optiMEET – Optimisation of mediated extracellular electron transfer for Cupriavidus necator

铜绿假单胞菌是电微生物生产增值化合物的理想候选者，因为它结合了高效电微生物过程所需的所有优点：i)作为兼性化学自养细菌，它在包括CO2/H2在内的各种底物上快速生长，达到高细胞密度。二)其完整基因组序列和遗传工具的可获得性使基因工程变得容易。Iii)C.Necator既执行好氧呼吸又执行无氧呼吸。在目前的项目中，申请者完成了以下任务：·开发并验证了一种基于八个试管的微生物电化学调查筛选工具。·建立了四个平行控制的电生物反应器，以进行可扩展的电微生物过程。·基于介导性电子转移的阳极呼吸作用已经被建立，可能参与这种电子转移的蛋白质已经被确定。·纳氏弧菌已被确认为另一种能够利用人工氧化还原介体进行阳极呼吸的生物。·人工氧化还原介质在活细胞培养中的毒性程度已经确定。·研究了Cyma/NAPC类金属蛋白在细胞质膜上的同源和异源过量生产，以增强从苯二酚库到(人工)周质电子受体的电子转移。·SpyTag/SpyCatcher系统已被设计用于在体外和体内合成周质氧化还原酶的共价偶联。因此，该项目的主要目标已经实现，到目前为止共出版了四份经同行审查的出版物。另一份手稿正在审查中，另外两份正在准备中。在前期工作的基础上，在后续项目中，申请者计划完成以下任务：·对基于基因敲除和过表达的链球菌的介导性电子转移有扎实的了解。·计算流体力学辅助的电生物反应器的放大。·以模型为基础优化电极、人工氧化还原介体和活微生物之间的相互作用。·将周质中氧化还原蛋白的中介电子转移和人工偶联的知识转让给其他生物(S.onedensis和V.Natriegens)。该项目的主要目标是深入了解阳极呼吸作用和链球藻的电合成特性，将知识转移到更大的反应器中，并将人工(胞外)电子转移的一般原理应用于其他生物。

Cupriavidus necator represents an ideal candidate for electro-microbial production of value-added compounds, as it combines all the advantages necessary for efficient electro-microbial processes: i) As a facultative chemolithoautotrophic bacterium, it grows rapidly to high cell densities on a variety of substrates, including CO2/H2. ii) The availability of of its complete genome sequence as well as genetic tools allow for easy genetic engineering. iii) C. necator performs both aerobic and anaerobic respiration. In the current project the applicants completed the following tasks: • An eight cuvette-based screening tool for electrochemical investigations on microbes has been developed and validated. • Four parallel-controlled electrobioreactors have been established to perform scalable electromicrobial processes. • Anodic respiration, based on a mediated electron transfer, has been established for C. necator, and proteins likely involved in this electron transfer have been identified. • Vibrio natriegens has been identified as another organism capable of utilizing artificial redox mediators for anodic respiration. • The extent of toxicity of artificial redox mediators has been determined in living cell cultures. • The homologous and heterologous overproduction of CymA/NapC-like metalloproteins in the cytoplasmic membrane was studied to enhance electron transfer from the quinone pool to (artificial) periplasmic electron acceptors. • The SpyTag/SpyCatcher system has been engineered for systhetic covalent coupling of periplasmic redox enzymes both in vitro and in vivo. Thus, main objectives of the project have already been achieved, leading to four peer-reviewed publications so far. Another manuscript is under review and two more are in preparation. Based on preliminary work, in the follow-up project the applicants are planning the following tasks: • Generate a solid understanding of mediated electron transfer based on gene knockout and overexpression strains of C. necator. • Computational fluid dynamics-assisted scale-up of electro-bioreactors. • Model-based optimization of the interaction between electrodes, artificial redox mediators and living microorganisms. • Transfer of knowledge of mediated electron transfer and artificial coupling of redox proteins in the periplasm to other organisms (S. oneidensis and V. natriegens). The main objectives of the project "Optimization of mediated electron transfer for C. necator" are to gain a deep understanding of anode respiration, as well as electrosynthetic properties of C. necator, to transfer the knowledge to larger reactors and the application of general principles of artificial (extracellular) electron transfer to other organisms.