In photosynthetic organisms, such as cyanobacteria, ATP and NADPH are generated through the light reaction, and then are used for CO2 fixation in the dark reaction. As light intensity always fluctuates under natural conditions, balancing the cofactor regeneration and consumption is essential to maintain active CO2 fixation as well as for metabolic engineering of strains that produce biochemicals. In this study, a time-resolved metabolome analysis of Synechocystis sp. PCC 6803 (PCC6803) was conducted to investigate a metabolic adaptation at 0-15 min after a sudden shift from light to dark conditions. Rapid accumulation of sedoheptulose 7-phosphate, ribulose 5-phosphate, xylulose 5-phosphate, and 6-phosphogluconate suggested that the central metabolism of PCC6803 was regulated by inactivation of phosphoribulokinase and activation of glucose-6-phosphate dehydrogenase (G6PDH) probably via the redox regulation. The culture and metabolic profile of the Delta zwf strain lacking G6PDH showed that the role of G6PDH in regeneration of NADPH could be complemented by the activation of isocitrate dehydrogenase in the TCA cycle, indicating the importance of the rapid regulation of NADPH regeneration after the shift to dark conditions. The mechanism underlying metabolic regulation is also useful for metabolic engineering of PCC6803, as the Delta zwf strain produced higher amount of organic acids than wild type. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.