A comprehensive time‐course metabolite profiling of the model cyanobacterium Synechocystis sp. PCC 6803 under diurnal light:dark cycles

Abstract

Cyanobacteria are a model photoautotroph and a chassis for the sustainable production of fuels and chemicals. Knowledge of photoautotrophic metabolism in the natural environment of day/night cycles is lacking, yet has implications for improved yield from plants, algae and cyanobacteria. Here, a thorough approach to characterizing diverse metabolites-including carbohydrates, lipids, amino acids, pigments, cofactors, nucleic acids and polysaccharides-in the model cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) under sinusoidal diurnal light:dark cycles was developed and applied. A custom photobioreactor and multiplatform mass spectrometry workflow enabled metabolite profiling every 30-120 min across a 24-h diurnal sinusoidal LD ('sinLD') cycle peaking at 1600 lmol photons m À2 sec À1 . We report widespread oscillations across the sinLD cycle with 90%, 94% and 40% of the identified polar/semi-polar, non-polar and polymeric metabolites displaying statistically significant oscillations, respectively. Microbial growth displayed distinct lag, biomass accumulation and cell division phases of growth. During the lag phase, amino acids and nucleic acids accumulated to high levels per cell followed by decreased levels during the biomass accumulation phase, presumably due to protein and DNA synthesis. Insoluble carbohydrates displayed sharp oscillations per cell at the day-to-night transition. Potential bottlenecks in central carbon metabolism are highlighted. Together, this report provides a comprehensive view of photosynthetic metabolite behavior with high temporal resolution, offering insight into the impact of growth synchronization to light cycles via circadian rhythms. Incorporation into computational modeling and metabolic engineering efforts promises to improve industrially relevant strain design.

Dynamic Inventory of Intermediate Metabolites of Cyanobacteria in a Diurnal Cycle

“…Other intermediates of the CBB cycle showed dampened oscillations in Synechocystis sp. 6803 compared with the present study (Werner et al, 2019). Another recent study reports much smaller oscillations in the levels of the CBB cycle metabolites in Synechocystis sp.…”

“…Next, it was of interest to compare our results with a recent study on metabolome profiling of Synechocystis sp. PCC 6803, in the diurnal cycle (Werner et al, 2019). Importantly, data on the key CBB cycle intermediates such as SBP, S7P, RuBP, and 3PGA were absent in that study.…”

“…iScience Article cause of such negative growth rate at dusk and dawn, a previous study on cyanobacteria suggests that the nighttime decline in OD 720 may be due to loss of biomass rather than due to a decrease in the cell number (Werner et al, 2019). In view of the observed diurnal growth pattern, the metabolome was profiled at 6:00 (morning), 12:00 (midday), 18:00 (evening), and 24:00 (midnight), in the second diurnal cycle after culture inoculation ( Figure 2A).…”

“…PCC 6803 by creating time point models spanning the light-dark cycle (Sarkar et al, 2019). More recently, Werner et al have reported the metabolite levels of that strain in a sinusoidal light-dark (sine LD) cycle and show differential accumulation of metabolites in certain phases of the day (Werner et al, 2019). This study, however, did not include some key intermediates of the Calvin-Benson-Bassham (CBB) cycle, such as ribulose-1,5-bisphosphate (RuBP), 3-phosphoglyceric acid (3PGA), sedoheptulose-1,7-bisphosphate (SBP), and sedoheptulose-7-phosphate (S7P) that are involved in the reactions mediated by carbon flux-controlling enzymes Liang and Lindblad, 2016).…”

Improving heterologous protein expression in Synechocystis sp. PCC 6803 for alpha-bisabolene production

“…Photobioreactor experiments were completed using 3 ​L photobioreactors (Allen Scientific Glass, Boulder, CO, USA) containing 1.5 ​L BG-11 (with 1 ​mM phosphate) buffered with 10 ​mM TES-NaOH, pH 8.0. An air stream enriched to 5% CO 2 was supplied to the bottom of the reactor at a rate of 200 ​mL ​min −1 , following the reactor setup and operation described by Werner et al. (2019).…”