Engineering of O2-tolerant hydrogenases and their physiological implications in recombinant bacteria in view of hydrogenase-driven NAD(P)H regeneration and H2 production

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The utilization of hydrogenases for H2-driven biotransformations and H2 production in living microbial cells is challenging, but bears huge potential for biotechnological applications towards a sustainable bioeconomy. In terms of structure and catalytic mechanism, hydrogenases are highly complex enzymes, which are expected to pose a metabolic burden when synthesized heterologously in living microbes. This research project focusses on O2-tolerant hydrogenases with high application potential for biocatalytic oxyfunctionalizations and photosynthesis driven H2 production. For this purpose, we aim at their genetic engineering, their implementation in whole-cell biocatalysts, and the elucidation of their interplay with cell physiology. The physiological response of cells on heterologous hydrogenase activity will be characterized via quantitative physiology studies and metabolic flux analyses. An existing Pseudomonas putida strain harboring a NADH-dependent P450 monooxygenase together with an O2-tolerant NAD+-reducing hydrogenase will serve as starting point. Moreover, highly active, styrene epoxidizing Pseudomonas and E. coli strains will be engineered to co-synthesize the same hydrogenase as cofactor regeneration catalyst. To enable NADPH-dependent Baeyer-Villiger oxidation based on highly active, recombinant Pseudomonas and E. coli strains, hydrogenase variants will be engineerd to accept NADP+ instead of NAD+. For efficient H2 production in vivo, we will develop hydrogenase variants with a preference for H+ reduction. For selection, screening, and characterization of the H2 formation capacity, suitable E. coli mutants and Pseudomonas strains will be used under mirco- and anaerobic conditions. These hydrogenase variants will be further characterized regarding the influence of H2 production as well as H2 oxidation on the metabolism of recombinant bacteria synthesizing them.The proposed project is expected to establish and promote the utilization of H2 as reductant in biotechnologically relevant in vivo biocatalysis and represents an important step towards the sustainable production of H2 as a biofuel.