Transcriptional landscapes of lipid producing microalgae Benoît M. Carrères 2019 Transcriptional landscapes of lipid producing microalgae Benoî[email protected]:~$ ▮ Transcriptional landscapes of lipid producing microalgae Benoît Manuel Carrères Thesis committee Promotors Prof. Dr Vitor A. P. Martins dos Santos Professor of Systems and Synthetic Biology Wageningen University & Research Prof. Dr René H. Wij$els Professor of Bioprocess Engineering Wageningen University & Research Co-promotors Dr Peter J. Schaa% Associate professor* Systems and Synthetic Biology Wageningen University & Research Dr Dirk E. Martens Associate professor* Bioprocess Engineering Wageningen University & Research ,ther mem-ers Prof. Dr Alison Smith* University of Cam-ridge Prof. Dr. Dic+ de Ridder* Wageningen University & Research Dr Aalt D.). van Di#+* Wageningen University & Research Dr Ga-ino Sanche/(Pere/* Genetwister* Wageningen This research 0as cond1cted under the auspices of the .rad1ate School V2A. 3Advanced studies in Food Technology* Agro-iotechnology* Nutrition and Health Sciences). Transcriptional landscapes of lipid producing microalgae Benoît Manuel Carrères Thesis su-mitted in ful8lment of the re9uirements for the degree of doctor at Wageningen University -y the authority of the Rector Magnificus, Prof. Dr A.P.). Mol* in the presence of the Thesis' ommittee a%%ointed by the Academic Board to be defended in pu-lic on Wednesday 2; Novem-er 2;<= at 1.>; p.m in the Aula. Benoît Manuel Carrères 5ranscriptional landsca%es of lipid producing microalgae* :AB pages PhD thesis, Wageningen University* Wageningen, The Netherlands (:;<=7 With references* with summary in English CSB6D 9EF(=G(B>=A(<F>(F D,CD 1;.<F<EGHA;GE:B Contents Chapter 1 Introduction and thesis outline 7 Chapter 2 Algal omics: the functional annotation challenge 21 Chapter 3 A systems approach to explore triacylglycerol production in Neochloris oleoabundans 39 Chapter 4 Neochloris oleoabundans is worth its salt: Transcriptomic analysis under salt and nitrogen stress 77 Chapter 5 Draft Genome Sequence of the Oleaginous Green Alga Tetradesmus obliquus UTEX 393 103 Chapter 6 The diurnal transcriptional landscape of the microalga Tetradesmus obliquus 107 Chapter 7 Evaluation of diurnal responses of Tetradesmus obliquus under nitrogen limitation 145 Chapter 8 Discussion 181 Summary 211 Citations 219 Acknowledgments 241 Curriculum vitae 251 Publication list 253 Overview of completed training activities 255 Chapter 1 Introduction and thesis outline Introduction and thesis outline 1.1 Microalgae to produce biofuels Microalgae are unicell1lar microscopic and photosynthetic organisms. Due to their long evolutionary history* their -iodiversity is very -road* -oth phenotypically and genetically I<J. They can live individually as single cells* in clusters or communities, or in sym-iosis 0ith -acteria. They are found in all sorts of environments ranging from fresh0ater to hy%er(saline conditions, nutrient rich and nutrient limited conditions, and in environments that di$er largely in tem%erature and %" I:J. )ust like other micro(organisms, their importance 0ithin the planet’s ecosystem 0as overlooked for a long time. Among the many im%ortant roles algae play and have played on the planet are that they are in the origin of the planet’s oLygen rich atmosphere that %aved the road for the evolution of other e1+aryotes* and they are currently still accounted for producing half of the atmospheric oLygen I>J. Microalgae have -een the main contri-utors of the fossil fuels 0e 1se in everyday life* and they are no0 seen as a hope to reduce our de%endence on these same fossil fuels I>MEJ. Cndeed* 0hile they have -een studied for their outstanding ca%acity to clean 0ater from eLcessive content in nitrogen, phosphorus, and metals, the interest has no0 shifted to0ards -iotechnological production of valua-le products IF* =J. Microalgae* and nota-ly green algae* are valua-le candidates for diverse -iotechnological applications -ecause they are fast gro0ing photoautotrophic organisms that re9uire minimal nutrition, do not re9uire ara-le land to gro0 and thus do not com%ete 0ith feed production, have s1%erior areal productivity* and are more efficient at converting photons into -iomass than plants I<;M<:J. Many microalgae are categorized as oleaginous -ecause they can accumulate a large proportion of their dry(0eight in fatty(acids 3>;(B;O7 I<>M<AJ. The two species studied in this thesis are the oleaginous microalgae named Ettlia oleoa-undans* formally +no0n as 6eochloris oleoa-undans* and 5etradesmus o-liq1us* formally +no0n as Scenedesmus obliquus and Acutodesm1s obliq1us. 2ike other oleaginous microalgae* they can produce triacylglycerides 35A.7* 0hich can -e eLtracted and easily converted into -iodiesel IG* E* <<J. Besides, 5A.s are free of un0anted chemical constituents unlike the phospholipids and the glycolipids, leading to purer -iofuel I<GJ. The main interests here are the rene0a-le and sustaina-le aspects of gro0ing microalgae* leading to a carbon- neutral -iofuel production. While promising, the current state of -iofuel prod1ction using microalgae is not economically feasible I<BJ. Besides -iofuels, other products 0ith higher = Chapter 1 added value can -e produced* such as pigments, unsaturated fatty acids* -ioplastics and proteins IB* 1EM:;J. Despite of its %otential* the costs of lipid production in algae are currently still too high to ma+e -iofuel production 0ith microalgae economically feasible considering the current oil prices. Ct 0as recently estimated that the cost of microalgal -iomass could -e as lo0 as ;.A; euros %er Pg, and at this price level* -iofuel production 0ould -e profita-le I<BJ. 4or the moment, commercial -ioproduction from microalgae is still limited to high(value products. Applications for commodities such as -ul+ chemicals and -iofuels re9uire su-stantial improvements, such as the increase of the total solar-to-product conversion efficiency. ,ne direct 0ay to achieve this is to optimize the eLternal conditions and to maintain these during scale(1%. The production scale(1p difficulties are studied at the Algae Production And Research Center 3AlgaePARC) I:<* ::J. Another 0ay to increase efficiency is to modify the meta-olism of the algae to reach a higher solar efficiency 1nder production conditions. 4or this, a -etter understanding of algal meta-olism and underlying mechanisms is needed. This is precisely the topic of this thesis, in 0hich C analy/e the internal phenoty%es of microalgae in response to a num-er of gro0th conditions relevant to industrial applications. S%ecifically* C studied the e$ect of light-dark cycles, salt water* intense light, and nitrogen limited or depleted conditions. 1.2 Limiting factors for profitable bioproduction Microalgae can -e gro0n in very diverse ty%e of -ioreactors* each coming 0ith their o0n advantages and disadvantages. The most used reactors are race(0ay %onds, diverse configurations of t1-ular systems, rectangular %anels, and plastic foil -ased -ags. Relying on freely availa-le sunlight is an im%ortant factor to minimize costs. "o0ever* outdoor cultivation is s1-#ect to variations in tem%erature* light intensity* and a light-dark cycle. During the dark %eriod* cellular respiration occurs and can lead 1% to :AO loss in daily produced -iomass I:>J. Commonly* this loss is due to the usage of a storage com%ound like starch that accumulates during the light %eriod. Thus, starch is a transient energy and carbon storage com%ound that is used in the night for maintenance purposes and for pre%aration of the cells for the neLt day. As a result, mutants that cannot %roduce starch sho0 a decrease in the efficiency in diurnal gro0th condition I:GJ. 2ipids, another possible storage com%ound* are hardly used during the night* even by starchless m1tants. <; Introduction and thesis outline 4ig1re <.<D Batch c1ltivation to increase 5A. yield for -iodiesel %rod1ction. Batch c1ltivation is done in t0o-step %rocess 0ith a Rgro0th %haseS and a Rstress %haseS. The stress %hase is ind1ced -y nitrogen depletion* res1lting in li%ids and 5A.s acc1m1lation. '1rrent sol1tions are not optimal for -iodiesel %rod1ction at a com%etitive %rice. Cn order to achieve high concentrations of lipids and 5A. 3>;(B;O7* microalgae need to -e eL%osed to adverse gro0th conditions. Commonly this is done -y applying nitrogen limitation or complete de%letion of nitrogen from the medium* 0hich results in overflo0 of the photon energy into carbon--ased molecules like starch and 5A. I:AJ. Starch and 5A. are the main forms of carbon and energy storage and serve as an electron sin+ 0hen the synthesis of reducing e9uivalents in the photosystems eLceeds the re9uirement for cell gro0th I<GJ. This mechanism has led to a simple t0o-ste% -atch strategy for 5A. production, starting 0ith a gro0th phase under replete conditions, follo0ed -y a stress phase during 0hich usually nitrogen is de%leted as displayed in 4igure <.< I:BJ. "o0ever* 1%on complete depletion of nitrogen the efficiency of photo(energy conversion decreases during the accumulation of 5A.* leading eventually to a decrease in the yield of TA. on photons. Therefore* instead of complete starvation* a less stressful condition of continuous nitrogen limitation 0as %roposed I:EJ. Cn such a system there is simultaneous gro0th and accumulation of 5A. for longer %eriods of time. 4urthermore* constant photosynthetic efficiency can -e maintained and the amount of do0n time for cleaning and restarting the system is much shorter. Additionally* such a condition allo0s more QeLibility to ad#1st the system 0ith the changing outdoor conditions. A recent com%arison against continuous production, -atch production displayed higher photon( to-5A. yield for -oth 0ild(ty%e and the starchless mutant 3slm<7. Even though the starchless mutant had :;O lo0er photo-to(5A. yield* it accumulated BAO more 5A. as %art of its dry 0eight than the wild(ty%e. Both strains were studied in this thesis in Chapters Error: Reference source not found and Error: Reference source not found I:FJ.