| Electroactive bacteria(EAB) are a group of microorganisms that can transfer intracellular electrons,generated from metabolism,through the cell membrane to the extracellular terminal electron acceptors,thereby finishing the respiration process,named“extracellular respiration”.Owing to the unique Extracellular Electron Transfer(EET)mechanism,EAB is holding a great potential in energy generation,wastewater treatment,and resource recycling.However,the native low EET efficiency of EAB limits their full potential to be fulfilled.In this dissertation,we harness synthetic biology approaches to engineer and reprogram Shewanella oneidensis MR-1,a model strain,in multiple angles and at different levels to realize the EET system refactoring,intelligent regulation and construction of electroactive living functional materials.Specifically,the T7 RNAP/PT7 system was developed to modularly refactor the EET encoding gene network;the Quorum Sensing(QS)components were used to develop the Population-State Decision(PSD)system to intelligently reprogram the EET;and the electroactive host was harnessed as cellular chassis to construct the multicellular engineered living materials.The main contents and results are as follows:1.System refactoring of EET encoding gene network of S.oneidensis MR-1.In this part of work,the T7 RNAP/PT7 system was developed for refactoring single module of EET network,and subsequently the modular assembly strategy was adopted to systematically enhancing the EET flux.First,the T7 RNAP/PT7 system for pathway refactoring was constructed in S.oneidensis.The "Plug-and-Play Scaffold" of T7 RNAP/PT7 was harnessed to refactor the EET encoding gene network,which was divided into three modules,including electron generation module,electron transport module and electron mediator module.All three modules were well characterized and verified for their functionalities.Then,a T7 promoter library of various intensities was constructed through coupling random mutations with high-throughput screenings.T7 promoter variants were used to tune the three modules to different output levels,which were subsequently assembled according to the orthogonal design table.The results show that the optimal combination strain exhbits a 16.6-fold improvement of peak current output,a 6.6-fold enhancement of power density,and a2-fold increase of Roxarsone bioreduction rate,comparing to the control.This part of the research not only demonstrates the power of systematic refactoring of the EET-encoding gene network,and the great potential of the engineered strains in energy recovery and pollution removal,but also provides an important fundamental toolkit for the genetic programming of environmental functional microorganisms.2.Developing a population-state decision(PSD)system for intelligently reprogramming extracellular electron transfer in S.oneidensis.The gene components of the lux quorum sensing(QS)system,i.e.,luxl and luxR,are used to construct the decision-making unit;the corresponding enzymes and the EET pathways were regulated by the promoter PLux as the PSD implementation unit.The signal molecule(AHL)is used to indicate the population status of EAB and intelligently regulate the strength of EET pathway.The PSD system can intelligently shift the metabolic flux from initial bacterial growth to subsequent EET enhancement(i.e.,after reaching a certain population-state threshold),thereby realizing the dynamic improvement of electron output.Powered by the PSD system,the engineered strains achieved up to 4.8-fold EET enhancement and exhibited a substantially improved pollutant reduction ability,increasing the reduction efficiencies of methyl orange and hexavalent chromium by 18.8-and 5.5-fold,respectively.After comparing with the constitutive expression systems and other typical synthetic biology strategies in EET regulation,the PSD-based cell resource allocation strategy exhibits the optimal results.The establishment of the PSD system provides a tool for intelligently regulating the EET ability,and will promote the development of new generation intelligent bio-electronic devices and functional materials in various fields.3.Developing engineered living materials(ELM)with S.oneidensis MR-1 for uranium resource recovery.First,the original promoters of biofilm promoting factor(bpfA)and TI-type secretion system(aggABC)was replaced with the constitutive PcN and inducible promoter lacl-PLac to enhance its biofilm formation ability;the Mtr pathway was enhanced to promote its direct EET ability;then,using the surface display technology,the super uranium binding protein(SUP)or metallothionein(MT)was displayed on the surface of MR-1,resepectively,to improve the adsorption and chelation ability of uranium ions.Finally,the resultant engineered strain was hybrid with base materials(such as nylon membrane or carbon felt)to prepare the ELM through direct precipitation.The developed ELM exhibits a 10-fold increase in the uranium recovery rate.This part of research not only demonstrates the significance of using EAB to develop ELM for recycling of heavy metals and radionuclide waste,but also provides a basis for broadening the application of ELM in environmental field. |
