Toward an effective use of microalgae: a study on Chlamydomonas reinhardtii to disentangle non photochemical quenching (NPQ) and to engineer ketocarotenoids biosynthesis

Photosynthetic organisms can use solar energy to produce organic biomass starting from simple elements as CO2 and water, releasing oxygen as side product. Algae are characterized by high growth rate, extremely rapid life cycle and intrinsic high photosynthetic efficiency. Moreover, microalgae can also be cultivated in a mixed autotrophic/heterotrophic condition, using reduced carbon sources. Several algal strains are characterized by high lipid accumulation or production of high value compounds. Thus, algae not only represent a valid alternative to plants, but they also play a central role considering the sustainability related to their cultivation. Wastewaters and flue gas can be used to ensure nutrients and CO2 for carbon fixation, and, after biomass harvesting, water can be reused leading to a far lower consumption with respect to plants (especially in closed photobioreactor in which the evaporation is low). Unfortunately, algae evolved in conditions extremely different compared to actual industrial ones which involves 24/24 hours of high irradiance, strong shaking as well as high CO2 concentration: all these elements ensure high photosynthetic rate and thus high biomass accumulation but make necessary a domestication of strains. Since this need became evident, engineers, biologists and biotechnologists had tried to overcome algae cultivation limitations in order to became it feasible and economically useful. From a biotechnological point of view several targets could be pointed. Optimization of absorption/dissipation of light energy is one of the most interesting and explored. This thesis reports the use of several approaches to investigate the heat dissipation mechanisms (NPQ) in green algae, mainly focusing on the model organism Chlamydomonas reinhardtii. The results obtained reveal the molecular mechanisms of energy conversion from excitation energy into heat by the activity of specific pigment binding proteins called LHCSR (Light Harvesting Stress Related), going deep into details of the protein domains and pigments involved in the quenching process and the protein interaction network necessary for NPQ. In particular, the regulation of the accumulation of LHCSR proteins in Chlamydomonas reinhardtii revealed to be a successful genetic engineering strategy to improve biomass productivity. Among the possible application of microalgae, one of the most promising one is their use as green factories to produce high value products: here, we report the metabolic engineering of Chlamydomonas reinhardtii as a bio-factory for ketocarotenoids production. The use of microalgae as host to produce high value metabolites, represents, indeed, an effective way to break down costs related to their cultivation with a potential high impact into the market. Astaxanthin is, currently, produce using Haematococcus. lacustris (recently renamed from Haematococcus pluvialis) in which, its accumulation causes a stop in growth. For that reason, in this thesis effects of astaxanthin accumulation of H. lacustris was investigated. This thesis presents, with different approaches, a leap forward in microalgae domestication both trough enrichment of knowledge about NPQ and trough application of metabolic engineering to develop green bio-factories

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

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

Functional analysis of photosynthetic pigment binding complexes in the green alga Haematococcus pluvialis reveals distribution of astaxanthin in Photosystems

Astaxanthin is a ketocarotenoid produced by photosynthetic microalgae. It is a pigment of high industrial interest in acquaculture, cosmetics, and nutraceutics due to its strong antioxidant power. Haematococcus pluvialis, a fresh-water microalga, accumulates high levels of astaxanthin upon oxidative stress, reaching values up to 5% per dry weight. H. pluvialis accumulates astaxanthin in oil droplets in the cytoplasm, while the chloroplast volume is reduced. In this work, we investigate the biochemical and spectroscopic properties of the H. pluvialis pigment binding complexes responsible for light harvesting and energy conversion. Our findings demonstrate that the main features of chlorophyll and carotenoid binding complexes previously reported for higher plants or Chlamydomonas reinhardtii are preserved under control conditions. Transition to astaxanthin rich cysts however leads to destabilization of the Photosystems. Surprisingly, astaxanthin was found to be bound to both Photosystem I and II, partially substituting β-carotene, and thus demonstrating possible astaxanthin biosynthesis in the plastids or transport from the cytoplasm to the chloroplast. Astaxanthin binding to Photosystems does not however improve their photoprotection, but rather reduces the efficiency of excitation energy transfer to the reaction centers. We thus propose that astaxanthin binding partially destabilizes Photosystem I and II

Current Nuclear Engineering Strategies in the Green Microalga Chlamydomonas reinhardtii

Perozeni F, Baier T. Current Nuclear Engineering Strategies in the Green Microalga Chlamydomonas reinhardtii. Life. 2023.The green model microalga Chlamydomonas reinhardtii recently emerged as a sustainable production chassis for the efficient biosynthesis of recombinant proteins and high-value metabolites. Its capacity for scalable, rapid and light-driven growth in minimal salt solutions, its simplicity for genetic manipulation and its “Generally Recognized As Safe” (GRAS) status are key features for its application in industrial biotechnology. Although nuclear transformation has typically resulted in limited transgene expression levels, recent developments now allow the design of powerful and innovative bioproduction concepts. In this review, we summarize the main obstacles to genetic engineering in C. reinhardtii and describe all essential aspects in sequence adaption and vector design to enable sufficient transgene expression from the nuclear genome. Several biotechnological examples of successful engineering serve as blueprints for the future establishment of C. reinhardtii as a green cell factory

In vitro and in vivo investigation of chlorophyll binding sites involved in non\u2010photochemical quenching in Chlamydomonas reinhardtii

Non-photochemical quenching (NPQ) of the light energy absorbed is one of the main photoprotective mechanisms evolved by oxygenic photosynthetic organisms to avoid photodamage, at a cost of reduced photosynthetic efficiency. Tuning of NPQ has been reported as a promising biotechnological strategy to increase productivity in both higher plants and unicellular microalgae. Engineering of NPQ induction requires the comprehension of its molecular mechanism(s), strongly debated in the last three decades with several different models proposed. In this work the molecular details of NPQ induction was investigated at intramolecular level by in vitro and in vitro site-specific mutagenesis on chlorophyll binding sites of the Light Harvesting Complex Stress-related 3 (LHCSR3) protein, the pigment binding complexes identified as the quencher during NPQ induction in the model organism for green algae Chlamydomonas reinhardtii. The results obtained demonstrate a correlation between the quenching activity of LHCSR3 variants in vitro and the NPQ phenotypes observed in vivo. In particular, multiple quenching sites in LHCSR3 cooperatively dissipating the excitation energy were revealed with a peculiar role of Chl 613, a chromophore located a close distance to carotenoid binding site L1

Chlamydomonas reinhardtii LHCSR1 and LHCSR3 proteins involved in photoprotective non-photochemical quenching have different quenching efficiency and different carotenoid affinity

Microalgae are unicellular photosynthetic organisms considered as potential alternative sources for biomass, biofuels or high value products. However, their limited biomass productivity represents a bottleneck that needs to be overcome to meet the applicative potential of these organisms. One of the domestication targets for improving their productivity is the proper balance between photoprotection and light conversion for carbon fixation. In the model organism for green algae, Chlamydomonas reinhardtii, a photoprotective mechanism inducing thermal dissipation of absorbed light energy, called Non-photochemical quenching (NPQ), is activated even at relatively low irradiances, resulting in reduced photosynthetic efficiency. Two pigment binding proteins, LHCSR1 and LHCSR3, were previously reported as the main actors during NPQ induction in C. reinhardtii. While previous work characterized in detail the functional properties of LHCSR3, few information is available for the LHCSR1 subunit. Here, we investigated in vitro the functional properties of LHCSR1 and LHCSR3 subunits: despite high sequence identity, the latter resulted as a stronger quencher compared to the former, explaining its predominant role observed in vivo. Pigment analysis, deconvolution of absorption spectra and structural models of LHCSR1 and LHCR3 suggest that different quenching efficiency is related to a different occupancy of L2 carotenoid binding site

Engineering astaxanthin accumulation reduces photoinhibition and increases biomass productivity under high light in Chlamydomonas reinhardtii

Cazzaniga S, Perozeni F, Baier T, Ballottari M. Engineering astaxanthin accumulation reduces photoinhibition and increases biomass productivity under high light in Chlamydomonas reinhardtii. Biotechnology for Biofuels and Bioproducts. 2022;15(1): 77.**Background** Astaxanthin is a highly valuable ketocarotenoid with strong antioxidative activity and is natively accumulated upon environmental stress exposure in selected microorganisms. Green microalgae are photosynthetic, unicellular organisms cultivated in artificial systems to produce biomass and industrially relevant bioproducts. While light is required for photosynthesis, fueling carbon fixation processes, application of high irradiance causes photoinhibition and limits biomass productivity. **Results** Here, we demonstrate that engineered astaxanthin accumulation in the green algaChlamydomonas reinhardtiiconferred high light tolerance, reduced photoinhibition and improved biomass productivity at high irradiances, likely due to strong antioxidant properties of constitutively accumulating astaxanthin. In competitive co-cultivation experiments, astaxanthin-richChlamydomonas reinhardtiioutcompeted its corresponding parental background strain and even the fast-growing green algaChlorella vulgaris. **Conclusions** Metabolic engineering inducing astaxanthin and ketocarotenoids accumulation caused improved high light tolerance and increased biomass productivity in the model species for microalgaeChlamydomonas reinhardtii. Thus, engineering microalgal pigment composition represents a powerful strategy to improve biomass productivities in customized photobioreactors setups. Moreover, engineered astaxanthin accumulation in selected strains could be proposed as a novel strategy to outperform growth of other competing microalgal strains

Heterologous expression of cyanobacterial Orange Carotenoid Protein (OCP2) as a soluble carrier of ketocarotenoids in Chlamydomonas reinhardtii

Photosynthetic organisms evolved different mechanisms to protect themselves from high irradiances and photodamage. In cyanobacteria, the photoactive Orange Carotenoid-binding Protein (OCP) acts both as a light sensor and quencher of excitation energy. It binds keto-carotenoids and, when photoactivated, interacts with phycobilisomes, thermally dissipating the excitation energy absorbed by the latter, and acting as efficient singlet oxygen quencher. Here, we report the heterologous expression of an OCP2 protein from the thermophilic cyanobacterium Fischerella thermalis (FtOCP2) in the model organism for green algae, Chlamydomonas reinhardtii. Robust expression of FtOCP2 was obtained through a synthetic redesigning strategy for optimized expression of the transgene. FtOCP2 expression was achieved both in UV-mediated mutant 4 strain, previously selected for efficient transgene expression, and in a background strain previously engineered for constitutive expression of an endogenous \u3b2-carotene ketolase, normally poorly expressed in this species, resulting into astaxanthin and other ketocarotenoids accumulation. Recombinant FtOCP2 was successfully localized into the chloroplast. Upon purification it was possible to demonstrate the formation of holoproteins with different xanthophylls and ketocarotenoids bound, including astaxanthin. Moreover, isolated ketocarotenoid-binding FtOCP2 holoproteins conserved their photoconversion properties. Carotenoids bound to FtOCP2 were thus maintained in solution even in absence of organic solvent. The synthetic biology approach herein reported could thus be considered as a novel tool for improving the solubility of ketocarotenoids produced in green algae, by binding to water-soluble carotenoids binding proteins

LHCSR3 is a nonphotochemical quencher of both photosystems in Chlamydomonas reinhardtii

Photosynthetic organisms prevent oxidative stress from light energy absorbed in excess through several photoprotective mechanisms. A major component is thermal dissipation of chlorophyll singlet excited states and is called nonphotochemical quenching (NPQ). NPQ is catalyzed in green algae by protein subunits called LHCSRs (Light Harvesting Complex Stress Related), homologous to the Light Harvesting Complexes (LHC), constituting the antenna system of both photosystem I (PSI) and PSII. We investigated the role of LHCSR1 and LHCSR3 in NPQ activation to verify whether these proteins are involved in thermal dissipation of PSI excitation energy, in addition to their well-known effect on PSII. To this aim, we measured the fluorescence emitted at 77 K by whole cells in a quenched or unquenched state, using green fluorescence protein as the internal standard. We show that NPQ activation by high light treatment in Chlamydomonas reinhardtii leads to energy quenching in both PSI and PSII antenna systems. By analyzing quenching properties of mutants affected on the expression of LHCSR1 or LHCSR3 gene products and/or state 1-state 2 transitions or zeaxanthin accumulation, namely, npq4, stt7, stt7 npq4, npq4 lhcsr1, lhcsr3-complemented npq4 lhcsr1 and npq1, we showed that PSI undergoes NPQ through quenching of the associated LHCII antenna. This quenching event is fast-reversible on switching the light off, is mainly related to LHCSR3 activity, and is dependent on thylakoid luminal pH. Moreover, PSI quenching could also be observed in the absence of zeaxanthin or STT7 kinase activity