The Construction And Optimization Of Enzyme-Assisted Microbial Electrosynthesis System

Excessive carbon dioxide emission would bring about climate change and environmental destruction.Microbial electrosynthes?MES?is a promising and sustainable technology to reduce CO₂ by feeding electricity to microorganisms to form intracellular reducing equivalents,thus synthesizing fuels and chemicals including hydrogen,alcohols and hydrocarbons.A number of acetogens could take up electrons directly from electrodes via direct contact-based extracellular electron transfer?EET?mechanims to form intracellular reducing equivalents,which reduced CO₂ to synthesize acetate.However,genome editing methods for these acetogens were yet established,which significantly hindered the spectrum of available products.To overcome this shortcoming,non-electroactive bacteria?e.g.,Ralstonia eutropha H16?were adopted to indirectly uptake electrons via electron shuttles?such as reduced redox dyes?.To avoid the impeding effect of reactive oxygen species?ROS?on cell growth,we firstly adopted a two-chamber bioelectrochemical reactor,in which R.eutropha in the cathode chamber was separated from the anode chamber by a proton exchange membrane.The ROS produced by the anode could not penetrate into the cathode.Secondly,We firstly constructed an enzyme-assisted MES by incorporating the formate dehydrogenase into NR-mediated MES system.On the one hand,the potential was applied on cathode only at-0.6 V,which much higher than that by James Liao et al.?-1.6 vs.Ag/AgCl?and tremendously saved energy.Both formate and NR as the mediators dramatically accelerated the elecron transport from the cathode electrode to R.eutropha.Thirdly,we effectively exploited the capacity of Calvin–Benson–Bassham cycle?CBB cycle?by heterologously expressing the key enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase from Synechococcus elongatus PCC7942.The rate of CO₂ fixation was promoted by engineering the metabolic pathways in R.eutropha.This study illuminated a novel strategy to efficiently convert CO₂ into PHB.One the one hand,we induced the elecron transport from the cathode electrode to microbes by incorporating the FDH into our MES system,which significantly boost the PHB production?378 mg/L?.On the other hand,we engineered the CBB cycle by introducing the key enzyme Rubisco from Synechococcus elongatus PCC7942 into R.eutropha,achieving a maximum PHB productivity of 472 mg/L.As a result,the PHB production was 1.9 times higher than the control?164 mg/L?.