Quantitative proteomics analysis reveals the metabolic response of H2 photoproduction by Synechocystis sp. PCC 6803 under different anaerobic sulfur-deprived substrate stress conditions

Synechocystis sp. PCC 6803, a model cyanobacterium, produces H2 by photosystem II-independent pathway under sulfur-deprived anaerobic conditions by inactivating photosystems II. To study anaerobic H2 photoproduction, it was grown aerobically until late exponential phase, then switched to four anaerobic substrate-stress conditions of sulfur-deprived media (SDM) only (control) or supplemented with a carbon source in autotrophic CO2 or fermentation (either glucose or acetate) conditions. Changes in H2 photoproduction, cell growth (OD), pH, and chlorophyll content (Chl a) were monitored. Key metabolic changes and altered protein expressions were identified using an 8-plex iTRAQ-based proteomics method. Acetate as a carbon source significantly increased H2 yield to 605.8 μL mg Chl a−1, but stabilized pH, and inhibited cell growth. Control conditions yielded 279 μL mg Chl a−1, while CO2 and glucose conditions had different yields, 72.6 and 35.7 respectively. Under (glucose) conditions, cell growth increased, but pH dropped to 4.9, reducing H2 photoproduction. Proteomic analysis identified 1713 proteins at a false discovery rate (FDR) of ≤1 %. In acetate conditions, proteins related to glycolysis, Calvin cycle, photosystems, and pyruvate metabolism were down-regulated, suggesting inactivation of photosystems (including D1 protein (PsbA)). Notably, the absence of detectable D1 protein (a core component of PSII) in the differential expression analysis across all conditions suggests functional deactivation of PSII, leading to increased H2 production via PSII-independent pathway and relatively stabilized Chl a content after 24 h. Glucose conditions down-regulated glycolysis and TCA cycle but upregulated photosynthesis through PSI, PSII, and RuBisCo. Despite the upregulation of H2-inducing proteins (such as lexA, sll1626, sll0359, Ferredoxin-1, petF, and Flavodoxin) in CO2 and glucose conditions, H2 production was lower than acetate conditions. The study offers the first comprehensive view of the molecular mechanisms of H2 regulation, providing a list of potential gene targets for further engineering of H2 photoproduction by Synechocystis sp. PCC 6803.