Systems metabolic engineering of Hydrogenophaga pseudoflava for aerobic biosynthesis of fatty acids using CO2 and electron carriers in a novel bioelectrochemical system

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Electrobiotechnology is a promising technology at the interface of electrochemistry and biotechnology to use CO2 and electricity for the microbial biosynthesis of chemicals and fuels in bioelectrochemical systems (BES). An appealing approach in this regard is the conversion of CO2 to CO and the formation of syngas (H2, CO, CO2) electrochemically; the latter is then biologically converted into chemicals or liquid fuels. Although the decoupled bioconversion of syngas has made impressive progresses recently, this technology still has several inherent obstacles such as limited mass transfer of gases into culture medium, low uptake or inefficient transfer of electrons or electron carriers to the microbial host, and safety issues regarding toxicity and explosiveness of the substrates. Furthermore, acetogens as the mostly used microbial hosts for syngas bioconversion have a limited spectrum of products since the production of more complex molecules is outside the metabolic capacity. In this project, the great potential of Hydrogenophaga pseudoflava in the aerobic utilization of syngas and the capacity of engineered H. pseudoflava for the production of fatty acids will be explored in a novel direct electromicrobial production system with in situ and on demand production of H2 and O2 (from water) and CO (from CO2). To develop a systemic and quantitative understanding of the electron transfer and its impact on redox and energy metabolism of this carboxydotrophic bacterium, we will apply metabolomics, flux analysis and quantify kinetic parameters of the wild type and engineered mutants defective in electron transfer, energy and redox metabolism under given gas compositions provided by an optimized BES. The BES will address current limitations of bio-electrochemical systems and gas fermentations as already mentioned above. The gained knowledge will be utilized to set up a first metabolic and electron transfer model of the autotrophic metabolism of H. pseudoflava, especially regarding uptake of the different electron carriers, energy and redox balances. Furthermore, we will engineer H. pseudoflava for the production of fatty acids which represent an ATP and NADPH intensive product class and therefore its overproduction will challenge the metabolism of H. pseudoflava especially under autotrophic conditions. A quantitative analysis of the developed strains will be used to evaluate and refine the metabolic and electron transfer model. This project will open up new possibilities to engineer efficient electromicrobial production strains and to develop improved electro-fermentation.