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Organization of glycerolipids in thylakoid membranes
The dense protein loading of thylakoid membranes requires a controlled and dynamic lipid matrix. One feature of thylakoids is the high abundance of polyunsaturated fatty acids that fill the grooves of membrane spanning proteins (Garab et al. 2016). While in plants the highest desaturation degree is found at 18:4, Heterokont chloroplast lipids are rich in 20:4 and 20:5 (Abida et al. 2015, Dolch & Maréchal 2015, Simionato et al. 2013) (figure 2.2).
An open question is whether the specific enrichment in VLC-PUFAs in thylakoid lipids have contributed to the success of the red lineage in marine systems that have higher photosynthetic and photoprotection capacities compared to the most green algae allowing higher biomass production (Kuczynska et al. 2015, Goss and Lepetit 2014, Chevalier- Smith 2003). Desaturation levels are dynamically changed in response to environmental factors as growth phase (Meï et al. 2015), temperature (see 2.7.3) and light quality. Blue light causes the absence of 20:0 and an increase in 20:5 in Nannochloropsis (Chen et al. 2013, Teo et al. 2014). This finding is especially interesting considering the opposing effects of blue and red light on photosynthesis, which indicates that a higher desaturation level is maintained when photoprotection is induced. The Heterokont fatty acid profiles of the four chloroplast lipids vary with the highest desaturation levels in MGDG, followed by DGDG based on the predominance of 20:5 in these lipids (Abida et al. 2015, Simionato et al. 2013).
The model organisms Phaeodactylum tricornutum and Nannochloropsis gaditana
More than 23,000 Heterokont species are described (Chapman 2009, (Riisberg et al. 2009). They make the major part of marine phytoplankton and are estimated to contribute to 20% of the worlds’ primary production (Falkowski et al. 1998, Vardi et al. 2008b). Indeed, per year around 45 gigatons of organic carbon are produced by the oceans (Falkowski et al. 1998).
Heterokonts include both photoautotroph and heterotroph species. Photosynthetic Heterokonts group into the Phaeista including the Eustigmatophyceae class and Khakista including Bacillariophyceae (diatoms) that are differentiated based on sequence comparisons and morphologies (Riisberg et al. 2009). Diatoms possess frustules, which are silica-containing cell walls. This is a unique feature of this phylogenetic group and the use in defence of desiccation are discussed (Sims 2006). While Bacillariophyceae forms the largest group with 4,256 accepted species, only 35 Eustigmatophyceae are known (Chapman 2009). The group of diatoms is furthermore split into centric and pennate species that have diverged 90 million years ago, but their genomes have strongly evolved so that less than 60% of the genes are shared (Bowler et al. 2008).
Morphological characteristics of pennate diatoms are presented in figure 2.5 (Falciatore & Bowler 2002). The cell contains a single photosynthetic plastid, the phaeoplast (called chloroplast in the following), and all common eukaryotic organs including lipid droplets. A silica containing frustule encloses the cell and consists of two engaged halves, the larger epitheka and the smaller hypotheca. The larger outer surface is called valve and is engraved by circular silica bands, the girdle. The raph slit allows secretion of mucus for motility purposes (Falciatore & Bowler 2002).
The interest in very long chain polyunsaturated fatty acids (VLC-PUFAs)
Lipids in the Chromalveolata kingdom are enriched in VLC-PUFAs with four or more desaturations, whereas in the green lineage linoleic acid (18:2 n-6, LA) and α-linolenic acid (18:3 n-3, ALA) are the most dominant VLC-PUFA species (Lang et al. 2011).
EPA is enriched in Chlorophyceae, Chrysophyceae, Prasinophyceae, Eustigmatophyceae and Bacillariophyceae (Wen & Chen 2003). Fatty acid profiles reflect phylogenetic relations (Lang et al. 2011). VLC-PUFAs are differentiated into the ω-3 and ω-6 series according to the position of the first double bond counted from the carboxyl-end of the fatty acid (figure 2.2). These are in the ω-3 series ALA, stearidonic acid 18:4 n-3, eicosatetraenoic acid 20:4 n-3, eicosapentaenoic acid 20:5 n-3 (EPA) and docosahexaenoic acid 22:6 n-3 (DHA). Marine ω-6 VLC-PUFAs are LA, γ-linolenic acid 18:3 n-6 (GLA) and arachidonic acid 20:4 n-6 (ARA). While C18-PUFAs are widely spread among the kingdoms, species that produce EPA and DHA except for microalgae are rare. Some bacteria and Mucorales fungi have the capacities of VLC-PUFA production (Bajpai & Bajpai 1993). Marine Shewanella and similar bacteria may contain up to 40% EPA (Yazawa 1996). Those species are found in the intestines of several fishes (Dailey et al. 2015). A lower plant, the liverwort Marchantia polymorpha contains up to 6% EPA and 3% ARA (Kajikawa et al. 2008). While most eukaryotic heterotrophs obtain ω-3 VLC-PUFAs via diet, scallop has the ability to synthesize EPA (Hall et al. 2002). For mammals, VLC-PUFAs are essential but production of EPA and DHA can be achieved in transgenic lines (Lee et al. 2016).
The interest in VLC-PUFAs for human nutrition is high. Mammals lack the Δ12FAD and are unable to synthesize the ω-6 fatty acid LA (18:2Δ9,12) and the ω-3 fatty acid ALA (18:3Δ9,12, 15) (Lee et al. 2016, Sayanova & Napier 2004). The dietary intake of these essential fatty acids determines the ratio of synthesized PUFAs of the ω-3 and ω-6 series. LA is being elongated and desaturated by substrate unspecific enzymes into ARA (20:4Δ5,8,11,14) and ALA into EPA (20:5Δ5,8,11,14,17) and DHA (22:5Δ4,7,10,13,16,19) (Dolch & Maréchal 2015, Sayanova & Napier 2004). However, the human capacity of elongation and desaturation of C18 species into EPA and DHA is only 8% and 0-4%, respectively, and thus a dietary intake is required to meet the demand of ω-3 VLC-PUFA (Ruiz-Lopez et al. 2015, Sayanova & Napier 2004). It is believed that human physiology has adapted to an ω-6:ω-3-intake ratio of about 1 but in todays’ Western diet the ratio is 15-17:1, thus enriched in pro-inflammatory fatty acids that may have a negative health impact (Ruiz-Lopez et al. 2015). VLC-PUFAs serve as substrates for hormone-like regulatory molecules, eicosanoids, namely prostaglandins, thromboxanes and leukotrienes, which depending on their ω-3 or ω-6 structure act as antagonists in different processes. They regulate inflammation, immune-reactivity, blood pressure, and platelet aggregation; and function in the prevention of atherosclerosis, arthritis, arrhythmia, heart attack, and thrombosis, TAG, metabolic disorders, diabetes, some cancer, multiple sclerosis, Alzheimer’s disease and asthma (Funk 2001, Ji et al. 2015, Lee et al. 2016, Ruiz-Lopez et al. 2015, Wen & Chen 2003). Docosanoids including resolvins and protectins are all anti-inflammatory and involved in brain development and neurodegeneration and eye function (Lee et al. 2016). Furthermore, DHA is present in structural lipids in human membranes, where it is involved in lipid raft formation (Ruiz-Lopez et al. 2015).
The worlds’ annual demand of EPA for medical applications alone is about 300 tons and the fatty acid must be refined from fish oil (Wen & Chen 2003). Herring, mackerel, sardine and salmon are the greatest dietary VLC-PUFA sources (Ruiz-Lopez et al.
2015). However, consumption of high quantities of fish may have negative health impacts due to the pollution of the oceans with cadmium, lead, mercury, dioxins, furans, polychlorinated biphenyls and polybrominated diphenyl ethers that accumulate in fish (Domingo et al. 2007). To reduce toxin indigestion, purified oils could serve as an alternative. Today, the major industrial source for EPA is oil from cod, menhaden, herring and krill (Robles Medina et al. 1998). Commercialized fish oil EPA pills contain 9-26% EPA and 9-17% DHA. Microalgae may contain twice as much of these fatty acid
species (Robles Medina et al. 1998). Furthermore, cultivation of algae does not interfere with the marine ecosystem that suffers from man-made loss of equilibrium and biodiversity (Worm et al. 2006). The conclusion on a need for industrialization of microalgae for EPA and DHA production is thus obvious.
Although Phaeodactylum wild type does not have the highest VLC-PUFA proportions, early studies on microalgae revealed the highest EPA productivity with up to 47.8 mg.L-1.d-1 (Robles Medina et al. 1998). Application of Heterokonts for food and feed does not necessarily involve TAG purification since the biochemical composition of Nannochloropsis spp. fulfils human nutrition demands. Protein, fibre, nucleotide, ash, nitrate and toxic heavy metal contents are low, but antioxidant, calcium, magnesium and zinc levels are high. Lipid and carbohydrate make more than 50% of biomass (Rebolloso-Fuentes et al. 2001). Microalgae and especially diatoms are already used in wastewater treatment, and the production of biofuel, fertilizers and secondary metabolites for medical compounds and pharmaceutics, as well as for food and feed for example in commercial aquaculture (Levitan et al. 2014, Vinayak et al. 2015). However, it is likely that gene engineered strains will be commercialized. In order to avoid having gene modified organisms in the food chain, EPA-rich oils could be extracted and used as feedstock for fish (Sayanova & Napier 2004).
For now, the only commercialized biotechnological resource providing an alternatively to fish are the yeast Yarrowia lipolytica and Thraustochytrids species that are related to Heterokonts (Gupta et al. 2012a, Ji et al. 2015, Lewis et al. 1999). A recent study compared an engineered Phaeodactylum strain that accumulates DHA (Pt_Elo5, overexpressing the Δ5-ELO from Ostreococcus tauri) to the wild type in which DHA is barely detected in different culture systems with volumes up to 1250 L. By modelling various growth factors (UV irradiation, light intensity, light phase duration, CO2 availability and mixotroph growth), the transgenic line yielded 6.4 µg.mg-1 dry weight DHA during mid-log growth, a value that would allow commercialization (Hamilton et al. 2015). Notably, these authors found mixotroph growth to not only to enhance the biomass yield in each growth system tested but to uncouple biomass production from the culture volume (Hamilton et al. 2015). Expression of the O. tauri Δ5-ELO in a trophically converted P. tricornutum strain cultivated on glucose in the light increased biomass production by about 20% compared to photoautotroph Pt_Elo5. Allowing mixotroph growth, EPA and DHA accumulated best. On the other hand, fatty acid profiles changed in the heterotroph strain when grown in darkness with reduced EPA and DHA accumulation (Hamilton et al. 2016). This case study indicates a high potential of the diatom for VLC-PUFA production. VLC-PUFA are dominant in membrane lipids, while medium fatty acid chains with low desaturation degrees allocate in neutral lipids and may be used as biofuels.
The interest in biofuels produced by microalgae
Worldwide, 96% of transportations rely on fossil fuels and cause 36% of the rise in atmospheric CO2 (Levitan et al. 2014). With the background of global warming and fossil resource exhaustion, the need for stronger implementation of alternative diesel is striking. In 2010, 18.2 billion liters of biodiesel have been produced (Liang & Jiang 2013). However in the same year, the petroleum consumption was about 3,830 billion liters, numbers still rising in spite of the Kyoto protocol (www.eia.gov/cfapps/ipdbproject/iedindex3.cfm?tid=5&pid=54&aid=2; 18.06.2016). Thus biodiesel compensated only about 0.5% of total fuel consumption. The yearly exploitation of fossil petroleum is worth about one million years of algal oil accumulation (Levitan et al. 2014).
Those species that stand at the base of fossil petroleum are now of interest for third generation biofuel production. High biomass producing oleaginous microorganisms in which 20-50% of dry weight is lipids have the highest potential to entirely replace fossil energy (Liang & Jiang 2013). Importantly, microalgae cultivation does not compete with agriculture. Indeed, since 22% of soy beans cultivated in the USA are used for biodiesel production the soybean oil price has increased by 100% (Durrett et al. 2008). In addition to using neutral lipids that accumulate in lipid droplets for diesel production, the algal biomass may also be used for the production of other energy carriers as hydrogen, ethanol, gas and oil (Paul Abishek et al. 2014). Notably, although environmental advantages of biodiesel are obvious, negative health impacts are likely to be similar to fossil fuels (Madden 2016).
Methods to enhance TAG production on this purpose can involve biochemical and genetic engineering. In the biochemical approach, cells are stressed to remodel fluxes into lipid synthesis (Courchesne et al. 2009). Most studies on Nannochloropsis or Phaeodactylum use this strategy but the rising availability of tools for genetic engineering might shift the research focus towards targeted gene or transcription factor modelling (Li et al. 2014).
Genetic engineering possibilities of Nannochloropsis and Phaeodactylum
The genomes of three model species have been sequenced (Bowler et al. 2008, Corteggiani Carpinelli et al. 2014, Radakovits et al. 2012, Vieler et al. 2012). The genome of N. oceanica contains 28.7 Mb encoding about 12,000 genes (Vieler et al. 2012). The N. gaditana genome is of similar size but comprises only 9,052 gene models. Despite a low total gene number compared to other microalgae, the N. gaditana genome is enriched in lipid metabolism associated genes that represent almost 50% of all ESTs (Radakovits et al. 2012). P. tricornutum has a genome size of 27.4 Mb containing 10,402 predicted genes (Bowler et al. 2008).
Targeted gene insertion to remove or substitute an endogene, silencing of protein translation of a specific mRNA, or the overexpression of a desired sequence provide powerful tools to study protein functions. Breeding could also contribute to the establishment of heterozygous mutants in case null-mutation of the desired gene is lethal. This requires sexual reproduction which for now is not reported to occur under lab conditions in Phaeodactylum or Nannochloropsis although the required genes might be encoded in the genome (Patil et al. 2015, Hibberd 1981). Nonetheless, several methods are described for genetic engineering of both Phaeodactylum and Nannochloropsis, but only a few studies using these techniques are published (Weeks 2011). A summary of gene engineered Phaeodactylum strains is given in table 2.1.
Table of contents :
1 Abstract
2 Literature Review
2.1 Heterokonts are secondary endosymbionts and important model organisms for glycerolipid research
2.1.1 Endosymbiosis
2.1.2 Structure and composition of chloroplast membranes
2.1.3 The model organisms Phaeodactylum tricornutum and
Nannochloropsis gaditana
2.2 Photochemistry
2.2.1 Light perception and pigments
2.2.2 Photochemistry
2.3 Photoprotection
2.3.1 The role of the proton gradient for NPQ
2.3.2 Function and regulation of the xanthophyll cycle
2.3.3 Chlorophyll and xanthophyll quenching mechanism
2.3.4 Photoinhibition
2.3.5 State transitions
2.3.6 Light reactions in Heterokonts differ from vascular plants
2.4 Carbon metabolism
2.4.1 Carbon concentration mechanism
2.4.2 Carbon fixation
2.4.3 The role of pyruvate in the production of carbon precursors for glycerolipid synthesis
2.5 Fatty acid synthesis
2.6 Fatty acid trafficking between the chloroplast and the ER
2.6.1 Preparation for the export
2.6.2 Export across the chloroplast limiting membranes to the cytosol
2.6.3 Traffic into the ER and back to the cytosol
2.6.4 Import across the chloroplast limiting membranes into the stroma
2.6.5Interaction of the chloroplast outer envelope membrane with the ER membrane
2.7 Fatty acid elongation and desaturation
2.7.1 Fatty acid elongation
2.7.2 Fatty acid desaturation
2.7.3 Homeoviscous adaptation
2.8 Lipid synthesis
2.8.1 Membrane lipid synthesis
2.8.2 TAG synthesis
2.9 Nitrogen metabolism and NO• signalling
2.9.1 Nitrogen fixation in Heterokonts
2.9.2 Nitric oxide production and possible function in lipid metabolism
3 Lipid and fatty acid profiles in Phaeodactylum tricornutum under different growth regimes
3.1 Introduction and overview
3.2 Determination of culture conditions for comparative studies using P. tricornutum
3.2.1 Long term cultivation of P. tricornutum
3.2.2 Short term cultivation of P. tricornutum
3.3 Membrane glycerolipid remodelling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum
3.3.1 Identification of phosphorous starvation-responsive genes in Phaeodactylum tricornutum by comparative analyses of lipid profiles and transcriptomic data from Thalassiosira pseudonana
3.3.2 Membrane Glycerolipid Remodeling Triggered by Nitrogen and P h os p ho r us Starvation Phaeodactylum tricornutum (published article) ..
3.3.3 Characterization of DGDG synthases in Phaeodactylum
4 Nitric oxide signalling in lipid accumulation
4.1 Introduction and overview
4.2 Small molecule screen
4.3 Oxylipin induced cell death does not trigger TAG accumulation in Phaeodactylum
4.4 NOA-dependent nitric oxide activates the transcription of nitrogen assimilation genes and triggers a glycerolipid remodelling in Phaeodactylum tricornutum (article in preparation)
4.4.1 Supplementary figures
4.5 The NO• production site is important for its signalling function in Phaeodactylum tricornutum neutral lipid metabolism
4.5.1 The location of NO• production is important for the TAG accumulation function
4.5.2 Nitrite triggers NO• but not TAG production in Phaeodactylum
4.5.3 Expression of a putative N. gaditana NOA in P. tricornutum triggers NO• but not TAG production
5 EPA synthesis and trafficking in P. tricornutum and N. gaditana via the elusive “omega pathway”
5.1 Introduction and overview
5.2 EPA biosynthesis
5.2.1 The desaturase equipment of P. tricornutum
5.2.2 Functional analysis of a P. tricornutum Δ-6 elongase in yeast heterologous system
5.2.3 A palmitic acid elongase controls eicosapentaenoic acid and plastid MGDG levels in Nannochloropsis (submitted article)
5.3 The “omega pathway” addressed by reverse genetics of PLDζ, AAS, ATS1 and ATS2 (Preliminary data)
5.3.1 Identification of knock down lines
5.3.2 Characterization of PLDas lines
5.3.3 Characterization of AASas lines
5.3.4 Characterization of ATS1as lines
5.3.5 Characterization of ATS2as lines
5.4 The role of EPA-rich MGDG in homeoviscous adaptation in Nannochloropsis gaditana
5.4.1 Choice of culture conditions
5.4.2 The role of qI in NPQ relaxation in N. gaditana under cold stress conditions
5.4.3 NPQ in the cold-stress response of N. gaditana grown at 10°C
5.4.4 Glycerolipid profiles in N. gaditana cultures grown at 10°C
5.4.5 Glycerolipid profiles in N. gaditana cultures grown at 15°C
6 Discussion and Conclusion
7 Material and methods
7.1 Phaeodactylum and Nannochloropsis strains and culture conditions
7.1.1 Strains
7.1.2 Culture conditions
7.2 Molecular biology
7.2.1 Genomic DNA extraction from microalgae
7.2.2 Generation of constructs for transformation of P. tricornutum and N. gaditana
7.2.3 Relative gene expression quantification by quantitative real time PCR 339
7.3 Cell transformation of Phaeodactylum and Nannochloropsis
7.3.1 Biolistic transformation of Phaeodactylum tricornutum
7.3.2 Electropulse transformation of Nannochloropsis gaditana
7.4 Physiological analyses of microalgae cultures
7.4.1 Determination of cell concentration
7.4.2 Detection of neutral lipids using Nile red staining
7.4.3 Relative chlorophyll fluorescence determination
7.4.4 Chlorophyll a fluorescence spectroscopy at room temperature .
7.4.5 Detection of nitric oxide using 4-amino-5-methylamino-2′,7′- difluororescein diacetate (DAF-FM)
7.5 Imaging
7.5.1 Confocal laser scanning microscopy (CLSM)
7.5.2 Transmission electron microscopy (TEM)
7.6 Lipodomic analyses
7.6.1 Glycerolipid extraction
7.6.2 Gas chromatography-ion flame detection (GC-FID) of fatty acid methyl esters (FAMEs)
7.6.3 Thin layer chromatography (TLC)
7.6.4 Mass Spectrometric (MS) analyses
7.6.5 Yeast transformation and substrate feeding experiments
7.7 Biochemistry
7.7.1 Protein extraction and quantification using Lowry reagent
7.7.2 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) and Western Blot analyses
7.7.3 Galactolipid synthase assay
7.8 In silico analyses
7.8.1 Retrieval of Phaeodactylum and Nannochloropsis Gene Sequences
7.8.2 Sequence analyses and phylogenetic reconstruction
7.8.3 Prediction of Subcellular Localization
8 Appendix
8.1 Additional publication
8.2 Additional results
8.2.1 DGD isoforms in Arabidopsis and Phaeodactylum
8.3 Insert sequences
8.3.1 NgNOA (Naga_100007g10)
8.3.2 Codon optimized NgNOA
8.3.3 Condon optimized NgΔ0-ELO1
8.4 Vector sequences
8.4.1 pH4-GUS-AS
8.4.2 UEP-p35S-loxP BSD FL1-FL2 526
8.4.3 pYES
8.5 Codon usage tables
8.5.1 Phaeodactylum tricornutum and Nannochloropsis gaditana
8.5.2 Saccharomyces cerevisae
9 Acknowledgements
10 References
