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

鉴于氢化酶驱动的 NAD(P)H 再生和 H2 生产，耐 O2 氢化酶的工程及其在重组细菌中的生理学意义

• 批准号: 405325648
• 负责人: Professor Dr. Bruno Bühler
• 金额: --
• 依托单位: Helmholtz-Zentrum für Umweltforschung (UFZ)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/405325648?language=en
• 关键词: Engineering; O2; tolerant; hydrogenases; their

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.

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生产是具有挑战性的，但在生物技术应用中具有巨大的潜力，可以实现可持续的生物经济。就结构和催化机理而言，氢化酶是一种高度复杂的酶，在生物体内异种合成时可能会造成代谢负担。本项目重点研究在生物催化氧化功能化和光合作用驱动制氢方面具有很高应用潜力的耐o2氢化酶。为此，我们的目标是它们的基因工程，它们在全细胞生物催化剂中的实现，以及它们与细胞生理学相互作用的阐明。细胞对外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nadh依赖的P450单加氧酶和耐受o2的NAD+还原氢化酶将作为起点。此外，高活性的苯乙烯环氧假单胞菌和大肠杆菌菌株将被设计成共同合成相同的氢化酶作为辅助因子再生催化剂。为了使nadph依赖的Baeyer-Villiger氧化基于高活性的重组假单胞菌和大肠杆菌菌株，将设计氢化酶变体以接受NADP+而不是NAD+。为了在体内高效生产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生成能力，将在微观和厌氧条件下使用合适的大肠杆菌突变体和假单胞菌菌株。这些氢化酶变体将进一步表征H2生成和H2氧化对合成它们的重组菌代谢的影响。该项目预计将建立和促进H2作为还原剂在生物技术相关的体内生物催化中的应用，并代表着H2作为生物燃料可持续生产的重要一步。