Physiological basis of photosynthetic hydrogen production in the cyanobacterium Synechocystis

Photosynthetic hydrogen (photoH ₂ ) production by the cyanobacterium Synechocystis sp. PCC 6803 is an attractive means for storing solar energy. However, photoH ₂ yields remain limited by competing electron flux pathways. Recent in vitro characterization suggests that photoH ₂ production requires electrons from both carbohydrate oxidation and photosynthesis. Engineered fusions between photosystem I (PSI) and hydrogenase (PSI-H ₂ ase) aim to divert electrons toward H ₂ production and rely exclusively on photosynthesis. Thus, photoH ₂ production differs fundamentally between wildtype (WT) and PSI-H ₂ ase fusion mutants. Here, we show that photoH ₂ production in WT is enhanced by supplemented glucose, consistent with the recently reported confurcating nature of HoxEFUYH H ₂ ases. PhotoH ₂ production was further studied in the new psaE-hoxUYH mutant by simultaneously monitoring electron flux through PSI alongside with turnover rates of O ₂ , CO ₂ and H ₂ . PsaE-hoxUYH achieved the highest photoH ₂ yield and longest production period among the currently available PSI-H ₂ ase mutants in Synechocystis , prolonged by removing O ₂ . Upon illumination, psaE-hoxUYH exhibited high initial photoH ₂ production rates, which decreased in parallel with CO ₂ fixation and ceased immediately in the presence of O ₂ . In absence of O ₂ , photoH ₂ production still declined slowly. Therefore, in addition to CO ₂ fixation and O ₂ , other yet unknown factors might limit photoH ₂ production under these conditions. Moreover, we traced a previously observed high H ₂ production phase of unclear origin in psaD-hoxYH cultures to contaminating [FeFe]-H ₂ ases from Clostridium intestinale rather than genuine photoH ₂ production by the mutant. Together, these findings indicate a complex metabolic interplay tuning photoH ₂ production in Synechocystis WT and PSI-H ₂ ase fusion mutants.