The application of hydrogenases for H2-driven biotransformations or H2 production in living microorganisms is a major challenge, but shows enormous potential for biotechnological applications towards a sustainable bioeconomy. Both structurally and mechanistically, hydrogenases are highly complex enzymes which, when heterologously synthesized in living cells, can represent a metabolic burden. This research project together with OFZ Leipzig (Prof. Dr. Bruno Bühler) focuses on O2-tolerant hydrogenases with high application potential for biocatalytic oxyfunctionalization and photosynthesis-driven H2 production. For this, we aim at their improvement at the molecular level and the elucidation of their interplay with cell physiology, for which quantitative physiology studies and metabolic flux analyses are planned. An existing Pseudomonas putida strain containing a P450 monooxygenase together with the O2-tolerant NAD+-reducing hydrogenase (SH) from Ralstonia eutropha H16 will serve as a starting point. In addition, SH is introduced into highly active styrene epoxidizing Escherichia coli and Pseudomonas strains for NADH regeneration. For NADPH-dependent Baeyer-Villiger oxidation based on highly active recombinant Pseudomonas and E. coli strains, NADP(H) acceptance by the SH is targeted by protein engineering. With the goal of H2 formation in vivo, SH variants with a preference for H+ reduction will also be generated. Suitable E. coli mutants and pseudomonads will be used as selection/screening tools and to characterize H2 formation capacity under microbial and anaerobic conditions. These hydrogenase variants will then be characterized with respect to the influence of H2 production as well as H2 oxidation on the metabolism of recombinant hydrogenase-containing bacteria. This research project will advance the use of H2 as a cheap and "green" reducing agent in biotechnological whole-cell processes and provide an important step towards the sustainable production of H2 as a biofuel.