Study on the molecular mechanisms at the basis of photosynthesis toward microalgae domestication

Oxygenic photosynthesis sustains life on Earth, being the process through which sunlight is used to drive carbon fixation into biomass releasing oxygen as by-product. Photosynthetic organisms convert light energy into chemical energy thanks to the activity of pigment-binding multiproteic complexes known as photosystems and their relative light-harvesting antenna complexes. More than often, light availability is non-optimal, either too scarce or in excess compared to the photosynthetic capacity of the organism operating photosynthesis, to the point it may result dangerous. Among the most sensitive complexes of the photosynthetic chain, photosystem II is the main site of Reactive Oxygen Species (ROS) formation. These detrimental compounds may lead to a compromised growth and losses in biomass productivity. For this reason, photosynthetic organisms evolved different photoprotection mechanisms, among which Non-Photochemical Quenching (NPQ) is one of the most important and investigated. Microalgae dissipate most of the absorbed energy through this safe-valve mechanism, even when conditions are not dangerous, determining a consequent reduction of biomass accumulation. Moving towards an optimized photosynthetic yield is one of the main objectives in microalgae domestication, which itself is an ever-growing interest, because sustainable microalgal industrial applications have the potential to satisfy many global demands. Among the most important, is the use of algal biomass as food and feed, bio-fuels production, extraction of high-value nutraceuticals and pharmaceuticals, recovery of wastewaters, and carbon capture. However, realization of this potential requires a decrease of the current production costs. To achieve profitability, identification of limiting factors is fundamental. Particularly, light-to-biomass conversion efficiency is a key bottleneck that needs to be addressed to achieve domestication. With this focus, the aim of this thesis was to study microalgal photosynthetic behavior upon different growth conditions and the relative activated energy dissipation processes as a possible target to improve productivity, thus enabling the use of microalgae as green cell factories. All the studies herein presented were conducted on the model organism for green algae, Chlamydomonas reinhardtii, for which extensive literature, mutants\u2019 libraries, and genetic tools are available

Increased biomass productivity in green algae by tuning non-photochemical quenching

Photosynthetic microalgae have a high potential for the production of biofuels and highly valued metabolites. However, their current industrial exploitation is limited by a productivity in photobioreactors that is low compared to potential productivity. The high cell density and pigment content of the surface layers of photosynthetic microalgae result in absorption of excess photons and energy dissipation through non-photochemical quenching (NPQ). NPQ prevents photoinhibition, but its activation reduces the efficiency of photosynthetic energy conversion. In Chlamydomonas reinhardtii, NPQ is catalyzed by protein subunits encoded by three lhcsr (light harvesting complex stress related) genes. Here, we show that heat dissipation and biomass productivity depends on LHCSR protein accumulation. Indeed, algal strains lacking two lhcsr genes can grow in a wide range of light growth conditions without suffering from photoinhibition and are more productive than wild-type. Thus, the down-regulation of NPQ appears to be a suitable strategy for improving light use efficiency for biomass and biofuel production in microalgae

LHCSR Expression under HSP70/RBCS2 Promoter as a Strategy to Increase Productivity in Microalgae

Microalgae are unicellular photosynthetic organisms considered as potential alternative sources for biomass, biofuels or high value products. However, limited biomass productivity is commonly experienced in their cultivating system despite their high potential. One of the reasons for this limitation is the high thermal dissipation of the light absorbed by the outer layers of the cultures exposed to high light caused by the activation of a photoprotective mechanism called non-photochemical quenching (NPQ). In the model organism for green algae Chlamydomonas reinhardtii, NPQ is triggered by pigment binding proteins called light-harvesting-complexes-stress-related (LHCSRs), which are over-accumulated in high light. It was recently reported that biomass productivity can be increased both in microalgae and higher plants by properly tuning NPQ induction. In this work increased light use efficiency is reported by introducing in C. reinhardtii a LHCSR3 gene under the control of Heat Shock Protein 70/RUBISCO small chain 2 promoter in a npq4 lhcsr1 background, a mutant strain knockout for all LHCSR genes. This complementation strategy leads to a low expression of LHCSR3, causing a strong reduction of NPQ induction but is still capable of protecting from photodamage at high irradiance, resulting in an improved photosynthetic efficiency and higher biomass accumulation

Antenna complexes protect Photosystem I from Photoinhibition

Background Photosystems are composed of two moieties, a reaction center and a peripheral antenna system. In photosynthetic eukaryotes the latter system is composed of proteins belonging to Lhc family. An increasing set of evidences demonstrated how these polypeptides play a relevant physiological function in both light harvesting and photoprotection. Despite the sequence similarity between antenna proteins associated with the two Photosystems, present knowledge on their physiological role is mostly limited to complexes associated to Photosystem II. Results In this work we analyzed the physiological role of Photosystem I antenna system in Arabidopsis thaliana both in vivo and in vitro. Plants depleted in individual antenna polypeptides showed a reduced capacity for photoprotection and an increased production of reactive oxygen species upon high light exposure. In vitro experiments on isolated complexes confirmed that depletion of antenna proteins reduced the resistance of isolated Photosystem I particles to high light and that the antenna is effective in photoprotection only upon the interaction with the core complex. Conclusions We show that antenna proteins play a dual role in Arabidopsis thaliana Photosystem I photoprotection: first, a Photosystem I with an intact antenna system is more resistant to high light because of a reduced production of reactive oxygen species and, second, antenna chlorophyll-proteins are the first target of high light damages. When photoprotection mechanisms become insufficient, the antenna chlorophyll proteins act as fuses: LHCI chlorophylls are degraded while the reaction center photochemical activity is maintained. Differences with respect to photoprotection strategy in Photosystem II, where the reaction center is the first target of photoinhibition, are discussed

Microalgae Cultivation on Anaerobic Digestate of Municipal Wastewater, Sewage Sludge and Agro-Waste

Microalgae are fast-growing photosynthetic organisms which have the potential to be exploited as an alternative source of liquid fuels to meet growing global energy demand. The cultivation of microalgae, however, still needs to be improved in order to reduce the cost of the biomass produced. Among the major costs encountered for algal cultivation are the costs for nutrients such as CO2, nitrogen and phosphorous. In this work, therefore, different microalgal strains were cultivated using as nutrient sources three different anaerobic digestates deriving from municipal wastewater, sewage sludge or agro-waste treatment plants. In particular, anaerobic digestates deriving from agro-waste or sewage sludge treatment induced a more than 300% increase in lipid production per volume in Chlorella vulgaris cultures grown in a closed photobioreactor, and a strong increase in carotenoid accumulation in different microalgae species. Conversely, a digestate originating from a pilot scale anaerobic upflow sludge blanket (UASB) was used to increase biomass production when added to an artificial nutrient-supplemented medium. The results herein demonstrate the possibility of improving biomass accumulation or lipid production using different anaerobic digestates. \ua9 2016 by the authors; licensee MDPI, Basel, Switzerland

Contrasting behavior of higher plant photosystem I and II antenna systems during acclimation

In this work we analyzed the photosynthetic apparatus in Arabidopsis thaliana plants acclimated to different light intensity and temperature conditions. Plants showed the ability to acclimate into different environments and avoid photoinhibition. When grown in high light, plants had a faster activation rate for energy dissipation (qE). This ability was correlated to higher accumulation levels of a specific photosystem II subunit, PsbS. The photosystem II antenna size was also regulated according to light exposure; smaller antenna size was observed in high light-acclimated plants with respect to low light plants. Different antenna polypeptides did not behave similarly, and Lhcb1, Lchb2, and Lhcb6 (CP24) are shown to undergo major levels of regulation, whereas Lhcb4 and Lhcb5 (CP29 and CP26) maintained their stoichiometry with respect to the reaction center in all growth conditions. The effect of acclimation on photosystem I antenna was different; in fact, the stoichiometry of any Lhca antenna proteins with respect to photosystem I core complex was not affected by growth conditions. Despite this stability in antenna stoichiometry, photosystem I light harvesting function was shown to be regulated through different mechanisms like the control of photosystem I to photosystem II ratio and the association or dissociation of Lhcb polypeptides to photosystem I

The Association of the Antenna System to Photosystem I in Higher Plants COOPERATIVE INTERACTIONS STABILIZE THE SUPRAMOLECULAR COMPLEX AND ENHANCE RED-SHIFTED SPECTRAL FORMS

We report on the association of the antenna system to the reaction center in Photosystem I. Biochemical analysis of mutants depleted in antenna polypeptides showed that the binding of the antenna moiety is strongly cooperative. The minimal building block for the antenna system was shown to be a dimer. Specific protein-protein interactions play an important role in antenna association, and the gap pigments, bound at the interface between core and antenna, are proposed to mediate these interactions Gap pigments have been characterized by comparing the spectra of the Photosystem I to those of the isolated antenna and core components. CD spectroscopy showed that they are involved in pigment-pigment interactions, supporting their relevance in energy transfer from antenna to the reaction center. Moreover, gap pigments contribute to the red-shifted emission forms of Photosystem I antenna. When compared with Photosystem II, the association of peripheral antenna complexes in PSI appears to be more stable, but far less flexible and functional implications are discussed

Molecular basis of autotrophic vs mixotrophic growth in Chlorella sorokiniana

In this work, we investigated the molecular basis of autotrophic vs. mixotrophic growth of Chlorella sorokiniana, one of the most productive microalgae species with high potential to produce biofuels, food and high value compounds. To increase biomass accumulation, photosynthetic microalgae are commonly cultivated in mixotrophic conditions, adding reduced carbon sources to the growth media. In the case of C. sorokiniana, the presence of acetate enhanced biomass, proteins, lipids and starch productivity when compared to autotrophic conditions. Despite decreased chlorophyll content, photosynthetic properties were essentially unaffected while differential gene expression profile revealed transcriptional regulation of several genes mainly involved in control of carbon flux. Interestingly, acetate assimilation caused upregulation of phosphoenolpyruvate carboxylase enzyme, enabling potential recovery of carbon atoms lost by acetate oxidation. The obtained results allowed to associate the increased productivity observed in mixotrophy in C. sorokiniana with a different gene regulation leading to a fine regulation of cell metabolism

Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii.

SummaryThe green alga Chlamydomonas reinhardtii does not synthesize high-value ketocarotenoids like canthaxanthin and astaxanthin, however, a β-carotene ketolase (CrBKT) can be found in its genome. CrBKT is poorly expressed, contains a long C-terminal extension not found in homologues and likely represents a pseudogene in this alga. Here, we used synthetic re-design of this gene to enable its constitutive overexpression from the nuclear genome of C. reinhardtii. Overexpression of the optimized CrBKT extended native carotenoid biosynthesis to generate ketocarotenoids in the algal host causing noticeable changes the green algal colour to a reddish-brown. We found that up to 50% of native carotenoids could be converted into astaxanthin and more than 70% into other ketocarotenoids by robust CrBKT overexpression. Modification of the carotenoid metabolism did not impair growth or biomass productivity of C. reinhardtii, even at high light intensities. Under different growth conditions, the best performing CrBKT overexpression strain was found to reach ketocarotenoid productivities up to 4.5 mg L-1 day-1. Astaxanthin productivity in engineered C. reinhardtii shown here is competitive with that reported for Haematococcus lacustris (formerly pluvialis) which is currently the main organism cultivated for industrial astaxanthin production. In addition, the extractability and bio-accessibility of these pigments was much higher in cell wall deficient C. reinhardtii than the resting cysts of H. lacustris. Engineered C. reinhardtii strains could thus be a promising alternative to natural astaxanthin producing algal strains and may open the possibility of other tailor-made pigments from this host