Discovery and characterisation of new enzymes for hydrogen production and green chemistry

使用氢气作为一种可持续的燃料和能源载体需要解决几个技术挑战，包括利用可再生资源实施清洁和高效的生产技术。在自然界中，氢气是由几种依赖氢酶的微生物产生的，氢酶是一大类复杂的金属酶，催化氢气代谢中的关键反应。在三类系统发育不同的氢酶中，[FeFe]-氢酶在生理性氢气生产中起着至关重要的作用。因此，它们因可能的生物技术应用而受到广泛关注。例如，[FeFe]-氢酶已经被设计并在合适的细胞底盘中过度表达，分别用于从废弃的生物质或水中暗驱动和光驱动地生产氢气。此外，提纯的[FeFe]-氢酶已被固定在半导体纳米粒子上，并被用于直接将太阳能储存到H2中。限制[FeFe]-氢酶广泛应用的主要因素之一是它们对大气氧的敏感性：O2是一种强大的抑制剂，抑制通常是不可逆的，但最近报道了一些O2稳定的[FeFe]-氢酶的例子。寻找性能更好的新型[FeFe]-氢酶(例如，更快的周转速度和/或更高的稳定性)将对在工业过程中开发它们至关重要。目前，由于这些酶中只有一小部分被实验地详细描述了这一事实，阻碍了实际开发，这限制了可用于任何给定应用的酶的“目录”。该博士项目的核心目标是：1)提供对[FeFe]-氢酶多样性的更全面的看法；2)显著扩展可用于制氢技术的可用酶的工具箱；3)探索[FeFe]-氢酶在绿色化学过程中的任何非常规反应性。在项目的初始阶段，学生将通过进行序列研究和建立结构模型，使用生物信息学工具在硅胶中识别新的[FeFe]-氢酶。这一阶段的目标是识别具有非常规特征的基因，这些基因可能会对酶的性能产生积极的影响，例如存在其他可能影响催化的酶亚基或特定残基。随后，在轮换项目中获得的专业知识的基础上，学生将转移到湿实验室并产生最佳靶标的选择(通过克隆选定的基因、重组过表达和纯化)。将使用已建立的方法在体内(通过天然/异源HydEFG成熟系统)或体外(通过预制的合成辅因子2FeMIM)整合催化簇。新酶的特征将是它们的比活性(氢气产生和氢氧化的反应速率)和它们的稳定性(特别是对温度、溶剂和氧气的稳定性)。还将与外部合作伙伴斯蒂芬·卡尔博士(哈威尔研究综合体)合作进行生物物理表征，包括光谱学、结晶学和X射线衍射。还将测试新酶(S)还原/氧化有用辅因子的能力，如NAD(P)+/NAD(P)H，或非天然底物，这些底物可以在绿色化学过程中突出这些酶的工业开发的新天地。蛋白质工程方法将被考虑来改善性能和/或将反应性重新定向到人工底物上。

Using hydrogen as a sustainable fuel and energy carrier requires tackling several technical challenges, including the implementation of clean and efficient production technologies from renewable resources. H2 is produced in nature by several microorganisms that rely on hydrogenases, a vast group of complex metallo-enzymes that catalyse the key reaction in H2 metabolism.Between the three phylogenetically distinct classes of hydrogenases, [FeFe]-hydrogenases are crucial in physiological H2 production. Consequently, they received extensive attention for possible biotechnological applications. For example, [FeFe]-hydrogenases have been engineered and overexpressed in suitable cellular chassis for both dark and light-driven H2 production from waste biomass or water, respectively. Moreover, purified [FeFe]-hydrogenases have been immobilised on semiconductor nanoparticles and utilised to direct storage of solar energy into H2.One of the main factors limiting a broader exploitation of [FeFe]-hydrogenases is their sensitivity to atmospheric oxygen: O2 is a powerful inhibitor and inhibition is usually irreversible, but some examples of O2-stable [FeFe]-hydrogenases have recently been reported.Identifying novel [FeFe]-hydrogenases with improved performances (for example, faster turnover rates and/or higher stability) will be crucial to exploit them in industrial processes. Currently, practical exploitation is hindered by the fact that only a small number of these enzymes have been experimentally characterised in detail, and this limits the "catalogue" of enzymes available for any given application.The core aims of this PhD project are: 1) to provide a more comprehensive view of the diversity of [FeFe]-hydrogenases; 2) to significantly expand the toolbox of available enzymes to be exploited for H2 production technologies; 3) to explore any unconventional reactivity of [FeFe]-hydrogenases to be exploited in green chemistry processes.In the initial phase of the project, the student will use bioinformatics tools to identify novel [FeFe]-hydrogenases in silico, by performing sequence studies and building structural models. This phase will aim at identifying genes with unconventional features that could positively influence the enzyme performances, such as the presence of additional enzyme subunits or specific residues that may allegedly influence catalysis.Subsequently, building on the expertise acquired during the rotation project, the student will move to the wet lab and produce a selection of the best targets (via cloning of the selected genes, recombinant overexpression and purification). Established methods to incorporate the catalytic cluster in vivo (via the native/heterologous HydEFG maturation system) or in vitro (via the pre-made synthetic cofactor 2FeMIM) will be used.The new enzymes will be characterised for their specific activity (reaction rate for H2 production and H2 oxidation) and for their stability (particularly towards temperature, solvents and O2). Biophysical characterisation will also be performed including spectroscopy, crystallography, and X-ray diffraction in collaboration with an external partner, Dr Stephen Carr (Research Complex at Harwell).The new enzyme(s) will also be tested for the ability to reduce/oxidise useful cofactors, such as NAD(P)+/NAD(P)H, or unnatural substrates that can highlight new horizons in the industrial exploitation of these enzymes within green chemistry processes. Protein engineering approaches will be considered to improve the performances and/or to redirect the reactivity towards artificial substrates.