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

电生物技术是介于电化学和生物技术之间的一种很有前途的技术，它利用二氧化碳和电力在生物电化学系统(BES)中利用微生物生物合成化学品和燃料。在这方面，一个有吸引力的方法是将二氧化碳转化为一氧化碳，并通过电化学形成合成气(氢气、一氧化碳、二氧化碳)，然后将后者生物转化为化学品或液体燃料。尽管近年来合成气的去偶联生物转化取得了令人印象深刻的进展，但该技术仍然存在一些固有的障碍，如气体进入培养介质的传质有限，电子或电子载体对微生物宿主的摄取低或转移效率低，以及底物的毒性和爆炸性等安全问题。此外，作为合成气生物转化最常用的微生物宿主，乙酸菌的产物谱有限，因为更复杂的分子的产生超出了代谢能力。在这个项目中，将在一种新的直接电微生物生产系统中探索假黄氢杆菌在好氧利用合成气方面的巨大潜力和工程假黄氢杆菌生产脂肪酸的能力，该系统可以原位和按需生产H2和O2(从水中)和CO(从CO2中)。为了系统和定量地了解电子转移及其对该羧基营养细菌氧化还原和能量代谢的影响，我们将应用代谢组学、通量分析和量化在优化的BES提供的给定气体组成下存在电子转移、能量和氧化还原代谢缺陷的野生型和工程突变体的动力学参数。BES将解决目前生物电化学系统和气体发酵的局限性，如上所述。所获得的知识将被用来建立假黄曲霉自养代谢的第一代谢和电子转移模型，特别是关于不同电子载体、能量和氧化还原平衡的吸收。此外，我们将改造假黄曲霉来生产代表ATP和NADPH密集型产品类别的脂肪酸，因此它的过量生产将挑战假黄曲霉的新陈代谢，特别是在自养条件下。将对开发的菌株进行定量分析，以评估和完善代谢和电子转移模型。该项目将为设计高效的电微生物生产菌株和开发改进的电发酵开辟新的可能性。

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.