END_REF]) source of renewable biodiesel capable of meeting future demands.

However, the present high cost makes the use of algae for high-value oils rather than biodiesel currently more attractive and economic [START_REF] Ratledge | Single Cell Oils : Microbial and algal oils[END_REF] (see later discussion). For more information about the use of microalgae to make biodiesel, see [560, 561] [562-564].

Many algae can produce substantial quantities of TAG (up to 80% of total lipids which can be up to 70% dry weight) [START_REF] Borowitzka | 13 -Algae oils for biofuels: chemistry, physiology, and production[END_REF]. However, this usually occurs in response to nutrient deprivation (especially N) [START_REF] Brodie | The algal revolution[END_REF]. Nevertheless, the green alga, Botryococcus braunii, produces >60% of its lipid as hydrocarbons. The bulk of these are accumulated outside cells [START_REF] Borowitzka | 13 -Algae oils for biofuels: chemistry, physiology, and production[END_REF],

making recovery somewhat easier. There are different types of B. braunii; the A-race, B-race and L-race strains [START_REF] Metzger | Botryococcus braunii: a rich source for hydrocarbons and related ether lipids[END_REF].

Clearly, a key consideration in choosing appropriate algae for biofuel production will be the nature of the oil produced (fatty acid composition), as well as the productivity of the strains selected. Such considerations are discussed in [START_REF] Griffiths | Lipid productivity as a key characteristic for choosing algal species for biodiesel production[END_REF][START_REF] Mutanda | Bioprospecting for hyperlipid producing microalgal strains for sustainable biofuel production[END_REF][START_REF] Sobczuk | Potential fuel oils from the microalga Choricystis minor[END_REF].

As mentioned above, TAG is the lipid normally accumulated in algae and aspects of its biosynthesis are discussed in section 4.5. The accumulation of TAG following nutrient stress (see section 6) is a major hurdle to be overcome, because growth ceases leading to poor overall productivity [START_REF] Georgianna | Exploiting diversity and synthetic biology for the production of algal biofuels[END_REF][START_REF] Scott | Biodiesel from algae: challenges and prospects[END_REF]. In efforts to address this problem, research has concentrated on enzymes important for TAG synthesis, such as DGAT [START_REF] Chen | A look at diacylglycerol acyltransferases (DGATs) in algae[END_REF] and seeing which of its isoforms were upregulated on N starvation [START_REF] Miller | Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism[END_REF]. Follow-up experiments to use a strong light-responsive promoter of the DGAT genes, however, failed to increase TAG levels, pointing to tight control of lipid synthesis (in C. reinhardtii) [START_REF] Brodie | The algal revolution[END_REF]. In comparison, expression in Phaeodactylum tricornutum under control of the light-responsive FCPC promoter increased neutral lipid levels [START_REF] Niu | Improvement of neutral lipid and polyunsaturated fatty acid biosynthesis by overexpressing a type 2 diacylglycerol acyltransferase in marine diatom Phaeodactylum tricornutum[END_REF]. Somewhat unexpectedly, it also changed the fatty acid composition of membrane as well as storage (TAG) lipids [START_REF] Brodie | The algal revolution[END_REF]. Other methods to attempt to overcome the nutritional stress problem are discussed in [START_REF] Brodie | The algal revolution[END_REF].

Further aspects of the biosynthesis of hydrocarbons [START_REF] Baba | Biosynthesis of lipids and hydrocarbons in algae[END_REF] and of TAGs are given in [START_REF] Trentacoste | Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth[END_REF][573][START_REF] Sharma | High lipid induction in microalgae for biodiesel production[END_REF][START_REF] Zhu | Growth and biomass characteristics of Picochlorum oklahomensis and Nannochloropsis oculata[END_REF][START_REF] Wase | Innovations in improving lipid production: Algal chemical genetics[END_REF]. This includes the use of multiple carbon fixation pathways [START_REF] Valenzuela | Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum[END_REF], reduction of competing catabolism [START_REF] Trentacoste | Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth[END_REF] and the recent use of a transcriptional regulator [START_REF] Ajjawi | Lipid production in Nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator[END_REF][START_REF] Posewitz | Algal oil productivity gets a fat bonus[END_REF]. Please refer to sections 4.6 and 6 for additional discussion of the regulation of TAG biosynthesis and accumulation. A recent review has focussed on the use of small molecules (through the process of 'chemical genetics') to improve algal lipid production [START_REF] Wase | Innovations in improving lipid production: Algal chemical genetics[END_REF].

Once TAG has been harvested from algae, it has to be converted into fatty acid methyl esters (FAMEs) for biodiesel. Four methods (base-catalysed transesterification, acid-catalysed transesterification, non-catalytic conversion, lipase-catalysed techniques) can be used [START_REF] Borowitzka | 13 -Algae oils for biofuels: chemistry, physiology, and production[END_REF].

The properties of the biodiesel produced are largely dependent on the fatty acid composition of the original TAG [START_REF] Knothe | Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters[END_REF] and this is very important for the final standard of the finished product [START_REF] Knothe | Analyzing biodiesel: standards and other methods[END_REF]. Thus, strain selection [START_REF] Rodolfi | Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor[END_REF] and growth conditions (e.g. temperature, light, salinity) [START_REF] Borowitzka | 13 -Algae oils for biofuels: chemistry, physiology, and production[END_REF] are vital considerations.

In terms of technical aspects, the use of Fourier transform infrared spectroscopy to monitor lipid production under various combinations of temperature and cell densities has been reported [START_REF] Liu | Optimization of lipid production for algal biodiesel in nitrogen stressed cells of Dunaliella salina using FTIR analysis[END_REF] while the use of various photobioreactors [START_REF] Ugwu | Photobioreactors for mass cultivation of algae[END_REF] and lipid synthesis in an open pond simulating reactor have been published [START_REF] Bellou | Biochemical activities in Chlorella sp. and Nannochloropsis salina during lipid and sugar synthesis in a lab-scale open pond simulating reactor[END_REF]. The influence of the growth medium has been studied with regard to the use of oil crop biomass residues [START_REF] Wang | A flexible culture process for production of the green microalga Scenedesmus dimorphus rich in protein, carbohydrate or lipid[END_REF] or palm oil mill effluent [START_REF] Idris | Cultivation of microalgae in medium containing palm oil mill effluent and its conversion into biofuel[END_REF]. Finally, new developments in biodiesel conversion technology have been reviewed [START_REF] Kim | Methods of downstream processing for the production of biodiesel from microalgae[END_REF].

As mentioned earlier, the main problem with algal-derived biodiesel currently is its cost versus petroleum-based products. A comprehensive evaluation of the economic (and environmental) impacts of microbial biodiesel has been made, including net energy balance, cost of goods sold etc. [START_REF] Caspeta | Economic and environmental impacts of microbial biodiesel[END_REF]. Their evaluation is based on current crops but highlights the necessity to mitigate against greenhouse gas emission. Chisti [START_REF] Chisti | Biodiesel from microalgae beats bioethanol[END_REF] argued strongly that microalgae are better than crops (as used for bioethanol) in terms of their smaller impact on the environment and their efficiency in producing biodiesel. His arguments were disputed by [START_REF] Reijnders | Do biofuels from microalgae beat biofuels from terrestrial plants?[END_REF] but then further countered by Chisti [START_REF] Chisti | Response to Reijnders: Do biofuels from microalgae beat biofuels from terrestrial plants?[END_REF]. With our present technology, algae seem to offer a realistic solution to replacing petroleum and, even more so, as a source of high-value products.

For calculation of the theoretical maximum algal oil production (at different global sites) see [START_REF] Weyer | Theoretical maximum algal oil production[END_REF].

Further general aspects of biodiesel production are covered by Ratledge and Cohen [START_REF] Ratledge | Single Cell Oils : Microbial and algal oils[END_REF] and economic analysis by Davis et al. [START_REF] Davis | Techno-economic analysis of autotrophic microalgae for fuel production[END_REF] while the future of algal biofuels has been discussed [START_REF] Jones | Algae biofuels: versatility for the future of bioenergy[END_REF]. All the subjects covered in section 7.2 are included in the comprehensive review by [START_REF] Unkefer | Review of the algal biology program within the National Alliance for Advanced Biofuels and Bioproducts[END_REF]. Although this is focussed on a particular programme, the review encompasses the same general area as this current article.

Research on the commercial production of biofuels from algae has been carried out for three decades but, to date, the high cost of production (over an order of magnitude) [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF][START_REF] Ratledge | Microbial and algal oils: Do they have a future for biodiesel or as commodity oils?[END_REF] compared to petroleum supplies, means that economic viability is not yet possible. Reducing costs remains the most important target and, until that has been done significantly, then highvalue products (such as VLCPUFA) will remain much more attractive [START_REF] Borowitzka | 13 -Algae oils for biofuels: chemistry, physiology, and production[END_REF].

Other useful products

There are a number of other valuable chemicals produced by algae which have a commercial niche.

Carotenoids

Carotenoids have utility in the food, cosmetic and pharmaceutical industries [START_REF] Henriquez | Carotenoids in microalgae[END_REF]. Different algae accumulate various pigments of which the most important commercially are astaxanthin, beta-carotene, phycobiliproteins, phycocyanin and phycoerythrin [START_REF] Zhang | Enhanced fatty acid accumulation in Isochrysis galbana by inhibition of the mitochondrial alternative oxidase pathway under nitrogen deprivation[END_REF]. Beta-carotene is a useful food supplement and is produced by Dunaliella salina at over 10% of its dry mass [START_REF] Sathasivam | Modified medium for enhanced growth of Dunaliella strains[END_REF].

Astaxanthin is commercially valuable and it is produced by Haematococcus pluvialis at 4-5% dry mass [START_REF] Sathasivam | Microalgae metabolites: A rich source for food and medicine[END_REF]. Phycobiliprotein pigments are fluorescent agents [START_REF] Perez-Garcia | Heterotrophic cultures of microalgae: Metabolism and potential products[END_REF] while phycocyanin and other pigments from red algae are used in both the food and cosmetic industries [START_REF] Kumar | Extraction and purification of C-phycocyanin from Spirulina platensis (CCC540)[END_REF]. See [START_REF] Khan | The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products[END_REF][START_REF] Borowitzka | 11 -Carotenoid production using microorganisms[END_REF] for further discussion.

Sterols

Phytosterols are used in the pharmaceutical industry and as nutraceuticals [START_REF] Khan | The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products[END_REF]. Pavlova and Thalassiosira genera are rich in sterols [START_REF] Luo | Advances in microalgae-derived phytosterols for functional food and pharmaceutical applications[END_REF][START_REF] Volkman | A review of sterol markers for marine and terrigenous organic matter[END_REF][START_REF] Volkman | Microalgal biomarkers: A review of recent research developments[END_REF]. These microalgae have been found to produce up to nearly 3% dry mass as sterols [START_REF] Ahmed | Pavlova lutheri is a high-level producer of phytosterols[END_REF]. Some 40 different sterols have been reported

in over 100 species of diatoms. Major sterols in Glaucocystophyta are sitosterol, campesterol and stigmasterol, dinoflagellates produce mostly 4α-methyl sterols while 24-propylidenecholesterol is mainly accumulated in Pelagophyceae [START_REF] Khan | The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products[END_REF].

Proteins and enzymes

Microalgae produce 2-8 tonnes/hectare/year of proteins [START_REF] Bleakley | Algal Proteins: Extraction, application, and challenges concerning production[END_REF] and a number of algae, such as Chlorella, produce marketable material [START_REF] Khan | The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products[END_REF]. Recently, there has been an increasing interest in many algal enzymes for the genetic manipulation of plants, particularly in order to produce

VLCPUFAs [START_REF] Vaezi | Identification and functional characterization of genes encoding omega-3 polyunsaturated fatty acid biosynthetic activities from unicellular microalgae[END_REF]. A considerable number of genes for such enzymes had already been isolated when we last reviewed the area [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF]. Nevertheless, there seems to be constant improvements in the conversion rates of ALA to EPA and DHA by employing newly characterised desaturases and elongases. For example, front-end delta4 and delta6 desaturases from the green alga Ostreococcus RCC809 gave 15% desaturation of 22:5 and 54% desaturation of ALA respectively. A 6 elongase from the cold-water diatom Fragilariopsis cylindrus gave 38% elongation of gamma-18:3. These genes allowed an expansion of activities available for the potential commercial production of EPA and DHA [START_REF] Vaezi | Identification and functional characterization of genes encoding omega-3 polyunsaturated fatty acid biosynthetic activities from unicellular microalgae[END_REF].

Any of the enzymes mentioned in sections 3, 4 and 5 could, potentially, be utilised for commercial purposes. Early work in this area included enzymes useful for the conversion of EPA into DHA [START_REF] Pereira | Identification of two novel microalgal enzymes involved in the conversion of the omega3-fatty acid, eicosapentaenoic acid, into docosahexaenoic acid[END_REF] and front-end desaturases to produce unusual fatty acids (pinolenic and coniferonic acids) [START_REF] Kajikawa | A front-end desaturase from Chlamydomonas reinhardtii produces pinolenic and coniferonic acids by omega 13 desaturation in methylotrophic yeast and tobacco[END_REF]. Another example would be the use of three front-end desaturases from P. salina for DHA biosynthesis which could be expressed in higher plants [START_REF] Zhou | Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis[END_REF]. More information on useful algal lipid enzymes and their utilisation will be found in [START_REF] Unkefer | Review of the algal biology program within the National Alliance for Advanced Biofuels and Bioproducts[END_REF].

Conclusions

It should be clear from the preceding text and accompanying references, that significant advances have taken place in the dozen years since the last time this topic has been reviewed intensively for eukaryotic algae [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF]. These advances have taken place in all aspects but with an increasing use of molecular biology to facilitate progress. Much of the research has been driven by the heightened interest in using algae for industrial purposes such as for nutraceuticals or biofuel.

Over the last decade, C. reinhardtii has become established as a model organism although, of course, given the diversity of algae this green microalga is not always a good substitute for specific organisms or situations. Nannochloropsis spp. and Phaeodactylum tricornutum have also been well studied because of their identification as algae of interest for commercialization.

We look forward to future advances in our knowledge which undoubtedly will take place. Perhaps these may eventually include the utilization of algae for biofuels---an area which is urgently needed within the background of climate change. This is just one area where the fascinating and diverse biochemistry of algae can have a global impact. This schema is made based on that of [245] (with permission).

Abbreviations: G3P, glycerol-3-phosphate; GPAT, glycerol 3-phosphate acyltransferase;

LPAAT, lysophosphatidic acid acyltransferase; PA, phosphatidic acid; DAG, diacylglycerol;

PDAT, phospholipid:diacylglycerol acyltransferase; PAP, phosphatidic acid phosphatase; DGAT, diacylglycerol acyltransferase; CoA, Coenzyme A; TAG, triacylglycerol. Table 1. The acyl lipid compositions of some algae.

Data taken from Harwood and Jones [START_REF] Harwood | Lipid metabolism in algae[END_REF], where original references will be found.

Table 2. Fatty acid composition of selected algae from the SAG culture collection.

The major fatty acids are shown as the % composition recalculated from data in Lang et al 2011 [START_REF] Lang | Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection[END_REF] where other compounds (eg phytol) are sometimes listed. 16:1 (9z), 16:2 (9z, 12z), 16:3 (7z, 10z, 13z), 18:1 (9z), *18:1 (11z), 18:2 (9z, 12z), 18:3 (9z, 12z, 15z), 18:4 (6z, 9z, 12z, 15z), 20:4 is ARA, 20:5 is EPA and 22:6 is DHA. Although all the algae analysed [START_REF] Lang | Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection[END_REF] were in the stationary phase, it should be borne in mind that their fatty acid composition can alter significantly with culture conditions (see Section 6). Therefore, this table should be used as a guide. Only commonly occurring major fatty acids are listed.

Table 3. Total fatty acid compositions of some algae.

Data taken from Harwood and Jones [START_REF] Harwood | Lipid metabolism in algae[END_REF] where original sources are listed. Although the original papers did not always fully define the double bond configuration and position, it can be assumed that these were probably as indicated in Table 2, with 16:4 being (6z, 9z, 12z, 15z) and 18:1 being oleic acid, 18:1 (9z). tr = trace (<0.5%).

Table 4. Total fatty acid composition of some marine algae.

Taken from Harwood and Jones [START_REF] Harwood | Lipid metabolism in algae[END_REF]. See Tables 2 and3 for information about the unsaturated fatty acids. tr = Trace (<0.5%).

Table 5. Very long chain PUFA produced by different algae of industrial interest. For further details see [START_REF] Khozin-Goldberg | Microalgae as a source for VLC-PUFA production[END_REF]. Taken from information in [START_REF] Ratledge | Single Cell Oils : Microbial and algal oils[END_REF].

Abbreviations: 3 . 5

 35 Abbreviations: (as a footnote) α-CT, α-carboxyltransferase; ACCase, acetyl-CoA carboxylase; ACK, acetate kinase; ACAD, acyl-CoA dehydrogenase; ACOX, acyl-CoA oxidase; ACP, acyl carrier protein; ACS, acetyl-CoA synthetase; AOX, alternative oxidase; APX, ascorbate peroxidase; ASC, ascorbate; β-CT, β-carboxyltransferase; BADC, biotin attachment domain-containing protein; BC, biotin carboxylase; BCAA, branched chain amino acid; BCCP, biotin carboxyl carrier protein; CAT, catalase; CEF, cyclic electron flow; CoA, coenzyme A; CTS1, comatose 1; COX, cytochrome oxidase; DAG, diacylglycerol; DGAT, diacylglycerol acyltransferase; DGDG, digalactosyldiacylglycerol; DHA, docosahexaenoic acid; DH, dehydrogenase; DGTA, 1,2diacylglyceryl-3-O-2'-(hydroxymethyl)-(N,N,N-trimethyl)-β-alanine; DGTS, diacylglyceryl-3-O-4'-(N,N,N-trimethyl)-homoserine; DYRK, dual-specificity tyrosine-phosphorylationregulated kinase; EPA, eicosapentaenoic acid; ER, enoyl-ACP reductase; FA, fatty acid; FAD, fatty acid desaturase; FAT, fatty acyl-ACP thioesterase; FAX1, fatty acid export1; KAS, 3ketoacyl-ACP synthase; G3P, glycerol-3-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase; HAD, hydroxyacyl-ACP dehydrase; HL, high light; KAR, ketoacyl-ACP reductase; LACS, long chain acyl-CoA synthetase; LEF, linear electron flow; Lyso-PA, lysophosphatidic acid; LPAAT, lysophosphatidic acid acyltransferase; MCMT, malonyl-CoA:ACP malonyltransferase; Mal, malate; MDA, malondialdehyde; ME, malic enzyme; MFP, multi-functional protein; MGDG, monogalactosyldiacylglycerol; NO, nitric oxide; NRR1, nitrogen response regulator1; OAA, oxaloacetate; PA, phosphatidic acid; PAT, phosphate acetyltransferase; PAP, phosphatidic acid phosphatase; PtdCho, phosphatidylcholine; PDAT, phospholipid:diacylglycerol acyltransferase; PDH, pyruvate dehydrogenase; PDK, pyruvate dehydrogenase kinase; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdSer, phosphatidylserine; PtdIns, phosphatidylinositol; PtdGro, phosphatidylglycerol; PGRL1; proton gradient regulation 5 like 1; PL, phospholipid; PLA2, phospholipase A2; PXN, peroxisomal NAD + carrier; PPP, pentose phosphate pathway; PUFA, polyunsaturated fatty acid; SAD, stearoyl-ACP desaturase; SQDG, sulfoquinovosyldiacylglycerol; TAG, triacylglycerol; TAR1, triacylglycerol accumulation regulator1; TCA, tricarboxylic acid; TF, transcription factor; TOR, target of rapamycin; VLCPUFA, very long chain polyunsaturated fatty acid.

Following their release from

  a membrane lipid or TAG, non-esterified FAs are first activated to their CoA esters by members of the long chain acyl-CoA synthetase (LACS) family. LACS proteins belong to a multi-protein family, and are ubiquitously present in numerous algal lineages. The resultant activated FA in the form of acyl-CoA is then ready for oxidative attack at the C-3 or β-carbon position, giving rise to the name β-oxidation. An acetyl-CoA (C2) is cleaved off the acyl-CoA (Cn) with each round of the β-oxidation spiral, and the remaining acyl-CoA (Cn-2) re-enters the spiral to repeat this process until acyl-CoA is completely converted to acetyl-CoA.

  CoA dehydrogenases/oxidases FA β-oxidation begins with the enzymes acyl-CoA dehydrogenase (ACAD) or acyl-CoA oxidase (ACOX), which catalyze the dehydrogenation of acyl-CoA to trans-2-enoyl-CoA either via the reduction of O2 to generate peroxide (ACOX) or via the reduction of FAD to FADH2

(

  with two exceptions, perhaps due to sequence contamination or lateral gene transfer) possess members of only a single ACOX subfamily (O-III), making them potentially interesting candidates for the engineering of enhanced lipid accumulation through tightened control of βoxidation. Similarly, Arabidopsis does not contain any ACADs and an absence of ACADs may explainthe exclusive peroxisomal localization of β-oxidation in higher plants. However, the lower plants Physcomitrella patens and Marchantia polymorpha do retain members of the D-X ACAD subfamily, which is also present in green algae (in addition to subfamilies D-II, D-VII, and DXI); it is unknown whether these enzymes are of peroxisomal or mitochondrial localization. By far the widest array of ACADs are present in the heterokont algae, which possess members of up to 10 separate ACAD subfamilies, one of which (D-I) is unique to heterokont and cryptomonad algae. Much work remains to be done in the study of ACOXs/ACADs, with the localization and enzymatic properties of many algae-containing eukaryotic subfamilies remaining completely unexplored.

Figure 1 .

 1 Figure 1. De novo fatty acid synthesiscarbon and energy sources. Abbreviations: ACP, acyl-CoA binding protein; ACCase, acetyl-CoA carboxylase; MCMT, malonyl-CoA: ACP malonyltransferase; Dof-type TF, DNA binding with one finger type transcription factor; bHLH, a basic helix-loop-helix; bZIP, a basic leucine zipper-domain containing TF; ER, enoyl-ACP reductase; KAS, keto-acyl-CoA synthase; KAR, ketoacyl-ACP reductase; HAD, hydroxyacyl-ACP dehydrase; ME, malic enzyme; FatA/B, fatty acid thioesterase A/B; PDH, pyruvate dehydrogenase complex; PSR1, Pi Starvation Response 1; SAD, Stearoyl-ACP Desaturase; FAX1, fatty acid export 1; TF, transcription factor.

Figure 2 .

 2 Figure 2. Maximum likelihood phylogenetic tree of ACCase negative regulators. (A). BADC/BCCP tree.(B). PII tree.Known Arabidopsis sequences were used as PSI-BLAST queries to comprehensively identify eukaryotic algal orthologs from the NCBI non-redundant database, or, for algal species/genes not present on NCBI, from the JGI algal genome database. Representative non-algal sequences of interest were also identified and included. For red algal BCCPs/BADCs, only a single representative sequence per family was retained for the final phylogenetic tree to avoid overcrowding, except in the case of the Cyanidiaceae. Amino acid sequences were aligned with the MAAFT (v. 7.308) plugin in Geneious (v. 11.1.4) using the E-LNS-I option. Target peptides and non-homologous or erroneous sequence regions were manually trimmed, alignments were further refined with MAAFT, and sites with 50% or more gaps were removed from the alignments. Trees were constructed with the RAxML 8.2.11 plugin using the Gamma JTT protein model and 100 rapid bootstrap replicates and were plotted with FigTree 1.4.3 followed by manual formatting in Adobe Illustrator. Bootstrap support is indicated by the darkness of branch lines while clades are colored by taxonomic group.

Figure 3 .

 3 Figure 3. Fatty acid desaturations in diatoms.The biosynthesis of LC-PUFAs in diatoms. Schematic representation of Δ6-and Δ8-pathways for LC-PUFAs biosynthesis. Diagram were taken from Sayanova et al[START_REF] Sayanova | Modulation of lipid biosynthesis by stress in diatoms[END_REF] (with permission).

Figure 4 .

 4 Figure 4. Glycerolipid synthesis in Chlamydomonas reinhardtii.This schema is made partly based on that ofKim et al ([249]. It is worth noting here that PDAT has been shown to use in vitro phosphalipids and galactolipids as acyl donors[START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF], however the situation in vivo is not clear. Abbreviations: CoA, Coenzyme A; DAG, diacylglycerol; DGAT, diacylglycerol acyltransferase; DGDG, digalactosyldiacylglycerol; DGTS, diacylglyceryl-3-O-4'-(N,N,Ntrimethyl)-homoserine; FAX1, fatty acid export 1; G3P, glycerol-3-phosphate; GPAT, glycerol 3-phosphate acyltransferase; LPAAT, lysophosphatidic acid acyltransferase; MGDG, monogalactosyldiacylglycerol; PDAT, phospholipid:diacylglycerol acyltransferase; PA, phosphatidic acid; PAP, phosphatidic acid phosphatase; TGD, trigalactosyldiacylglycerol; TAG, triacylglycerol.

Figure 5 .

 5 Figure 5. A simplified Kennedy pathway for lipid biosynthesis.

Figure 6 .

 6 Figure 6. Pathways of synthesis of phosphatidylcholine (PtdCho) in various plants and algae. Diagram were taken from Sato et al., 2016 [288] (with permission).

Figure 7 .

 7 Figure 7. Fatty acid and glycerolipid molecular species distribution in Chlamydomonas. Data are taken from Li-Beisson et al., 2015 [57] (with permission).

Figure 8 .

 8 Figure 8. Changes in lipid content in response to changes in nitrogen status in the media. (A). Changes in cellular TAG levels as quantified by Flow cytometry after cells being stained with Nile red.(B). Changes in polar membrane lipid content.Abbreviations: A.U.: artificial unit; MM, minimal media; N, nitrogen; TAG, triacylglycerol; TAP, tri-acetate-phosphate media; PL, polar lipids. Data are modified based on[START_REF] Siaut | Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves[END_REF].

Figure 9 .

 9 Figure 9. Maximum likelihood phylogenetic tree of eukaryotic algal acyl-CoA oxidases/dehydrogenases. Phylogenetic analysis was performed as in Figure 2, except that PSI-BLAST queries were supplemented with known human and Ustilago mays acyl-CoA oxidases/dehydrogenases (ACOXs/ACADs) and sites with 10% of more gaps were removed from the alignment. Dotted lines surround distinct eukaryotic ACOX/ACAD subgroups, with subgroups being designated by a letter indicating their provenance (D = ACAD, O = ACOX, and G = Glutaryl-CoA dehydrogenase) followed by a dash and a roman numeral (e.g. D-XI). The thicker dashed line in the middle of the figure indicates the approximate division between ACOXs and ACADs based on enzyme activities of known members. Note that ACOXs are paraphyletic, with group O-IX, which contains Arabidopsis acyl-CoA oxidase 4, having likely arisen via horizontal gene transfer of an early eukaryotic glutaryl-CoA dehydrogenase into proteobacteria, followed by a horizontal transfer of the resultant proteobacterial glutaryl-CoA dehydrogenase back into the

Figure 10 .

 10 Figure 10. Lipolysis and -oxidation of fatty acids in microalgaecarbon and energetic aspects. Abbreviations: TAG, triacylglycerol; LCS, long chain acyl-CoA synthetase; CTS1, comatose 1; ACOX, acyl-CoA oxidase; CoA, coenzyme A; MFP, multi-functional protein; DH, dehydrogenase; ASC, ascorbate; MDA, malondialdehyde; APX, ascorbate peroxidase; CAT, catalase; Mal, malate; PXN, peroxisomal NAD carrier translocator; OAA, oxaloacetate.

Table 6 .

 6 Comparison of the fatty acid composition of Crypthecodinium cohnii andSchizochytrium sp. and commercial oils produced from them.