Photosynthesis is nature’s primary CO₂ capture and utilization (CCU) process, in which solar energy is captured by light harvesting pigments and stored in high-energy metabolites such as ATP and NADPH, which in turn drive CO₂ fixation and energy storage in carbon-carbon bonds. Production and consumption of high-energy metabolites dictate productivity, thus understanding their management is central to understanding photosynthetic energy conversion process, as well as the rational engineering of microalgal chassis cells for the synthesis of fuels and chemicals.

Since photosynthesis occurs in natural environments with dynamic light, temperature, and nutrient conditions, synthesis and consumption of ATP and NADPH must respond dynamically to changes in the environment. This drives the evolution of an abundance of mechanisms that help balance energy production with demands. This balance can be achieved by modulation of electron transport—either through dissipation by non-photochemical quenching or re-routing of electron transport via cyclic electron transport and alternative electron transport pathways (Allahverdiyeva et al., 2015). Biosynthetic pathways can also act as sinks for excess ATP and NADPH, and can serve to optimize metabolism in response to environmental conditions (Cano et al., 2018).

Synthesis of carbon reserves, such as glycogen in cyanobacteria as well as starch and lipids in eukaryotic algae, provides a sink for energy during nutrient limitation and serves as a reserve for cellular maintenance at night or during acclimation to changing light and nutrient availability (Vitova et al., 2015; Luan et al., 2019; Ran et al., 2019). In the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis), fixed carbon is committed to glycogen synthesis by ADP-glucose pyrophosphorylase (AGPase) encoded by the glgC gene (Figure 1; Luan et al., 2019). Previous characterization of a ΔglgC mutant that can no longer synthesize glycogen showed that the glycogen synthesis/degradation cycle acts as an energy management mechanism (Cano et al., 2018). Loss of this mechanism leads to a higher energy charge (EC; [ATP]/([ATP + ADP])) in the light, and to the excretion of organic acids under high light, mixotrophic conditions, or nitrogen deprivation (Carrieri et al., 2012; Grundel et al., 2012; Cano et al., 2018). Additionally, blocking glycogen synthesis could provide a means for increasing carbon partitioning into target molecules like mannitol and ethanol (Jacobsen and Frigaard, 2014; Namakoshi et al., 2016). However, a drawback of blocking glycogen synthesis is the reduced fitness under saturating and diurnal light conditions even in the presence of alternative sinks (Suzuki et al., 2010; Grundel et al., 2012).

Sucrose synthesis has been studied regarding its role in protection against increases in salinity. Aquatic environments face dynamic changes in salinity with depth and at mixing zones that drives osmotic loss of water, disrupting ion homeostasis and loss of cellular turgor pressure (Hagemann, 2011). To combat dynamic changes in salinity, cyanobacteria have evolved a suite of mechanisms that can rapidly adjust ion homeostasis. This involves transcriptional reprogramming, effectively increasing active export of Na⁺ and Cl^–, and triggers the accumulation of osmo-protective organic compounds (Marin et al., 2004; Hagemann, 2011). In response to salt stress, Synechocystis accumulates primarily sucrose and glucosylglycerol (Kirsch et al., 2018, 2019). Salt shock is immediately followed by the rapid accumulation of sucrose within the first few hours, which is then followed by the slower accumulation of glucosylglycerol. After 24 h, sucrose content drops, and glucosylglycerol dominates as the primary osmolyte for long term accumulation in response to salt stress (Desplats et al., 2005). Sucrose accumulation is a product of salt ion activation of the sucrose phosphate synthetase (SPS; sll0045) and inactivation of a sucrose invertase (INV; sll0626) (Hagemann and Marin, 1999; Kirsch et al., 2018). Kirsch et al. (2018) demonstrated that the sucrose synthesis and degradation cycle is also active under non-stressed conditions, with Δinv mutants displaying 10 times the sucrose accumulation as compared to wild-type. To our knowledge, sucrose synthesis has not been studied as an energy management mechanism.

In this study, we show genetic and physiological evidence to support the hypothesis that cyanobacteria use the sucrose synthesis and degradation cycle in addition to the glycogen synthesis and degradation cycle to regulate ATP. In addition, we tested the hypothesis that a partial inhibition of glycogen synthesis would improve strain robustness while increasing cellular energy supply toward the synthesis of biomass. Disruption of such energy management mechanisms led to higher cellular energy levels and faster growth under a range of light conditions including simulated outdoor light conditions. These findings suggest a new approach to manipulate cellular energy metabolism toward higher photosynthetic productivity.

Understanding the regulation of high energy molecules such as ATP and NADPH is central in understanding biological processes such as photosynthesis, and in engineering microalgal chassis cells for carbon sequestration and utilization. This work expanded our prior findings on mechanisms for intracellular energy management in Synechocystis. Previous work demonstrated a high energy charge in a mutant that can no longer accumulate glycogen (ΔglgC). However, this strain had reduced fitness under saturating light and diurnal conditions (Grundel et al., 2012; Cano et al., 2018). Therefore, we hypothesized that a strain with low glycogen content may display high EC and improved photosynthetic rates without reducing fitness. Here we found that the complemented ΔglgC mutant strain [LGS (Carrieri et al., 2012)] displayed increased EC as well as growth rates.

Glycogen plays an essential role in both balancing energy homeostasis and initiation of photosynthetic metabolism. Shinde et al. (2020) demonstrated that glycogen degradation is required for initiating photosynthesis upon transition from dark to light conditions. Furthermore, they showed that absence of glycogen synthesis in the ΔglgC mutant or the oxidative pentose phosphate pathway in the Δgnd mutant led to reduced photosynthetic efficiency and slower PSI (P700⁺) re-reduction rates. This was used to support the hypothesis that glycogen degradation through the OPP pathway is essential for initiation of photosynthesis by generating the NADPH needed to activate key Calvin-Benson cycle enzymes (Shinde et al., 2020). We previously showed that the ΔglgC mutant under photoautotrophic conditions experiences metabolite overflow, with both increased EC and organic acid excretion to the extracellular medium (Carrieri et al., 2012, 2015; Cano et al., 2018). Taken together, these results indicate that glycogen synthesis and degradation facilitate metabolic homeostasis by either generating NADPH when reducing energy is low or can act as a sink for carbon and ATP when energy is in excess. Here we found that faster growth in our LGS was correlated with higher energy charge (Figures 3, 6) and a lower glycogen content (Figure 2F and Supplementary Figure 4). In contrast to moderate and diurnal light levels, our LGS displayed comparable growth rates and glycogen levels to wild-type under constant saturating light intensities (500 μmol photon m^–2 s^–1), reflecting light induction of the psbA2 promoter used for complementation. This suggests that even under non-stress conditions, cyanobacteria use a glycogen synthesis/degradation “futile cycle” to help maintain energy balance and that reducing glycogen storage under non-saturating light conditions can enhance biomass productivity.

Sucrose synthesis is essential for adaption to salt stress. Previous characterization of the Δsps mutant demonstrated its importance for growth under salt stress, with Δsps displaying lower growth in BG-11 supplemented with 684 mM NaCl. In the wild-type, despite a consistent growth reduction, 684 mM NaCl leads to a transient sucrose accumulation that peaks at 4 h and then drops off significantly after 12 h (Desplats et al., 2005). This decrease of sucrose levels reflects the activity of invertase, with the Synechocystis invertase mutant displaying no drop off in sucrose under salt stress and ∼9 times the sucrose concentration in standard BG-11 (Kirsch et al., 2018). Here we demonstrate that without inducing salt stress (growth in normal BG-11), the Δsps mutant displays increased EC and improved growth under continuous moderate light and simulated outdoor light conditions (diurnal sinusoidal fluctuating light with high maximum intensity) (Figures 3, 5, 6). The higher EC in this case reflects the savings of two ATP per sucrose synthesis cycle and indicates that the sucrose cycle plays a role in energy regulation under constant light conditions. The observations of higher EC and faster growth in the Δsps mutant indicate that faster growth is not just a consequence of carbon redirection, as there is minimal sucrose accumulation under these conditions.

Sucrose and glycogen synthesis reflect metabolically linked cycles and their reduction increases fitness under moderate light. Previous efforts to modify carbon partitioning in cyanobacteria have highlighted the interconnection of the sucrose and glycogen cycles. Sucrose and glycogen synthesis both involve glucose-6-phosphate generated from the Calvin Benson cycle or through regeneration of glucose through glycogen or sucrose catabolism. Sucrose and glycogen pools appear linked, though flux between them is dependent on environmental conditions. Under salt stress, knockdown of glycogen synthesis (glgC) limited sucrose production in Synechococcus PCC7942, indicating that under environmental stress glycogen acts as a carbon pool for sucrose synthesis (Qiao et al., 2018). This is supported by the observations that overexpression of both glgC and sps can significantly increase sucrose accumulation, and by similar observations on the dependence of sucrose synthesis on the glycogen pool in Synechococcus UTEX 2973 under salt stress (Qiao et al., 2018; Lin et al., 2020). In contrast, under non-salt stress conditions, deletion of glgC in Synechococcus PCC7942 was shown to increase sucrose accumulation rates by 10–15% (Ducat et al., 2012). This suggests that under normal conditions, sucrose, glycogen synthesis and anabolic metabolism all compete for glucose-6-phosphate. We found that either knocking out sucrose synthesis or reducing glycogen synthesis led to similar phenotypes. While both appears to be impacted in their acclimation state based on their chlorophyll content under low and moderate constant light, each mutant displayed increases in growth rates, especially under diurnal conditions (Figure 5). These increases were correlated with a high EC, which may also facilitate improved photosynthetic function observed at constant irradiances of 200 μmol photons m^–2 s^–1 (Figures 3, 4).

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

MiC and JY designed the study. MiC, TP, and MeC collected the data. MiC, WX, KC, and JY analyzed the data. MiC drafted the manuscript. All authors edited manuscript and agreed on the submitted manuscript.