optiMEET – Optimisation of mediated extracellular electron transfer for Cupriavidus necator

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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.