The flavodiiron proteins (FDPs) Flv1 and Flv3 in cyanobacteria function in photoreduction of O2 to H2O, without concomitant formation of reactive oxygen species, known as the Mehler-like reaction. Both Flv1 and Flv3 are essential for growth under fluctuating light (FL) intensities, providing protection for PSI. Here we compared the global transcript profiles of the wild type (WT), Δflv1 and Δflv1/Δflv3 grown under constant light (GL) and FL. In the WT, FL induced the largest down-regulation in transcripts involved in carbon-concentrating mechanisms (CCMs), while those of the nitrogen assimilation pathways increased as compared with GL. Already under GL the Δflv1/Δflv3 double mutant demonstrated a partial down-regulation of transcripts for CCM and nitrogen metabolism, while in FL conditions the transcripts for nitrogen assimilation were strongly down-regulated. Many alterations were specific only for Δflv1/Δflv3, and not detected in Δflv1, suggesting that certain transcripts are affected primarily because of the lack of flv3. By constructing the strains overproducing solely either Flv1 or Flv3, we demonstrate that the homo-oligomers of these proteins also function in acclimation of cells to FL, by catalyzing reactions with as yet unidentified components, while the presence of both Flv1 and Flv3 is a prerequisite for the Mehler-like reaction and thus the electron transfer to O2. Considering the low expression of flv1, it is unlikely that the Flv1 homo-oligomer is present in the WT.

Flavodiiron proteins (FDPs) constitute a group of modular enzymes widespread in Bacteria, Archaea and Eukarya. Synechocystis sp. PCC 6803 has four FDPs (Flv1-4), which are essential for the photoprotection of photosynthesis. A direct comparison of light-induced O2 reduction (Mehler-like reaction) under high (3% CO2, HC) and low (air level CO2, LC) inorganic carbon conditions demonstrated that the Flv1/Flv3 heterodimer is solely responsible for an efficient steady-state O2 photoreduction under HC, with flv2 and flv4 expression strongly down-regulated. Conversely, under LC conditions, Flv1/Flv3 acts only as a transient electron sink, due to the competing withdrawal of electrons by the highly induced NDH-1 complex. Further, in vivo evidence is provided indicating that Flv2/Flv4 contributes to the Mehler-like reaction when naturally expressed under LC conditions, or, when artificially overexpressed under HC. The O2 photoreduction driven by Flv2/Flv4 occurs down-stream of PSI in a coordinated manner with Flv1/Flv3 and supports slow and steady-state O2 photoreduction.

Summary In oxygenic photosynthetic organisms, excluding angiosperms, flavodiiron proteins (FDPs) catalyze light‐dependent reduction of O2 to H2O. This alleviates electron pressure on the photosynthetic apparatus and protects it from photodamage. In Synechocystis sp. PCC 6803, four FDP isoforms function as hetero‐oligomers of Flv1 and Flv3 and/or Flv2 and Flv4. An alternative electron transport pathway mediated by the NAD(P)H dehydrogenase‐like complex (NDH‐1) also contributes to redox hemostasis and the photoprotection of photosynthesis. Four NDH‐1 types have been characterized in cyanobacteria: NDH‐11 and NDH‐12, which function in respiration; and NDH‐13 and NDH‐14, which function in CO2 uptake. All four types are involved in cyclic electron transport. Along with single FDP mutants (∆flv1 and Δflv3) and the double NDH‐1 mutants (∆d1d2, which is deficient in NDH‐11,2 and ∆d3d4, which is deficient in NDH‐13,4), we studied triple mutants lacking one of Flv1 or Flv3, and NDH‐11,2 or NDH‐13,4. We show that the presence of either Flv1/3 or NDH‐11,2, but not NDH‐13,4, is indispensable for survival during changes in growth conditions from high CO2/moderate light to low CO2/high light. Our results show functional redundancy between FDPs and NDH‐11,2 under the studied conditions. We suggest that ferredoxin probably functions as a primary electron donor to both Flv1/3 and NDH‐11,2, allowing their functions to be dynamically coordinated for efficient oxidation of photosystem I and for photoprotection under variable CO2 and light availability.

The value and efficiency of microalgal biofuel production can be improved in an integrated system using waste streams as feed-stock, with fuel-rich biomass and treated wastewater being key end-products. We have evaluated seven native cyanobacterial isolates and one native green alga for their nutrient removal, biomass accumulation and lipid production capacities. All native isolates were successfully grown on synthetic wastewater mimicking secondary treated municipal wastewater (without organic carbon). Complete phosphate removal was achieved by the native green alga, isolated from Tvärminne (SW Finland). Optimisation of the C:N ratio available to this strain was achieved by addition of 3% CO 2 and resulted in complete ammonium removal in synthetic wastewater. The native green alga demonstrated similar nutrient removal rates and even stronger growth in screened municipal wastewater, which had double the ammonium concentration of the synthetic media and also contained organic carbon. Sequencing of the genes coding for 18S small rRNA subunit and the ITS1 spacer region of this alga placed it in the Scenedesmaceae family. The lipid content of native isolates was evaluated using BODIPY (505/515) staining combined with high-throughput flow cytometry, where the native green alga demonstrated significantly greater neutral lipid accumulation than the cyanobacteria under the conditions studied.

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The ability to capture and convert sunlight, water and nutrients into useful compounds make photosynthetic microbes ideal candidates for the bio-industrial factories of the future. However, the suitability of isolates from temperate regions to grow under Nordic conditions is questionable. In this work, we explore the chemotaxonomy of Nordic strains of cyanobacteria and one green alga and evaluate their potential as raw materials for the production of lipid-based bio-industrial compounds. Thin-layer chromatography was used to identify the presence of triacylglycerol, which were detected in the majority of strains. Fatty acid methyl ester profiles were analysed to determine the suitability of strains for the production of biodiesel or the production of polyunsaturated fatty acids for the nutraceutical industry. The Nordic Synechococcus strains were unique in demonstrating fatty acid profiles comprised mostly C14:0, C16:0 and C16:1 and lacking polyunsaturated fatty acids. These properties translated to superior predicted biodiesel qualities, including cetane number, cold filter plugging point and oxidative stability compared to the other evaluated strains. Polyunsaturated fatty acids were detected at high levels (38-53%), with Calothrix sp. 336/3 being abundant in two essential fatty acids, linoleic and alpha-linolenic acid (21 and 17%, respectively). Gamma-linoleic acid was the predominant polyunsaturated fatty acid for the remaining strains (13-21%). In addition to assessing the potential of Nordic strains for bio-industrial production, this work also discusses issues such as taxonomy and predictive modelling, which can affect the identification of prospective highperforming strains. | INTRODUCTIONCyanobacteria and green algae are oxygenic photosynthetic microorganisms able to adapt to and flourish in a diversity of environments.They utilise many protective mechanisms, such as lipid unsaturation, to adjust to changing environments. Lipids, being major components of membranes, play a key role in protecting the photosynthetic machinery against environmental stresses such as strong light, salt, and extremes in temperature (Los et al., 2013). The Nordic region is generally characterised by low temperatures, with long, dark winters contrasting short, bright summers (Ferro et al., 2020). Using strains adapted to such unique conditions may be advantageous in the pursuit of robust algal production systems (Cheregi et al., 2019). Indeed, Long-chain polyunsaturated fatty acids (PUFAs) have been found to Anita Santana-S anchez and Fiona Lynch contributed equally to this study.

Flavodiiron proteins (FDPs) catalyse light-dependent reduction of oxygen to water in photosynthetic organisms such as cyanobacteria, creating a protective electron sink that alleviates electron pressure on the photosynthetic apparatus. However, the electron donor to FDPs and the molecular mechanism regulating FDP activity have remained elusive. To address these questions, we employed spectroscopic and gas flux analysis of photosynthetic electron transport, bimolecular fluorescence complementation assays for in vivo protein-protein interactions in the model cyanobacterium Synechocystis sp. PCC 6803, as well as in silico surface charge modelling. We confirmed Ferredoxin-1 as the main electron donor to FDP heterooligomers and revealed that association of FDP heterooligomers with thylakoid membranes is promoted by dissipation of trans-thylakoid proton motive force. We propose a self-feedback mechanism to dynamically control FDP activity. Our findings elucidate the regulatory mechanisms of photosynthesis and have implications for rationally directing electron flux toward desired reactions in photosynthesis-based biotechnological applications.

22 23 Flavodiiron proteins (FDPs) constitute a group of modular enzymes widespread in all 24 life Domains. Synechocystis sp. PCC 6803 has four FDPs (Flv1-4) essential for 25 photoprotection of photosynthesis. A direct comparison of the Mehler-like reaction (O2 26 photoreduction) in high Ci (3% CO2, HC) and low Ci (air level CO2, LC) acclimated 27 cells demonstrated that the Flv1/Flv3 heterodimer is responsible for an efficient steady-28 state O2 photoreduction under HC, with flv2 and flv4 expression strongly down-29 regulated. Conversely, under LC conditions Flv1/Flv3 acts only as a transient electron 30 sink due to competing withdrawal of electrons by the highly induced NDH-1 complex. 31 Further, in vivo evidence is provided indicating that Flv2/Flv4 contributes to the 32 Mehler-like reaction when naturally expressed under LC conditions, or when 33 artificially overexpressed under HC. The O2 photoreduction driven by Flv2/Flv4 occurs 34 down-stream of PSI in a coordinated manner with Flv1/Flv3 and supports slow and 35 steady-state O2 photoreduction. 36 37 38 39 40 Recently resolved crystal structure of truncated Flv1 from Synechocystis revealed a 59 monomeric form with a 'bent' configuration (Borges et al. 2019). Photosynthetic FDPs 60 gained attention first in 2002 when recombinant Synechocystis Flv3 protein was shown 61 to function in O2 reduction to water without producing ROS (Vicente et al., 2002). 62Later it was demonstrated that Synechocystis Flv1 and Flv3 proteins function in vivo 63 in the photoreduction of O2 downstream to PSI (Helman et al., 2003). Since then, 64 extensive research has been pursued to reveal the crucial function of Flv1 and Flv3 65As reported earlier, the Ci level has a remarkable effect on the expression of FDPs at 131 transcript and protein level: Flv2, Flv4 and Flv3 have been strongly upregulated under 132 LC (Zhang et al., 2009;Wang et al., 2004; Battchikova et al., 2010), and down-133 regulated upon a shift to HC (Zhang et al., 2009; Hackenberg et al., 2012; Figure 1C). 134Nevertheless, a direct comparison of the efficiency and kinetics of the Mehler-like 135 reaction in HC-and LC-acclimated cells has not been reported, thus making the 136 contribution of different FDPs to O2 photoreduction difficult to assess. Therefore, as 137 an initial experiment, we evaluated the activity of the Mehler-like reaction in 138Synechocystis cells grown at LC and HC (3% CO2) conditions, at pH 8.2. 139