Cyanobacteria experience drastic changes in their carbon metabolism under daily light/dark cycles. During the day, the Calvin-Benson cycle fixes CO 2 and diverts excess carbon into glycogen storage. At night, glycogen is degraded to support cellular respiration. The dark/light transition represents a universal environmental stress for cyanobacteria and other photosynthetic lifeforms. Recent studies revealed the essential genetic background necessary for the fitness of cyanobacteria during diurnal growth. However, the metabolic processes underlying the dark/light transition are not well understood. In this study, we observed that glycogen metabolism supports photosynthesis in the cyanobacterium Synechococcus elongatus PCC 7942 when photosynthesis reactions start upon light exposure. Compared with the wild type, the glycogen mutant ΔglgC showed a reduced photosynthetic efficiency and a slower P700 1 rereduction rate when photosynthesis starts. Proteomic analyses indicated that glycogen is degraded through the oxidative pentose phosphate (OPP) pathway during the dark/light transition. We confirmed that the OPP pathway is essential for the initiation of photosynthesis and further showed that glycogen degradation through the OPP pathway contributes to the activation of key Calvin-Benson cycle enzymes by modulating NADPH levels. This strategy stimulates photosynthesis in cyanobacteria following dark respiration and stabilizes the Calvin-Benson cycle under fluctuating environmental conditions, thereby offering evolutionary advantages for photosynthetic organisms using the Calvin-Benson cycle for carbon fixation.

Terpenoids are a large group of secondary metabolites with broad industrial applications. Engineering cyanobacteria is an attractive route for the sustainable production of commodity terpenoids. Currently, a major obstacle lies in the low productivity attained in engineered cyanobacterial strains. Traditional metabolic engineering to improve pathway kinetics has led to limited success in enhancing terpenoid productivity. In this study, we reveal thermodynamics as the main determinant for high limonene productivity in cyanobacteria. Through overexpressing the primary sigma factor, a higher photosynthetic rate was achieved in an engineered strain of S ynechococcus elongatus PCC 7942. Computational modeling and wet lab analyses showed an increased flux toward both native carbon sink glycogen synthesis and the non-native limonene synthesis from photosynthate output. On the other hand, comparative proteomics showed decreased expression of terpene pathway enzymes, revealing their limited role in determining terpene flux. Lastly, growth optimization by enhancing photosynthesis has led to a limonene titer of 19 mg/L in 7 days with a maximum productivity of 4.3 mg/L/day. This study highlights the importance of enhancing photosynthesis and substrate input for the high productivity of secondary metabolic pathways, providing a new strategy for future terpenoid engineering in phototrophs.

1Cyanobacteria experience drastic changes in their carbon metabolism under daily light-2 dark cycles. In the light, the Calvin-Benson cycle fixes CO 2 and divert excess carbon into 3 glycogen storage. At night, glycogen is degraded to support cellular respiration. Dark-light 4 transition represents a universal environmental stress for cyanobacteria and other photosynthetic 5 lifeforms. Recent studies in the field revealed the essential genetic background necessary for the 6 fitness of cyanobacteria during diurnal growth. However, the metabolic engagement behind the 7 dark-light transition is not well understood. In this study, we discovered that glycogen 8 metabolism can jump-start photosynthesis in the cyanobacterium Synechococcus elongatus PCC 9 7942 when photosynthesis reactions start upon light. Compared to the wild type, the glycogen 10 mutant (∆glgC) showed much lower photosystem II efficiency and slower photosystem I-11 mediated cyclic electron flow rate when photosynthesis starts. Proteomics analyses indicated that 12 glycogen is degraded through the oxidative pentose phosphate pathway (OPPP) during dark-light 13 transition. We confirmed that the OPPP is essential for the initiation of photosynthesis, and 14 further showed that glycogen degradation through the OPPP is likely to contribute to the 15 activation of key Calvin-Benson cycle enzymes by modulating NADPH levels during the 16 transition period. This ingenious strategy helps jump-start photosynthesis in cyanobacteria 17 following dark respiration, and stabilize the Calvin-Benson cycle under fluctuating 18 environmental conditions. It has evolutionary advantages for the survival of photosynthetic 19 organisms using the Calvin-Benson cycle for carbon fixation. 20