Improving the sunlight-to-biomass conversion efficiency in microalgal biofactories.

Journal of Biotechnology 201 (2015) 28–42

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Improving the sunlight-to-biomass conversion efficiency in microalgal biofactories Lutz Wobbe a,∗ , Claire Remacle b a b

Bielefeld University, Faculty of Biology, Center for Biotechnology (CeBiTec), Universitätsstrasse 27, 33615 Bielefeld, Germany University of Liège, Genetics and Physiology of Microalgae, Phytosystems, Boulevard du Rectorat 27, 4000 Liège, Belgium

a r t i c l e

i n f o

Article history: Received 22 May 2014 Received in revised form 31 July 2014 Accepted 18 August 2014 Available online 24 August 2014 Keywords: Microalgae Light conversion efficiency Truncated antenna mutants Strain engineering Calvin cycle

a b s t r a c t Microalgae represent promising organisms for the sustainable production of commodities, chemicals or fuels. Future use of such systems, however, requires increased productivity of microalgal mass cultures in order to reach an economic viability for microalgae-based production schemes. The efficiency of sunlight-to-biomass conversion that can be observed in bulk cultures is generally far lower (35–80%) than the theoretical maximum, because energy losses occur at multiple steps during the light-driven conversion of carbon dioxide to organic carbon. The light-harvesting system is a major source of energy losses and thus a prime target for strain engineering. Truncation of the light-harvesting antenna in the algal model organism Chlamydomonas reinhardtii was shown to be an effective way of increasing culture productivity at least under saturating light conditions. Furthermore engineering of the Calvin–Benson cycle or the creation of photorespiratory bypasses in A. thaliana proved to be successful in terms of achieving higher biomass productivities. An efficient generation of novel microalgal strains with improved sunlight conversion efficiencies by targeted engineering in the future will require an expanded molecular toolkit. In the meantime random mutagenesis coupled to high-throughput screening for desired phenotypes can be used to provide engineered microalgae. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Phototrophic microbes have a great potential for the sustainable generation of bulk products such as food, feed, materials, chemicals and fuels (Wijffels et al., 2013). These organisms can be of the prokaryotic (e.g. Cyanobacteria) or eukaryotic type (e.g. microalgal species) and especially microalgae, which represent a diverse group of unicellular photosynthetic organisms that can be found in distinct freshwater as well as marine environments, possess traits making them highly attractive for green biotechnology. Compared to terrestrial plants, the efficiency of solar energy to biomass conversion is significantly higher in phototrophic microbes including unicellular microalgae (Dismukes et al., 2008), where in contrast to higher plants every cell is photosynthetic.

Abbreviations: LHCBM/LHCII, major light-harvesting protein of photosystem II; PCE, photon conversion efficiency; PBR, photobioreactor; NPQ, non-photochemical quenching; PAR, photosynthetically active radiation; CCM, carbon-concentrating mechanism. ∗ Corresponding author at: Department of Biology, Algae Biotechnology & Bioenergy – Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany. Tel.: +49 0 521 106 12260; fax: +49 0 521 106 12290. E-mail address: [email protected] (L. Wobbe). http://dx.doi.org/10.1016/j.jbiotec.2014.08.021 0168-1656/© 2014 Elsevier B.V. All rights reserved.

Unlike terrestrial energy crops, microalgae can be cultivated on non-arable land, thereby saving valuable agricultural resources and avoiding a direct competition with food production (Georgianna and Mayfield, 2012). Depending on the cultivation system, the overall water supply needed to produce microalgal biomass can be substantially lower than the supply required for higher plantderived biomass (Wijffels and Barbosa, 2010) and microalgae can thrive in nutrient-rich wastewater streams or nourish on feed sources such as anaerobic digester waste effluents (Yang et al., 2011; Georgianna and Mayfield, 2012). Some green algae are extremely halotolerant and can also be cultivated in saline water (Tafreshi and Shariati, 2009), which reduces the diversion of freshwater from other critical applications like human consumption and irrigation. Importantly, algal biomass is rich in valuable components like neutral lipids as a feedstock for biodiesel production (Wijffels and Barbosa, 2010; Bondioli et al., 2012) or compounds of high value for human nutrition including long-chain polyunsaturated fatty acids (Draaisma et al., 2013). Starch produced in a photoautotrophic regime might be converted into value-added products like bioethanol via fermentation (Markou et al., 2012) and residual biomass (e.g. after biohydrogen production) can be integrated into bio-refinery concepts by its use as a substrate for biomethane production (Mussgnug et al., 2010). In addition, several

L. Wobbe, C. Remacle / Journal of Biotechnology 201 (2015) 28–42

proteins for medical (Rasala and Mayfield, 2014) and industrial (Lauersen et al., 2013) use have already been produced in microalgae. Compared to higher plants, biomass processing should in general be easier if algal-derived material is used, since microalgae do not contain recalcitrant “woody” matter due to their inability to synthesize lignin (Domozych et al., 2012). Although unicellular photosynthetic organisms convert solar energy more efficiently into biomass than multi-cellular phototrophs containing non-green tissues, several factors limit conversion rates and hence the productivity of algal mass cultures. The present review aims to address the main sources of energy loss during the conversion of sunlight into biomass and to summarize the past effort taken to improve photosynthetic efficiency.

1.1. The light reactions of photosynthesis The process of oxygenic photosynthesis can be divided into light reactions, which comprise all steps needed to provide energy (ATP) and reducing equivalents (NADPH) by the light-driven process of water-splitting, and the so-called “dark reactions” that consume the produced ATP and NADPH to convert inorganic carbon into glucose via the Calvin–Benson cycle (Fig. 1A). In the linear mode of photosynthetic electron transport (PET), electrons resulting from the splitting of water molecules into protons and oxygen at photosystem II (PSII) reduce NADP+ as the terminal acceptor of an electron transport chain. To connect the final acceptor and PSII as the site of water photolysis, the membrane-soluble electron carrier plastoquinone (PQ), a Cytb6 f complex, the soluble electron carrier plastocyanin (PC), photosystem I (PSI) and ferredoxin (Fd) are employed. The oxidation of PC and subsequent reduction of Fd is like water splitting, a light-driven reaction catalyzed by PSI and the ultimate step of NADPH formation requires the enzyme FNR. The release of protons into the thylakoid lumen by PSII as well as the electron transfer to PQ, that is combined with proton translocation via the Cytb6 f complex (Q cycle), creates a proton gradient across the thylakoid membrane, whose dissipation is coupled to ATP synthesis (Mitchell and Moyle, 1967). A cyclic mode of photosynthetic electron transport (CEF) also exists in addition to the linear mode (LET), which contributes to the build-up of the proton gradient but does not result in NADPH accumulation. In the CEF mode, electrons are recycled from either reduced Fd or NADPH to plastoquinone, and subsequently to the Cytb6 f complex (Munekage et al., 2004). The energy needed for charge separation reactions in the reaction centres of PSII and PSI is provided by the adsorption of light energy and distinct light-harvesting systems emerged during evolution. In contrast to cyanobacteria, which use extrinsic phycobilisomes to harvest light energy, green algae and higher plants collect photons via intrinsic light-harvesting complexes (LHC) located at both photosystems and spanning the thylakoid membrane (Ballottari et al., 2012). Pigment-binding LHC proteins are designated LHCI (LHCA) or LHCII depending on their predominant location at PSI or PSII and for LHCII proteins two types can be distinguished (Fig. 1A). The less abundant monomeric LHCII proteins (CP26/CP29) are located in close proximity to the PSII core complex, whereas major LHCII proteins (LHCBM) are far more abundant and form the peripheral antenna of PSII (Kouril et al., 2012). The chlorophyll a and b molecules associated with LHCII participate in the energy transfer towards the special pair chlorophyll of the PSII reaction centre (P680), whose excitation is followed by charge separation reactions and electron transfer to PQ (Barber and Archer, 2001). In addition, lutein, neoxanthin, and xanthophyll cycle carotenoids are associated with LHCII, being involved in energy transfer and/or dissipation as well as scavenging of reactive oxygen species (ROS) upon excess irradiation (Niyogi et al., 1997; Ballottari et al., 2012).

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2. The theoretical upper limits and observed photon conversion efficiencies Several previous publications have dealt with an estimation of the theoretical maximum efficiency of light-to-biomass conversion in distinct phototrophic organisms (Gordon and Polle, 2007; Kruse et al., 2005a, 2005b; Robertson et al., 2011; Weyer et al., 2010; Zhu et al., 2008, 2010). If sunlight is considered as the source of energy used to drive photosynthesis, it has to be taken into account that only a fraction (≈46% (Weyer et al., 2010) – 49% (Zhu et al., 2008)) of the incident light can actually be absorbed by the photosystems. This is the so-called PAR (photosynthetically active radiation) range (400–700 nm; McCree, 1971) of the solar spectrum and the amount of light ultimately reaching the photosynthetic apparatus is further reduced by factors such as imperfect absorbance/reflectance in terrestrial plant vegetation (≈10%; Zhu et al., 2008) or light attenuation and light-scattering effects in algal mass cultures (Formighieri et al., 2012). Published maximum sunlight conversion efficiencies (PCE rates) calculated for the combined operation of light and dark reactions and yielding in the conversion of water and CO2 to glucose are in the range of 8–13% (Bolton and Hall, 1991; Kruse et al., 2005a, 2005b; Weyer et al., 2010; Zhu et al., 2008). Such numbers can, for instance, be calculated (Eq. (1); adapted from Weyer et al., 2010) by taking into account the minimal photon requirement (np ) to provide sufficient ATP/NADPH for carbon fixation as well as their average energy content (E(p)av ). This defines an average light energy input which can be set in relation to the chemical energy stored in formed glucose (standard free energy change (G) of reaction (1); Emerson, 1958). For the conversion efficiency of sunlight to glucose, it has to be considered that only the PAR fraction of light can be used (XPAR ). Reaction (1): CO2 + H2 O → O2 + CH2 O Eq. (1):

PCEsunlight =

np

G mol photons mol CH2 O

kj mol CH2 O

× E(p)av

kj mol photons

× XPAR

Eq. (1) can be used to describe the intrinsic quantum efficiency of photosynthesis and defines a theoretical upper efficiency limit and the minimal energy losses of photosynthetic carbohydrate synthesis. Differences in the published calculated values are partly based on distinct assumptions regarding the number of photons required to provide the 3 ATP and 2 NADPH, which exactly satisfy the Calvin cycle demand. Photon numbers higher than eight are usually taken under consideration that additional electron recycling at PSI (CEF) is needed to create a proton gradient (proton motive force) sufficient for the production of the third ATP (Allen, 2002). 2.1. Photorespiration Extra ATP provision by photosynthetic light reactions is of special relevance in C4 plants and for the NADP-malic enzyme (ME) subtype an ATP demand of five ATP in addition to two NADPH for the assimilation of one carbon dioxide molecule has been predicted, which would reduce the PCE rate according to Eq. (1) due to a higher photon requirement. The extra investment of two ATP molecules, however, pays off because energy equivalents are used to concentrate CO2 at the site of Rubisco which is utilized by all photosynthetic eukaryotes in the first step of inorganic carbon assimilation (Zhu et al., 2008). Especially at elevated temperatures and high O2 /CO2 ratios this enzyme accepts oxygen as a substrate instead of carbon dioxide and triggers a reaction cycle known as photorespiration. In contrast to the carboxylation reaction, which

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Fig. 1. (A) Photosynthetic electron transport pathways in the thylakoid membrane of C. reinhardtii and related green algae at subsaturating light intensities. Shown are PSII, cytb6 f, PSI, and the ATP synthase. Linear electron transport (LEF) starts with the light-induced oxidation of water catalyzed by the oxygen-evolving complex (OEC) of PSII followed by electron transfer from PSII through the plastoquinone (PQ) pool to cytb6 f, which oxidizes plastoquinol (PQH2 ) thereby reducing the soluble electron carrier plastocyanin (PC). This so-called Q cycle pumps protons from the stromal to the luminal side of the thylakoid membrane (black dotted lines). PSI acts as light-driven plastocyanin ferredoxin (Fd) oxidoreductase and reduced ferredoxin is re-oxidized by another oxidoreductase (FNR) which converts NADP+ to NADPH. The energy needed for charge separation in PSII and water splitting is provided via light absorbtion in the peripheral antenna of PSII (LHCBM) and transfer of excitation energy (EET) to the special pair chlorophyll in the PSII reaction centre (P680). In the cyclic electron flow (CEF) mode, electrons are recycled from either reduced Fd or NADPH to plastoquinone, and subsequently to the cytochrome b6 f complex. The Fd-dependent pathway requires the proteins PGR5 and PGRL1 while the NADPH-dependent pathway uses the enzyme NDA2 to re-inject electrons from NADPH into the PQ pool. Like linear electron flow, the cyclic mode is coupled to proton translocation and formation of a pH gradient that drives ATP production via ATP synthase, but in contrast to LEF, the operation of CEF does not result in NADPH accumulation. The ATP and NADPH generated by the photosynthetic light reactions is consumed by the Calvin–Benson cycle to produce carbohydrate from fixed CO2 . (B) Oversaturation of photosynthetic electron transport at saturating light intensities when the rates of ATP/NADPH supply exceed their consumption by the Calvin–Benson cycle. Together with the accumulation of NADPH, kinetic bottlenecks such as slow PQH2 oxidation by the cytb6 f complex cause an overreduction of the electron transport chain. In this overexcited state of PSII a large fraction of absorbed light is re-emitted as fluorescence and singlet oxygen production (not shown) is increased. The reduced backflow of protons into the stroma caused by ATP accumulation leads to an acidification of the thylakoid lumen. This activates energy-dependent quenching (qE) as one of the major photoprotective mechanisms by protonation of LHCSR3 and subsequent conformational changes in the antenna. In this quenched state, the yield of chlorophyll-excited states and energy transfer to the reaction centre are reduced, because light energy is mainly dissipated as heat in the antenna and not used for photochemistry. This prevents singlet oxygen formation but lowers photosynthetic efficiency.


results in the formation of two molecules of 3-phosphoglycerate, oxygenation of Rubisco produces 2-phosphoglycolate in addition to one molecule of 3-phosphoglycerate. The conversion of 2phosphoglycolate to 3-phosphoglycerate that re-enters the Calvin cycle requires ATP besides reducing power and during the cycle fixed carbon is lost as CO2 , yielding in an overall energy loss that reduces light-to-biomass conversion efficiency. These energy and carbon losses have been estimated to significantly reduce the theoretical photon conversion efficiency of C3 plants grown with air-concentrations of CO2 by about 49% and a similar loss in efficiency does not occur in C4 plants, which largely suppress Rubisco oxygenation (Zhu et al., 2008). Many microalgae are equipped with carbon-concentrating mechanisms (CCMs) that reduce photorespiratory losses by pumping bicarbonate into the cell and converting it back to CO2 in a sub-cellular structure called pyrenoid, where Rubisco is located (Moroney and Ynalvez, 2007). This is an ATPdriven process that requires at least one extra molecule of ATP in addition to the 3 ATP minimally needed to fix one molecule of CO2 and the further ATP demand is met by cyclic electron flow (Cardol et al., 2009; Duanmu et al., 2009). Cultivation of microalgae in photobioreactors (PBRs), however, can be accompanied by the accumulation of oxygen leading to high O2 /CO2 ratios that favour the oxygenation reaction catalyzed by Rubisco and result in energy losses (Kliphuis et al., 2011). 2.2. Mitorespiration Besides photorespiration, respiration in mitochondria is a source of energy loss that must be considered, since photosynthate can be either converted into biomass components or consumed via the combined operation of glycolysis, citric acid cycle and mitochondrial oxidative phosphorylation, which results in the release of fixed carbon as carbon dioxide. For the minimal energy loss caused by respiration a value of 30% has been estimated for higher plants (Zhu et al., 2008) and a 35% respiratory loss was determined for the microalga Isochrysis galbana (Sukenik et al., 1991), which was based on measured ratios of the respiratory CO2 loss as a fraction of CO2 uptake. Estimation of the energy losses associated with mitochondrial respiration is inherently difficult and different approaches for the integration of mitorespiration into metabolic models have been proposed (Amthor, 2000). Importantly, the release of carbon dioxide by mitorespiration is accompanied by the synthesis of large ATP amounts, which can be consumed to support anabolic reactions leading to the formation of biomass. This “growth respiration” can therefore not be simply viewed as a mechanism reducing the overall light-to-biomass conversion efficiency. On the other hand “maintenance respiration” that provides the ATP needed to sustain basic cellular functions, not connected to the formation of new biomass (e.g. macromolecular turnover and maintenance of ion gradients), represents a source of energy loss. Metabolic modelling of Chlamydomonas reinhardtii cells grown under photoautotrophic conditions with different light intensities (Kliphuis et al., 2012), predicted that maintenance respiration only constitutes a smaller fraction of total mitorespiration at high growth rates and underscored the growth-supporting role of mitochondrial ATP synthesis. 2.3. Sunlight-to-biomass conversion efficiencies By taking into account the aforementioned intrinsic minimal energy losses, maximal sunlight-to-biomass conversion efficiencies of 4.6% (C3) and 6% (C4) were estimated for higher plants (Zhu et al., 2008) and a value of 6.3% for microalgae (Weyer et al., 2010). Such estimations can be further refined by including additional photon requirements for biomass production such as nitrate assimilation (Zijffers et al., 2010). Experimentally determined conversion

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efficiencies of light to biomass that have been reported for green algae and C4 plants are 35–80% lower than the predicted theoretical maximum (Beale and Long, 1995; Burlew, 1953; Monteith and Moss, 1977; Thomas et al., 1984; Zijffers et al., 2010). The theoretical upper conversion efficiency limits have been calculated based on minimal energy losses occurring during the conversion of sunlight to biomass. As indicated by the discrepancy between theoretical and practical values many different factors lower photosynthetic conversion efficiencies. In the case of microalga large-scale cultivation, growth regimes using low light intensities are associated with unsatisfactory culture productivities. Higher light intensities, however, easily over-saturate photosynthesis due to different bottlenecks within the light and dark reactions of photosynthesis. This over-saturated state of photosynthesis has distinct origins, but can be regarded as one of the main reasons for subsequent energy losses including quenching of excited chlorophyll states or alternative routes of electron flow to avoid the accumulation of harmful reactive oxygen species (ROS). 3. Engineering of the light-harvesting antenna to reduce quenching losses and light attenuation in microalgal mass cultures Green algae evolved large photosynthetic antenna systems to maximize light-harvesting efficiency as an evolutionary adaptation to a natural environment where light availability might be a growth-limiting factor and cell density is very low (Kirk, 1994). Exploitation of microalgae for the sustainable production of biomass and value-added compounds, however, requires largescale cultivation with dense cultures to increase the productivity per volume. Under such conditions a high pigment optical density, resulting from the large antenna systems assembled by green algae, and light-scattering effects at high cell densities limit the penetration of light into mass cultures grown in open ponds or closed photobioreactors (PBRs). In these cultivation systems the outmost layers receive light quantities saturating photosynthesis, while cells in the centre of the reactor are exposed to virtual darkness, performing respiration instead of photosynthesis (Formighieri et al., 2012). This over-saturation of photosynthesis causes energy losses in the antenna and it has been estimated that at full sunlight intensity about 75% of the energy captured by the LHC antenna complex is dissipated as heat or re-emitted as fluorescence and not used to drive photochemistry (Melis, 2009). Especially in the outer layers of PBRs or top layers of open pond systems, cells are exposed to saturating photon flux densities, where the activation of non-photochemical quenching (NPQ) mechanisms protects photosynthetic reaction centres from over-excitation (Finazzi et al., 2010), but simultaneously reduces photon conversion efficiencies caused by the loss of absorbed light energy as dissipated heat. 3.1. Non-photochemical quenching mechanisms reduce the photon conversion efficiency of plants and microalga If over-excitation is not prevented by NPQ mechanisms the halflife of singlet excited Chl states (1 Chl*) increases and spin inversion resulting in the formation of 3 Chl* becomes more likely. Triplett excited states of chlorophyll can in turn act as a photosensitizer converting harmless triplet oxygen (3 O2 ; ground state oxygen) into the potent reactive oxygen species singlet oxygen (1 O2 ), that causes damage to the photosynthetic apparatus, especially to PSII (KriegerLiszkay, 2005). The major NPQ component under elevated light conditions is the feedback-regulated de-excitation of chlorophyll molecules in PSII that operates on a second timescale and is induced by a drop in thylakoid lumen pH when light excitation flux exceeds the rates of

32


carbon dioxide fixation (de Bianchi et al., 2010). This fast-reversible component is also referred to as energy-dependent quenching (qE; Fig. 1B) and shows little activity under low light conditions in green microalgae such as C. reinhardtii, but can be activated by prolonged exposure to high light (Bonente et al., 2012). In contrast to unicellular phototrophic eukaryotes, higher plants exhibit a constitutive qE that is active immediately after exposure to high light and requires the protein PsbS (Li et al., 2000; Niyogi et al., 2005; Bonente et al., 2008) in addition to a functional xanthophyll cycle (Niyogi et al., 1998). Within the xanthophyll cycle excess light causes an acidification of the thylakoid lumen that in turn activates the enzyme violaxanthin de-epoxidase (VDE) leading to the formation of the carotenoid zeaxanthin (Arnoux et al., 2009), which binds to specific antenna proteins and enhances photoprotection by modulating the yield of potentially dangerous chlorophyll-excited states in vivo and preventing the production of singlet oxygen (Dall’Osto et al., 2012). The protein PSBS is thought to act as a pH-sensitive trigger (Li et al., 2002; Bonente et al., 2008) that induces a conformational change of PSII antenna proteins. This qE-active state promotes a charge transfer mechanism that involves energy transfer from chlorophylls in the PSII-LHCII complex to a zeaxanthin–chlorophyll heterodimer followed by charge separation and subsequent recombination to the ground state, which dissipates excited state energy into heat (Ahn et al., 2008). Green microalgae, like C. reinhardtii, use a qE mechanism that differs from the one exploited by higher plants in regard to the participating components (Fig. 1B). Although the nuclear genome of C. reinhardtii contains a PSBS gene, this gene is not expressed (Bonente et al., 2008) and the formation of zeaxanthin is not absolutely required for non-photochemical quenching in this organism (Niyogi et al., 1997). In C. reinhardtii a functional qE mechanism requires the protein LHCSR3 (Peers et al., 2009), which is present in microalgae and the moss Physcomitrella patens (Alboresi et al., 2010), but not in vascular plants. LHCSR3 accumulates in high-light exposed C. reinhardtii cells (Richard et al., 2000) and NPQ capacity is directly correlated to the amount of available LHCSR3 (Bonente et al., 2012). Binding of LHCSR3 to PSII-LHCII supercomplexes in high-light-treated C. reinhardtii cells (Tokutsu and Minagawa, 2013) and its protonation (Bonente et al., 2011a, 2011b) in an acidified lumen modifies LHC antenna conformation, thereby creating quenching centres. The strong proton gradient needed to trigger qE depends on cyclic electron flow around PSI via the Ferredoxindependent pathway (Munekage et al., 2002) and accordingly C. reinhardtii knock-out mutants lacking PGRL1 (Tolleter et al., 2011) or PGR5 (Johnson et al., 2014) as key components of this pathway exhibit severely reduced non-photochemical quenching. Besides qE, state transitions (qT) represent another major NPQ component in green algae, with the latter reducing PSII excitation pressure by a physical displacement of associated antenna proteins (Bonaventura and Myers, 1969; Allen, 1992). Especially with regard to higher plants this process was for a long time believed to play only a minor role under high light conditions (Rintamaki et al., 2000), but recent data obtained with mutants affected in qT as well as qE, suggested that state transitions control excitation pressure during the time needed for LHCSR3 accumulation and full induction of qE (Allorent et al., 2013). 3.2. Manipulation of NPQ mechanisms to improve light conversion efficiency In the surface exposed areas of PBRs non-photochemical quenching mechanism reduce photon conversion efficiencies, because absorbed light energy is dissipated as heat or re-emitted as fluorescence and not used to drive photosynthetic electron transport. Therefore these processes restrict the productivity of mass cultures, but are also required to protect photosynthetic cells from

irreversible damage caused by excess light. The manipulation of NPQ mechanisms as part of genetic engineering concepts seems to be counterintuitive, since strain optimization often aims to create robust traits including high-light tolerance. It might, however, be a strategy worth considering in the case of higher plants, which posses a constitutively active qE and a slowly relaxing NPQ component (qZ) based on the formation of zeaxanthin (Murchie and Niyogi, 2011). Such a constitutive activation of qE could limit growth rate at sub-saturating light intensity due to energy losses via thermal dissipation (Formighieri et al., 2012). In accordance with this notion, the Arabidopsis npq4 mutant lacking PSBS, showed improved growth in low light, while it was sensitive to high light (Dall’Osto et al., 2005). The situation in many lower photosynthetic eukaryotes such as green alga is different. In C. reinhardtii the qE capacity at sub-saturating light intensity is low and requires activation by high light, so that removal of qE (e.g. by a knock-down of the LHCSR3 gene) should only result in a reduced high-light tolerance without improving growth under limited light conditions. In higher plants the slowly relaxing NPQ component qZ (Nilkens et al., 2010) depends on the accumulation of zeaxanthin, while this component contributes little to NPQ in C. reinhardtii (Niyogi et al., 1997). For higher plants genetic engineering strategies might therefore include manipulations affecting the xanthophyll cycle to decrease the time required for qZ relaxation, which could improve photosynthetic efficiency during low light phases in a fluctuating light environment (Niyogi et al., 1997). If adverse growth environments are considered, even an enhancement of NPQ mechanisms could improve the overall photosynthetic efficiency (Horton, 2000), by reducing the susceptibility of photosynthetic cells to high-lightinduced damage. 3.3. Truncation of the light-harvesting antenna and its effect on light use efficiency in C. reinhardtii The energy losses resulting from the activation of NPQ mechanisms are a direct consequence of the oversaturation of photosynthesis, which could be circumvented by reducing the energy input at PSII. The generation of strains with size-reduced antenna systems at PSII has been applied as a strategy to increase light use efficiency in C. reinhardtii cells by avoiding over-saturation of photosynthesis and by decreasing light attenuation in dense cultures (Table 1). Besides the losses of energy based on NPQ processes, which are active in the light-penetrated bioreactor zones, light attenuation along the light path limits the overall culture productivity by causing light-limitation in the inner cell layers (Formighieri et al., 2012). This is caused by a high pigment optical density (Kok, 1953; Melis, 2009) and light-scattering effects (Berberoglu et al., 2008; Formighieri et al., 2012). The generation of mutants with truncated antenna systems at PSII and PSI is deemed a reasonable strategy, since only 37 and 95 of the 350 and 300 Chl molecules found in PSII or PSI, respectively, are needed for the assembly of core complexes as the minimum photosynthetic unit size. Residual chlorophylls associated with the light-harvesting complex (LHC) are, in theory, dispensable (Glick and Melis, 1988). Forward genetic approaches were taken to identify truncated antenna mutants, after random insertion of DNA into the nuclear genome, based on altered chlorophyll fluorescence characteristics at room temperature (Polle et al., 2003; Bonente et al., 2011a, 2011b; Kirst et al., 2012). Insertional mutagenesis of the C. reinhardtii TLA1 gene led to a chlorophyll deficient phenotype, with lower amounts of light-harvesting proteins and a functional chlorophyll antenna size of PSI and PSII being about 50% and 65% of that of the wild type, respectively. In addition to the lower level of chlorophyll per cell, tla1 mutant has a smaller number of electron-transport chains (30–35% reduction compared to Wt) in


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Table 1 Summarizing table containing the phenotypic characteristics of different truncated antenna mutants in regard to total cellular Chl contents, a/b ratios, functional antenna sizes and growth rates. All values are given in percent with the respective parental strain set to 100%. Growth rate increase (%)

PSII antenna size (%)

PSI antenna size (%)

Ref.

270

65

50

Polle et al. (2003)

30

231

74

50

Mitra et al. (2012)

16

444

38

52

Kirst et al. (2012)

237

≈20

Bonente et al. (2011a, 2011b)

130

≈80

Bonente et al. (2011a, 2011b)

Mutant

Affected gene

Genetic mod.

Chl total (%)

Chl a/b ratio (%)

tla1

GenBank: AF534570 GenBank: AF534570 GenBank: XP 001703704

Random DNA insertion RNAi-mediated knock-down Random DNA insertion Random DNA insertion Random DNA insertion RNAi-mediated knock-down of all LHCBM genes

37.5

32

208

185

Mussgnug et al. (2007)

RNAi-mediated knock-down LHCBM1/2/3 genes

50

114

165

Oey et al. (2013)

Site-directed mutagenesis

80

106

153

ri6 tla3 as1 as2 stm3LR3

stm6Glc4L01

stm6Glc4T7

Antisense RNA probe derived from LHCBM1 GenBank: AAM18057 LHCBM1 GenBank: AAM18057 LHCBM2 GenBank: XP 001693987 LHCBM3 GenBank: XP 001703699 NAB1 GenBank: AY157846.2

its thylakoid membranes. Saturation of photosynthesis in the tla1 strain required higher light intensities and under mass culture conditions solar conversion efficiencies and photosynthetic productivity were higher than in the wild type (Polle et al., 2003). Recently, it was shown that TLA1 can be used as a tool for antenna engineering by applying RNAi-mediated knock-down and overexpression of the TLA1 gene (Mitra et al., 2012). Within the screen for truncated light-harvesting antenna size (tla) mutants another insertion mutant tla3 displaying a “pale green” phenotype was recently identified (Kirst et al., 2012). A knock-out of the gene encoding the chloroplast-localized SRP43 signal recognition particle in the tla3 mutant led to a lower chlorophyll per cell content and higher chlorophyll a/b ratio compared corresponding wildtype strains. Similar to the tla1 mutant, cells of the tla3 strain contained less electron transport chains per cell (only 35% of the number found in Wt). As already seen for the tla1 mutant, the light-saturation curve of tla3 dramatically differed from the wildtype curve (half saturation intensity >1000 ␮E in tla3 vs. 210 ␮E in Wt cells) and the light-saturated rate of photosynthesis (Pmax ) was two-fold higher in tla3. In addition to strategies employing the construction and screening of mutant libraries, targeted genetic engineering was successfully used to obtain novel strains with a “pale green” phenotype (Mussgnug et al., 2007; Beckmann et al., 2009; Oey et al., 2013). A simultaneous knock-down of all LHCII (LHCBM) genes encoding components of the major PSII-associated antenna in C. reinhardtii via RNA interference resulted in a strong reduction of total chlorophyll contents and a/b ratios (Mussgnug et al., 2007). Compared to its parental strain, the knock-down strain stm3LR3 displayed an improved photon conversion efficiency, which was seen as a higher (by about 81%) photochemical quantum yield (PSII), which is a measure for the fraction of photons actually used to drive photosynthetic electron transport. Furthermore stm3LR3 was less prone to photoinhibiton than its parental counterpart and grew faster under high-light conditions. A more recent approach was taken by Oey et al. and consisted of the combined knock-down of three distinct LHCBM isoforms (LHCBM1/2/3) in the strain stm6Glc4 (Oey et al., 2013), which is an excellent photosynthetic hydrogen producer (Doebbe et al., 2007). The resultant strain stm6Glc4L01 mutant showed improved light to H2 (180%) and biomass (165%)

83–90

Beckmann et al. (2009)

conversion efficiencies at high photon flux densities. Antenna size reduction probably increased the hydrogen production capacity by a combination of different factors including reduced energy losses due to non-photochemical quenching, improved light penetration of cultures and a lower photosynthetic oxygen production, that resulted in an earlier onset of anaerobiosis as a prerequisite for induction of the hydrogen production pathway. In C. reinhardtii the accumulation of LHCBM proteins is regulated via translation control and the protein NAB1 is a key factor implicated in this process (Mussgnug et al., 2005). In its active state the protein binds to LHCBM transcripts in the cytosol and represses their translation, while inactivation of the protein via cysteine modification relieves repression (Wobbe et al., 2009). A mutant of NAB1 lacking both cysteines crucial for the deactivation mechanism was expressed in the C. reinhardtii strain stm6Glc4 (Beckmann et al., 2009). Expression of the permanently active repressor variant of NAB1 caused a pale green phenotype with reduced total chlorophyll levels and increased a/b ratios. The functional PSII antenna size in strain stm6Glc4T7 was reduced by 10–17% and the strain showed an about 50% higher photosynthetic quantum yield (PSII) together with an increased growth rate under high-light conditions. In summary, experience with antenna truncated mutants of C. reinhardtii within short-term batch cultivations showed that such strains grow faster at higher light intensities on a lab-scale. At these light intensities, which are over-saturating for wild-type strains, energy losses by non-photochemical quenching are lower in antenna mutants. In order to assess the true potential of antenna truncated mutants for biotechnological applications more data in regard to their growth within long-term chemostat cultivations are urgently needed. Depending on the cultivation condition microalgae might not always be exposed to sufficient light and especially if outdoor cultivation is considered periods of limited light availability could become an issue. If light availability in the system is low, truncated antenna mutants can be expected to grow at lower rate than the corresponding wild-type strains, because the respiration rate per Chl is much higher in antenna mutants (Formighieri et al., 2012). Thus higher light intensities are needed to overcome the compensation point and to create a situation where photosynthetic oxygen

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production exceeds dark respiration resulting in a photosynthate production sufficient for biomass formation. The growth of antenna mutants under outdoor conditions should therefore be addressed by future research. Light scattering is another aspect that has to be accounted for in mass cultures and cells of antenna mutants that show decreased light absorption by the lack of chlorophyll might scatter light more intensively (Berberoglu et al., 2008; Formighieri et al., 2012). Future research should address the question, if truncated antenna mutants indeed display an increased productivity under large-scale conditions and prolonged cultivation, especially outdoors. Targeted engineering of the light-harvesting antenna in future approaches instead of simply truncating large parts could also help to achieve the desired increase in light use efficiency. 3.4. Isoform-specific engineering of the light-harvesting antenna The peripheral antenna of green algae is composed of distinct LHCII (LHCBM) isoforms, which show a high degree of identity if their amino acid sequences are inspected, but whose function is not redundant. Most of the available antenna mutants show an isoformunspecific general reduction of LHCBM protein levels. Especially in C. reinhardtii the molecular function of individual isoforms has already been addressed by several past studies (Elrad et al., 2002; Ferrante et al., 2012; Grewe et al., 2014). It seems that these isoforms have two additional functions besides their primary role as efficient light-harvesting devices. One function is to promote energy-dependent quenching as a photoprotective mechanism in high light, for instance via the direct interaction with LHCSR3 (Fig. 1B), as was proposed for LHCBM1 (Ferrante et al., 2012). Another function of special relevance under excess light conditions is direct ROS scavenging by antenna proteins like LHCBM1/2/7 containing the carotenoid neoxanthin (Dall’Osto et al., 2007; Ferrante et al., 2012). Recently, it has been reported that isoform LHCBM9, which is only expressed under specific stress-conditions such as nutrient-deprivation, also plays a crucial role in photoprotection via light-energy dissipation (Grewe et al., 2014). In addition it has been demonstrated that LHCBM isoforms play distinct roles during the state-transitions process (Ferrante et al., 2012). This improved knowledge about the specific functions of individual isoforms could be exploited as part of a rational antenna design that aims for creating novel strains with truncated antennas but relatively intact photoprotection mechanisms. As already mentioned a constitutive truncation of the antenna could be problematic, if microalgae are cultivated outside in closed PBRs. It might thus be reasonable to generate strains with a conditionally truncated antenna, which could be achieved by using high-light inducible promoters in conjunction with the amiRNA technology (Molnar et al., 2009). C. reinhardtii is an ideal system to gain further experience on the upand down-sides of antenna truncation as a strategy to increase PCE rates. Ultimately this knowledge has to be transferred to industrially more relevant species, which display for instance an oleaginous phenotype. 3.5. Engineering antennas with an expanded wavelength range of photosynthetically active radiation Another approach, completely distinct from the antenna truncation concept, would be to expand the wavelength range that can be utilized by a photosynthetic antenna. As mentioned before only a fraction of the incident sunlight can be used to drive photosynthesis in phototrophic organisms. In green algae and higher plants which use chlorophyll subtypes a and b in their photosynthetic apparatus light exceeding a wavelength greater than 700 nm cannot be used to drive PET. It was recently proposed to extend the PAR region in eukaryotic phototrophs by implementing pathways for the

production of chlorophyll types d and f, which can be found in certain cyanobacteria and absorb above 700 nm (Chen and Blankenship, 2011). Chen and Blankenship estimated that an introduction of these chlorophyll sub-types into microalgae or higher plants could increase the overall availability of PAR photons by 19%. 3.6. Improvement of photobioreactor design A complementary approach to genetic engineering could be an improvement of photobioreactor design to reduce photosynthesis saturation at high sunlight intensities in surface exposed layers (Morweiser et al., 2010). Cultures in PBRs are usually mixed and cells shuttle between the light-penetrated “photic” zones and dark areas. Depending on the PBR design cultivated cells experience distinct light/dark cycle durations and medium duration cycles that occur in tubular and flat panel PBRs decrease light use efficiency in comparison to continuous illumination (Janssen et al., 2000). Improved mixing regimes could therefore reduce the time cells are exposed to saturating light while decreasing the time spent in dark zones, where respiration dominates (Sforza et al., 2012; Vejrazka et al., 2012). 4. Engineering of the PET chain to overcome kinetic constraints? Some of the already discussed light energy losses are directly connected to different kinetic bottlenecks within the photosynthetic electron transport chain (Subramanian et al., 2013). Light absorption and energy transfer in the light-harvesting antenna ultimately leading to primary charge separation reactions in PSII is a relatively fast process, but already for these early events of photosynthesis kinetic differences between participating reactions exist. Primary charge transfer in the reaction centre of PSII occurs in the 1–3 ps range and energy transfer from the peripheral antenna to the reaction centre requires 20–30 ps (Pawlowicz et al., 2007). The major bottleneck of photosynthetic electron transport, however, is the coupled electron transfer and proton translocation catalyzed by the Cytb6 f complex (Fig. 1) whose rate-limiting step is in the millisecond range, whereas rate-limiting PSII and PSI reactions require micro- and nanoseconds, respectively (Harris and Königer, 1997; Govindjee et al., 2001; Johnson and Alric, 2012). Other kinetic constraints imposed on PET include slow diffusion rates of the lipidsoluble electron carrier PQ in the thylakoid membrane (Kirchhoff et al., 2002) and relatively slow turnover rates of the ATP synthase complex (Junesch and Gräber, 1985). The impact of reduced Cytb6 f vs. reduced ATP synthase levels on photosynthetic electron transport rates has been analyzed via individual knock-down of both genes in tobacco and these analyses demonstrated that a lowered availability of Cytb6 f complexes affects electron transport more strongly than a reduction in the number of ATP synthase complexes (Yamori et al., 2011). It might therefore be a reasonable strategy to increase the number or activity of Cytb6 f complexes present in photosynthetic cells in order to overcome kinetic bottlenecks that result in energy losses (Subramanian et al., 2013). Furthermore increasing the PQ pool size by genetic engineering might be a worthwhile approach (Subramanian et al., 2013), since the overall capacity for electron and proton charge storage is positively correlated with the photochemical quantum yield of PSII (Kruse et al., 2005a, 2005b) as a measure for the fraction of photons actually used to drive photochemistry. An increased PQ pool size might thus reduce energy losses (as heat or re-emitted fluorescence) in the PSII reaction centre resulting from charge recombination events. While an increased allocation of resources to the synthesis of PQ or Cytb6 f might help to overcome the kinetic bottlenecks of photosynthetic light reactions, this could create other metabolic bottlenecks


because intersystem electron carriers or Cytb6 f components will be produced at the expense of other building blocks needed for biomass formation. It remains to be shown whether the expected enhanced capacity for proton/electron charge storage results in a net gain of light to biomass conversion efficiency.

5. Rubisco engineering and the construction of synthetic photorespiratory bypasses Under conditions when the provision of ATP/NADP by photosynthetic light reactions exceeds their consumption by the Calvin–Benson cycle an over-reduction of the PET chain and overacidification of the thylakoid lumen result in heat as well as fluorescence losses of absorbed light energy. The rate-limiting step of the Calvin cycle is the fixation of inorganic carbon catalyzed by the enzyme Rubisco as the initial reaction of photosynthetic carbohydrate synthesis. In addition to its slow turn-over rate Rubisco has low specificity and accepts oxygen as substrate, which leads to the loss of fixed carbon as part of a photorespiratory cycle and reduces the overall light-to-biomass conversion efficiency. In regard to catalytic rates and substrate specificity substantial variations exist in nature, with Rubisco from red algae having a specificity three times higher than that of C3 crop species (Uemura et al., 1997; Zhu et al., 2010). This natural diversity prompted extensive research on the feasibility of creating improved Rubisco versions e.g. by combining amino acid sequence elements derived Rubisco enzymes expressed in evolutionary distinct organisms (Karkehabadi et al., 2005). An inherent problem of these approaches is that an increased specificity and lower oxygenation potential is usually also accompanied by a low turn-over rate (Whitney et al., 2011), meaning that any strategy to improve Rubisco might suffer from the trade-off between specificity and catalytic activity (Zhu et al., 2010). Against this background it might be more prudent to reduce the energetic costs of Rubisco‘s promiscuity rather than to engineer the enzyme itself. Oxygenation of Ribulose-1,5-bisphosphate by Rubisco leads to the formation of 2-Phosphoglycolate, which has to be converted into 3-phosphoglycerate, the sole product of the carboxylation reaction, in a photorespiratory (C2) cycle (Fig. 2A). This is a costly cascade of reactions from an energetic perspective (Fig. 2B; C2 cycle) that occurs in two different compartments (chloroplast, mitochondria) in the green alga C. reinhardtii (Atteia et al., 2009) or even in a third compartment (peroxisome) if higher plants are considered (Tolbert, 1997). Besides the loss of fixed carbon as part of the glycine decarboxylase reaction (Fig. 1A; GDC), whose re-fixation requires energy and reducing equivalents, the loss of organicallybound ammonia and its re-fixation (Fig. 2A; GOGAT; GS2) require ATP and reducing power (Fdred ). The release of carbon dioxide via GDC takes places in mitochondria of green alga and higher plants and hence at a site remote from Rubisco, where the carbon has to be re-fixed. In order to re-locate carbon dioxide release from the mitochondrion to the chloroplast and to prevent ammonia release completely, transgenic A. thaliana plants with a plastid-located photorespiratory bypass were generated (Kebeish et al., 2007; Maier et al., 2012). Kebeish et al. introduced a glycolate catabolic pathway from Escherichia coli (Fig. 2B; brown pathway) into A. thaliana chloroplasts and increased biomass formation and higher apparent rates of CO2 assimilation were observed in plants producing glyoxylate by over-expression of the first enzyme and those plants producing glycerate by expression of the whole pathway (Kebeish et al., 2007). In another approach taken by Maier et al. enhanced CO2 fixation and growth improvement were noted when A. thaliana plants expressed an alternative glycolate catabolic cycle in the chloroplast (Fig. 2B; blue pathway) in which glycolate is completely oxidized within the chloroplast to two molecules of

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CO2 (Maier et al., 2012). In both cases, transgenic plants showed enhanced biomass formation exclusively when grown in short days and displayed a changed leaf morphology (Kebeish et al., 2007; Maier et al., 2012). For both pathways the total costs in terms of energy and reducing equivalents that need to be invested to form triose phosphate under conditions when oxygenation of Rubisco occurs are lower than those associated with the C2 cycle (Fig. 2B). Recently, a synthetic photorespiratory bypass based on the 3-hydroxypropionate bi-cycle was engineered into the cyanobacterium Synechococcus elongatus sp. PCC 7942 (Shih et al., 2014). In contrast to the synthetic pathways constructed in A. thaliana, the heterologously expressed 3-hydroxypropionate bi-cycle is designed to function as both a photorespiratory bypass and an additional CO2 -fixing pathway, supplementing the Calvin–Benson cycle. Although all six enzymes could be successfully overexpressed in S. elongatus, engineered strains did not show any obvious phenotype, which might be explained by a low activity of introduced enzymes. As proposed by the authors future optimization steps should include testing of alternative enzymes from mesophilic organisms, since the enzymes used in the study were taken from the thermophile Chloroflexus aurantiacus and evolved to function at temperatures not experienced by S. elongatus. The strategy suggested by Shih et al. is challenging from a technical point of view and its use restricted to organisms highly amenable to metabolic engineering, because six distinct enzymes including a ≈600 kDa enzyme have to be expressed simultaneously. If the active pathway can be introduced into photosynthetic cells, it has an excellent energy balance especially in regard to the light-driven synthesis of biomass components derived from pyruvate as a precursor (Shih et al., 2014). Many green algae concentrate CO2 in close proximity to Rubisco in a CCM process. As mentioned above high oxygen concentrations in closed PBRs cause a high oxygenase activity of Rubisco (up to 20.5% of the carboxylase activity) despite the presence of an active CCM, that contributes significantly to the reduction in light-tobiomass conversion efficiency (Kliphuis et al., 2011). The creation of photosynthetic bypasses might thus be a worthwhile strategy to increase microalgal PCE rates in closed PBRs. It should, however, be taken into account that photorespiration is a mechanism crucial for the dissipation of reducing equivalents accumulating in the chloroplast under certain conditions such as excess light availability (Raghavendra and Padmasree, 2003) and some synthetic photorespiratory bypasses generate additional reducing equivalents in the chloroplast (Fig. 2B; blue pathway).

6. Enhancing the energy/reductant sink capacity of the Calvin–Benson cycle The Calvin cycle is initiated by the enzyme Rubisco that catalyzes the carboxylation of the CO2 acceptor molecule ribulose-1,5bisphopsphate (RuBP) leading to the formation of 3-PGA which is converted into the triose phosphates glyceraldehydes-3-phosphate (GAP) and dihydroxyacetone phosphate via two reactions that consume ATP and NADPH. In the regenerative phase of the cycle a series of reactions convert triose phosphates into the CO2 acceptor molecule RuBP and the majority (five-sixths) of the triose phosphate produced in the Calvin cycle remain within the cycle to regenerate RuBP, while one-sixth of the carbon exits the cycle for biosynthesis of compounds such as starch. This acceptor regeneration cycle involves eight distinct enzymes and the phenotypic analysis of antisense plants with reduced levels of individual enzymes identified those enzymes which have significant control over carbon flux through the Calvin cycle (Raines, 2011). Small reductions in the enzyme sedoheptulose-1,7-bisphosphatase (SBPase) resulted in a decreased CO2 fixation and growth rate,

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Fig. 2. (A) Overview of the photorespiratory process (C2 cycle) occurring in the chloroplast, cytosol and mitochondria of a Chlamydomonas reinhardtii cell. The investment of ATP or NADPH and carbon losses is highlighted in red. Enzymes: rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; PGP, phosphoglycolate phosphatase; GYD, glycolate dehydrogenase; AAT, alanine aminotransferase; AGT, alanine-glyoxylate transaminase; GDC, glycine decarboxylase; SHMT, serine hydroxymethyl transferase; SGA, serine-glyoxylate aminotransferase; HPR, hydroxypyruvate reductase; GYK, glycerate kinase; GS, glutamine synthetase; GOGAT, glutamine-oxoglutarate aminotransferase. Compound abbreviations: RuBP, ribulose-1,5-bisphosphate; 2-PGL, 2-phosphoglycolate; 3-PGA, 3-phosphoglycerate; eared/ox , reduced/oxidized electron acceptor; Pyr, pyruvate; Ala, alanine; Glu, glutamate; 2-OG, 2-oxoglutarate; Gly, glycine; CH2 -THF, 5,10-methylene-tetrahydrofolate; Ser, serine; Pyr-OH, 3-hydroxypyruvate; GA, glycerate; Gln, glutamine. (B) Energy balance of photorespiratory bypasses vs. the classical C2 pathway (see (A)) if they were introduced into the chloroplast of C. reinhardtii. A glycolate catabolic pathway from E. coli (Kebeish et al., 2007; brown reaction arrows) and a complete glycolate catabolic cycle (Maier et al., 2012; blue reaction arrows) were considered. Enzymes: GYD, glycolate dehydrogenase; GCL, glyoxylate carboligase; TSR, tartronate semialdehyde reductase; GOX, glycolate oxidase; CAT, catalase; MS, malate synthase; NADP-ME, NADP-malic enzyme; PDH, pyruvate dehydrogenase. The energy and reducing equivalent requirements for the individual pathways were estimated based on a demand of 3 ATP + 2 NAD(P)H per molecule of GAP (glyceraldehyde-3-phosphate) produced via oxygenation of RuBP in addition to the requirements for 3-PGA regeneration from 2-PGL. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

identifying this enzyme as a major control point in the C3 cycle under certain conditions (Harrison et al., 1997). In addition, carbon flux through the C3 cycle was also lowered in plants with small reductions in the activity of either transketolase (TK) or aldolase, although these enzymes are not highly regulated and operate close to equilibrium (Haake et al., 1998; Henkes et al., 2001). Overexpression of either a bifunctional SBPase/FBPase from

a cyanobacterium or plant SBPase in tobacco led to increased photosynthetic CO2 fixation and growth besides higher photosynthetic capacities as revealed by chlorophyll fluorescence imaging of young plants (Miyagawa et al., 2001; Lefebvre et al., 2005). Recently, the C. reinhardtii SBPase gene was transformed into the green alga Dunaliella bardawil, which resulted in enhanced photosynthetic activity and increased total organic carbon content


(Fang et al., 2012). Another interesting strategy to increase the rates of photosynthetic carbon fixation was recently applied to the cyanobacterium S. elongatus (Ducat et al., 2012). Heterologous expression of a symporter of protons and sucrose yielded cyanobacterial strains exporting sucrose irreversibly to media concentrations of >10 mM. Biomass production rates, PSII activity and carbon fixation rates were enhanced relative to wild-type strains and it was proposed that sucrose export provides an expanded photosynthetic sink, helping maintain a relatively oxidized PET chain and suppressing photoinhibition.

7. Mitorespiration as a target for strain engineering The mitochondrial electron transport chain is composed of four main complexes (complexes I, II, III and IV), which catalyze electron transfer from reducing equivalents to molecular oxygen (Fig. 3). This process is coupled to synthesis of ATP catalyzed by complex V (or ATP synthase) through an electrochemical transmembrane gradient. In higher plants and green algae, following electron transfer from NADH and succinate to ubiquinone by complex I and II respectively, two pathways drive the electrons from ubiquinol to molecular oxygen: the cytochrome pathway which comprises complex III and IV and the alternative pathway which is a monomeric alternative oxidase (AOX) (Fig. 3), encoded by a multigene family in higher plants. Together with complex I, the cytochrome pathway leads to ATP synthesis by contributing to the electrochemical transmembrane gradient. In contrast AOX, as well as type II NAD(P)H dehydrogenases, located in the outer (NDe) or the inner (NDi) mitochondrial membrane, do not contribute to the trans-membrane gradient (Fig. 3). They thus lower ATP production per O2 consumed. These enzymes can function when the proton pumping enzymes are inhibited by a large electrochemical proton gradient, and can thus prevent an over-reduction of the electron transport chain, for example in case of high light or active photorespiration (Rasmusson and Wallström, 2010; Schertl and Braun, 2014), contributing very efficiently to crosstalk with chloroplasts and thus the whole cell metabolism. As a matter of fact, inactivation, down-regulation or overexpression of type II NAD(P)H dehydrogenases in higher plants can affect vegetative growth, reproduction, and central carbon metabolism (Liu et al., 2008, 2009; Rasmusson and Wallström, 2010; Wallström et al., 2014). In accordance with its role in ROS scavenging, AOX inactivation can lead to high-light sensitivity and ROS production (Vishwakarma et al., 2014) while its overexpression can confer aluminium tolerance in tobacco cells (Panda et al., 2013), cold tolerance in rice (Li et al., 2013) or salt tolerance in Arabidopsis thaliana (Smith et al., 2009). At last, uncoupling proteins (UCP) are also present and uncouple electron transport from ATP synthesis by mediating a fatty acid-dependent, proton leak across the inner mitochondrial membrane (Fig. 3). Like the AOX and type II NAD(P)H dehydrogenases, they also form a multigene family and in higher plants, overexpression of UCP1 has been shown to increase tolerance to drought and salinity (Begcy et al., 2011). On the contrary, inactivation of UCP1 decreases photosynthetic carbon assimilation rates (Sweetlove et al., 2006). Therefore alternative modes of respiration, which efficiently dissipate excess reducing equivalents without producing ATP, are crucial for the acclimation to various types of abiotic stress. Mitochondrial mutants in the green microalga Chlamydomonas have been isolated at the levels of both the cytochrome pathway and the alternative pathway as well as in complex I (NADH:ubiquinone oxidoreductase), the main entry point of the electrons in the respiratory chain (reviewed in Salinas et al., 2014). Mitochondrial mutants (dum mutants) affected in cytochrome pathway present alteration of complex III (ubiquinol:cytochrome c oxidoreductase) or complex IV (cytochrome c oxidase) activities.

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They display reduced dark respiration, reduced growth rate when grown under mixotrophic conditions (light + acetate) and null growth under heterotrophic conditions (dark + acetate). Complex I mutants grow in the dark but much slower than the wild-type strain and do not display a marked phenotype in the light with acetate. In contrast, growth on minimal medium is usually not affected, except for mutants altered in both complex I and complex III activities (Duby and Matagne, 1999). The dum mutants have been characterized in detail for their photosynthetic performances when grown under mixotrophic conditions and low light (acetate and 50 ␮M m−2 s−1 ). Light-saturation curves of photosynthetic oxygen evolution are lower and their apparent yield of photosynthetic linear electron transport is dependent on the number of active proton-pumping sites in mitochondria (2 for complex I mutants, 1 for mutants in complex III or IV, 0 for mutants in both complex I and the cytochrome pathway of respiration). In addition, they are in state 2, which favours cyclic over linear electron transport in the chloroplast and ATP synthesis at the expense of NAD(P)H (Cardol et al., 2003). Mutants affected in both complex I and complex III also show decreased photosynthetic performances when grown under phototrophic conditions (Cardol et al., 2009). Recently, it was shown that a complex III mutant does not activate programmed cell death after H2 O2 treatment (Murik et al., 2014), suggesting an increased tolerance to oxidative stress. A mutant defective for the alternative oxidase pathway was also isolated by RNA interference in Chlamydomonas (Mathy et al., 2010). Dark respiration rate and light-saturation curve of photosynthetic oxygen evolution are the same as in wild type but the AOX1-RNAi mutant displays increased levels of H2 O2 and superoxide anion. Interestingly, the AOX1-RNAi mutant also shows an increased cell size and cell biomass, suggesting that the inactivation of AOX1 might lead to an overproduction of reduced cofactors that could block catabolic reactions and induce a shift towards anabolic reactions. From results presented above, the question arises whether these mutants would be interesting for biomass production in PBRs. The mutants affected in the ‘classical’ respiratory complexes (I, III or IV) present decreased photosynthetic activity, which is clearly not in favour of their use compared to wild-type strains, but could be more resistant to high light. On the contrary, the AOX1-deficient line does not present altered photosynthetic activity but produces more ROS and increased biomass per cell, which could be an interesting point to investigate if this cell line is not light sensitive because of its ROS-production phenotype. Like most of these parameters have been determined under low light conditions and in the presence of acetate, it is difficult to predict their behaviour in other growth conditions and additional studies should be realized, in particular to define their phototrophic productivity under different controlled conditions. In conclusion, future research aiming at an improved understanding of the factors limiting microalgal productivities in PBRs should also address the interactions between mitorespiration and photosynthesis by using mutants differentially affected in mitorespiration. The dum mutants can be used along with mutant stm6, which is inactivated for a mitochondrial transcription termination factor (Schönfeld et al., 2004; Wobbe and Nixon, 2013) and shows in contrast to dum mutants increased rates of dark respiration, which lead to altered PET (Kruse et al., 2005b) and a light sensitive phenotype (Schönfeld et al., 2004; Nguyen et al., 2011). At last, a mutant defective for isocitrate lyase, a key enzyme of the glyoxylate cycle, a shunt of the Krebs cycle allowing growth on C2 compounds like acetate, has been recently isolated. The mutant shows decreased growth rates in the presence of acetate, altered rate of dark respiration and modified central carbon metabolism when grown under mixotrophic conditions (Plancke et al., 2014). In contrast to the dum and stm6 mutants, the isocitrate lyase mutant is not affected in

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Fig. 3. Mitochondrial electron transport chain in C. reinhardtii and related green algae. CI, complex I (NADH:ubiquinone oxidoreductase); CII, complex II (succinate dehydrogenase); CIII, complex III (ubiquinol:cytochrome c oxidoreductase); cyt c, cytochrome c; CIV, complex IV (cytochrome c oxidase); NDe, external type II NAD(P)H dehydrogenase; NDi, internal type II NAD(P)H dehydrogenase; AOX, alternative oxidase; UCP, uncoupling proteins; UQ, ubiquinone pool. Classical components of the respiratory chain are shown in dark grey and alternative enzymes in light grey. In C. reinhardtii, 6 type II NAD(P)H dehydrogenases (Nda1, 2, 3, 5, 6 and 7) are found, of which one (Nda1) has been experimentally located on the inner side of the inner mitochondrial membrane (Lecler et al., 2012); 2 alternative oxidases (Aox1 and 2) are found, the function of one of them (Aox1) has been determined experimentally (Mathy et al., 2010); and at least 3 UCP (Ucp1, 2 and 3) are found.

photosynthesis and does not show a light sensitive phenotype. It could thus also represent a useful strain for determining limiting factors specifically linked to dark respiration and glyoxylate cycle activities. 8. Outlook 8.1. Expanding the molecular toolbox for industrially relevant microalgal species Engineering of photosynthesis to avoid energy losses and thereby improving light to biomass conversion efficiency can be done within knowledge-based, targeted approaches including for instance synthetic biology. This, however, requires the availability of sequenced genomes and genetic tools such as transformation techniques, constitutive/inducible promoters and established methods for the knock-down/out of individual genes. Several nuclear genome-sequencing projects have now been completed and the availability of rapid large-scale sequencing technology represents a revolution in microalga research (Radakovits et al., 2010). Successful genetic transformation has been reported for more than 30 different strains of microalgae including green (Chlorophyta), red (Rhodophyta), and brown (Phaeophyta) algae; diatoms; euglenids; and dinoflagellates (Radakovits et al., 2010). To date the genetic toolbox is most advanced for the green algal model organism C. reinhardtii. Several constitutive (Schroda et al., 2000; Fischer and Rochaix, 2001) and inducible (Ohresser et al., 1997; Ferrante et al., 2008) nuclear promoters are available and recently novel nuclear expression vectors were constructed, which improved robustness and levels of target gene expression (Rasala et al., 2012). Importantly, these novel vectors can also be used for gene stacking by using a multi-cistronic transformation vector to express three distinct genes of interest simultaneously from one nuclear promoter (Rasala et al., 2014). Poor expression levels of nuclear transgenes is a phenomenon frequently encountered by molecular biologist working with C. reinhardtii, but new expression strains are now

available, which show enhanced transgene expression in terms of attainable levels and robustness (Neupert et al., 2009). Although targeted gene knock-out has still not been established for C. reinhardtii, the applicability of amiRNA to knock-down individual genes in an either constitutive or conditional fashion (Molnar et al., 2009; Schmollinger et al., 2010), enables metabolic engineering in combination with the opportunity to overexpress certain enzymes of a pathway. Considered that an already advanced repertoire of genetic techniques exists for C. reinhardtii, it might be possible to establish targeted genome editing methods such as the CRISPR technology in this organism, which has recently been applied with success to higher plants (Jiang et al., 2013). Especially for the engineering of photosynthetic processes chloroplast transformation techniques, applicable to C. reinhardtii, open up additional opportunities. A multitude of heterologous proteins has already been expressed in Chlamydomonas chloroplasts (Rasala and Mayfield, 2014), but in contrast to higher plant chloroplasts (Lu et al., 2013) there is currently no precedence for the successful expression of polycistronic mRNAs driven by a single plastid promoter in C. reinhardtii chloroplasts. Development of such systems would greatly facilitate metabolic engineering approaches, which often require the concerted expression of several enzymes. Besides C. reinhardtii, the diatom Phaeodactylum tricornutum can be regarded as an established microalgal model organism, which is amenable to strain engineering (Hamilton et al., 2014) due to the existence of a sequenced genome (Bowler et al., 2008) and an advanced molecular toolbox (Siaut et al., 2007; De Riso et al., 2009). The genus Nannochloropsis, which is only distantly related to Chlamydomonas, comprises several oleaginous microalgal species of industrial relevance and members such as N. gaditana (Radakovits et al., 2012) or N. oceanica (Vieler et al., 2012) are currently emerging as novel model organisms. Due to its expanded molecular toolbox C. reinhardtii would be an excellent system to test and validate targeted strain engineering strategies before applying them to green alga with higher robustness and oilcontent or other attributes that define their industrial potential.


This, however, demands for the development of necessary genetic tools in a tedious and time-consuming process. Ultimately the generation of green cell factories that convert light energy efficiently into biomass might require the identification of species with an intrinsic high conversion efficiency in a first step (Bogen et al., 2013a) that is followed by further strain engineering based on available genome information (Bogen et al., 2013b). 8.2. Alternatives to the use of targeted strain engineering strategies An alternative to knowledge-based targeted engineering approaches could be the use of random mutagenesis in conjunction with a strong selection pressure to identify strains which show increased biomass productivity. Chlamydomonas mutants with an increased tolerance towards excess light were for instance obtained by prolonged cultivation of wild-type cells under extreme highlight conditions, which gave rise to spontaneous mutations (Förster et al., 1999). These “gain-of-function” mutations were shown to provide enhanced resistance to high light and/or oxidative stress without reducing culture productivity. The resistance to high-light was not connected to an alteration in electron transport rate, photosystem functionality or enhanced photochemical quenching. A reduced excitation pressure by down-regulation of the lightharvesting antennae or increased non-photochemical quenching was also excluded as the origin of light tolerance in several of the strains. A combination of traits including an enhanced capacity to tolerate reactive oxygen species and increased zeaxanthin accumulation as well as a higher D1 repair cycle activity were instead proposed to create the phenotype (Förster et al., 2005). Such high-light resistant strains could also be generated from wildtype strains of microalgae that are not amenable to sophisticated molecular engineering strategies due to the lack of necessary tools. If a sequenced genome is available, UV-induced or spontaneous mutations conferring high-light resistance can be further characterized by whole-genome-sequencing, as was recently done with very high-light resistant mutants derived from a C. reinhardtii wild type (Schierenbeck et al., 2014). Schierenbeck et al. developed an advanced method that enables rapid identification of mutations causing the phenotype of interest without the need for crossing steps, meiotic mapping or pooled progeny. The low generation time of photosynthetic microbes could also be exploited by applying techniques such as Adaptive Laboratory Evolution (ALE), which is also based on the occurrence of spontaneous mutations that confer a growth advantage under defined conditions (Ibarra et al., 2002). To date there is only few examples for the successful use of ALE aiming at a higher biomass productivity of microalgae (Fu et al., 2012). In this case, a Chlorella vulgaris strain was iteratively adapted to an LED light source resulting in enhanced biomass productivity. ALE could probably be applied to adapt any microalgal strain to the preferred cultivation setting. Acknowledgements CR is supported by Fonds National de la Recherche Scientifique (FRFC 2.4567.11) and LW and CR were supported by the European Union (FP7-No 245070 KBBE SUNBIOPATH). References Ahn, T.K., Avenson, T.J., Ballottari, M., Cheng, Y.C., Niyogi, K.K., Bassi, R., Fleming, G.R., 2008. Architecture of a charge-transfer state regulating light harvesting in a plant antenna protein. Science 320, 794–797. Alboresi, A., Gerotto, C., Giacometti, G.M., Bassi, R., Morosinotto, T., 2010. Physcomitrella patens mutants affected on heat dissipation clarify the evolution of photoprotection mechanisms upon land colonization. Proc. Natl. Acad. Sci. U.S.A. 107, 11128–11133.

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