Final Report Summary - SUNBIOPATH (Towards a better sunlight to biomass conversion efficiency in microalgae)
Executive summary:
Culturing algal cells in photobioreactors requires high-density cells at high light intensities in order to get the maximum photosynthesis efficiency and biomass productivity. However, whereas photosynthesis efficiency is directly proportional to photon flux in low light, it rapidly decreases in high light intensities and excess light energy is dissipated to avoid oxidative damage. Several mechanisms exist to dissipate excess light energy, including the use a variety of exciton traps, which are collectively referred as non-photochemical quenchers (NPQ).
The SUNBIOPATH consortium investigated the high light response of algal cells (Chlamydomonas) and established a comprehensive list of genes and photosynthetic mechanisms that are crucial for growth in these conditions.
Project Context and Objectives:
SUNBIOPATH - towards a better sunlight to biomass conversion efficiency in microalgae - was an integrated program of research aimed at improving biomass yields and valorization of biomass for two Chlorophycean photosynthetic algae, Chlamydomonas reinhardtii and Dunaliella salina. For that purpose, the scientific workpackages were organized in order to get step by step insight into how algal cells use light energy, beginning with the primary reactions of photosynthesis and going deeper into the biochemistry of the cell to identify key components for biomass productivity. Then the general knowledge coming from these researches would be used to engineer cells for efficient transgenic expression and biomass production. In parallel, optimization of algal culture in photobioreactors was performed.
The linear photosynthetic electron transfer chain is composed of well known enzymes that use light to make the basic photochemistry involving extraction of electrons from water and transfer to NADP+, to generate NADPH. A proton-gradient is built up during electron transport and used by an ATP-synthase to generate ATP. Both NADPH and ATP are needed for CO2 fixation in the Calvin cycle for carbon compound synthesis. Coupled with the linear transfer of electrons, the auxiliary routes of electron flow (cyclic and chlororespiration), and changes in energy distribution between the two photosystems (state transitions) participate in the adaptation of photosynthesis to contrasting conditions. Another layer of control is performed by the antennas (made of pigments and proteins of the light harvesting complexes or Lhc) which not only collect energy and play a role in state transition but also dissipate energy under the form of heat or fluorescence in conditions of excess of light. For more efficient algal growth, it is important to understand the role of these alternative routes and discover ways for controlling energy dissipation.
Furthermore, efficient photosynthesis relies on mitochondrial respiration in the light. The rates of ATP and NADPH generation by the photosynthetic light reaction differ greatly from the rates of consumption by downstream pathways such as the Calvin cycle under certain conditions, so that accumulation of excess reducing equivalents in the plastid has to be prevented by valve systems. In order to ensure that photosynthesis is not perturbed by these imbalances, several strategies were evolved by photosynthetic cells that dissipate excess reducing power via the export of reducing equivalents which can be eventually used by the mitochondrial respiratory chain. Photosynthesis and photosynthetic acclimation are therefore dependent on a functional mitochondrial respiratory chain.
Lastly, the ability to manipulate chloroplast genes of Chlamydomonas represents an important molecular tool for dissecting and manipulating the process of photosynthesis and its regulation. Furthermore, the insertion of genes for novel enzymes provides the opportunity to alter chloroplast metabolism and increase the synthesis of high-energy fuel molecules or high value molecules. However, manipulation of the chloroplast is still at in infancy because of a tight system of expression regulation.
Our main objectives were
(1) to improve algal growth in photobioreactors and identify key components for production of biomass,
(2) to manipulate chloroplast gene expression with the aim of producing biologicals,
(3) to optimize and establish models for growth in photobioreactors.
Objective 1: Improving algal growth in photobioreactors and identifying key components for production of biomass
a) Improving algal growth in high light
The productivity of phototrophic cultivation is highly dependent on the quantum yield of photosynthesis, the process by which primarily carbon (CO2) is assimilated into organic matter. Whereas photosynthesis efficiency is directly proportional to photon flux in low light, it rapidly decreases in high light intensities and excess light energy is dissipated to avoid oxidative damage. However, maximum photosynthesis efficiency in high light would be desirable for culturing the algal cells, in order to get higher cell concentrations and higher biomass yields. Several mechanisms exist to dissipate excess light energy, among which are the use of electron sinks or a variety of exciton traps, which are collectively referred as nonphotochemical quenchers (NPQ). The consortium focused its researches on both of these ways of energy dissipation. Special attention was given to the role of the different proteins from the light harvesting complexes from Photosystems I and II during acclimation to different light intensities.
The consortium demonstrated that LhcSR3, a light harvesting protein, is able to sense the luminal pH of the chloroplast and to act as a quencher of excitation energy, the correlation between LhcSR3 and NPQ being clearly shown. This strict relationship demonstrated that LhcSR3 is a key protein involved in biomass production. The consortium also showed that induction of LhcSR3 by high light is depending on a Ca++ sensor protein (CAS), an important finding, which opens new insights into the regulation of the high light response of Chlamydomonas. To get a complete view of light harvesting efficiency, the roles of the other light harvesting proteins (Lhc) of the antenna in light capture and energy transfer to photosystem reaction centers as well as in NPQ have also been established by selectively inactivating the expression of the corresponding genes.
In parallel, the manipulation of the size of the light harvesting complex antenna has been the subject of a lot of attention with the aim of improving growth in photobioreactors. It is indeed thought that a smaller light-harvesting antenna size would increase light penetrance in deep layers of photobioreactors and reduce Lhc-dependent heat dissipation of absorbed light energy, thereby increasing photosynthetic efficiency in high light and high cell density.
To this aim, a library of mutants was built and screened with a computer-assisted micro-spectrophotometer device, designed by partners of the consortium. 10 mutants with lower chlorophyll content were isolated. Mutations responsible for this phenotype were identified and concern either chloroplast biogenesis, insertion of the light harvesting complexes in the thylakoid membranes or chlorophyll biosynthesis. These mutants were tested for growth first in small-scale photobioreactors (500 mL to 1.5 L) and then in large-scale photobioreactors (25 L). Three mutants presented higher photoconversion efficiency than the corresponding wild type in high light (500 μE/m2/s), with one of the mutants even doubling it (from 4% in wild type to 8% in the mutant). That corresponded to a productivity of 0.6 g/L.Day for the wild type and 1.2 g /L.Day for the mutant respectively. Simulation calculation suggested that this increase productivity could be the result of both less quenching at high light intensities due to lower chlorophyll content and less high light saturation.
Besides the light harvesting complexes whose primary role is to collect light energy, cyclic electron flow is an important player in the control of chloroplast energy and redox metabolism. One of the objectives of the consortium was thus to investigate cyclic electron flow by several means including genetic, proteomic and biophysical experiments. This allowed to demonstrate a Ca++-dependent regulation of cyclic electron flow, via the combined action of CAS, ANR1 and PGLR1, with ANR1 being important for the acclimation to high light, and thus crucial for efficient sunlight to biomass conversion.
In addition, efficient high light growth also depends on the subsequent reactions of the Calvin cycle for CO2 fixation. The aim of the consortium in that field was to find candidate rate-limiting enzymes in photosynthesis in order to manipulate them for increasing biomass. The consortium focused on sedoheptulose-1,7-bisphosphatase (SBPase). Chlamydomonas mutant lines expressing a synthetic codon-optimized SBPase gene from Dunaliella in the chloroplast were isolated. Several mutant lines that express the Dunaliella SBPase gene were found to have elevated biomass and higher starch levels.
Efficient photosynthesis relies on mitochondrial respiration, since excess reducing equivalents accumulating in the plastid can be exported and consumed by mitorespiration acting as a valve system. One of the aims of the consortium in that specific field was to find such actors and to demonstrate their role in high light growth. The consortium demonstrated that mTERF proteins, which are key regulators of mitochondrial expression, play a role in high light acclimation, by 'preparing' mitochondria for its function as electron sink under conditions where reducing equivalents accumulate in the plastid. Thus these proteins are crucial for efficient sun to biomass conversion in fluctuating conditions such as outdoors cultivation. Strains overexpressing one of the mTERF proteins (MOC1) have been constructed and will constitute valuable tools for photobioreactor growth.
b) Understanding algal growth in outdoor conditions
When cultivated in outdoors systems, algal cells are subject to dark periods during which they consume their carbon resources (starch) accumulated during the day. An additional exogenous carbon source such as acetate for Chlamydomonas can be added to the medium and used as substrate for respiration to boost growth during the night. One of the aims of the project was to study the role of mitochondria in biomass conversion and assimilation of acetate in the cell. The consortium demonstrated that the isocitrate lyase enzyme from the glyoxylate cycle is essential for acetate utilization and that its lack has manifold consequences at the proteome and metabolome levels.
The consortium also focused attention on the mechanisms that occur during transition from dark to light. The comprehension of these processes is indeed important to facilitate the transition and avoid oxidative stresses. The consortium demonstrated the crucial role of the Mehler reaction during a dark-light transition in aerobic condition. However, when cells encounter anaerobic condition (due to sustained respiration in the dark), the consortium demonstrated that state transitions and the induction of a chloroplast hydrogenase independently promote the activation of photosynthesis.
Objective 2. Manipulating chloroplast gene expression with the aim of producing biologicals
Production of high value products in the chloroplast is still at in infancy. Transgene expression in Chlamydomonas can be limited due to negative feedback regulation that is exerted by unassembled proteins on the translation of their own mRNA (control by epistasy of synthesis, CES). The consortium put a lot of effort to the discovery of new regulators that negatively control chloroplast gene expression. Additionally, transgene expression can also be limited by proteolytic degradation. One of the objectives in that field was thus to find and characterize proteases from the chloroplast and to test transgene expression in a protease deficient context. The consortium achieved these aims by characterizating 4 factors that controls chloroplast mRNA genes and one protease (ftsH).
For efficient production of biologicals, it is often required to express more than one transgene. Efforts were thus made to develop trans-operons that could be co-transcribed and co-translated from a single transcription unit. The consortium demonstrated that expression of operons is possible.
In addition, an essential requirement for any recombinant platform is that transgene expression can be tightly regulated such that production of recombinant metabolites that are toxic to the host can be tightly controlled. This allows the uncoupling of host growth from production whereby the biomass is produced first and then it is induced to produce metabolites. Three strategies were thus investigated to establish an inducible expression system but none could be as efficient as the one, which is actually in use in the laboratory of one of the partners (riboswitch technique).
Objective 3. Optimizing and modelling algal growth in photobioreactors
When culturing cells in photobioreactors, the determination of the different parameters like temperature, pH, medium composition, CO2 supply (concentration/ partial pressure) and light intensity is critical for efficient growth. The first aim of the consortium was thus to determine the process conditions and the possible ranges of control. Once the best conditions for growth would be established, then growth kinetics of the wild type and different mutants generated by the consortium, would be determined to calculate the photoconversion efficiencies and biomass productivities.
The consortium demonstrated that the antenna reduced mutants showed higher photoconversion efficiency at middle and high light intensities. To bring these advantages into action, a wave-surface-reactor was developed to maximize the growth of the mutants. The material costs for this photobioreactor could be cut by half, and cheaper materials could be used because of reduced hydrodynamic pressure. Techno economic analysis revealed a reduction of the CO2 footprint for algal biomass production by about 30% using these mutants.
In addition, as an improvement for growth, one objective in that field was also to build a dynamic model to predict the response of the microalgae to various light regimes. The consortium reached that objective and provided a model which a good basis to simulate growth in real photobioreactors, which are characterized by mixing induced light/dark cycles in the range of 1 to 25 Hz.
In order to maximize valorization of the whole biomass, the consortium also looked for the potential of several microalgae as alternate substrate for biogas production focusing on the biorefinery concept. We concluded that selected algae species can be good substrates for biogas production and that anaerobic fermentation can seriously be considered as final step in future microalgae-based biorefinery concepts.
Project Results:
Main S and T results/foreground
Project objectives
The general aim of the project was to improve biomass yields and valorization of biomass for two Chlorophycean photosynthetic microalgae, Chlamydomonas reinhardtii and Dunaliella salina. For that purpose, the work was organized into 5 scientific workpackages (WP) and one management WP that will be described elsewhere The scientific WP were organized in order to get step by step insight into how algal cells use light energy, beginning with the primary reactions of photosynthesis (WP1 and WP2) and going deeper into the biochemistry of the cell (WP3, WP4). Then the general knowledge coming from these WP is used to engineer cells for efficient transgenic expression and biomass production (WP2, WP3, and WP4). In parallel, optimization of algal culture in photobioreactors has been performed (WP5).
Titles of WP
WP1: Photochemistry and light capture efficiency
WP2: Light harvesting complex engineering
WP3: Identification and functional analysis of novel limiting enzymatic steps for biomass production
WP4: Control of chloroplast gene expression and metabolic engineering of the chloroplast
WP5: Optimization and valorization of algal culture in photobioreactors
General introduction
Optimal performance of photosynthesis requires a perfect balance between reactions of light capture and conversion into reducing equivalents and metabolic reactions involved into the utilization of energy. During linear electron flow, electrons are extracted from water at the level of photosystem II (PSII) and transferred to photosystem I (PSI) via plastoquinone (PQ), the cytochrome b6f complex (cyt b6f) and plastocyanin (PC). PSI donates electrons to NADP+ via ferredoxin (PetF) and ferredoxin-NADP+ reductase (FNR). A trans-thylakoid proton-gradient is built up during the electron transport and used by an ATP-synthase (ATPsynth) to generate ATP. Both NADPH and ATP are needed for CO2 fixation by ribulose-bisphosphate carboxylase oxygenase (Rubisco) and triose-phosphate (Trio-P) generation by the Calvin cycle for carbon compound synthesis (starch, protein, lipids). Linear electron flow is associated with state I conditions, in which the light harvesting complexes (LHC) of PSII (LHCII) are associated with PSII. Electron transport can also be cyclic, generating a trans-thylakoid proton gradient and ATP. During this process, electrons are reinjected from PSI either to the plastoquinone pool via a NADH dehydrogenase (Ndh) or to cyt b6f via a supercomplex including PSI, cyt b6f, FNR and PGR5/PGRL1. In C. reinhardtii, cyclic electron transport is associated with state II (LHCII associated with PSI). PSI and PSII collect energy via their light harvesting complexes (LHCI and LHCII), comprising several antenna proteins (Lhc) and pigments (chlorophyll a, chlorophyll b and carotenoids). Photosynthetic antennae are devoted not only to light harvesting but also to photoprotection. For an applied point of view, it would interesting to obtain algae with diminished size of their antenna for allowing a better penetrance of light in the photobioreactors, without disturbing the photoprotective effect.
Non-photochemical reduction of PQ involves Ndh (the Nda2 protein). In association with a plastid plastoquinol terminal oxidase (PTOX), Nda2 would perform chlororespiration, which is a respiratory-like electron transport chain.
Together with mechanisms allowing dissipation of energy at the level of the antenna (qE), the auxiliary routes of electrons (cyclic and chlororespiration), and changes in energy distribution between the two photosystems (state transitions) participate in the adaptation of photosynthesis to contrasting conditions (light modifications, nutrient and CO2 availability, excess of salts, etc.) and it is important to understand their role and discover ways of control for a more efficient algal growth.
Moreover, ATP is also produced in two other places, the cytosol via glycolysis and mitochondria via the Krebs cycle and respiration. Metabolic exchanges occur between these compartments, which also contributes to algal adaptation to changing environments.
WP1: Photochemistry and light capture efficiency
General objective. The productivity of phototrophic cultivation is highly dependent on the quantum yield of photosynthesis, the process by which primarily carbon, but also nitrogen and sulphur are assimilated into organic matter. In the first WP, we aimed to better understand the array of mechanisms that regulates the utilization of absorbed light by the photosynthetic apparatus as a function of incident photon flux or as a function of the cellular energetic status. This knowledge is the base for biotechnological improvement of solar light energy conversion into chemical energy to be used for industrial energetic purposes.
Objectives and achievements
For the whole period, our objectives were to build a high-performance micro-spectrophotometer device for computer-assisted detection of mutant colonies (goal 1, D1-1); to use it to generate an insertional mutant library affected in photosynthetic traits (goal 2, D1-2); to isolate mutants affected in state transition (goal 3, D1-3); to analyse protein members of the light harvesting antenna (Lhc) family (goal 4, D1-4); and to investigate the response to changing light (goal 5, D1-5).
D1-1: Micro-spectrophotometer device for computer-assisted detection of mutant colonies
The unicellular green alga Chlamydomonas reinhardtii is suitable for random nuclear genetic transformation and the availability of nuclear genome sequence information (Merchant et al., 2007) makes it an organism of choice for both forward and reverse genetic studies. We developed a strategy to identify mutants induced by random insertion mutagenesis with reduction in pigment content by fluorescence measurement using video-imaging devices coupled with spectrophotometer-fluorimeter. A new imaging set up was devised by partner 4, which is particularly suited to assess in vivo photosynthetic activity. The system specifically measures time-resolved chlorophyll fluorescence in response to light. It is composed of a fast digital camera equipped with a wide-angle lens for the analysis of samples up to 10 x 10 cm, i.e. entire plants or petri dishes. Together with new image processing techniques, it has proven most useful to screen large numbers of unicellular algal mutant colonies to identify those with subtle changes in photosynthetic electron flow (Johnson et al., 2009).
D1-2: Library of insertion mutants affected in photosynthetic traits.
Among a library of 10,000 mutants in Chlamydomonas obtained by insertional mutagenesis by using a device as described in the D1-1 section, partner 5 identified three mutants severely affected in chlorophyll content, about 8%, 20% and 50% of the wild-type level, named gun4, as1 and as2 (Bonente et al., 2011). As described in WP2, as1 and as2 mutants proved affected in chloroplast biogenesis mechanisms, while gun4 is affected chlorophyll biosynthetic pathway, resulting in either cases in a reduction of pigmentation per cell. A number of other insertional mutants potentially altered in pathways for recycling reducing power have been produced and analysed by partner 4, with the set up described above in D1-1. Among these were two mutants altered in the MRL1 gene controlling RbcL expression, two mutants altered in the biogenesis of the ATP synthase and two mutants in the PTOX2 gene (see below). In addition, a dozen of mutants altered in PSI were sent for further characterization to partner 8, and a dozen of mutants altered in PSII biogenesis were sent to a colleague working in Munich on these issues.
D1-3: Identification of novel genes involved in state transitions
The process of state transitions involves the dynamic allocation of the mobile part of the LHCII antenna to PSII or PSI. This allows an appropriate redox balance of the photosynthetic electron transfer chain in response to changes in light or in the metabolic demands of the alga. State II, where the LHCII antenna is phosphorylated and associates with PSI, favors cyclic electron flow and the production of ATP. Partner 4 identified two of the mutants mentioned in D1-2 as altered in state transitions because of an increased reduction level of the PQ pool in darkness due to a mutation of the PTOX2 gene (see D3-1). Partner 4 also analysed several mutants from partner 5 that were potentially state transition mutants but proved unaffected in this process. Partner 1 investigated prolonged anaerobiosis in Chlamydomonas that leads to the expression of enzymes belonging to various fermentative pathways. Among them, oxygen-sensitive hydrogenases (HydA1/2) catalyze the synthesis of molecular hydrogen from protons and reduced ferredoxin (by PSI) in the stroma. Based on chlorophyll fluorescence induction kinetics typical of hydrogenase-deficient mutants, partner 1 set up an in vivo fluorescence imaging screening protocol allowing the isolation of mutants impaired in hydrogenase expression or activity, or altered in related metabolic pathways required for energy production in anaerobiosis.
D1-4: Purified Light-harvesting antenna apoproteins
Various members of the Light harvesting proteins, Lhca and Lhcb, were produced in E.coli by partner 5 and reconstituted in vitro by adding specific pigments. In the case of antenna proteins of PSI, the Lhca proteins, their biochemical and spectroscopic characterization in vitro has been reported in (Mozzo et al., 2010). Pigment binding and spectroscopic properties were conserved among the nine Lhca subunits, with enrichment in red forms in Lhca2, Lhca4 and Lhca9. The main biochemical and spectroscopic properties of Lhca complexes showed good conservation through evolution from green algae to higher plants in contrast to Lhcb proteins that diverged more strongly. In particular recombinant Lhcbm1 and Lhcbm9 (which in vivo is expressed only under stress condition) have a lower fluorescence yield, with Lhcbm9 being particularly efficient in reducing singlet oxygen formation upon exposure to strong light. These results suggest that Lhcbm1 and 9 have a critical role in photoprotection of PSII.
D1-5: Dynamics of light-energy dissipation in response to changing light intensities in Chlamydomonas
Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, among which are the use of electron sinks or of a variety of exciton traps which are collectively referred to as nonphotochemical quenchers (Cardol et al., 2011). The latter are part of at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of PSII and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. Partners1, 2 and 4 participated in a large international collaboration to investigate the respective roles of qE and qT in photoprotection. To this end a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition-deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4).
WP2: Light harvesting complex engineering
General objective. Light harvesting complexes (LHC) comprise several polypeptides that bind photosynthetic pigments such as chlorophyll and carotenoids. They have a dual function in respect to photosynthesis: not only do they capture light and transfer energy to photosystem reaction centres but they also dissipate excess energy to avoid oxidative stress. This work package aims at the generation of microalgal strains displaying improved growth properties in photobioreactors. One strategy to achieve this goal is to engineer the light-harvesting antenna by genetic means yielding strains with size-reduced antenna systems at PSII. A smaller light-harvesting antenna increases light penetrance in deep layers of photobioreactors and reduces LHC-dependent heat dissipation of absorbed light energy thereby increasing photosynthetic efficiency.
Objectives and achievements
For the whole period, our objectives were to determine function of genes affecting light-harvesting process efficiency (goal 1, D2-1); to obtain mutants affected in the expression of the light-harvesting antenna proteins (goal 2, D2-2); to select mutant strains efficient for growth in photobioreactors with either low or high optical length (goal 3, D2-3).
D2-1: Function of genes affecting light-harvesting process efficiency
Biochemical and physiologic characterization of mutants screened in WP1 and described in D2-2, allowed to determine the functions of the different genes affected by mutation on light-harvesting process efficiency and chloroplast biogenesis. Silencing of Lhcb2+7 resulted in a clear decrease in the abundance of trimeric LHCII band in native green gel electrophoresis, implying that Lhcbm2+7 is an essential component of trimeric LHCII complex. Instead, small or even no decrease in LHCII trimers was observed in the npq5 mutant or in the mutants in which Lhcbm1 was silenced, implying that Lhcbm1, although a component of LHCII trimers, can be substituted by other gene products. Silencing of both LHCBM1 and LHCBM2+8 was efficient in decreasing the functional antenna size of PSII.
D2-2: Mutant strains affected in the expression of the light-harvesting antenna proteins
The antenna moiety of Photosystem II in Chlamydomonas reinhardtii is composed of LHCB4, LHCB5 proteins encoding the monomeric components CP29 and CP26 and by a number of LHCBM genes encoding the major LHCII complex. In order to gain knowledge on the structural organization of antenna system in green unicellular algae, an artificial microRNA (amiRNA) silencing technology was used (Molnar et al., 2009), which allows discriminating between different genes and to coordinately silence genes sharing identical regions, while keeping unaltered the level of expression of other genes in the family.
D2-3: Mutant strains efficient for growth in photobioreactors with either low or high optical length
Mutants as1 and as2 described in the previous section were tested for growing in photobioreactor (Task 2-3). At the beginning partner 5 tested the growth of these mutants in small-scale photobioreactor (400 ml) and in a home-made system in which different small flasks (20 ml) were shading each other, in order to artificially recreate the shading effect of different layer in a large scale photobioreactor.
WP3: Identification and functional analysis of novel limiting enzymatic steps for biomass production
General objective. WP3 was dedicated to investigate in novel metabolic pathways and key enzyme components of energetic metabolism that are critical for biomass yield with micro algae. It was intended
-to modify the expression of proteins identified by comparing the proteome adaptation in cells of Dunaliella and Chlamydomonas metabolic mutants,
-to screen for new Chlamydomonas mutants affected in light-independent electron transfer pathways and
-to investigate in the function of specific enzymes known to play a key role either in the interplay between respiration and photosynthesis or to control respiratory activity.
Objectives and achievements
For the whole period, our objectives were to identify key proteins involved in biomass production in Chlamydomonas and Dunaliella (goal 1, D3-1); to build vectors for silencing growth rate-limitating enzymes in Chlamydomonas and Dunaliella (goal 2, D3-2); to deliver a proteotypic peptide profiling platform available for further analysis of Chlamydomonas and Dunaliella mutant strains (goal 3, D3-3) ; to build vectors for overexpressing growth rate-limiting enzymes in Chlamydomonas and Dunaliella (goal 4, D3-4) ; to produce TILLING (Targeting Induced Local Lesions In Genomes) resources in Chlamydomonas and Dunaliella (goal 5, D3-5) ; to isolate Chlamydomonas and Dunaliella RNAi/miRNA mutants affected in metabolic processes (goal 6, D3-6) ; to obtain proteotypic peptide profiles of engineered mutant strains (goal 7, D3-7) ; to isolate Chlamydomonas and Dunaliella mutants with enhanced carbon assimilation rates and biomass accumulation in photobioreactors (goal 8, D3-8).
D3-1: Key proteins involved in biomass production in Chlamydomonas and Dunaliella
(a) Enzymes implicated in the utilization of acetate
Partner 1 identified several enzymes implicated in biomass production in Chlamydomonas when cells are grown in the presence of acetate either in the light (mixotrophic conditions) or in the dark (heterotrophic conditions) such as the Pdsw and nd4 subunits of the mitochondrial complex I of the respiratory chain (Barbieri et al., 2011; Larosa et al., 2012). Nda1 is a type II NADH dehydrogenase located on the inner side of the inner mitochondrial membrane.
(b) Enzymes implicated in the acclimation to high light and/or chlororespiration
Metabolic processes in chloroplasts and mitochondria are tightly linked by an intense inter-organellar crosstalk involving metabolite exchange. Efficient photosynthesis relies on mitochondrial respiration, since excess reducing equivalents accumulating in the plastid can be exported and consumed by mitorespiration acting as a valve system. Organellar gene expression (OGE) is among the candidate signal sources thought to initiate and modulate retrograde signalling events. Apart from an intense communication between organelles and nucleus the acclimation of photosynthetic eukaryotes to environmental changes critically depends on a tightly coordinated expression of the nuclear and organellar genomes.
D3-2: Vectors for silencing growth rate-limitating enzymes in Chlamydomonas and Dunaliella
Several vectors have been constructed during the course of the project for silencing the expression of different genes: NDA1, PETO (encoding a phosphoprotein from cytb6f), CAS, ANR1, MOC1, LHCSR3, sedoheptulose-1,7-bisphosphatase (SBPase).
D3-3: Proteotypic peptide profiling platform available for further analysis of Chlamydomonas and Dunaliella mutant strains. Partner 6 developed a proteotypicprofiling platform and successfully analyzed protein dynamics in relation to biomass yield in wild-type that led to identification of CAS and ANR1 proteins (see D3-1). In addition, partner 6 employed quantitative proteomics to analyze photo-heterotrophic versus photo-autotrophic grown cells. These experiments allowed the quantitation of 1608 proteins and give detailed insights into how cell metabolism differs between the different trophic states (Hoehner et al., manuscript submitted).
D3-4: Vectors for overexpressing growth rate-limiting enzymes in Chlamydomonas and Dunaliella
Over-expression of MOC1 was performed to improve growth under conditions which are known to cause an accumulation of excess reducing equivalents (e.g. high-light, low CO2). Comparison between wild type strains and transformants revealed that the expression level of MOC1 cannot be further increased by employing the tested promoter systems and further indicated a high activity of the endogenous MOC1 promoter. These strains will represent valuable tools in future analyses aiming at the detailed characterization of MOC1 promoter regulation. In addition, two other vectors (gift of partner 3) for over-expression of the isocitrate lyase gene in Chlamydomonas have been constructed by partner 1. These vectors will be used for selection of transformants with better acetate utilization. Partner 7 constructed a synthetic codon-optimized sedoheptulose-1,7-bisphosphatase (SBPase) gene from Dunaliella tertiolecta for expression in the chloroplast of Chlamydomonas.
D3-5: TILLING (Targeting Induced Local Lesions In Genomes) resource in Chlamydomonas and Dunaliella
One of the roles of partner 5 was assessed as to construct a TILLING (Targeting Induced Local Lesions In Genomes) library in Chlamydomonas and Dunaliella (Task 3.5). This kind of resource has been previously applied to crop plants but not yet to algae. After the approval of project however, several problems arose with this technique, mainly due to difficulty in making reverse genetics in algae. Moreover being Chlamydomonas a haploid organism for most of its life cycle, the possibility to rescue mutants with mutations in essential genes is null. A similar tilling resource was produced in Berkeley by the lab of Kris Niyogi with rather limited success. In a meeting of the consortium in December 2010, it was suggested to change the method for obtaining a library of mutants in Chlamydomonas and to build a large insertional mutant library where each mutant could be identified by a Flanking Sequence Tag (FST).
D3-6: Chlamydomonas and Dunaliella RNAi/miRNA mutants affected in metabolic processes.
Partner 1 has investigated Chlamydomonas mutants affected in enzymes responsible for growth on acetate. Respiratory-deficient mutants affected in different enzymes of the respiratory chain have been analyzed in mixotrophic conditions. They all show decreased respiration and photosynthesis rates. In addition, whereas protein content does not vary, starch and neutral lipids contents per cell are strongly decreased. This suggests that the respiratory deficient mutants present reduced metabolic activities, responsible for decreased accumulation of carbon resources (Lecler et al., 2011).
D3-7: Proteotypic peptide profiles of engineered mutant strains
In collaboration with partner 1, partner 6 analyzed an isocitrate-lyase knockout mutant by quantitative proteomics using differential 15N/14N metabolic labeling. Between 1684 and 1792 proteins have been identified in the four datasets, respectively and allowed in-depth characterization of metabolic remodeling due to ICL deficiency (Plancke et al., manuscript submitted). Comparative quantitative analysis of a CAS mutant strain unable to acclimate to high light revealed among 423 quantified proteins, 31 proteins that are repressed in the mutant versus the wild-type and might be important for the high-light response in C. reinhardtii. In a new set of quantitative proteomics experiments partner 6 analysed the highlight response of C. reinhardtii using differential 15N/14N metabolic labelling.
D3-8: Chlamydomonas and Dunaliella mutants with enhanced carbon assimilation rates and biomass accumulation in photobioreactors
The strict relationship between NPQ and productivity (see D3-1) and NPQ and LhcSR3, demonstrate that LhcSR is a key protein involved in biomass production in Chlamydomonas. Partner 5 tested this hypothesis by measuring PCE in npq4 mutant compared to WT and the light use efficiency and productivity in npq4 is 22% higher than WT at 200 μmol m-2s-1 under L/D cycles of 1s. Together with the increase PCE described for mutants with reduced pigmentation, this result finally suggests that the quality and quantity of Lhc and Lhc-like protein is crucial to determine biomass productivity in Chlamydomonas.
WP4: Control of chloroplast gene expression and metabolic engineering of the chloroplast
The overall goal of this work package was to build on the existing expertise in chloroplast molecular-genetic to gain a fully understanding of the molecular mechanisms underlying the expression and regulation of chloroplast genes in the green alga Chlamydomonas. This knowledge would then allow the exploitation of the algal chloroplast as an expression platform in which transgenes are introduced into the chloroplast genome with a view to making novel proteins and metabolites. Below is a summary of the research work carried out by partners 4, 8 and 9.
Objectives and achievements
For the whole period, our objectives were to obtain an optimal inducible system for transgene expression in the chloroplast (goal 1, D4-1); to identify factors controlling the epistasy of synthesis (CES) process, which controls in turn the synthesis rate of key chloroplast proteins (goal 2, D4-2); to obtain a modular system in which multiple genes can be assembled together in an operon that is co-transcribed and co-translated (goal 3, D4-3); to isolate Chlamydomonas nuclear mutants affected in chloroplast proteases (goal 4, D4-4); to obtain Chlamydomonas strains with enhanced chloroplast transgene expression (goal 5, D4-5); to obtain Chlamydomonas strains expressing biosynthetic genes for C10 monoterpenoids (goal 6, D4-6).
D4-1: Optimal inducible system for transgene expression in the chloroplast
An essential requirement for any recombinant platform is that transgene expression can be tightly regulated such that production of recombinant metabolites that are toxic to the host can tightly controlled. This allows the uncoupling of host growth from production whereby the biomass is produced first and then it is induced to produce the metabolite. Several strategies were investigated.
i) Temperature-regulated control of transgene expression using the E. coli lac repressor (partner 8). An attempt was made to create an inducible system based on the expression of a temperature sensitive variant of the lac repressor in the chloroplast, coupled to a modified chloroplast promoter element containing binding sites for the E. coli lac repressor. In this system, expression of a gene-of-interest linked to the promoter would be prevented by repressor binding at the non-permissive temperature. However, a rise in temperature would result in a lack of binding and induction of expression. Earlier, we had shown the successful expression in the chloroplast of a synthetic gene encoding the lac repressor.
ii) Use of the E. coli FimE DNA recombinase as a molecular switch for induction of transgene expression (partner 8). The aim of this project was to express the FimE gene in the Chlamydomonas nucleus under the control of an inducible promoter (from CYC6) such that the FimE protein would be targeted into the chloroplast. Once in the chloroplast, the recombinase would recognise short target sequences flanking a promoter element and 'flip' its orientation, thereby turning on expression of a downstream transgene.
iii) Riboswitches to control transgenes (partner 9). To investigate the usefulness of bacterial riboswitches for the experimental manipulation of chloroplast gene expression, two riboswitches were tested. The first is the synthetic theophyllin-responsive 'on' riboswitch that was previously shown to function in tobacco chloroplasts (Verhounig et al., 2010). The second is a putative 'off' riboswitch from a cyanobacterial thiC gene. These riboswitches were placed between the psaA promoter /5'UTR and the lucCP reporter, and homoplasmic transformed lines were obtained. However with either riboswitch, luciferase activity was very low, was strongly dependent on the physiological state of the culture, and was not markedly responsive to theophyllin or thiamine respectively.
D4-2: Factors controlling the epistasy of synthesis (CES) process, which in turn controls the synthesis rate of key chloroplast proteins
i) Nuclear factors mediating CES (partners 4 and 9). Previous work in these partners labs had shown that transgene expression in the Chlamydomonas chloroplast can be limited due to the negative feedback regulation that is exerted by unassembled subunits on the translation of their own mRNA (control by epistasy of synthesis (CES); reviewed by (Choquet and Vallon, 2000). The expression of a transgene placed under the control of the psaA promoter and 5'UTR is enhanced in a nuclear mutant background that prevents trans-splicing of psaA and hence accumulation of the PsaA protein (Wostrikoff et al., 2004; Michelet et al., 2011). However with only one exception (Boulouis et al., 2011), the identity of the nuclear-encoded proteins that mediate this type of negative feedback regulation has not been elucidated. In an effort to identify such regulators three trans-acting factors were investigated; namely, Taa1, Mac1 and Mbb1.
ii) The role of the TDA1 factor (partner 4). The TDA1 nuclear gene controlling the expression of subunit a of the chloroplast ATP synthase in Chlamydomonas, which is critical for biomass production in phototrophic conditions, has been fully characterized. It is specifically required for translation of the chloroplast atpA transcript. The sequence of TDA1 contains eight copies of a degenerated 38 residues motif, or Octotrico Peptide Repeats (OPR), previously described in a few other trans-acting factors targeted to the Chlamydomonas chloroplast.
iii) The role of Mca1 in the CES process (partner 4). The role of the Mca1 factor as a translational enhancer for cytochrome f expression has been further documented by its action in strains altered for the CES process, i.e. the assembly-dependent regulation of cytochrome f translation. As a major conclusion drawn from these experiments, MCA1 turns out to be the nuclear trans-acting factor that controls the CES process for cytochrome f expression. Attempts to probe a direct molecular interaction between cytochrome f and MCA1 are currently being developed in two hybrid experiments.
iv) A genetic screen for the identification of mutants in the CES process (partner 4). An experimental strategy has been devised for isolating mutant strains escaping the CES process, which negatively down-regulates the expression of most chloroplast genes, thereby preventing high accumulation of their products. As a first attempt, Chlamydomonas mutants with impaired auto-regulation of cytochrome f synthesis, the best characterised CES protein, were constructed. In strains defective for cytochrome b6f assembly, expression of the cytochrome f gene, petA and of 5'petA-driven reporters is reduced. In particular, the 5'petA-aadA chimera only confers resistance to low levels of aminoglycosides.
D4-3: Modular system in which multiple genes can be assembled together into a trans-operon that is co-transcribed and co-translated (partner 9).
The goal of this project was to develop a simple operon assembly system that allows the efficient translation of multiple transgenes in the chloroplast from a single transcription unit. The design strategy was based on the hypothesis that the overlap (or close association) of the stop codon of the upstream ORF with the start codon of the next ORF should facilitate 'translational coupling' where the termination of translation and its re-initiation at the next translation start are intermittently coupled. Evidence of such coupling is found in both plant and algal chloroplast genomes, as well as bacterial and viral systems. We therefore used a synthetic biology approach to create as series of two-ORF constructs in our chloroplast expression vector pASapI and introduce these into the Chlamydomonas chloroplast genome. The ORFs encode an endolysin (cpl1) and the kanamycin resistance gene aphA6 and were coupled in this order but in three different ways (a 5bp gap between the TAA and ATG; a 1bp gap; a 1bp overlap).
D4-4: Chlamydomonas nuclear mutants affected in chloroplast proteases
i) The role of FtsH (partner 4). FtsH is the major transmembrane protease in the thylakoids, and an FtsH mutant was isolated by a suppressor approach. This mutant has been used extensively to study the action spectrum of FtsH. It was found that the primary roles of FtsH are (i) the recycling of photosynthesis proteins in stress conditions and (ii) the quality control of misassembled protein complexes. The substrate spectrum of the FtsH protease is currently under investigation. Besides PSII degradation in photoinhibitory conditions, FtsH proved to be involved in PSII degradation in nutrient starvation conditions, whether it being the absence of phosphate or of sulphur.
ii) Altered expression of the RNAse J, a chloroplast-based 5'-exo/endonuclease (partner 4). RNAi vectors for attenuating RNAseJ expression, a major 5'-3' chloroplast exonuclease have been devised and were used in nuclear gene transformation assays. Altered patterns of maturation of polycistronic transcripts were observed. How these alterations reflect on the level of expression of the corresponding protein products remains to be investigated.
iii) Remodeling of the photosynthetic apparatus upon nitrogen starvation (partner 4). We showed that cytochrome b6f and most cytochrome b6f-related proteins involved in its biogenesis are selectively lost when Chlamydomonas is starved of a nitrogen source. This nutrient stress response was investigated further because it is the most commonly used procedure with Chlamydomonas for biotechnological approaches aimed at biofuel production. It was shown that a deep remodelling of the photosynthetic membrane develops with the time of nitrogen deprivation. The membrane loses its ability to contribute to CO2 fixation, not only because of the selective degradation of cyt b6f complexes but also because of the degradation of stromal RubisCO. In addition, prolonged illumination under nitrogen stress leads to the photo-destruction of the two photosystems.
D4-5 (a): Chlamydomonas strains with enhanced chloroplast transgene expression
In this project partners 8 and 9 have developed complementary genetic screens to isolate mutant strains that show either enhanced transgene expression or improve stability/accumulation of recombinant proteins in the chloroplast.
i) Chlamydomonas strains with enhanced chloroplast transgene expression (partner 8). The levels of transgene expression obtained in the chloroplast of Chlamydomonas have been highly variable depending on the protein of interest and on the regulatory elements used for its expression. Although the limiting factors are not well understood, it is clear that negative feedback regulation of gene expression at the post-transcriptional level plays a role, and at least in some cases, proteolytic degradation of the protein product (Specht et al., 2010; Michelet et al., 2011).
ii) Chlamydomonas strains with enhanced chloroplast transgene expression (partner 9). This alternate screen involved first creating a transformant line in which the accumulation of the kanamycin-resistance protein AphA6 is compromised by the addition of an N-terminal extension, leading to a lower resistance level compared to the native AphA6. This transformant was then used to make a double transformant in which a HA-tagged version of the aadA gene encoding spectinomycin resistance was placed at a neutral locus downstream of psaA-3. This double transformant is resistance to both kanamycin (100 μg/ml) and spectinomycin (1000 μg/ml), and was used in a UV-mutagenesis screen in which the aim is to select for increased kanamycin resistance and then score for increased AadA protein by western blot (the high level of spectinomycin-resistance conferred by the highly active AadA precludes scoring for any increased resistance).
D4-5 (b): Transgene expression in protease-defective mutant strains
i) Expression in an FtsH protease deficient mutant (partners 4 and 9). To investigate whether the FtsH protease may degrade transgenic proteins and hence lower their level of expression, the ftsh1 mutant was crossed with lines expressing transgenic reporter proteins in the chloroplast. The following were used as reporters: the vapA gene encoding a surface antigen of Aeromonas salmonicida, the aacC gene that confers moderate levels of resistance to gentamycin, recently developed in this project, and the firefly luciferase gene lucCP (Matsuo et al., 2006). Proteolytic degradation was previously shown to limit VapA levels.
D4-6: Chlamydomonas strains with efficient expression of biosynthetic genes for a C10 biofuel (partner 9).
As a proof-of-concept for the utilization of the chloroplast genetic engineering tools developed during the SUNBIOPATH project, it was planned to manipulate the terpenoid biosynthesis pathway with a view to producing the novel C10 terpenoid, geraniol.
This work is still in progress, but the following has been achieved so-far:
a) A cDNA clone encoding the plant enzyme geraniol synthase (GES) was obtained from Dr Eran Pichersky (Iijima et al., 2004), modified to remove the 5' coding region for the chloroplast targeting sequence and to add an HA-tag sequence at the 3' end, and then cloned into our chloroplast expression vector, pASapI.
b) The gene encoding the cyanobacterial enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS) was amplified from genomic DNA of Synechocystis PCC6803, modified to encode a C-terminal HA tag and cloned into a second chloroplast expression vector pSRSapI.
c) Homoplasmic chloroplast transformants have been generated for both constructs and western analysis has confirmed the presence of the DXS protein, but not the GES protein. An assay for DXS activity it currently being established.
WP5: Optimization and valorization of algal culture in photobioreactors
General objective. The optimized cultivation of algae in photobioreactors relies not only on efficient strains but also on precisely controlled physico-chemical parameters. Currently, there is a need for the critical evaluation of relevant parameters that control growth in photobioreactors. This has been achieved in this WP through the analysis of photosynthetic rates and macroscopic kinetic studies in lab-scale reactors. For the most interesting mutants that show a significantly improvement of growth rates and biomass yield (see WP2 and 3), growth in indoor reactors has been examined. Data obtained have been used to design a reactor built with cheap materials. Algal have been tested for biomethane production. Finally a techno economic and environmental analysis has been performed from the obtained results taking into account the C-credit of the whole process.
Objectives and achievements for the period
For the whole period, our objectives were to establish optimized parameters for algal growth in photobioreactors (goal 1, D5-1); to develop theoretical models describing the algal response to various light regimes (goal 2, D5-2); to design of a reactor that is based on cheap materials (goal 3, D5-3); to determine the efficiency of algal strains obtained by the consortium for biogas production (goal 4, D5-4); to realize a techno economic and environmental analysis of algal strains obtained by the consortium (goal 5, D5-5) .
D5-1: Optimized parameters for algal growth in photobioreactors
The influence of variations in different parameters like temperature, pH, medium composition, CO2 supply (concentration/ partial pressure) and light intensity were investigated and optimized to determine the process conditions and possible ranges for control. For two wild types and different antenna-reduced mutants of Chlamydomonas reinhardtii (wt13, wt8b+, T7, as1, as2, bf4) growth kinetics was determined. The algae grow light limited up to intensities of about 300-500 μE/m2/s. Then photosynthesis becomes saturated at higher light intensities and could be even inhibited. 300-500 μE/m2/s light should be supplied to all algae cells within the whole reactor volume to operate production reactors with maximum efficiency.
D5-2:Theoretical development of a dynamic model describing the response of algae to various light regimes
A photosynthesis-irradiance curve was measured under continuous illumination and used to calculate the net oxygen yield on light energy, which was maximal between a PFD of 100 and 200 μmol m-2 s 1. Net oxygen yield under flashing light was proven to be duty cycle dependent: the highest yield was observed at a duty cycle of 0.1 which corresponds to a time-averaged PFD of 115 μmol m-2 s-1. At lower duty cycles maintenance respiration reduced net oxygen yield. At higher duty cycles the specific photon absorption rate exceeded the maximal photon utilization rate and, as a result, surplus light energy was dissipated as heat. This behaviour was identical with the observation under continuous light.
D5-3: Design of a mass reactor that is based on cheap materials
To bring these advantages into action, a wave-surface-reactor has been developed. The reactor is a surface structured horizontal reactor. Horizontal reactors have different advantages but the basic disadvantage is that light dilution is only possible to a limited extend. The antenna-reduced mutants however, allow for this new reactor design.
D5-4: Efficiency of algal strains obtained by the consortium for biogas production
In order to maximize valorization of the whole biomass, the consortium also looked for the potential of several microalgae (Chlamydomonas, Scenedesmus, Euglena, Dunaliella, ) as alternate substrate for biogas production. The potential of these species to serve as a substrate for biomethane production differs greatly and is for instance dependent on the rigidity of the cell wall as a determinant of digestibility. Fermentation of the green alga Chlamydomonas was most efficient. We conclude from our results that selected algae species represent good substrates for biogas production. In addition, to evaluate integrative biorefinery concepts, hydrogen production in Chlamydomonas prior to anaerobic fermentation of the algae biomass was measured and resulted in a 20% increase of biogas generation. We conclude that selected algae species can be good substrates for biogas production and that anaerobic fermentation can seriously be considered as final step in future microalgae-based biorefinery concepts.
D5-5: Techno economic and environmental analysis of algal strains obtained by the consortium
A reduction of specific absorption by the biomass leads to a reduction of non-photochemical quenching at the front side of the reactor, what is the first and main reason for light saving. Along the light path the light intensity is higher compared to the wild type case also leading to better growth and a reduced fraction of respiration. Experiments as given in deliverable 5-1 showed that these effects have been accumulated to a doubling of PCE for the antenna reduced mutant bf4 under high light conditions and high biomass concentration. This is a clear prove that the concept of antenna reduction leads to a direct effect in saving energy for biomass production. A reduction of the CO2 footprint for algal biomass production is possible by about 30%.
Potential Impact:
1. Strategic impacts
Potential impact for culturing algae
Culturing algal cells in photobioreactors requires high-density cells at high light intensities in order to get the maximum photosynthesis efficiency and biomass productivity. The consortium demonstrated that small antenna size mutants represent good options to maximize growth in these conditions since photoconversion efficiency and biomass productivity was doubled in high light and high density in the mutants compared to the corresponding wild type. The SUNBIOPATH consortium thus contributed very efficiently to the improvement of algal growth in photobioreactors, and allowed an evaluation as to whether metabolic engineering of Chlamydomonas can result in superior strains for solar-to-fuel energy conversion. This knowledge could be applied to Dunaliella, another microalgal species, used in the consortium and interesting for the production of glycerol and beta-carotenes.
2. Wider societal implications
By its large audience dissemination activities including journal articles and interviews on public radio chains (see below), the SUNBIOPATH project contributed to a better knowledge of the 'algal world' in the public and by this way, the concept of using algae for energy, food and feed, reached a large audience, from very young people to adults.
3. Main dissemination activities
Dissemination to scientific audience
Peer-reviewed papers
Members of the SUNBIOPATH consortium published an impressive number of scientific articles in international peer-reviewed journals, such as Plos Biology, Plant Cell, Proc Natl Acad Sci (USA), Plant J, Plos One, and applied and industrial journals such as Journal of Biotechnology, Engineering of Life Sciences, Biotechnology Bioengineering. Altogether, these articles contributed very efficiently to the dissemination of the knowledge of SUNBIOPATH to the scientific community.
Book chapter
In addition, the members of the SUNBIOPATH consortium participated to the writing of a book chapter (Bassi et al. 2011, Finding the bottle-neck: A research strategy for improved biomass production, in Microalgal Biotechnology: Integration and Economy, C. Posten and C. Walter, Editors. 2012, De Gruyter: Berlin. p. 227-52), in which the reader will find the main achievements of SUNBIOPATH. Written in a book co-edited by one of the members of SUNBIOPATH (Clemens Posten) and covering the present day microalgal biotechnology knowledge, this chapter represents a very efficient way to reach scientific from different fields, as well as economists and stakeholders.
Participation to scientific meetings
In addition, members of the SUNBIOPATH consortium including PI and potsdocs hired by the project participated to numerous international scientific conferences all over the world and organized meetings and workshops, and this contributed very efficiently to the dissemination of the project.
Participation to industrial meetings
In addition, the SUNBIOPATH project was presented at industrial events such as the European Biomass Conference held in Berlin in June 2011 or the Algae Europe held in Milano in October 2011.
Organization of an international meeting at the end of the project
One of the achievements of SUNBIOPATH is the organization of a joint international meeting with two other KBBE-funded projects, BAMMBO and GIAVAP. The meeting was held in Brussels on January 21-23 2013.
GIAVAP (Genetic Improvement of Algae for Value Added Products) and BAMMBO (Sustainable production of Biologically Active Molecules of Marine Origin) are two European projects funded in 2010 by KBBE. Together with Stefan Leu from GIAVAP and Daniel Walsh from BAMMBO, a joint meeting was organized that gathered around 60 people from the three consortia as well as Tomasz Calikowski, project officer of GIAVAP and BAMMBO. As described below, the meeting was organized in twelve sessions that combined the results of the three projects. In total, 42 presentations were shown. In addition, external speakers and members of the advisory boards of the projects (Prof R Bock from Max Planck Institute of Golm, Prof Arthur Grossman from the Carnegie Institution for Science - Standford, Dr Thomas Kyi Global Head Strategic Business Development - Lonza) also gave plenary lectures and provided valuable feedbacks about the meeting and the future of the algal research.
Dissemination to public audience
The SUNBIOPATH project was presented in a Belgian weekly newspaper (Le Vif l'Express) and in the FNRS-News edited by the Fonds de la Recherche Scientifique from Belgium. The project was also presented at the occasion of an interview of C Remacle by Veronique Thyberghien, about the utilization of microalgae to produce electricity [27/03/13, Program 'O positif' of the RTBF (Radio Television Belge Francophone)].
Oral presentations
Several oral presentations to public audience have contributed to the dissemination of the SUNBIOPATH project. The target audience was stakeholders of regional governments or general audience.
The project also made synergies with Science Education by presenting the SUNBIOPATH project at the occasion of the 'Plant Fascination Day' organized by EPSO in the universities of Geneva and Liege on May 18 2012 as well as during the 'Researcher's Night' in September 2011 in Liege.
4. Exploitation of results
To use the specific advantages of antenna-reduced mutants, specific reactor designs are possible. Light dilution for the developed mutants can be reduced by a factor 2, which allows for the reduction of the surface to footprint area by this factor. Therefore, the material costs can be cut by half. In addition, cheaper materials are possible in thinner layers by reduced hydrodynamic pressure.
List of Websites:
http://www2.ulg.ac.be/genemic/SUNBIOPATH/
Culturing algal cells in photobioreactors requires high-density cells at high light intensities in order to get the maximum photosynthesis efficiency and biomass productivity. However, whereas photosynthesis efficiency is directly proportional to photon flux in low light, it rapidly decreases in high light intensities and excess light energy is dissipated to avoid oxidative damage. Several mechanisms exist to dissipate excess light energy, including the use a variety of exciton traps, which are collectively referred as non-photochemical quenchers (NPQ).
The SUNBIOPATH consortium investigated the high light response of algal cells (Chlamydomonas) and established a comprehensive list of genes and photosynthetic mechanisms that are crucial for growth in these conditions.
Project Context and Objectives:
SUNBIOPATH - towards a better sunlight to biomass conversion efficiency in microalgae - was an integrated program of research aimed at improving biomass yields and valorization of biomass for two Chlorophycean photosynthetic algae, Chlamydomonas reinhardtii and Dunaliella salina. For that purpose, the scientific workpackages were organized in order to get step by step insight into how algal cells use light energy, beginning with the primary reactions of photosynthesis and going deeper into the biochemistry of the cell to identify key components for biomass productivity. Then the general knowledge coming from these researches would be used to engineer cells for efficient transgenic expression and biomass production. In parallel, optimization of algal culture in photobioreactors was performed.
The linear photosynthetic electron transfer chain is composed of well known enzymes that use light to make the basic photochemistry involving extraction of electrons from water and transfer to NADP+, to generate NADPH. A proton-gradient is built up during electron transport and used by an ATP-synthase to generate ATP. Both NADPH and ATP are needed for CO2 fixation in the Calvin cycle for carbon compound synthesis. Coupled with the linear transfer of electrons, the auxiliary routes of electron flow (cyclic and chlororespiration), and changes in energy distribution between the two photosystems (state transitions) participate in the adaptation of photosynthesis to contrasting conditions. Another layer of control is performed by the antennas (made of pigments and proteins of the light harvesting complexes or Lhc) which not only collect energy and play a role in state transition but also dissipate energy under the form of heat or fluorescence in conditions of excess of light. For more efficient algal growth, it is important to understand the role of these alternative routes and discover ways for controlling energy dissipation.
Furthermore, efficient photosynthesis relies on mitochondrial respiration in the light. The rates of ATP and NADPH generation by the photosynthetic light reaction differ greatly from the rates of consumption by downstream pathways such as the Calvin cycle under certain conditions, so that accumulation of excess reducing equivalents in the plastid has to be prevented by valve systems. In order to ensure that photosynthesis is not perturbed by these imbalances, several strategies were evolved by photosynthetic cells that dissipate excess reducing power via the export of reducing equivalents which can be eventually used by the mitochondrial respiratory chain. Photosynthesis and photosynthetic acclimation are therefore dependent on a functional mitochondrial respiratory chain.
Lastly, the ability to manipulate chloroplast genes of Chlamydomonas represents an important molecular tool for dissecting and manipulating the process of photosynthesis and its regulation. Furthermore, the insertion of genes for novel enzymes provides the opportunity to alter chloroplast metabolism and increase the synthesis of high-energy fuel molecules or high value molecules. However, manipulation of the chloroplast is still at in infancy because of a tight system of expression regulation.
Our main objectives were
(1) to improve algal growth in photobioreactors and identify key components for production of biomass,
(2) to manipulate chloroplast gene expression with the aim of producing biologicals,
(3) to optimize and establish models for growth in photobioreactors.
Objective 1: Improving algal growth in photobioreactors and identifying key components for production of biomass
a) Improving algal growth in high light
The productivity of phototrophic cultivation is highly dependent on the quantum yield of photosynthesis, the process by which primarily carbon (CO2) is assimilated into organic matter. Whereas photosynthesis efficiency is directly proportional to photon flux in low light, it rapidly decreases in high light intensities and excess light energy is dissipated to avoid oxidative damage. However, maximum photosynthesis efficiency in high light would be desirable for culturing the algal cells, in order to get higher cell concentrations and higher biomass yields. Several mechanisms exist to dissipate excess light energy, among which are the use of electron sinks or a variety of exciton traps, which are collectively referred as nonphotochemical quenchers (NPQ). The consortium focused its researches on both of these ways of energy dissipation. Special attention was given to the role of the different proteins from the light harvesting complexes from Photosystems I and II during acclimation to different light intensities.
The consortium demonstrated that LhcSR3, a light harvesting protein, is able to sense the luminal pH of the chloroplast and to act as a quencher of excitation energy, the correlation between LhcSR3 and NPQ being clearly shown. This strict relationship demonstrated that LhcSR3 is a key protein involved in biomass production. The consortium also showed that induction of LhcSR3 by high light is depending on a Ca++ sensor protein (CAS), an important finding, which opens new insights into the regulation of the high light response of Chlamydomonas. To get a complete view of light harvesting efficiency, the roles of the other light harvesting proteins (Lhc) of the antenna in light capture and energy transfer to photosystem reaction centers as well as in NPQ have also been established by selectively inactivating the expression of the corresponding genes.
In parallel, the manipulation of the size of the light harvesting complex antenna has been the subject of a lot of attention with the aim of improving growth in photobioreactors. It is indeed thought that a smaller light-harvesting antenna size would increase light penetrance in deep layers of photobioreactors and reduce Lhc-dependent heat dissipation of absorbed light energy, thereby increasing photosynthetic efficiency in high light and high cell density.
To this aim, a library of mutants was built and screened with a computer-assisted micro-spectrophotometer device, designed by partners of the consortium. 10 mutants with lower chlorophyll content were isolated. Mutations responsible for this phenotype were identified and concern either chloroplast biogenesis, insertion of the light harvesting complexes in the thylakoid membranes or chlorophyll biosynthesis. These mutants were tested for growth first in small-scale photobioreactors (500 mL to 1.5 L) and then in large-scale photobioreactors (25 L). Three mutants presented higher photoconversion efficiency than the corresponding wild type in high light (500 μE/m2/s), with one of the mutants even doubling it (from 4% in wild type to 8% in the mutant). That corresponded to a productivity of 0.6 g/L.Day for the wild type and 1.2 g /L.Day for the mutant respectively. Simulation calculation suggested that this increase productivity could be the result of both less quenching at high light intensities due to lower chlorophyll content and less high light saturation.
Besides the light harvesting complexes whose primary role is to collect light energy, cyclic electron flow is an important player in the control of chloroplast energy and redox metabolism. One of the objectives of the consortium was thus to investigate cyclic electron flow by several means including genetic, proteomic and biophysical experiments. This allowed to demonstrate a Ca++-dependent regulation of cyclic electron flow, via the combined action of CAS, ANR1 and PGLR1, with ANR1 being important for the acclimation to high light, and thus crucial for efficient sunlight to biomass conversion.
In addition, efficient high light growth also depends on the subsequent reactions of the Calvin cycle for CO2 fixation. The aim of the consortium in that field was to find candidate rate-limiting enzymes in photosynthesis in order to manipulate them for increasing biomass. The consortium focused on sedoheptulose-1,7-bisphosphatase (SBPase). Chlamydomonas mutant lines expressing a synthetic codon-optimized SBPase gene from Dunaliella in the chloroplast were isolated. Several mutant lines that express the Dunaliella SBPase gene were found to have elevated biomass and higher starch levels.
Efficient photosynthesis relies on mitochondrial respiration, since excess reducing equivalents accumulating in the plastid can be exported and consumed by mitorespiration acting as a valve system. One of the aims of the consortium in that specific field was to find such actors and to demonstrate their role in high light growth. The consortium demonstrated that mTERF proteins, which are key regulators of mitochondrial expression, play a role in high light acclimation, by 'preparing' mitochondria for its function as electron sink under conditions where reducing equivalents accumulate in the plastid. Thus these proteins are crucial for efficient sun to biomass conversion in fluctuating conditions such as outdoors cultivation. Strains overexpressing one of the mTERF proteins (MOC1) have been constructed and will constitute valuable tools for photobioreactor growth.
b) Understanding algal growth in outdoor conditions
When cultivated in outdoors systems, algal cells are subject to dark periods during which they consume their carbon resources (starch) accumulated during the day. An additional exogenous carbon source such as acetate for Chlamydomonas can be added to the medium and used as substrate for respiration to boost growth during the night. One of the aims of the project was to study the role of mitochondria in biomass conversion and assimilation of acetate in the cell. The consortium demonstrated that the isocitrate lyase enzyme from the glyoxylate cycle is essential for acetate utilization and that its lack has manifold consequences at the proteome and metabolome levels.
The consortium also focused attention on the mechanisms that occur during transition from dark to light. The comprehension of these processes is indeed important to facilitate the transition and avoid oxidative stresses. The consortium demonstrated the crucial role of the Mehler reaction during a dark-light transition in aerobic condition. However, when cells encounter anaerobic condition (due to sustained respiration in the dark), the consortium demonstrated that state transitions and the induction of a chloroplast hydrogenase independently promote the activation of photosynthesis.
Objective 2. Manipulating chloroplast gene expression with the aim of producing biologicals
Production of high value products in the chloroplast is still at in infancy. Transgene expression in Chlamydomonas can be limited due to negative feedback regulation that is exerted by unassembled proteins on the translation of their own mRNA (control by epistasy of synthesis, CES). The consortium put a lot of effort to the discovery of new regulators that negatively control chloroplast gene expression. Additionally, transgene expression can also be limited by proteolytic degradation. One of the objectives in that field was thus to find and characterize proteases from the chloroplast and to test transgene expression in a protease deficient context. The consortium achieved these aims by characterizating 4 factors that controls chloroplast mRNA genes and one protease (ftsH).
For efficient production of biologicals, it is often required to express more than one transgene. Efforts were thus made to develop trans-operons that could be co-transcribed and co-translated from a single transcription unit. The consortium demonstrated that expression of operons is possible.
In addition, an essential requirement for any recombinant platform is that transgene expression can be tightly regulated such that production of recombinant metabolites that are toxic to the host can be tightly controlled. This allows the uncoupling of host growth from production whereby the biomass is produced first and then it is induced to produce metabolites. Three strategies were thus investigated to establish an inducible expression system but none could be as efficient as the one, which is actually in use in the laboratory of one of the partners (riboswitch technique).
Objective 3. Optimizing and modelling algal growth in photobioreactors
When culturing cells in photobioreactors, the determination of the different parameters like temperature, pH, medium composition, CO2 supply (concentration/ partial pressure) and light intensity is critical for efficient growth. The first aim of the consortium was thus to determine the process conditions and the possible ranges of control. Once the best conditions for growth would be established, then growth kinetics of the wild type and different mutants generated by the consortium, would be determined to calculate the photoconversion efficiencies and biomass productivities.
The consortium demonstrated that the antenna reduced mutants showed higher photoconversion efficiency at middle and high light intensities. To bring these advantages into action, a wave-surface-reactor was developed to maximize the growth of the mutants. The material costs for this photobioreactor could be cut by half, and cheaper materials could be used because of reduced hydrodynamic pressure. Techno economic analysis revealed a reduction of the CO2 footprint for algal biomass production by about 30% using these mutants.
In addition, as an improvement for growth, one objective in that field was also to build a dynamic model to predict the response of the microalgae to various light regimes. The consortium reached that objective and provided a model which a good basis to simulate growth in real photobioreactors, which are characterized by mixing induced light/dark cycles in the range of 1 to 25 Hz.
In order to maximize valorization of the whole biomass, the consortium also looked for the potential of several microalgae as alternate substrate for biogas production focusing on the biorefinery concept. We concluded that selected algae species can be good substrates for biogas production and that anaerobic fermentation can seriously be considered as final step in future microalgae-based biorefinery concepts.
Project Results:
Main S and T results/foreground
Project objectives
The general aim of the project was to improve biomass yields and valorization of biomass for two Chlorophycean photosynthetic microalgae, Chlamydomonas reinhardtii and Dunaliella salina. For that purpose, the work was organized into 5 scientific workpackages (WP) and one management WP that will be described elsewhere The scientific WP were organized in order to get step by step insight into how algal cells use light energy, beginning with the primary reactions of photosynthesis (WP1 and WP2) and going deeper into the biochemistry of the cell (WP3, WP4). Then the general knowledge coming from these WP is used to engineer cells for efficient transgenic expression and biomass production (WP2, WP3, and WP4). In parallel, optimization of algal culture in photobioreactors has been performed (WP5).
Titles of WP
WP1: Photochemistry and light capture efficiency
WP2: Light harvesting complex engineering
WP3: Identification and functional analysis of novel limiting enzymatic steps for biomass production
WP4: Control of chloroplast gene expression and metabolic engineering of the chloroplast
WP5: Optimization and valorization of algal culture in photobioreactors
General introduction
Optimal performance of photosynthesis requires a perfect balance between reactions of light capture and conversion into reducing equivalents and metabolic reactions involved into the utilization of energy. During linear electron flow, electrons are extracted from water at the level of photosystem II (PSII) and transferred to photosystem I (PSI) via plastoquinone (PQ), the cytochrome b6f complex (cyt b6f) and plastocyanin (PC). PSI donates electrons to NADP+ via ferredoxin (PetF) and ferredoxin-NADP+ reductase (FNR). A trans-thylakoid proton-gradient is built up during the electron transport and used by an ATP-synthase (ATPsynth) to generate ATP. Both NADPH and ATP are needed for CO2 fixation by ribulose-bisphosphate carboxylase oxygenase (Rubisco) and triose-phosphate (Trio-P) generation by the Calvin cycle for carbon compound synthesis (starch, protein, lipids). Linear electron flow is associated with state I conditions, in which the light harvesting complexes (LHC) of PSII (LHCII) are associated with PSII. Electron transport can also be cyclic, generating a trans-thylakoid proton gradient and ATP. During this process, electrons are reinjected from PSI either to the plastoquinone pool via a NADH dehydrogenase (Ndh) or to cyt b6f via a supercomplex including PSI, cyt b6f, FNR and PGR5/PGRL1. In C. reinhardtii, cyclic electron transport is associated with state II (LHCII associated with PSI). PSI and PSII collect energy via their light harvesting complexes (LHCI and LHCII), comprising several antenna proteins (Lhc) and pigments (chlorophyll a, chlorophyll b and carotenoids). Photosynthetic antennae are devoted not only to light harvesting but also to photoprotection. For an applied point of view, it would interesting to obtain algae with diminished size of their antenna for allowing a better penetrance of light in the photobioreactors, without disturbing the photoprotective effect.
Non-photochemical reduction of PQ involves Ndh (the Nda2 protein). In association with a plastid plastoquinol terminal oxidase (PTOX), Nda2 would perform chlororespiration, which is a respiratory-like electron transport chain.
Together with mechanisms allowing dissipation of energy at the level of the antenna (qE), the auxiliary routes of electrons (cyclic and chlororespiration), and changes in energy distribution between the two photosystems (state transitions) participate in the adaptation of photosynthesis to contrasting conditions (light modifications, nutrient and CO2 availability, excess of salts, etc.) and it is important to understand their role and discover ways of control for a more efficient algal growth.
Moreover, ATP is also produced in two other places, the cytosol via glycolysis and mitochondria via the Krebs cycle and respiration. Metabolic exchanges occur between these compartments, which also contributes to algal adaptation to changing environments.
WP1: Photochemistry and light capture efficiency
General objective. The productivity of phototrophic cultivation is highly dependent on the quantum yield of photosynthesis, the process by which primarily carbon, but also nitrogen and sulphur are assimilated into organic matter. In the first WP, we aimed to better understand the array of mechanisms that regulates the utilization of absorbed light by the photosynthetic apparatus as a function of incident photon flux or as a function of the cellular energetic status. This knowledge is the base for biotechnological improvement of solar light energy conversion into chemical energy to be used for industrial energetic purposes.
Objectives and achievements
For the whole period, our objectives were to build a high-performance micro-spectrophotometer device for computer-assisted detection of mutant colonies (goal 1, D1-1); to use it to generate an insertional mutant library affected in photosynthetic traits (goal 2, D1-2); to isolate mutants affected in state transition (goal 3, D1-3); to analyse protein members of the light harvesting antenna (Lhc) family (goal 4, D1-4); and to investigate the response to changing light (goal 5, D1-5).
D1-1: Micro-spectrophotometer device for computer-assisted detection of mutant colonies
The unicellular green alga Chlamydomonas reinhardtii is suitable for random nuclear genetic transformation and the availability of nuclear genome sequence information (Merchant et al., 2007) makes it an organism of choice for both forward and reverse genetic studies. We developed a strategy to identify mutants induced by random insertion mutagenesis with reduction in pigment content by fluorescence measurement using video-imaging devices coupled with spectrophotometer-fluorimeter. A new imaging set up was devised by partner 4, which is particularly suited to assess in vivo photosynthetic activity. The system specifically measures time-resolved chlorophyll fluorescence in response to light. It is composed of a fast digital camera equipped with a wide-angle lens for the analysis of samples up to 10 x 10 cm, i.e. entire plants or petri dishes. Together with new image processing techniques, it has proven most useful to screen large numbers of unicellular algal mutant colonies to identify those with subtle changes in photosynthetic electron flow (Johnson et al., 2009).
D1-2: Library of insertion mutants affected in photosynthetic traits.
Among a library of 10,000 mutants in Chlamydomonas obtained by insertional mutagenesis by using a device as described in the D1-1 section, partner 5 identified three mutants severely affected in chlorophyll content, about 8%, 20% and 50% of the wild-type level, named gun4, as1 and as2 (Bonente et al., 2011). As described in WP2, as1 and as2 mutants proved affected in chloroplast biogenesis mechanisms, while gun4 is affected chlorophyll biosynthetic pathway, resulting in either cases in a reduction of pigmentation per cell. A number of other insertional mutants potentially altered in pathways for recycling reducing power have been produced and analysed by partner 4, with the set up described above in D1-1. Among these were two mutants altered in the MRL1 gene controlling RbcL expression, two mutants altered in the biogenesis of the ATP synthase and two mutants in the PTOX2 gene (see below). In addition, a dozen of mutants altered in PSI were sent for further characterization to partner 8, and a dozen of mutants altered in PSII biogenesis were sent to a colleague working in Munich on these issues.
D1-3: Identification of novel genes involved in state transitions
The process of state transitions involves the dynamic allocation of the mobile part of the LHCII antenna to PSII or PSI. This allows an appropriate redox balance of the photosynthetic electron transfer chain in response to changes in light or in the metabolic demands of the alga. State II, where the LHCII antenna is phosphorylated and associates with PSI, favors cyclic electron flow and the production of ATP. Partner 4 identified two of the mutants mentioned in D1-2 as altered in state transitions because of an increased reduction level of the PQ pool in darkness due to a mutation of the PTOX2 gene (see D3-1). Partner 4 also analysed several mutants from partner 5 that were potentially state transition mutants but proved unaffected in this process. Partner 1 investigated prolonged anaerobiosis in Chlamydomonas that leads to the expression of enzymes belonging to various fermentative pathways. Among them, oxygen-sensitive hydrogenases (HydA1/2) catalyze the synthesis of molecular hydrogen from protons and reduced ferredoxin (by PSI) in the stroma. Based on chlorophyll fluorescence induction kinetics typical of hydrogenase-deficient mutants, partner 1 set up an in vivo fluorescence imaging screening protocol allowing the isolation of mutants impaired in hydrogenase expression or activity, or altered in related metabolic pathways required for energy production in anaerobiosis.
D1-4: Purified Light-harvesting antenna apoproteins
Various members of the Light harvesting proteins, Lhca and Lhcb, were produced in E.coli by partner 5 and reconstituted in vitro by adding specific pigments. In the case of antenna proteins of PSI, the Lhca proteins, their biochemical and spectroscopic characterization in vitro has been reported in (Mozzo et al., 2010). Pigment binding and spectroscopic properties were conserved among the nine Lhca subunits, with enrichment in red forms in Lhca2, Lhca4 and Lhca9. The main biochemical and spectroscopic properties of Lhca complexes showed good conservation through evolution from green algae to higher plants in contrast to Lhcb proteins that diverged more strongly. In particular recombinant Lhcbm1 and Lhcbm9 (which in vivo is expressed only under stress condition) have a lower fluorescence yield, with Lhcbm9 being particularly efficient in reducing singlet oxygen formation upon exposure to strong light. These results suggest that Lhcbm1 and 9 have a critical role in photoprotection of PSII.
D1-5: Dynamics of light-energy dissipation in response to changing light intensities in Chlamydomonas
Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, among which are the use of electron sinks or of a variety of exciton traps which are collectively referred to as nonphotochemical quenchers (Cardol et al., 2011). The latter are part of at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of PSII and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. Partners1, 2 and 4 participated in a large international collaboration to investigate the respective roles of qE and qT in photoprotection. To this end a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition-deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4).
WP2: Light harvesting complex engineering
General objective. Light harvesting complexes (LHC) comprise several polypeptides that bind photosynthetic pigments such as chlorophyll and carotenoids. They have a dual function in respect to photosynthesis: not only do they capture light and transfer energy to photosystem reaction centres but they also dissipate excess energy to avoid oxidative stress. This work package aims at the generation of microalgal strains displaying improved growth properties in photobioreactors. One strategy to achieve this goal is to engineer the light-harvesting antenna by genetic means yielding strains with size-reduced antenna systems at PSII. A smaller light-harvesting antenna increases light penetrance in deep layers of photobioreactors and reduces LHC-dependent heat dissipation of absorbed light energy thereby increasing photosynthetic efficiency.
Objectives and achievements
For the whole period, our objectives were to determine function of genes affecting light-harvesting process efficiency (goal 1, D2-1); to obtain mutants affected in the expression of the light-harvesting antenna proteins (goal 2, D2-2); to select mutant strains efficient for growth in photobioreactors with either low or high optical length (goal 3, D2-3).
D2-1: Function of genes affecting light-harvesting process efficiency
Biochemical and physiologic characterization of mutants screened in WP1 and described in D2-2, allowed to determine the functions of the different genes affected by mutation on light-harvesting process efficiency and chloroplast biogenesis. Silencing of Lhcb2+7 resulted in a clear decrease in the abundance of trimeric LHCII band in native green gel electrophoresis, implying that Lhcbm2+7 is an essential component of trimeric LHCII complex. Instead, small or even no decrease in LHCII trimers was observed in the npq5 mutant or in the mutants in which Lhcbm1 was silenced, implying that Lhcbm1, although a component of LHCII trimers, can be substituted by other gene products. Silencing of both LHCBM1 and LHCBM2+8 was efficient in decreasing the functional antenna size of PSII.
D2-2: Mutant strains affected in the expression of the light-harvesting antenna proteins
The antenna moiety of Photosystem II in Chlamydomonas reinhardtii is composed of LHCB4, LHCB5 proteins encoding the monomeric components CP29 and CP26 and by a number of LHCBM genes encoding the major LHCII complex. In order to gain knowledge on the structural organization of antenna system in green unicellular algae, an artificial microRNA (amiRNA) silencing technology was used (Molnar et al., 2009), which allows discriminating between different genes and to coordinately silence genes sharing identical regions, while keeping unaltered the level of expression of other genes in the family.
D2-3: Mutant strains efficient for growth in photobioreactors with either low or high optical length
Mutants as1 and as2 described in the previous section were tested for growing in photobioreactor (Task 2-3). At the beginning partner 5 tested the growth of these mutants in small-scale photobioreactor (400 ml) and in a home-made system in which different small flasks (20 ml) were shading each other, in order to artificially recreate the shading effect of different layer in a large scale photobioreactor.
WP3: Identification and functional analysis of novel limiting enzymatic steps for biomass production
General objective. WP3 was dedicated to investigate in novel metabolic pathways and key enzyme components of energetic metabolism that are critical for biomass yield with micro algae. It was intended
-to modify the expression of proteins identified by comparing the proteome adaptation in cells of Dunaliella and Chlamydomonas metabolic mutants,
-to screen for new Chlamydomonas mutants affected in light-independent electron transfer pathways and
-to investigate in the function of specific enzymes known to play a key role either in the interplay between respiration and photosynthesis or to control respiratory activity.
Objectives and achievements
For the whole period, our objectives were to identify key proteins involved in biomass production in Chlamydomonas and Dunaliella (goal 1, D3-1); to build vectors for silencing growth rate-limitating enzymes in Chlamydomonas and Dunaliella (goal 2, D3-2); to deliver a proteotypic peptide profiling platform available for further analysis of Chlamydomonas and Dunaliella mutant strains (goal 3, D3-3) ; to build vectors for overexpressing growth rate-limiting enzymes in Chlamydomonas and Dunaliella (goal 4, D3-4) ; to produce TILLING (Targeting Induced Local Lesions In Genomes) resources in Chlamydomonas and Dunaliella (goal 5, D3-5) ; to isolate Chlamydomonas and Dunaliella RNAi/miRNA mutants affected in metabolic processes (goal 6, D3-6) ; to obtain proteotypic peptide profiles of engineered mutant strains (goal 7, D3-7) ; to isolate Chlamydomonas and Dunaliella mutants with enhanced carbon assimilation rates and biomass accumulation in photobioreactors (goal 8, D3-8).
D3-1: Key proteins involved in biomass production in Chlamydomonas and Dunaliella
(a) Enzymes implicated in the utilization of acetate
Partner 1 identified several enzymes implicated in biomass production in Chlamydomonas when cells are grown in the presence of acetate either in the light (mixotrophic conditions) or in the dark (heterotrophic conditions) such as the Pdsw and nd4 subunits of the mitochondrial complex I of the respiratory chain (Barbieri et al., 2011; Larosa et al., 2012). Nda1 is a type II NADH dehydrogenase located on the inner side of the inner mitochondrial membrane.
(b) Enzymes implicated in the acclimation to high light and/or chlororespiration
Metabolic processes in chloroplasts and mitochondria are tightly linked by an intense inter-organellar crosstalk involving metabolite exchange. Efficient photosynthesis relies on mitochondrial respiration, since excess reducing equivalents accumulating in the plastid can be exported and consumed by mitorespiration acting as a valve system. Organellar gene expression (OGE) is among the candidate signal sources thought to initiate and modulate retrograde signalling events. Apart from an intense communication between organelles and nucleus the acclimation of photosynthetic eukaryotes to environmental changes critically depends on a tightly coordinated expression of the nuclear and organellar genomes.
D3-2: Vectors for silencing growth rate-limitating enzymes in Chlamydomonas and Dunaliella
Several vectors have been constructed during the course of the project for silencing the expression of different genes: NDA1, PETO (encoding a phosphoprotein from cytb6f), CAS, ANR1, MOC1, LHCSR3, sedoheptulose-1,7-bisphosphatase (SBPase).
D3-3: Proteotypic peptide profiling platform available for further analysis of Chlamydomonas and Dunaliella mutant strains. Partner 6 developed a proteotypicprofiling platform and successfully analyzed protein dynamics in relation to biomass yield in wild-type that led to identification of CAS and ANR1 proteins (see D3-1). In addition, partner 6 employed quantitative proteomics to analyze photo-heterotrophic versus photo-autotrophic grown cells. These experiments allowed the quantitation of 1608 proteins and give detailed insights into how cell metabolism differs between the different trophic states (Hoehner et al., manuscript submitted).
D3-4: Vectors for overexpressing growth rate-limiting enzymes in Chlamydomonas and Dunaliella
Over-expression of MOC1 was performed to improve growth under conditions which are known to cause an accumulation of excess reducing equivalents (e.g. high-light, low CO2). Comparison between wild type strains and transformants revealed that the expression level of MOC1 cannot be further increased by employing the tested promoter systems and further indicated a high activity of the endogenous MOC1 promoter. These strains will represent valuable tools in future analyses aiming at the detailed characterization of MOC1 promoter regulation. In addition, two other vectors (gift of partner 3) for over-expression of the isocitrate lyase gene in Chlamydomonas have been constructed by partner 1. These vectors will be used for selection of transformants with better acetate utilization. Partner 7 constructed a synthetic codon-optimized sedoheptulose-1,7-bisphosphatase (SBPase) gene from Dunaliella tertiolecta for expression in the chloroplast of Chlamydomonas.
D3-5: TILLING (Targeting Induced Local Lesions In Genomes) resource in Chlamydomonas and Dunaliella
One of the roles of partner 5 was assessed as to construct a TILLING (Targeting Induced Local Lesions In Genomes) library in Chlamydomonas and Dunaliella (Task 3.5). This kind of resource has been previously applied to crop plants but not yet to algae. After the approval of project however, several problems arose with this technique, mainly due to difficulty in making reverse genetics in algae. Moreover being Chlamydomonas a haploid organism for most of its life cycle, the possibility to rescue mutants with mutations in essential genes is null. A similar tilling resource was produced in Berkeley by the lab of Kris Niyogi with rather limited success. In a meeting of the consortium in December 2010, it was suggested to change the method for obtaining a library of mutants in Chlamydomonas and to build a large insertional mutant library where each mutant could be identified by a Flanking Sequence Tag (FST).
D3-6: Chlamydomonas and Dunaliella RNAi/miRNA mutants affected in metabolic processes.
Partner 1 has investigated Chlamydomonas mutants affected in enzymes responsible for growth on acetate. Respiratory-deficient mutants affected in different enzymes of the respiratory chain have been analyzed in mixotrophic conditions. They all show decreased respiration and photosynthesis rates. In addition, whereas protein content does not vary, starch and neutral lipids contents per cell are strongly decreased. This suggests that the respiratory deficient mutants present reduced metabolic activities, responsible for decreased accumulation of carbon resources (Lecler et al., 2011).
D3-7: Proteotypic peptide profiles of engineered mutant strains
In collaboration with partner 1, partner 6 analyzed an isocitrate-lyase knockout mutant by quantitative proteomics using differential 15N/14N metabolic labeling. Between 1684 and 1792 proteins have been identified in the four datasets, respectively and allowed in-depth characterization of metabolic remodeling due to ICL deficiency (Plancke et al., manuscript submitted). Comparative quantitative analysis of a CAS mutant strain unable to acclimate to high light revealed among 423 quantified proteins, 31 proteins that are repressed in the mutant versus the wild-type and might be important for the high-light response in C. reinhardtii. In a new set of quantitative proteomics experiments partner 6 analysed the highlight response of C. reinhardtii using differential 15N/14N metabolic labelling.
D3-8: Chlamydomonas and Dunaliella mutants with enhanced carbon assimilation rates and biomass accumulation in photobioreactors
The strict relationship between NPQ and productivity (see D3-1) and NPQ and LhcSR3, demonstrate that LhcSR is a key protein involved in biomass production in Chlamydomonas. Partner 5 tested this hypothesis by measuring PCE in npq4 mutant compared to WT and the light use efficiency and productivity in npq4 is 22% higher than WT at 200 μmol m-2s-1 under L/D cycles of 1s. Together with the increase PCE described for mutants with reduced pigmentation, this result finally suggests that the quality and quantity of Lhc and Lhc-like protein is crucial to determine biomass productivity in Chlamydomonas.
WP4: Control of chloroplast gene expression and metabolic engineering of the chloroplast
The overall goal of this work package was to build on the existing expertise in chloroplast molecular-genetic to gain a fully understanding of the molecular mechanisms underlying the expression and regulation of chloroplast genes in the green alga Chlamydomonas. This knowledge would then allow the exploitation of the algal chloroplast as an expression platform in which transgenes are introduced into the chloroplast genome with a view to making novel proteins and metabolites. Below is a summary of the research work carried out by partners 4, 8 and 9.
Objectives and achievements
For the whole period, our objectives were to obtain an optimal inducible system for transgene expression in the chloroplast (goal 1, D4-1); to identify factors controlling the epistasy of synthesis (CES) process, which controls in turn the synthesis rate of key chloroplast proteins (goal 2, D4-2); to obtain a modular system in which multiple genes can be assembled together in an operon that is co-transcribed and co-translated (goal 3, D4-3); to isolate Chlamydomonas nuclear mutants affected in chloroplast proteases (goal 4, D4-4); to obtain Chlamydomonas strains with enhanced chloroplast transgene expression (goal 5, D4-5); to obtain Chlamydomonas strains expressing biosynthetic genes for C10 monoterpenoids (goal 6, D4-6).
D4-1: Optimal inducible system for transgene expression in the chloroplast
An essential requirement for any recombinant platform is that transgene expression can be tightly regulated such that production of recombinant metabolites that are toxic to the host can tightly controlled. This allows the uncoupling of host growth from production whereby the biomass is produced first and then it is induced to produce the metabolite. Several strategies were investigated.
i) Temperature-regulated control of transgene expression using the E. coli lac repressor (partner 8). An attempt was made to create an inducible system based on the expression of a temperature sensitive variant of the lac repressor in the chloroplast, coupled to a modified chloroplast promoter element containing binding sites for the E. coli lac repressor. In this system, expression of a gene-of-interest linked to the promoter would be prevented by repressor binding at the non-permissive temperature. However, a rise in temperature would result in a lack of binding and induction of expression. Earlier, we had shown the successful expression in the chloroplast of a synthetic gene encoding the lac repressor.
ii) Use of the E. coli FimE DNA recombinase as a molecular switch for induction of transgene expression (partner 8). The aim of this project was to express the FimE gene in the Chlamydomonas nucleus under the control of an inducible promoter (from CYC6) such that the FimE protein would be targeted into the chloroplast. Once in the chloroplast, the recombinase would recognise short target sequences flanking a promoter element and 'flip' its orientation, thereby turning on expression of a downstream transgene.
iii) Riboswitches to control transgenes (partner 9). To investigate the usefulness of bacterial riboswitches for the experimental manipulation of chloroplast gene expression, two riboswitches were tested. The first is the synthetic theophyllin-responsive 'on' riboswitch that was previously shown to function in tobacco chloroplasts (Verhounig et al., 2010). The second is a putative 'off' riboswitch from a cyanobacterial thiC gene. These riboswitches were placed between the psaA promoter /5'UTR and the lucCP reporter, and homoplasmic transformed lines were obtained. However with either riboswitch, luciferase activity was very low, was strongly dependent on the physiological state of the culture, and was not markedly responsive to theophyllin or thiamine respectively.
D4-2: Factors controlling the epistasy of synthesis (CES) process, which in turn controls the synthesis rate of key chloroplast proteins
i) Nuclear factors mediating CES (partners 4 and 9). Previous work in these partners labs had shown that transgene expression in the Chlamydomonas chloroplast can be limited due to the negative feedback regulation that is exerted by unassembled subunits on the translation of their own mRNA (control by epistasy of synthesis (CES); reviewed by (Choquet and Vallon, 2000). The expression of a transgene placed under the control of the psaA promoter and 5'UTR is enhanced in a nuclear mutant background that prevents trans-splicing of psaA and hence accumulation of the PsaA protein (Wostrikoff et al., 2004; Michelet et al., 2011). However with only one exception (Boulouis et al., 2011), the identity of the nuclear-encoded proteins that mediate this type of negative feedback regulation has not been elucidated. In an effort to identify such regulators three trans-acting factors were investigated; namely, Taa1, Mac1 and Mbb1.
ii) The role of the TDA1 factor (partner 4). The TDA1 nuclear gene controlling the expression of subunit a of the chloroplast ATP synthase in Chlamydomonas, which is critical for biomass production in phototrophic conditions, has been fully characterized. It is specifically required for translation of the chloroplast atpA transcript. The sequence of TDA1 contains eight copies of a degenerated 38 residues motif, or Octotrico Peptide Repeats (OPR), previously described in a few other trans-acting factors targeted to the Chlamydomonas chloroplast.
iii) The role of Mca1 in the CES process (partner 4). The role of the Mca1 factor as a translational enhancer for cytochrome f expression has been further documented by its action in strains altered for the CES process, i.e. the assembly-dependent regulation of cytochrome f translation. As a major conclusion drawn from these experiments, MCA1 turns out to be the nuclear trans-acting factor that controls the CES process for cytochrome f expression. Attempts to probe a direct molecular interaction between cytochrome f and MCA1 are currently being developed in two hybrid experiments.
iv) A genetic screen for the identification of mutants in the CES process (partner 4). An experimental strategy has been devised for isolating mutant strains escaping the CES process, which negatively down-regulates the expression of most chloroplast genes, thereby preventing high accumulation of their products. As a first attempt, Chlamydomonas mutants with impaired auto-regulation of cytochrome f synthesis, the best characterised CES protein, were constructed. In strains defective for cytochrome b6f assembly, expression of the cytochrome f gene, petA and of 5'petA-driven reporters is reduced. In particular, the 5'petA-aadA chimera only confers resistance to low levels of aminoglycosides.
D4-3: Modular system in which multiple genes can be assembled together into a trans-operon that is co-transcribed and co-translated (partner 9).
The goal of this project was to develop a simple operon assembly system that allows the efficient translation of multiple transgenes in the chloroplast from a single transcription unit. The design strategy was based on the hypothesis that the overlap (or close association) of the stop codon of the upstream ORF with the start codon of the next ORF should facilitate 'translational coupling' where the termination of translation and its re-initiation at the next translation start are intermittently coupled. Evidence of such coupling is found in both plant and algal chloroplast genomes, as well as bacterial and viral systems. We therefore used a synthetic biology approach to create as series of two-ORF constructs in our chloroplast expression vector pASapI and introduce these into the Chlamydomonas chloroplast genome. The ORFs encode an endolysin (cpl1) and the kanamycin resistance gene aphA6 and were coupled in this order but in three different ways (a 5bp gap between the TAA and ATG; a 1bp gap; a 1bp overlap).
D4-4: Chlamydomonas nuclear mutants affected in chloroplast proteases
i) The role of FtsH (partner 4). FtsH is the major transmembrane protease in the thylakoids, and an FtsH mutant was isolated by a suppressor approach. This mutant has been used extensively to study the action spectrum of FtsH. It was found that the primary roles of FtsH are (i) the recycling of photosynthesis proteins in stress conditions and (ii) the quality control of misassembled protein complexes. The substrate spectrum of the FtsH protease is currently under investigation. Besides PSII degradation in photoinhibitory conditions, FtsH proved to be involved in PSII degradation in nutrient starvation conditions, whether it being the absence of phosphate or of sulphur.
ii) Altered expression of the RNAse J, a chloroplast-based 5'-exo/endonuclease (partner 4). RNAi vectors for attenuating RNAseJ expression, a major 5'-3' chloroplast exonuclease have been devised and were used in nuclear gene transformation assays. Altered patterns of maturation of polycistronic transcripts were observed. How these alterations reflect on the level of expression of the corresponding protein products remains to be investigated.
iii) Remodeling of the photosynthetic apparatus upon nitrogen starvation (partner 4). We showed that cytochrome b6f and most cytochrome b6f-related proteins involved in its biogenesis are selectively lost when Chlamydomonas is starved of a nitrogen source. This nutrient stress response was investigated further because it is the most commonly used procedure with Chlamydomonas for biotechnological approaches aimed at biofuel production. It was shown that a deep remodelling of the photosynthetic membrane develops with the time of nitrogen deprivation. The membrane loses its ability to contribute to CO2 fixation, not only because of the selective degradation of cyt b6f complexes but also because of the degradation of stromal RubisCO. In addition, prolonged illumination under nitrogen stress leads to the photo-destruction of the two photosystems.
D4-5 (a): Chlamydomonas strains with enhanced chloroplast transgene expression
In this project partners 8 and 9 have developed complementary genetic screens to isolate mutant strains that show either enhanced transgene expression or improve stability/accumulation of recombinant proteins in the chloroplast.
i) Chlamydomonas strains with enhanced chloroplast transgene expression (partner 8). The levels of transgene expression obtained in the chloroplast of Chlamydomonas have been highly variable depending on the protein of interest and on the regulatory elements used for its expression. Although the limiting factors are not well understood, it is clear that negative feedback regulation of gene expression at the post-transcriptional level plays a role, and at least in some cases, proteolytic degradation of the protein product (Specht et al., 2010; Michelet et al., 2011).
ii) Chlamydomonas strains with enhanced chloroplast transgene expression (partner 9). This alternate screen involved first creating a transformant line in which the accumulation of the kanamycin-resistance protein AphA6 is compromised by the addition of an N-terminal extension, leading to a lower resistance level compared to the native AphA6. This transformant was then used to make a double transformant in which a HA-tagged version of the aadA gene encoding spectinomycin resistance was placed at a neutral locus downstream of psaA-3. This double transformant is resistance to both kanamycin (100 μg/ml) and spectinomycin (1000 μg/ml), and was used in a UV-mutagenesis screen in which the aim is to select for increased kanamycin resistance and then score for increased AadA protein by western blot (the high level of spectinomycin-resistance conferred by the highly active AadA precludes scoring for any increased resistance).
D4-5 (b): Transgene expression in protease-defective mutant strains
i) Expression in an FtsH protease deficient mutant (partners 4 and 9). To investigate whether the FtsH protease may degrade transgenic proteins and hence lower their level of expression, the ftsh1 mutant was crossed with lines expressing transgenic reporter proteins in the chloroplast. The following were used as reporters: the vapA gene encoding a surface antigen of Aeromonas salmonicida, the aacC gene that confers moderate levels of resistance to gentamycin, recently developed in this project, and the firefly luciferase gene lucCP (Matsuo et al., 2006). Proteolytic degradation was previously shown to limit VapA levels.
D4-6: Chlamydomonas strains with efficient expression of biosynthetic genes for a C10 biofuel (partner 9).
As a proof-of-concept for the utilization of the chloroplast genetic engineering tools developed during the SUNBIOPATH project, it was planned to manipulate the terpenoid biosynthesis pathway with a view to producing the novel C10 terpenoid, geraniol.
This work is still in progress, but the following has been achieved so-far:
a) A cDNA clone encoding the plant enzyme geraniol synthase (GES) was obtained from Dr Eran Pichersky (Iijima et al., 2004), modified to remove the 5' coding region for the chloroplast targeting sequence and to add an HA-tag sequence at the 3' end, and then cloned into our chloroplast expression vector, pASapI.
b) The gene encoding the cyanobacterial enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS) was amplified from genomic DNA of Synechocystis PCC6803, modified to encode a C-terminal HA tag and cloned into a second chloroplast expression vector pSRSapI.
c) Homoplasmic chloroplast transformants have been generated for both constructs and western analysis has confirmed the presence of the DXS protein, but not the GES protein. An assay for DXS activity it currently being established.
WP5: Optimization and valorization of algal culture in photobioreactors
General objective. The optimized cultivation of algae in photobioreactors relies not only on efficient strains but also on precisely controlled physico-chemical parameters. Currently, there is a need for the critical evaluation of relevant parameters that control growth in photobioreactors. This has been achieved in this WP through the analysis of photosynthetic rates and macroscopic kinetic studies in lab-scale reactors. For the most interesting mutants that show a significantly improvement of growth rates and biomass yield (see WP2 and 3), growth in indoor reactors has been examined. Data obtained have been used to design a reactor built with cheap materials. Algal have been tested for biomethane production. Finally a techno economic and environmental analysis has been performed from the obtained results taking into account the C-credit of the whole process.
Objectives and achievements for the period
For the whole period, our objectives were to establish optimized parameters for algal growth in photobioreactors (goal 1, D5-1); to develop theoretical models describing the algal response to various light regimes (goal 2, D5-2); to design of a reactor that is based on cheap materials (goal 3, D5-3); to determine the efficiency of algal strains obtained by the consortium for biogas production (goal 4, D5-4); to realize a techno economic and environmental analysis of algal strains obtained by the consortium (goal 5, D5-5) .
D5-1: Optimized parameters for algal growth in photobioreactors
The influence of variations in different parameters like temperature, pH, medium composition, CO2 supply (concentration/ partial pressure) and light intensity were investigated and optimized to determine the process conditions and possible ranges for control. For two wild types and different antenna-reduced mutants of Chlamydomonas reinhardtii (wt13, wt8b+, T7, as1, as2, bf4) growth kinetics was determined. The algae grow light limited up to intensities of about 300-500 μE/m2/s. Then photosynthesis becomes saturated at higher light intensities and could be even inhibited. 300-500 μE/m2/s light should be supplied to all algae cells within the whole reactor volume to operate production reactors with maximum efficiency.
D5-2:Theoretical development of a dynamic model describing the response of algae to various light regimes
A photosynthesis-irradiance curve was measured under continuous illumination and used to calculate the net oxygen yield on light energy, which was maximal between a PFD of 100 and 200 μmol m-2 s 1. Net oxygen yield under flashing light was proven to be duty cycle dependent: the highest yield was observed at a duty cycle of 0.1 which corresponds to a time-averaged PFD of 115 μmol m-2 s-1. At lower duty cycles maintenance respiration reduced net oxygen yield. At higher duty cycles the specific photon absorption rate exceeded the maximal photon utilization rate and, as a result, surplus light energy was dissipated as heat. This behaviour was identical with the observation under continuous light.
D5-3: Design of a mass reactor that is based on cheap materials
To bring these advantages into action, a wave-surface-reactor has been developed. The reactor is a surface structured horizontal reactor. Horizontal reactors have different advantages but the basic disadvantage is that light dilution is only possible to a limited extend. The antenna-reduced mutants however, allow for this new reactor design.
D5-4: Efficiency of algal strains obtained by the consortium for biogas production
In order to maximize valorization of the whole biomass, the consortium also looked for the potential of several microalgae (Chlamydomonas, Scenedesmus, Euglena, Dunaliella, ) as alternate substrate for biogas production. The potential of these species to serve as a substrate for biomethane production differs greatly and is for instance dependent on the rigidity of the cell wall as a determinant of digestibility. Fermentation of the green alga Chlamydomonas was most efficient. We conclude from our results that selected algae species represent good substrates for biogas production. In addition, to evaluate integrative biorefinery concepts, hydrogen production in Chlamydomonas prior to anaerobic fermentation of the algae biomass was measured and resulted in a 20% increase of biogas generation. We conclude that selected algae species can be good substrates for biogas production and that anaerobic fermentation can seriously be considered as final step in future microalgae-based biorefinery concepts.
D5-5: Techno economic and environmental analysis of algal strains obtained by the consortium
A reduction of specific absorption by the biomass leads to a reduction of non-photochemical quenching at the front side of the reactor, what is the first and main reason for light saving. Along the light path the light intensity is higher compared to the wild type case also leading to better growth and a reduced fraction of respiration. Experiments as given in deliverable 5-1 showed that these effects have been accumulated to a doubling of PCE for the antenna reduced mutant bf4 under high light conditions and high biomass concentration. This is a clear prove that the concept of antenna reduction leads to a direct effect in saving energy for biomass production. A reduction of the CO2 footprint for algal biomass production is possible by about 30%.
Potential Impact:
1. Strategic impacts
Potential impact for culturing algae
Culturing algal cells in photobioreactors requires high-density cells at high light intensities in order to get the maximum photosynthesis efficiency and biomass productivity. The consortium demonstrated that small antenna size mutants represent good options to maximize growth in these conditions since photoconversion efficiency and biomass productivity was doubled in high light and high density in the mutants compared to the corresponding wild type. The SUNBIOPATH consortium thus contributed very efficiently to the improvement of algal growth in photobioreactors, and allowed an evaluation as to whether metabolic engineering of Chlamydomonas can result in superior strains for solar-to-fuel energy conversion. This knowledge could be applied to Dunaliella, another microalgal species, used in the consortium and interesting for the production of glycerol and beta-carotenes.
2. Wider societal implications
By its large audience dissemination activities including journal articles and interviews on public radio chains (see below), the SUNBIOPATH project contributed to a better knowledge of the 'algal world' in the public and by this way, the concept of using algae for energy, food and feed, reached a large audience, from very young people to adults.
3. Main dissemination activities
Dissemination to scientific audience
Peer-reviewed papers
Members of the SUNBIOPATH consortium published an impressive number of scientific articles in international peer-reviewed journals, such as Plos Biology, Plant Cell, Proc Natl Acad Sci (USA), Plant J, Plos One, and applied and industrial journals such as Journal of Biotechnology, Engineering of Life Sciences, Biotechnology Bioengineering. Altogether, these articles contributed very efficiently to the dissemination of the knowledge of SUNBIOPATH to the scientific community.
Book chapter
In addition, the members of the SUNBIOPATH consortium participated to the writing of a book chapter (Bassi et al. 2011, Finding the bottle-neck: A research strategy for improved biomass production, in Microalgal Biotechnology: Integration and Economy, C. Posten and C. Walter, Editors. 2012, De Gruyter: Berlin. p. 227-52), in which the reader will find the main achievements of SUNBIOPATH. Written in a book co-edited by one of the members of SUNBIOPATH (Clemens Posten) and covering the present day microalgal biotechnology knowledge, this chapter represents a very efficient way to reach scientific from different fields, as well as economists and stakeholders.
Participation to scientific meetings
In addition, members of the SUNBIOPATH consortium including PI and potsdocs hired by the project participated to numerous international scientific conferences all over the world and organized meetings and workshops, and this contributed very efficiently to the dissemination of the project.
Participation to industrial meetings
In addition, the SUNBIOPATH project was presented at industrial events such as the European Biomass Conference held in Berlin in June 2011 or the Algae Europe held in Milano in October 2011.
Organization of an international meeting at the end of the project
One of the achievements of SUNBIOPATH is the organization of a joint international meeting with two other KBBE-funded projects, BAMMBO and GIAVAP. The meeting was held in Brussels on January 21-23 2013.
GIAVAP (Genetic Improvement of Algae for Value Added Products) and BAMMBO (Sustainable production of Biologically Active Molecules of Marine Origin) are two European projects funded in 2010 by KBBE. Together with Stefan Leu from GIAVAP and Daniel Walsh from BAMMBO, a joint meeting was organized that gathered around 60 people from the three consortia as well as Tomasz Calikowski, project officer of GIAVAP and BAMMBO. As described below, the meeting was organized in twelve sessions that combined the results of the three projects. In total, 42 presentations were shown. In addition, external speakers and members of the advisory boards of the projects (Prof R Bock from Max Planck Institute of Golm, Prof Arthur Grossman from the Carnegie Institution for Science - Standford, Dr Thomas Kyi Global Head Strategic Business Development - Lonza) also gave plenary lectures and provided valuable feedbacks about the meeting and the future of the algal research.
Dissemination to public audience
The SUNBIOPATH project was presented in a Belgian weekly newspaper (Le Vif l'Express) and in the FNRS-News edited by the Fonds de la Recherche Scientifique from Belgium. The project was also presented at the occasion of an interview of C Remacle by Veronique Thyberghien, about the utilization of microalgae to produce electricity [27/03/13, Program 'O positif' of the RTBF (Radio Television Belge Francophone)].
Oral presentations
Several oral presentations to public audience have contributed to the dissemination of the SUNBIOPATH project. The target audience was stakeholders of regional governments or general audience.
The project also made synergies with Science Education by presenting the SUNBIOPATH project at the occasion of the 'Plant Fascination Day' organized by EPSO in the universities of Geneva and Liege on May 18 2012 as well as during the 'Researcher's Night' in September 2011 in Liege.
4. Exploitation of results
To use the specific advantages of antenna-reduced mutants, specific reactor designs are possible. Light dilution for the developed mutants can be reduced by a factor 2, which allows for the reduction of the surface to footprint area by this factor. Therefore, the material costs can be cut by half. In addition, cheaper materials are possible in thinner layers by reduced hydrodynamic pressure.
List of Websites:
http://www2.ulg.ac.be/genemic/SUNBIOPATH/