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

利用氢化酶在活微生物细胞中进行H2驱动的生物转化和H2生产具有挑战性，但在生物技术应用中具有巨大的潜力，可持续发展生物经济。就结构和催化机制而言，氢化酶是高度复杂的酶，当在活微生物中异源合成时，预期其会造成代谢负担。该研究项目的重点是耐氧氢化酶，具有很高的应用潜力，用于生物催化氧化功能化和光合作用驱动的H2生产。为此，我们的目标是他们的基因工程，他们在全细胞生物催化剂的实施，并阐明他们与细胞生理学的相互作用。细胞对异源氢化酶活性的生理反应将通过定量生理学研究和代谢通量分析来表征。现有的恶臭假单胞菌菌株携带的NADH依赖性P450单加氧酶与O2耐受NAD+还原氢化酶将作为起点。此外，还发现了高活性的苯乙烯环氧化假单胞菌和E.大肠杆菌菌株将被工程化以共合成与辅因子再生催化剂相同的氢化酶。为了使NADPH依赖的Baeyer-Villiger氧化能够基于高活性的重组假单胞菌和E.在大肠杆菌菌株中，氢化酶变体将被工程化以接受NADP+而不是NAD+。为了在体内有效地产生H2，我们将开发具有H+还原偏好的氢化酶变体。为了选择、筛选和表征H2形成能力，合适的E.大肠杆菌突变株和假单胞菌菌株将在微厌氧条件下使用。这些氢酶变体将进一步表征关于H2生产以及H2氧化对合成它们的重组细菌的代谢的影响。拟议的项目预计将建立和促进利用H2作为生物技术相关的体内生物催化的还原剂，并代表朝向可持续生产H2作为生物燃料的重要一步。

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.