Regulatory electron transport pathways of photosynthesis in cyanobacteria and microalgae: Recent advances and biotechnological prospects

Cyanobacteria and microalgae perform oxygenic photosynthesis where light energy is harnessed to split water into oxygen and protons. This process releases electrons that are used by the photosynthetic electron transport chain to form reducing equivalents that provide energy for the cell metabolism. Constant changes in environmental conditions, such as light availability, temperature, and access to nutrients, create the need to balance the photochemical reactions and the metabolic demands of the cell. Thus, cyanobacteria and microalgae evolved several auxiliary electron transport (AET) pathways to disperse the potentially harmful over-supply of absorbed energy. AET pathways are comprised of electron sinks, e.g. flavodiiron proteins (FDPs) or other terminal oxidases, and pathways that recycle electrons around photosystem I, like NADPH-dehydrogenase-like complexes (NDH) or the ferredoxin-plastoquinone reductase (FQR). Under controlled conditions the need for these AET pathways is decreased and AET can even be energetically wasteful. Therefore, redirecting photosynthetic reducing equivalents to biotechnologically useful reactions, catalyzed by i.e. innate hydrogenases or heterologous enzymes,


| INTRODUCTION
Cyanobacteria are a group of oxygenic photosynthetic Gram-negative bacteria that evolved at least 2.4 billion years ago. Importantly, they were the progenitors of chloroplasts, the endosymbiotic photosynthetic organelles in plants and algae, to which modern cyanobacteria still bear significant functional and structural similarities. In oxygenic photosynthesis, photons of light, harvested by protein complexes called phycobilisomes (PBS), excite P700 and P680 chlorophyll pigments at the reaction centers of photosystem (PS) I and II, respectively, embedded in the thylakoid membrane. This allows extraction of electrons from water, releasing oxygen as a byproduct. Electrons are conveyed via PSII, cytochrome (Cyt) b 6 f, plastocyanin (PC)/Cyt c 6 , and PSI to ferredoxin (Fd), which functions as a distribution hub for photosynthetic electrons. The photosynthetic electron transfer chain (PETC) also generates a difference in electrochemical potential over the thylakoid membrane, the proton motive force (pmf ), which drives the production of ATP from ADP and P i by the ATP synthase (Mullineaux, 2014).
Depending on environmental conditions and the metabolic state of the cell, electrons are conveyed from Fd via Fd-NAD(P)Hoxidoreductase (FNR), converting NADP+ to the electron carrier NADPH which is mainly used for CO 2 fixation in the Calvin-Benson-Bassham (CBB) cycle. However, if the sink capacity of the CBB cycle is saturated, the PETC may become excessively reduced, resulting in generation of reactive oxygen species (ROS). In natural environments of cyanobacteria and algae, this may occur during fluctuations in light intensity or nutrient availability. Over-reduction and massive production of ROS can severely damage the photosynthetic machinery, which has necessitated the evolution of photoprotective mechanisms and auxiliary electron transport (AET) pathways ( Figure 1). The photosystems are vulnerable to excessive reduction, and while PSII has a rapid repair cycle, recovery of PSI from damage is slow (Komenda et al., 2012;Sonoike, 2011). Photosynthetic organisms therefore invest heavily in protection of PSI by multiple mechanisms that aim at maintaining PSI and its P700 reaction center chlorophyll pair in an oxidized state.
F I G U R E 1 Schematics of photosynthetic light reactions and auxiliary electron transport pathways. The electron transport pathways in the thylakoids of cyanobacteria (A) and microalgae (B) branches into auxiliary electron transport routes, to distribute excess electrons when the sink capacity of the CBB cycle is insufficient. The major electron distribution hub is Fd, and the electron transport route toward the CBB-cycle converges there with auxiliary pathways driven by FDPs, RTOs, hydrogenases and in cyanobacteria, NDH-1. Electron fluxes and CO 2 fixation are adjusted to the redox state of the Fd-pool via Trx, which conveys redox signals to enzymes of the CBB-cycle, ATP synthase, and possibly, FDPs, PGRL1, and in cyanobacteria, NDH-1. In turn, the activity of these enzymes contributes to the pH gradient across the thylakoid membrane, a key regulatory component of photosynthesis, creating a dynamic system fine-tuned by feedback regulation. Dashed lines indicate putative electron transfer and dotted line marks proton transfer. HOX, Ni-Fe hydrogenase; HYD, Fe-Fe hydrogenase; LHCI and II, light harvesting complex; NDA2, NADH:Ubiquinone reductase; SDH, succinate dehydrogenase The basic composition of PETC has remained largely unchanged over the course of evolution, with the exception of the substituting light harvesting complex proteins in place of PBS as the outer antennae of the photosystems in algae and plants. A major difference between prokaryotic cyanobacteria and eukaryotic algae and plants however, is that spatial separation of photosynthetic and respiratory bioenergetic pathways in chloroplasts and mitochondria has evolved in eukaryotes, while in cyanobacteria all bioenergetic and metabolic pathways exist in the same compartment ( Figure 1). This has created distinct needs for coordination of the pathways by a variety of regulatory mechanisms. In this review, we summarize the recent advances in understanding these mechanisms in the unicellular cyanobacterial model species Synechocystis sp. PCC 6803 (hereafter Synechocystis) and in the unicellular green alga Chlamydomonas reinhardtii.
We focus in particular on recent advances in understanding the mechanisms, physiological functions, and regulation of the Mehler-like reaction, catalyzed by flavodiiron proteins (FDPs), and the bioenergetic pathways mediated by NADH-dehydrogenase-like (NDH) complexes or the putative Fd-PQ-reductase (FQR)-mediated pathway.
Moreover, we discuss the significance of the various terminal oxidases in thylakoid membranes, as well as processes contributing to the generation and regulation of the pmf. The photoprotective and AET pathways allow photosynthetic cells to survive in natural habitats where environmental conditions fluctuate. We also review recent advances in biotechnological modification of these mechanisms aimed at directing photosynthetic electrons into desired processes producing solar driven biochemicals.

| FLAVODIIRON PROTEINS
In oxygenic photosynthetic organisms apart from red or brown algae and angiosperms, a crucial mechanism to alleviate excessive reduction of the electron transfer chain involves FDPs (Alboresi et al., 2019;Allahverdiyeva et al., 2015). FDPs function as efficient release valves for excessive electrons in the PETC, reducing O 2 to H 2 O-without the concomitant production of ROS-known as the Mehler-like reaction (Allahverdiyeva et al., 2013;Helman et al., 2003).
Cyanobacteria possess 2-6 isoforms of FDPs (Flv1-4, Flv1B,3B), Flv2 and Flv4 being unique to β cyanobacteria-such as Synechocystis-while Flv1B and Flv3B are found only in heterocysts of filamentous species (Allahverdiyeva et al., 2015). Based on the observation that Flv1 or Flv3 is unable to catalyze the Mehler-like reaction without the other in vivo, and the accumulation of these proteins are co-dependent at least to some extent (Allahverdiyeva et al., 2013;Mustila et al., 2016) a heterodimer is likely the active form of Flv1 and Flv3 in the cells (Allahverdiyeva et al., 2013;Helman et al., 2003).
However, as Flv3 is substantially more abundant than Flv1 in Synechocystis (Allahverdiyeva et al., 2013), alternative oligomerization is likely to occur. Indeed, Flv3 homodimers were detected in Synechocystis, but the existence of Flv3 homotetramers or Flv1/3 heterotetramers cannot be excluded either. Flv3 homooligomers, being unable to reduce O 2 to H 2 O in vivo, serve an as of yet unknown physiological function (Mustila et al., 2016). Moreover, the presence of Flv2/4 heterodimer in Synechocystis and Flv2 homodimer in the corresponding Δflv4 mutant was reported (P. Zhang et al., 2012 It has been established that Flv1/3 heterodimers operate on the acceptor side of PSI (Allahverdiyeva et al., 2013;Helman et al., 2003).
In contrast, it was initially proposed Flv2/4 hetero-oligomers may extract electrons from PSII or the PQ pool, alleviating excitation pressure on PSII in order to avoid production of ROS (P. Zhang et al., 2012). However, while Flv2/4 hetero-oligomers contribute to the Mehler-like reaction under steady state and low carbon conditions, light-induced O 2 reduction is inhibited when electron transfer at Cyt b 6 f is blocked by DBMIB in the mutant deficient in the cytochrome bd quinol oxidase (Cyd) (Santana-Sanchez et al., 2019). This indicates that the source of electrons for Flv2/4 induced Mehler-like reaction is not at PSII or at the plastoquinone pool, but rather downstream of the Cyt b 6 f complex.
The green alga C. reinhardtii possesses two FDPs (FLVA and FLVB) which possibly function as heterodimers and share higher homology with Flv1 and Flv3 than Flv2 or Flv4 (Allahverdiyeva et al., 2015). FLVA and FLVB catalyze O 2 photoreduction during dark to light transitions (Chaux et al., 2017). The estimated maximal rate of O 2 uptake (V max ), in the presence of a plastid terminal oxidase (PTOX) inhibitor, was attributed to FDPs and was reported as 45 μmol O 2 mg Chl −1 h −1 (Saroussi et al., 2019). This is in the same range reported for FDPs in Synechocystis (Santana-Sanchez et al., 2019), although direct comparison is difficult due to different experimental conditions. Under fluctuating light intensities from low to high light C. reinhardtii FDPs represent the major photoprotective mechanism enabling cell growth by acting as fast and strong electron sinks downstream of PSI . It is important to note that these studies were performed with flvB knockout mutants in which the accumulation of FLVA is also severely impaired (Chaux et al., 2017;Jokel et al., 2018), thus distinguishing between the function of homodimers or heterodimers is impossible. Interestingly, in addition to photoreduction of O 2 , recent data show the involvement of FDPs in light-induced reduction of NO to N 2 O, indicating a wider contribution of FDPs to the metabolism in C. reinhardtii (Burlacot et al., 2020).

| FLAVODIIRON PROTEINS ELECTRON DONOR(S)
The precise identities of the electron donors of FDPs have long remained uncertain. Class C FDPs contain a C-terminal NAD(P)H:flavin oxidoreductase-like domain and indeed, in vitro studies indicated NADH and NADPH-dependent O 2 reduction activity of Synechocystis Flv1 and Flv3 (Brown et al., 2019;Vicente et al., 2002) as well as Flv4 (Shimakawa et al., 2015), suggesting NAD(P)H as the electron donor to FDPs. It is important to note however, that these enzymatic assays were carried out with homo-oligomers of FDPs, which do not catalyze photoprotective reduction of O 2 in vivo like the hetero-oligomeric forms (Mustila et al., 2016;Santana-Sanchez et al., 2019). Moreover, both Vicente et al. (2002) and Shimakawa et al. (2015) reported low reaction rates and low or no affinity of FDPs to NADPH as compared with NADH, albeit higher rates and relative NADPH affinities for Flv1 and Flv3 were measured by Brown et al. (2019). Thus, it remains questionable whether NADPH is an electron donor to FDPs in vivo.
Furthermore, no in vitro enzymatic assays testing FDP activity with alternative electron donors, such as Fd or FNR, have been published.
Synechocystis Flv3 (Cassier-Chauvat & Chauvat, 2014) and its C. reinhardtii orthologue FLVB (Peden et al., 2013) have been shown to interact with Fd in two-hybrid tests, and Synechocystis Flv1 and Flv3 in a Fd-chromatographic assay (Hanke et al., 2011). Recently, it was also shown by near-infrared absorbance and fluorescence spectroscopy that absence of Flv1/3 results in impaired oxidation of the Fd pool in dark-adapted Synechocystis cells upon illumination, while NADPH redox kinetics are unaltered (Nikkanen et al., 2020;Sétif et al., 2020). These findings strongly support the hypothesis that Fd, instead of NADPH, is the primary electron donor to Flv1/3 heterooligomers in vivo, although possible involvement of F A F B iron-sulfur clusters of PSI cannot be excluded (Sétif et al., 2020). An intriguing possible explanation for the discrepancy between in vitro and in vivo results would be that the homo-oligomeric and hetero-oligomeric forms of FDPs would have distinct electron donors, allowing control between their distinct physiological functions based on the redox states of the cytosolic electron carriers. Identity of the electron donor to Flv2/4 hetero-oligomers also remains unresolved.
There are nine Fd isoforms in Synechocystis (Fed1-9) (Cassier-Chauvat & Chauvat, 2014). Little is known about the specific functions of or the potential redundancies between individual Fd isoforms, but Fed1 is likely the main isoform involved in photosynthetic electron transfer. Moreover, Fed2-9 are expressed at comparatively low levels, and even though Fed9 interacted with Flv3 in a two-hybrid test (Cassier-Chauvat & Chauvat, 2014), Fed2-9 are unlikely to be able to provide the reducing capacity to drive the large electron sink activity of the Mehler-like reaction. This leaves Fed1 as the most likely candidate to constitute the main electron donor to FDP hetero-oligomers.

| REGULATION OF FLAVODIIRON PROTEIN ACTIVITY
In Synechocystis, Flv1/3-mediated O 2 photoreduction is rapidly activated during the first seconds of illumination or upon sudden increases in light intensity, but diminished about 30 s thereafter (Nikkanen et al., 2020;Santana-Sanchez et al., 2019). In contrast, Flv2/4-dependent O 2 photoreduction is activated more slowly but persists at steady state at least for 5-10 min during illumination (Santana-Sanchez et al., 2019). Therefore, it is highly likely that the activities of FDP hetero-oligomers are subject to tight regulation, likely to avoid competition for reducing power with an activated CBB cycle.
Based on structural in silico modeling, it has been suggested that FLVA and FLVB hetero-oligomers of Physcomitrella patens could switch between an "open" active or a "closed" inactive conformation in response to a regulatory signal for example via redox regulation (Alboresi et al., 2019). Accordingly, Synechocystis, Anabaena sp. PCC7120, and C. reinhardtii FDPs all contain conserved cysteine residues (Alboresi et al., 2019;Jokel et al., 2018) that could potentially be targets to regulatory thiol-disulfide exchange, mediated by the thioredoxin (Trx) system. Interestingly, light-dependent redox modulation was observed in Cys33 and Cys226 of Synechocystis Flv1 and Cys207 of Flv3 in a redox proteomic assay, but no such changes were detected in Flv2 or Flv4 (Guo et al., 2014). The physiological significance of these redox exchanges is still being investigated, but coupling FDP activity to cellular thiol redox state could provide a rapid and reversible mechanism to coordinate the activity of the Mehler-like reaction according to environmental stimuli and the physiological state of the carbon metabolism, most importantly the CBB cycle.
While Flv2 and Flv4 contain no light-dependently redoxmodulated cysteine residues, the activity of Flv2/4 hetero-oligomers may be controlled via pmf and Mg 2+ -dependent association with the thylakoid membrane (P. Zhang et al., 2012), which would regulate the access of the hetero-oligomers to their photosynthetically produced reductant, be it Fd, NADPH, or FNR. Interestingly, regulation via the magnitude of the pmf could enable control of Flv2/4 via the activity of Flv1/3, given that during the first seconds of sudden light increases, up to 75% of pmf generation is dependent on Flv1/3 (Nikkanen et al., 2020). Moreover, the expression of Flv2 and Flv4 is strongly inhibited by elevated C i concentration as well as alkaline pH. Interestingly, in high C i where Flv2 and Flv4 are absent, the presence of Flv1/3 is sufficient to catalyze strong steady-state O 2 photoreduction (Santana-Sanchez et al., 2019).
Alternatively, FDP activity could be reversibly controlled by phosphorylation and de-phosphorylation, as a phosphoproteomic analysis of Synechocystis cells revealed phosphorylated serine residues in Flv3 and Flv4 (Angeleri et al., 2016). Besides, Cyd contributes to ΔpH formation by reducing O 2 to water, which alkalizes the cytosol. Although Cox primarily functions in the dark, it assists in the regulation of electron flow to PSI by transferring electrons from PC/Cyt c 6 to O 2 in light (Ermakova et al., 2016;Schmetterer, 2016). Cox is indispensable under low light (Kufryk & Vermaas, 2006) and contributes to photosynthetic control by likely pumping protons at a 4H + /2e − ratio. Mutant strains deficient in both Cox and Cyd cannot survive in a square-wave diurnal light regime (sharp alteration of 12 h high light and 12 h dark periods), but interestingly, are able to grow under sinusoidal diurnal regime (gradual changes in light intensity), under shorter square-wave light regime (5 min dark/5 min high light), or under constant illumination (Ermakova et al., 2016;Lea-Smith et al., 2013). The contribution of RTOs seems to depend on the length of the dark and length and intensity of light periods, and the amount of photodamage occurring during the light period. Importantly, light can induce O 2 uptake by RTOs when the linear electron transport is inhibited at Cyt b 6 f with DBMIB (Berry et al., 2002) or at PSI by cultivating the Flv1/3-deficient mutant under mild fluctuating light (Allahverdiyeva et al., 2013;Ermakova et al., 2016). This activity however cannot rescue the fatal loss of Flv1/3 under strong fluctuating light (Allahverdiyeva et al., 2013) due to low responsiveness and sink capacity of RTOs (Ermakova et al., 2016). While RTOs only have a minor effect on the redox poise of the PETC in steady-state light conditions (Helman et al., 2005), they appear to tune the redox poise of cyanobacterial thylakoids when illumination ceases. Dark respiratory rates in Synechocystis are reportedly higher after illumination than before (Nikkanen et al., 2020;Santana-Sanchez et al., 2019). It is possible that relaxing photosynthetic control and/or increased concentration of O 2 stimulates RTOs.

| PHOTOPROTECTIVE RESPIRATORY TERMINAL OXIDASES IN CYANOBACTERIAL AND ALGAL THYLAKOIDS
Besides RTOs, PTOX can be found in some cyanobacteria (Schmetterer, 2016) and green algae. In chloroplasts of C. reinhardtii, auxiliary electron pathways branch off the PQ-pool via PTOX1 and PTOX2 to O 2 . PTOX2 was shown to be the major plastidial terminal oxidase in C. reinhardtii preventing over-reduction of the PQ-pool in light, thus operating as a photoprotective electron sink (Houille-Vernes et al., 2011). Indeed, loss of PTOX2 in C. reinhardtii leads to excess excitation pressure and decreased growth rate under intermittent light periods but not under constant illumination (Nawrocki, Buchert, et al., 2019b).

| NADH DEHYDROGENASE-LIKE COMPLEXES
In cyanobacteria, the NADH dehydrogenase-like complex 1 (NDH-1) is a highly versatile multisubunit bioenergetic machinery. By incorporating specific subunits, NDH-1 functions in the main pathway of cyclic electron transport (CET) around PSI in cyanobacteria, respiratory electron transport, as well as the carbon-concentrating mechanism (CCM). Several cryo-EM structures of cyanobacterial NDH-1 complexes have been recently reported, providing detailed information on their composition (Laughlin et al., 2019(Laughlin et al., , 2020Schüller et al., 2019Schüller et al., , 2020. The core complex consists of membrane-embedded subunits and the peripheral arm on the cytosolic side of the thylakoid membrane. Addition of D1 and F1, or D2 and F1 subunits to the core creates the NDH-1 1 and NDH-1 2 isoforms, respectively, which both catalyze cyclic as well as respiratory electron transport. Whether NDH-1 1 and NDH-1 2 have distinct physiological roles remains to be investigated. Low concentration of C i triggers the expression of the D3/F3/CupA/CupS operon and the formation of the NDH-1 3 isoform, which functions in high-affinity conversion of CO 2 to HCO 3 − in the CCM. In contrast, the D4/F4/CupB operon is expressed constitutively, and incorporation of its protein products into the NDH-1 core creates the NDH-1 4 isoform that catalyzes CO 2 uptake but with low affinity (Peltier et al., 2016).
Functional and structural evidence has indicated that Fd (instead of NADPH) is the direct electron donor to photosynthetic NDH-1 complexes and binds to an oxidation site in the cytosolic peripheral arm of the complex likely formed by the O, I, H, V, and S subunits (Laughlin et al., 2020;Schüller et al., 2019;C. Zhang et al., 2020). A shared electron donor between FDPs and NDH-1 may, therefore, enable coordination of the two electron transfer pathways. Accordingly, we reported recently on partial redundancy between Flv1/3 and NDH-1 1/2 in protecting PSI by maintaining efficient oxidation of PSI in high light and air-level CO 2 concentration (Nikkanen et al., 2020).

Similar functional redundancy was observed between FDPs and
NDH-1 as well as PGRL1/PGR5 in the moss P. patens (Storti, Puggioni, et al., 2020a;Storti, Segalla, et al., 2020b). In Synechocystis, absence of both Flv1/3 and NDH-1 1/2 was even lethal when cells were moved from conditions of high CO 2 concentration and low light to air-level CO 2 concentration and high light, as in addition to exacerbated impairment of PSI oxidation, these mutants were unable to induce accumulation of proteins related to the CCM (Nikkanen et al., 2020).
Interestingly, however, simultaneous loss of Flv1/3 and NDH-1 3-4 , the forms associated with CCM, was not lethal in similar changes in growth conditions, suggesting that coordination of functions occurs specifically between Flv1/3 and NDH-1 1-2 .
There is clear evidence that NDH-1 contributes to oxidation of PSI in both cyanobacteria and plants (Nikkanen et al., 2018(Nikkanen et al., , 2020Shimakawa & Miyake, 2018;Storti, Puggioni, et al., 2020a). It is not immediately obvious why however, since cyclic and respiratory elec- NADPH consumption was not impaired, however, in Synechocystis cells lacking both Flv1/3 heterooligomer and NDH-1 1-2 (Δflv3d1d2), suggesting that an impairment of CBB cycle activity due to ATP deficiency is not the factor responsible for the diminished ability of these mutants to keep PSI oxidized. In contrast, in cells lacking only NDH 1-2 (Δd1d2), PSI oxidation and pmf generation are not impaired due to enhanced activity of FDPs, but activation of carbon fixation and NADPH consumption are delayed (Nikkanen et al., 2020).
Hyperactive FDPs in Δd1d2 cells, while maintaining effective oxidation of PSI, may be out-competing the Trx system, whose reduction is required for activation of the CBB cycle (Tamoi et al., 2005), for electrons. When the competitive electron sink of Flv1/3 is removed in Δflv3 d1d2 cells, the CBB cycle is activated normally but PSI oxidation is severely impaired, resulting in photodamage. These studies with Synechocystis knockout strains aptly demonstrate how intricate coordination of the auxiliary electron transfer pathways is required to maintain cellular redox balance and integrity of the photosynthetic machinery in changing environmental conditions.
Coupling the activity of NDH-1 to that of thylakoid RTOs, would also enhance oxidation of PSI, but it is unlikely to be a major factor due to the low electron transfer capacity of RTOs (Ermakova et al., 2016;Helman et al., 2005). Lastly, the CCM-associated NDH-1 3 conformation utilizes reduced Fd to convert CO 2 to HCO 3 − (Schüller et al., 2020), and should therefore also enhance oxidation of PSI when its expression is induced in low CO 2 conditions.
NDH-1 is also present in chloroplasts of most seed plants, but interestingly, it has been lost in unicellular green algae (Peltier et al., 2016). Instead, NDH-2 (called NDA2 in C. reinhardtii), a singlesubunit flavoenzyme, reduces PQ and mediates CET without pumping additional protons (Jans et al., 2008). Therefore, NDA2-mediated CET affects the NADPH/ATP ratio but is less effective in generating pmf than the cyanobacterial NDH-1 complex. The non-photochemical PQreduction via NDA2 is the point of entry for electrons that derive from glycolysis and other breakdown processes to PETC, and can subsequently feed alternative electron sinks such as hydrogenases (Mignolet et al., 2012;Milrad et al., 2021) or PTOX (Saroussi et al., 2016). subunits also contain several light-dependently redox modulated cysteine residues, which could potentially act as Trx targets. NdhK, NdhI, and NdhJ subunits all contained cysteines whose reduction was induced by light (Guo et al., 2014 The presence and nature of the FQR-pathway in cyanobacteria is still poorly characterized, but possible involvement of a Pgr5 homolog in CET has been suggested, although its physiological role would be minor (Yeremenko et al., 2005) with NDH-1 constituting the main CET pathway in cyanobacteria (Miller et al., 2021). Recently, the Synechocystis Sll1217 protein with low sequence similarity to PGRL1 was proposed to be a functional analog of PGRL1 (Dann & Leister, 2019).

| REGULATION OF NDH-1 ACTIVITY
The Sll1217 protein interacts in vitro with Pgr5 and its knockout mutant shows impaired P700 oxidation levels, which the authors suggested to be due to decreased CET. Although the physiological rel- ΔpH formation (Shikanai & Yamamoto, 2017). The ΔpH component also has a crucial regulatory role in inducing photosynthetic control, a mechanism that inhibits excessive electron transfer at the Cyt b 6 f complex in order to protect PSI. The physiological significance of photosynthetic control has been mostly discussed in plants and algae, but the mechanism most likely functions also in cyanobacteria, as evidenced by increased re-reduction rate of Cyt f in light due to addition of pmf uncouplers (Checchetto et al., 2012). It is likely, however, that high PSI/PSII ratio and the presence of FDPs makes photosynthetic control less essential for photoprotection of PSI in cyanobacteria than in angiosperms. In plants and algae ΔpH also induces non-photochemical quenching (NPQ), another photoprotective mechanism dissipating excessive excitation energy at PSII antennae as heat (Niyogi & Truong, 2013). In cyanobacteria NPQ induction does not depend on acidification of the lumen, but is induced by the orange carotenoid protein activated by strong light (Muzzopappa & Kirilovsky, 2020). Nonetheless, adjusting the magnitude of the pmf according to environmental conditions by controlling the influx and efflux of protons across the thylakoid membrane is of utmost importance for all photosynthetic organisms.
Flv1/3 is essential for effective generation of pmf at dark/light transitions. In Synechocystis at the onset of light up to 75% of thylakoid proton flux was attributable to the presence of Flv3 (Nikkanen et al., 2020), and similar albeit somewhat lower contributions to pmf have been reported for FLVA/B in C. reinhardtii (Chaux et al., 2017) and P. patens (Gerotto et al., 2016), and for Flv1 in the liverwort Marchantia polymorpha (Shimakawa et al., 2017). In part the FDP contribution to pmf is due to cytosolic consumption of H + in O 2 photoreduction, but perhaps even more importantly, FDPs enhance pmf generation by enabling a higher rate of linear electron transfer.
Accordingly Since dark to light transitions are rare in natural environments, contribution of FDPs could rather be significant when irradiation fluctuates and the generated pmf might contribute to photosynthetic control.
However, FDP-driven electron transfer as a means of pmf generation is inefficient as it wastefully consumes reducing power unlike CET.
As the transfer of 2e − from Fd to PQ by NDH-1 complexes is coupled to pumping of 3H + (NDH-1 3-4 ; Schüller et al., 2020) or 4H + (NDH-1 1-2 ; Schüller et al., 2019) into the lumen, as well as translocation of additional 4H + in the Q cycle, NDH-1 also has a role in controlling the magnitude of pmf, and as a consequence, ATP synthesis.
However, as high ΔpH inhibits the Fd-PQ reductase activity of NDH-1, its contribution to pmf has been suggested to be most significant under low irradiance (Shikanai & Yamamoto, 2017). It was recently shown that, in Synechocystis, NDH-1 1-2 complexes could maintain up to 40% of lumenal acidification rate at dark/light transitions when electron transfer from PSII was inhibited, while NDH-1 3-4 only contributed about 5% (Miller et al., 2021). This constitutes the first demonstration of NDH-1-mediated proton pumping in living cyanobacterial cells, and while these results were obtained in the presence of extensive inhibitor cocktails, which makes it difficult to assess their significance in more natural physiological conditions, they are in line with a recent report attributing a maximum of 35% of total electron transport at PSI to CET in living Synechocystis cells (Theune et al., 2021). Interestingly, in the M55 mutant (lacking all NDH-1 complexes) pmf generation was completely abolished at dark/light transitions (Miller et al., 2021). This suggests that the FQR-dependent CET pathway does not contribute to pmf formation at dark/light transitions in Synechocystis.
In order to adjust pmf during illumination and to prevent ATP hydrolysis in the dark, the pmf release rate must be controlled by regulating the activity of the ATP synthase. In plants and algae, including C. reinhardtii, reduction of a disulfide bond in the γ subunit of the chloroplast ATP synthase by Trx activates the ATP synthase at the onset of light. This mechanism is, however, absent in cyanobacteria (Hisabori et al., 2013). Instead, reverse-activity of the ATP synthase and ATP hydrolysis in the dark are inhibited by the ϵ subunit (Imashimizu et al., 2011). The mechanisms of regulation of the conductivity of the ATP synthase and thylakoid ion channels during changes in light conditions in cyanobacteria and algae remain largely uncharacterized and further studies are required to elucidate them.

| IMPROVING BIOPRODUCTION BY ENGINEERING AUXILIARY ELECTRON TRANSPORT PATHWAYS
Photosynthetic microbes are promising chassis for biotechnological applications, due to their ability to convert light energy into chemical potential ( Figure 2). However, economic feasibility of the system requires significant improvement. The theoretical photon-to-product conversion efficiency of photosynthesis is about 10-13%, whereas in reality efficiency barely exceeds 1-3% (Melis, 2009 (Appel et al., 2020;Kanygin et al., 2020). Alternatively, a "pulse illumination protocol" was developed recently, in which microalgae are exposed to a train of strong yet short light pulses superimposed on dark (or low light) background, channeling photosynthetic electrons originated from PSII water splitting to the hydrogenase instead of CO 2 fixation (Kosourov et al., 2018(Kosourov et al., , 2020.
Elimination of FDPs further increased H 2 production under "pulsing" light regime and constant illumination, possibly by providing more photosynthetic electrons for the hydrogenase (Chaux et al., 2017;Jokel et al., 2019). However, it is uncertain how significant the O 2 photoreduction activity of FDPs is under microoxic conditions. Since FDPs in C. reinhardtii contribute to photoreduction of NO to N 2 O (Burlacot et al., 2020), the loss of FLVB could affect NO homeostasis and/or signaling mediated by reactive nitrogen species (Jokel et al., 2019). Moreover, delayed ΔpH generation in cells lacking FLVB (Chaux et al., 2017) could also contribute to the elevated H 2 production during pulse illumination. Indeed, proton uncouplers or the loss of PGRL1 reportedly increase H 2 photoproduction in C. reinhardtii. Delay in ΔpH compromises photosynthetic control and thus, enables electrons to be transferred toward the hydrogenase (Tolleter et al., 2011).
Loss of FDPs or FQR function appears to have diverse effects on the metabolism that influences H 2 photoproduction. C. reinhardtii cells lacking FLVB, e.g. demonstrated higher respiratory activity compared to WT (Jokel et al., 2019), likely by providing a more optimal intracellular milieu for the [Fe-Fe]-hydrogenases that are reportedly O 2 sensitive.
The pgr5 and pgrl1 mutants showed an increase in respiratory processes as well, together with enhanced PSII stability, resulting in higher H 2 photoproduction compared with WT (Steinbeck et al., 2015).
F I G U R E 2 Harnessing photosynthetic reductants for production of targeted chemicals. Photosynthetic light reactions produce a large amount of reducing equivalents such as reduced Fd and NADPH. Photosynthetic microbes with engineered photosynthesis (eliminated competing routes) channel a majority of electrons to desired chemicals which are excreted from the cells. In this scenario the engineered photosynthetic microbes act as light-driven biocatalysts with a constantly recycled pool of reducing cofactors Light-driven whole-cell biotransformations, where heterologous oxidoreductases, imine reductases, mono-oxygenases, or ene-reductases are directly coupled to photosynthetic light reactions allow the sustainable utilization of photosynthetic reductants (NADPH or reduced Fd). Heterologous enzymes represent an additional intracellular electron sink, therefore an optimal balance between photosynthetic electron supply and consumption by the desired redox reactions is necessary for increased photosynthetic yield, optimal cell fitness and thus, prolonged production. Accordingly, a heterologously expressed Fd-dependent monooxygenase, Cyt P450, increased the photosynthetic capacity and ATP/NADPH ratio in Synechococcus sp. 7002 (Berepiki et al., 2018).
Elimination of photosynthetic competing pathways could positively impact productivity of the heterologous enzymes by providing an increased amount of photosynthetic reducing power ( Figure 2 CO 2 . However, ΔFlv1 and ΔFlv3 strains but not the WT underwent chlorosis during the production (Selão et al., 2020). Importantly, deletion of Pgr5 did not induce chlorosis and led to the highest D-lactate titer observed in the study. However, elevating either CO 2 level and/or temperature abolished the production advantage of each photosynthetic mutant over the D-lactate dehydrogenase-expressing reference strain (Selão et al., 2020).
An innate osmoprotectant, sucrose, was utilized as a marker product in salt-stressed Synechocystis overexpressing sucrose permease to evaluate whether more photosynthetic electrons could be channeled towards carbon-derived product in Flv3 deletion mutant of Synechocystis (Thiel et al., 2019). Indeed, sucrose titer and productivity under constant low or mild illumination and high carbon (1% CO 2 ) conditions was higher in the ΔFlv3 mutant compared to the reference strain. However, when higher light intensities were applied to increase the photosynthetic electron flux, the advantage of ΔFlv3 disappeared. The carbon flux under high light shifted toward polyhydroxybutyrate (PHB) instead of sucrose biosynthesis, and the loss of Flv3 exacerbated that shift. In silico model of the metabolism suggests that producing PHB is energetically more favorable than sucrose, thus carbon reallocation likely occurred as a compensatory mechanism (Thiel et al., 2019). Moreover, the alteration of the ATP/NADPH ratio can strongly affect carbon flux engineering in Synechocystis (Yao et al., 2020).
In conclusion, redirecting photosynthetic electrons toward carbon-derived products can be realized only when metabolic and photosynthetic engineering are combined and coupled with modeling and high-throughput omics studies on molecular mechanisms. Therefore, channeling photosynthetic electrons is more efficient towards enzymes associated directly with photosynthetic light reactions rather than the central carbon metabolic pathways.

| INTRODUCING FDPS TO CROPS
FDPs were lost in angiosperms during the course of evolution, presumably due to more sophisticated and energetically less costly regulatory mechanisms of photosynthesis. Introducing FDPs to chloroplasts is a tempting possibility to equip angiosperms with an additional photoprotective mechanism for improved robustness and ideally, higher crop yields.